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https://openalex.org/W2750847959	https://link.springer.com/content/pdf/10.1007%2Fs00280-017-3423-5.pdf	English	null	Superactive human leptin antagonist (SHLA), triple Lan1 and quadruple Lan2 leptin mutein as a promising treatment for human folliculoma	Cancer chemotherapy and pharmacology	2,017	cc-by	8,512	* E. L. Gregoraszczuk ewa.gregoraszczuk@uj.edu.pl 1 Department of Physiology and Toxicology of Reproduction, Institute of Zoology and Biomedical Research, Jagiellonian University, Gronostajowa 9, 30‑387 Kraków, Poland Cancer Chemother Pharmacol (2017) 80:815–827 DOI 10.1007/s00280-017-3423-5 Cancer Chemother Pharmacol (2017) 80:815–827 DOI 10.1007/s00280-017-3423-5 ORIGINAL ARTICLE ORIGINAL ARTICLE Superactive human leptin antagonist (SHLA), triple Lan1 and quadruple Lan2 leptin mutein as a promising treatment for human folliculoma E. Fiedor1 · E. L. Gregoraszczuk1 Received: 15 May 2017 / Accepted: 15 August 2017 / Published online: 31 August 2017 © The Author(s) 2017. This article is an open access publication two granulosa cancer cell lines: the juvenile form (COV434) and the adult form (KGN). Abstract two granulosa cancer cell lines: the juvenile form (COV434) and the adult form (KGN). Results ObR gene expression in cancer cell lines was 50% higher than in the non-cancer cells. Lan-1 and Lan-2 decreased ObR expression in COV434, while it had no effect in KGN cells. Higher ERβ expression in non-cancer and higher ERα expression in both cancer cell lines was noted. SHLA and Lan-1 changed the ratio towards greater expression of ERβ, characteristic of non-cancer granulosa cells. All ObR antagonists in HCrC1 and KGN but only Lan-2 in COV434 reversed leptin-stimulated proliferation. In both non-cancer and cancer granulosa cells, leptin acts as a cyclinD/cdk4, cyclin A/cdk2 and E2F inhibitor. Conclusion These results indicate that SHLA and Lan2 are promising leptin receptor inhibitors that can eliminate the negative effects of leptin. These compounds should be considered in further ex vivo studies on the cancer microenvironment. Purpose There are no data showing a direct correlation between obesity and increased blood leptin levels with folliculoma. Moreover, folliculoma is not the best studied among other ovarian cancer types. We investigated whether oestradiol can modulate ObR expression in some oestro- gen-responsive tissues and that leptin exerts its activity not only via the leptin receptor but also through cross talk with other signalling systems. We hypothesise that blocking ObR expression could be a novel treatment for gonadal ovarian cancer. Results ObR gene expression in cancer cell lines was 50% higher than in the non-cancer cells. Lan-1 and Lan-2 decreased ObR expression in COV434, while it had no effect in KGN cells. Higher ERβ expression in non-cancer and higher ERα expression in both cancer cell lines was noted. SHLA and Lan-1 changed the ratio towards greater expression of ERβ, characteristic of non-cancer granulosa cells. All ObR antagonists in HCrC1 and KGN but only Lan-2 in COV434 reversed leptin-stimulated proliferation. In both non-cancer and cancer granulosa cells, leptin acts as a cyclinD/cdk4, cyclin A/cdk2 and E2F inhibitor. Methods We evaluated the effect of SHLA, Lan1 and Lan2 blockers on cell proliferation (BrdU incorporation assay), ObR and ERα/β gene expression (qPCR), oestra- diol secretion (ELISA) and cell cycle protein expression (Western blot) in the non-cancerous cell line HGrC1 and y y Conclusion These results indicate that SHLA and Lan2 are promising leptin receptor inhibitors that can eliminate the negative effects of leptin. These compounds should be considered in further ex vivo studies on the cancer microenvironment. Abstract :(0123456789) 1 3 :(0123456789) 1 3 :(01234 1 3 23456789) 3 816 Cancer Chemother Pharmacol (2017) 80:815–827 Graphical abstract Graphical abstract Graphical abstract Keywords Folliculoma cells · Leptin receptor antagonists · ObR expression · ERα/ERβ expression · Cell cycle protein expression reported to be higher in overweight and obese women than in women with normal weight. In obese individuals, leptin levels can reach 40 ng/mL, which is up to ten times higher than in normal weight people [3, 4]. Cancer risk is higher among overweight and obese people, with an increased risk of 16 and 30%, respectively [5]. Moreover, leptin and its receptors are over-expressed in different human cancers [1]. Leptin has been proposed as one of the six markers of ovar- ian cancer [6]. Uddin et al. revealed a significant associa- tion between ObR overexpression and poor survival rates in 59.2% of epithelial ovarian cancer cases [7]. Introduction Leptin is a small (16 kDa) protein produced and secreted by adipose tissue, which is involved in appetite regulation, bone formation and reproductive function. Recent studies have demonstrated that this hormone stimulates growth, migra- tion, invasion and angiogenesis in tumour cell models, sug- gesting that leptin is capable of promoting an aggressive can- cer phenotype [1]. Epidemiological studies have indicated a positive correlation between obesity and an increased risk of several types of cancer [2]. Serum leptin levels have been Granulosa cell tumours constitute the second largest group of ovarian tumours (approximately, 25–30%), with tumours derived from epithelial cells accounting for approxi- mately 70% [8]. Granulosa cell tumours can be divided into two histopathological forms: a mature form, diagnosed most 1 3 817 Cancer Chemother Pharmacol (2017) 80:815–827 frequently in peri-menopausal women (95%), and a juvenile form, diagnosed in young women and girls who have aged prematurely (5% of cases) [9]. Despite the fact that granu- losa cell tumours can be successfully treated by surgery, relapses are often observed and further adjuvant treatment is still not possible. frequently in peri-menopausal women (95%), and a juvenile form, diagnosed in young women and girls who have aged prematurely (5% of cases) [9]. Despite the fact that granu- losa cell tumours can be successfully treated by surgery, relapses are often observed and further adjuvant treatment is still not possible. F41A mutant), Lan2 (L39A/D40A/F41A/I42A mutant) and SHLA (D23L/L39A/D40A/F41A mutant) on leptin and oestradiol receptor gene and protein expression, cell pro- liferation including cell cycle protein expression, caspase-3 activity and oestradiol secretion in two granulosa tumour cell lines. The two cell lines were COV434, representing the juvenile form of granulosa tumour, and the steroidogenic human ovarian granulosa-like tumour (KGN), representing the adult type of this cancer (corresponding to peri- to post- menopausal age). The human immortalised non-luteinised granulosa cell line HGrC1 was used as the control. To our knowledge, there are no data showing a direct correlation between obesity and folliculomas. Moreover, fol- liculoma is not well studied among the ovarian cancer types, although both the short (ObRa) and long (ObRb) forms of the leptin receptor are present in human granulosa cells [10, 11]. Löffler et al. [10] showed that, in polycystic ovaries, leptin-positive cells were noted both in the hypertrophied theca layer and in the luteinised granulosa layer. Reagents Dulbecco’s modified Eagle’s medium/Nutrient Mixture F-12 (DMEM/F-12) was obtained from Gibco by Thermo Fisher Scientific (Waltham, MA, USA). DMEM, foetal bovine serum (FBS, heat inactivated), penicillin and streptomycin were obtained from Sigma Chemical Co. (St. Louis, MO, USA). All applied media were oestradiol free. Leptin was obtained from Sigma Chemical Co. (St. Louis, MO, USA). Leptin receptor antagonists (SHLA, Lan1 and Lan2) were obtained from Protein Laboratories Rehovot (PLR) Ltd. (Rehovot, Israel). All antagonists have the same specificity. They interact with the cytokine homology domain 2 in the leptin receptor and do not interact with IGD (immunoglob- ulin-like domain) of the receptor, as the 39–40, 39–41 or 39–42 Ala mutation abolished this interaction [20]. Introduction Taking into consideration that leptin at a supraphysiological concentra- tion, as noted in obese women, has stimulatory effects on testosterone secretion characteristic of polycystic ovarian syndrome correlated with obesity [12], we hypothesised that, as in the case of epithelial ovarian cancer, ObR overexpres- sion in granulosa cell tumours could be correlated with the incidence of granulosa cell cancer, and leptin receptor block- ers might be used as an adjuvant therapy. Based on the fact that leptin exerts its activity not only through the leptin receptor (ObR), but also through cross talk with other signalling systems implicated in tumour genesis [18, 19], in this study we focused our attention on the relationship between the leptin/ObR axis and oestrogen receptors (ERα/β). Taking into consideration that oestradiol can modulate ObR expression in some oestrogen-responsive tissues, we hypothesised that blocking ObR expression could be a novel treatment for granulosa ovarian cancer. Currently, several groups of scientists are working on the synthesis of molecules that block ObR. A number of leptin receptor antagonists have been synthesised for thera- peutic use, with several completing pre-clinical testing [13], indicating their possible use in anticancer therapy. In previous studies using different epithelial cancer cell lines, we showed that SHLA and quadruple leptin mutein, Lan2 (L39A/D40A/F41A/I42A), had no effect on non-cancerous HOSEpiC cell proliferation [14]. However, both antagonists reversed the stimulatory effect of leptin on metastatic carci- noma CaOV-3 cell proliferation to control levels and even below control levels in chemoresistant OVCAR-3 cells. Lep- tin receptor antagonists have been investigated in breast and prostate cancers, which mainly are hormone dependent. It has been shown that Aca1, Allo-aca and D-ser can inhibit leptin-stimulated proliferation in MCF-7 breast cancer cells [15]. Another leptin receptor antagonist, LDFI (Leu-Asp- Phe-Ile), is also able to inhibit the proliferation of MCF-7 cells in vitro and in vivo [16]. The antagonist Lan1 is able to inhibit the phosphorylation of leptin-signalling proteins Jak2, ERK1/2 and Akt, in PC3 and DU145 prostate cancer cell lines [17]. In a previously published study using epithe- lial ovarian cancer cell lines, we investigated the effect of SHLA and Lan2 on the JAK/Stat3, MAPK/ERK and PI3K/ Akt pathways and showed an inhibitory effect of SHLA on all tested signalling proteins in OVCAR-3 cells and of Lan2 on Stat3 and ERK1/2 proteins in CaOV-3 cells [14]. These data point to a similar signalling pathway in the antagonistic effects of leptin receptor blockers.f Western blot analysis Cells were plated into 24-well plates at a density of 2.5 × 104 (HGrC1 cells), 3 × 104 (COV434 cells) and 6 × 104 (KGN cells) and allowed to attach overnight. The following day, the media were changed and cells were treated with 40 μg/ mL leptin alone or in combination with 1000 μg/mL SHLA, Lan1 or Lan2. To examine cell cycle protein expression, cells were incubated for 48 h. After incubation, cells were washed with ice-cold PBS and lysed with Laemmli lysis buffer (Sigma Chemical Co., St. Louis, MO, USA). The lysed cells were then scraped, transferred to microtubes and stored at −70 °C until analysis. Cells were plated into 24-well plates at a density of 2.5 × 104 (HGrC1 cells), 3 × 104 (COV434 cells) and 6 × 104 (KGN cells) and allowed to attach overnight. The following day, the media were changed and cells were treated with 40 μg/ mL leptin alone or in combination with 1000 μg/mL SHLA, Lan1 or Lan2. To examine cell cycle protein expression, cells were incubated for 48 h. After incubation, cells were washed with ice-cold PBS and lysed with Laemmli lysis buffer (Sigma Chemical Co., St. Louis, MO, USA). The lysed cells were then scraped, transferred to microtubes and stored at −70 °C until analysis. KGN cells were obtained from Masatoshi Nomura and Hajime Nawata, Kyushu University, Japan. With luteinisa- tion capability, they constitute a useful model for under- standing the regulation of steroidogenesis, cell growth and apoptosis in human granulosa cells [23]. HGrC1 and COV434 cells were routinely cultured in DMEM + 2 mM glutamine + 10% FBS. KGN cells were routinely cultured in DMEM/F-12 + 10% FBS. Cells were grown in 75 cm2 tissue culture dishes (Nunc, Denmark) in a 37 °C incubator with a humidified mixture of 5% CO2:95% air. Prior to analysis, samples were sonicated and centrifuged at 15,000×g for 15 min at 4 °C. The quantity of protein was determined using the Bradford method and the clear supernatant was used for electrophoresis. Equal amounts of protein (100 µg) from each treatment group were separated by SDS-PAGE and transferred to PVDF membranes using a Bio-Rad Mini-Protean 3 apparatus (Bio-Rad Laborato- ries Inc., Hercules, CA, USA). The blots were blocked for 1 h in 5% BSA with 0.1% Tween-20 in 0.02 M TBS buffer. Western blot analysis Blots were incubated overnight with primary antibodies spe- cific to ObR (ab5593, Abcam, Cambridge, Great Britain) at a dilution of 1:2000, cyclin D1 (#2978, Cell Signaling Technology Inc., Beverly, MA, USA), cdk4 (#12790), cdk2 (#2546), cyclin A2 (#4656) at a 1:1000 dilution and E2F-1 (sc-251 Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), E2F-2 (sc-633), ERα (sc-8002) and ERβ (sc-6822) at a dilution of 1:200. After incubation with the primary antibody, the membranes were washed three times with 0.1% Tween-20 in 0.02 M TBS buffer and incubated for 1 h with an appropriate horseradish peroxidase-conjugated secondary antibody (#7074 or #7076, Cell Signaling Technology Inc., Beverly, MA, USA; dilution 1:2000). air. Cell culture HGrC1 (human non-luteinised granulosa cell line) cells were a gift from Dr Ikara Iwase (Nagoya University, Japan) and cultured according to the protocol described by Baya- sula et al. [21]. HGrC1 may possess the characteristics of granulosa cells in early stage follicles. A human immor- talised non-luteinised granulosa cell line (HGrC1) origi- nally derived from mural granulosa cells expresses the FSH receptor and is responsive to the transforming growth factor (TGF)-β superfamily and FSH, retaining its original f In the present study, we evaluated the effect of leptin and three of its receptor antagonists: Lan1 (L39A/D40A/ 1 3 818 Cancer Chemother Pharmacol (2017) 80:815–827 at 60 °C). The relative expression of genes was normalised against the endogenous reference gene GAPDH (Human GAPD Endogenous Control, number 4333764F) (ΔCq) and converted to relative expression using the 2−ΔΔCq method. The results are expressed as relative values (RQ). granulosa cell character and function. HGrC1 might also be capable of growth transition from a gonadotrophin-inde- pendent status to gonadotrophin-dependent one, but they are not capable of undergoing luteinisation. COV434 cells were obtained from the Sigma Chemical Co. (St. Louis, MO, USA). The biological characteristics of this cell line include production of 17β-oestradiol in response to FSH, absence of the LH receptor, no luteinisa- tion capability and the presence of specific molecular mark- ers of apoptosis enabling the induction of follicular atresia [22]. qPCR analysis qPCR analysis Basal ObR gene expression and expression of the ObR gene under the influence of leptin and leptin antagonists was determined by qPCR. Cells were seeded into 96-well culture plates at a density of 5 × 103 cells/well (HGrC1), 8 × 103 cells/well (COV434) and 1.5 × 104 cells/well (KGN) taking into consideration the size of the cells and the popula- tion doubling time. The next day, the medium was changed and cells were treated with leptin at a dose of 40 ng/mL and SHLA, Lan1 and Lan2 at a dose of 1000 ng/mL with leptin at a dose of 40 ng/mL for 24 h. Doses of leptin were chosen based on literature data [3, 4]. Total RNA isolation and cDNA synthesis was performed using the TaqMan Gene Expression Cell-to-CT Kit (Applied Biosystems, Carlsbad, CA, USA) in accordance with the manufacturer’s proto- col. Amplifications were performed using the StepOnePlus system (Applied Biosystems, Carlsbad, CA, USA) and the TaqMan Leptin Receptor primer (Cat. No. Hs00174497_ m1), oestrogen receptor α primer (Hs00174860) and oestro- gen receptor β primer (Hs01100353) in combination with the TaqMan Gene Expression Master Mix (Applied Biosystems, Carlsbad, CA, USA), in accordance with the manufacturer’s instructions. β-Actin was used as an internal loading control; mem- branes were washed for 30 min in stripping buffer (0.25 M glycine, 1% SDS, pH 2) and reprobed by overnight incu- bation with primary antibodies specific to β-actin (A5316, Sigma Chemical Co., St. Louis, MO, USA; dilution 1:2000) and for 1 h with a horseradish peroxidase-conjugated sec- ondary antibody (P0447 DAKO, Glostrup, Denmark; dilu- tion 1:5000). A PCR was performed using a final volume of 20 μL, including 100 ng/reaction cDNA. PCR conditions were as follows: pre-incubation (2 min at 50 °C and 10 min at 95 °C), amplification for 40 cycles (15 s at 95 °C and 1 min Immunopositive bands were visualised using Western Blotting Luminol Reagent (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and ChemiDoc™ XRS+ System (Bio-Rad Laboratories Inc., Hercules, CA, USA). Relative 3 819 Cancer Chemother Pharmacol (2017) 80:815–827 cells/well. Leptin receptor antagonists were added at concen- trations of 10, 100 or 1000 ng/mL, with leptin at a concentra- tion of 40 ng/mL. Cells were cultured for 48 h with repeated exposure. The culture medium was changed daily and fresh compounds were added. Androstenedione (10−5 M) was used as a substrate for oestradiol production. The action of SHLA, Lan1 and Lan2 on leptin‑stimulated leptin receptor (ObR) gene and protein in different cell lines HGrC1 cells were seeded in 96-well culture plates at a density of 5 × 103 cells/well, COV434 cells at a density of 8 × 103 cells/well and KGN cells at a density of 1.2 × 104 cells per well. Leptin receptor antagonists were added at concentrations of 10, 100 or 1000 ng/mL, with leptin at a concentration of 40 ng/mL. Cells were cultured for 48 h with repeated exposure. The culture medium was changed daily and fresh compounds were added. After exposure, the medium was removed and the plates were stored at −70 °C. Cells were lysed in buffer containing 50 mM HEPES, 100 mM NaCl, 0.1% CHAPS, 1 mM EDTA, 10% glycerol and 10 mM DTT. The assay was carried out by adding 20 M of Ac DEVD-AMC, a substrate for the fluorometric determination of caspase-3 activity. Reaction mixtures were incubated at 37 °C. After 3 h, fluorescence was measured at 360 nm excitation and 460 nm emission using a micro- ELISA plate reader (Bio-Tek Instruments). The basal leptin receptor gene expression varied between different cell lines. Assuming the gene expression in HGrC1 cell to be 1, ObR gene expression in COV434 and KGN cells was 50% higher (Fig. 1a), while there were no differences in ObR protein expression (Fig. 1b). In non-cancerous HGrC1 cells, leptin (40 ng/mL) decreased ObR gene expression, had no effect on ObRb, but increased ObRa protein expression. In both cancer cell lines, COV434 and KGN, leptin slightly increased gene expression but had no effect on both ObRb and ObRa protein expression (Fig. 1c, d). In HGrC1 cells, all three antagonists increased ObR gene expression (Fig. 2a), reversing the inhibitory effect of leptin on ObR gene expression (Fig. 1c), but had no effect on pro- tein expression (Fig. 2b). None of the antagonists affected ObR gene expression in COV434 and KGN cells (Fig. 2c, e). The inhibitory effects of Lan1 and Lan2 on both forms of ObR protein expression were observed in COV434 cells (Fig. 2d). In KGN cells, none of the ObR antagonists inves- tigated had an effect on ObR protein expression (Fig. 2f). Cell proliferation BrdU assay DNA synthesis in proliferating cells was determined by measuring bromodeoxyuridine (BrdU) incorporation with the commercial Cell Proliferation ELISA System (Roche Molecular Biochemicals, Mannheim, Germany). The cells were seeded in 96-well culture plates at a density of 5 × 103 cells/well (HGrC1), 8 × 103 cells/well (COV434) and 1.2 × 104 cells/well (KGN). Leptin was added at a con- centration of 40 ng/mL. Leptin receptor antagonists were added at concentrations of 10, 100 or 1000 ng/mL, with leptin at a concentration of 40 ng/mL. Cells were cultured for 48 h with repeated exposure; the culture medium was changed daily and fresh compounds added. After 48 h, the medium was removed and cells were incubated for 3 h with a BrdU labelling solution, containing 10 µM BrdU. The assay was performed according to the manufacturer’s instruc- tions. Absorbance values were measured at 450 nm using an ELISA reader (ELx808 BIO-TEK Instruments, Vinooski, VT, USA). The culture medium alone was used as a control for non-specific binding. Statistical analysis Data were expressed as mean ± SEM from the four inde- pendent experiments performed in triplicate. Statistical anal- yses were performed using GraphPad Prism 5. Data were analysed using a one-way analysis of variance (ANOVA) followed by Tukey’s honestly significant difference (HSD) multiple range test. A value of P < 0.05 was considered to be statistically significant. Caspase‑3 activity assay The action of SHLA, Lan1 and Lan2 on leptin‑stimulated leptin receptor (ObR) gene and protein in different cell lines qPCR analysis After 48 h, the media were collected and stored at −20 °C until analysis. Oestradiol concentrations in the medium were measured by enzyme immunoassay (EIA) using a commercially available ELISA kit (DRG Diagnostic, Germany). All samples were run in duplicate in the same assay. The analytical sensitivity was 10.6 pg/mL. The intra-assay variation was 8.7–9.23%, and the inter-assay variation was 6.87–14.91%. The range of the oestradiol (E2) assay was 10.6–2000 pg/ml. levels of protein expression were determined using ImageJ software (US National Institutes of Health, Bethesda, MD, USA). Individual protein levels were normalised to β-actin controls and the ratio of protein to β-actin was normalised to 1 in the untreated control group. on leptin‑stimulated expression of oestrogen receptor (ER) gene and protein in different cell lines The basal expression of the ERβ form in HGrC1 cells was twofold greater than the ERα form at both the gene and pro- tein levels (Fig. 3a, b). In contrast, in the cancer cell lines, both gene and protein ERβ expressions were higher than those of the ERα form (Fig. 3a, b). Oestradiol secretion performed by the ELISA method HGrC1 cells were seeded in 96-well culture plates at a density of 5 × 103 cells/well, COV434 cells at a density of 8 × 103 cells/well and KGN cells at a density of 1.2 × 104 1 3 Cancer Chemother Pharmacol (2017) 80:815–827 820 The action of SHLA, Lan1 and Lan2 on leptin‑stimulated expression of oestrogen receptor (ER) gene and protein in different cell lines The basal expression of the ERβ form in HGrC1 cells was ERβ gene expression. Lan1 increased ERβ gene expression and decreased ERβ protein expression, while Lan2 had the opposite effect (Fig. 4c, d). In KGN cells, all three blockers failed to affect ERα gene expression (Fig. 4e) but reduced ERα protein level (Fig 4f) No effect on ERβ gene or protein Fig. 1 Basal expression of leptin receptor (ObR) a gene and b pro- tein, and leptin action on ObR c gene and d protein expression in dif‑ ferent cell lines. Basal mRNA was evaluated by qPCR after 24 h of cell culture and by Western blot after 48 h. All the results were nor- malised to HGrC1 (ObR expression) with a value equal to 1. Values are mean ± SEM. Statistically significant differences between groups are indicated by (p < 0.05) and (p < 0.01). Densitometry results were normalised to β-actin loading controls to obtain a band ratio. All values marked with (p < 0.01) are significantly different from the control Fig. 1 Basal expression of leptin receptor (ObR) a gene and b pro- tein, and leptin action on ObR c gene and d protein expression in dif‑ ferent cell lines. Basal mRNA was evaluated by qPCR after 24 h of cell culture and by Western blot after 48 h. All the results were nor- malised to HGrC1 (ObR expression) with a value equal to 1. Values are mean ± SEM. Statistically significant differences between groups are indicated by (p < 0.05) and (p < 0.01). Densitometry results were normalised to β-actin loading controls to obtain a band ratio. All values marked with (p < 0.01) are significantly different from the control The action of SHLA, Lan1 and Lan2 on leptin‑stimulated expression of oestrogen receptor (ER) gene and protein in different cell lines ERβ gene expression. Lan1 increased ERβ gene expression and decreased ERβ protein expression, while Lan2 had the opposite effect (Fig. 4c, d). In KGN cells, all three blockers failed to affect ERα gene expression (Fig. 4e) but reduced ERα protein level (Fig. 4f). No effect on ERβ gene or protein expression was observed in KGN cells (Fig. 4e, f). Effect of leptin receptor antagonists on proliferation, caspase‑3 activity and oestradiol secretion Leptin (40 ng/mL) decreased both ERα and ERβ gene expression in HGrC1 cells, but had no effect on protein lev- els. In cancer cells, the expression of ER gene (Fig. 3c) and protein (Fig. 3d) in COV434 and KGN cells was unchanged by the addition of leptin. In HGrC1 cells, leptin increased cell proliferation (80%) but had no effect on caspase-3 activity and slightly increased estradiol secretion (30%). All three leptin receptor blockers decreased BrdU incorporation (Fig. 5a), except that Lan2 at the highest concentration (1000 ng/mL; Fig. 5d) had no effect on caspase-3 activity. Lan1 (1000 ng/mL) and Lan2 (10 and 100 ng/mL) reduced oestradiol secretion (Fig. 5g). In HGrC1 cells, leptin increased cell proliferation (80%) but had no effect on caspase-3 activity and slightly increased estradiol secretion (30%). All three leptin receptor blockers decreased BrdU incorporation (Fig. 5a), except that Lan2 at the highest concentration (1000 ng/mL; Fig. 5d) had no effect on caspase-3 activity. Lan1 (1000 ng/mL) and Lan2 (10 and 100 ng/mL) reduced oestradiol secretion (Fig. 5g). In COV434 cells, SHLA and Lan2 at dose 1000 ng/mL decreased BrdU incorporation, while Lan1 had no effect (Fig. 5b). None of the blockers investigated had an effect on caspase-3 activity (Fig. 5e) or oestradiol secretion (Fig. 5h). In HGrC1 cells, all three leptin receptor blockers increased ERα gene expression, but had no effect on ERβ gene expression (Fig. 4a). None of the antagonists affected ERα protein expression, although an inhibitory effect of Lan1 and Lan2 on ERβ protein expression was observed (Fig. 4b). In COV434 cells, SHLA decreased ERα gene expression and ERβ protein expression, but had no effect on In COV434 cells, SHLA and Lan2 at dose 1000 ng/mL decreased BrdU incorporation, while Lan1 had no effect (Fig. 5b). None of the blockers investigated had an effect on caspase-3 activity (Fig. 5e) or oestradiol secretion (Fig. 5h). 3 Cancer Chemother Pharmacol (2017) 80:815–827 821 Fig. 2 Effect of leptin receptor antagonists (SHLA, Lan1 and Lan2) on leptin receptor (ObR) gene (a, c, e) and protein (b, d, f) expres- sion. All values marked with (p < 0.05) are significantly different from control values. Values are mean ± SEM. Densitometry results were normalised to β-actin loading controls from control values. Values are mean ± SEM. Densitometry results were normalised to β-actin loading controls Fig. Effect of leptin receptor antagonists on proliferation, caspase‑3 activity and oestradiol secretion 2 Effect of leptin receptor antagonists (SHLA, Lan1 and Lan2) on leptin receptor (ObR) gene (a, c, e) and protein (b, d, f) expres- sion. All values marked with (p < 0.05) are significantly different Fig. 3 Expression of the oestrogen receptor (ER) α and β forms at a the gene and b protein levels in different cell lines and the action of leptin on ER c gene and d protein expression. Each point represents the mean ± SEM from three independent experiments. All values marked with (p < 0.05) and (p < 0.01) are significantly different between ERα and ERβ (a, b) or from control values (c, d). All values marked with (p < 0.05) and ** (p < 0.01) are significantly different from control values. Densitometry results were normalised to β-actin loading controls between ERα and ERβ (a, b) or from control values (c, d). All values marked with (p < 0.05) and * (p < 0.01) are significantly different from control values. Densitometry results were normalised to β-actin loading controls Fig. 3 Expression of the oestrogen receptor (ER) α and β forms at a the gene and b protein levels in different cell lines and the action of leptin on ER c gene and d protein expression. Each point represents the mean ± SEM from three independent experiments. All values marked with (p < 0.05) and (p < 0.01) are significantly different 1 1 3 3 Cancer Chemother Pharmacol (2017) 80:815–827 822 822 Cancer Chemother Pharmacol (2017) 80:815–827 Fig. 4 Effect of leptin receptor antagonists (SHLA, Lan1 and Lan2) on the expression of the oestradiol receptor α and β forms on the gene (a, c, e) and protein (b, d, f) level. Values are mean ± SEM. All val- ues marked with (p < 0.05) and *(p < 0.01) are significantly differ- ent from control values Fig. 4 Effect of leptin receptor antagonists (SHLA, Lan1 and Lan2) on the expression of the oestradiol receptor α and β forms on the gene (a, c, e) and protein (b, d, f) level. Values are mean ± SEM. All val- ues marked with (p < 0.05) and *(p < 0.01) are significantly differ- ent from control values Effect on leptin receptor antagonists on selected cell cycle protein expression In KGN cells, SHLA at the highest dose and Lan1 and Lan2 at doses 100 and 1000 ng/mL decreased BrdU incor- poration (Fig. 5c). None of the three antagonists investi- gated affected caspase-3 activity (Fig. 5f), except for Lan2, which at the highest concentration decreased oestradiol levels. SHLA and Lan1 had no effect on oestradiol secre- tion (Fig. 5i). In HGrC1 cells, leptin increased cdk4 and cdk2 protein expression and had no effect on cyclin D1 and A2. No effect on E2F1 or E2 was observed. Of the investigated lep- tin receptor blockers, SHLA had no effect on selected cell cycle gene or protein expression. An inhibitory effect on 1 3 Cancer Chemother Pharmacol (2017) 80:815–827 823 Fig. 5 Effect of leptin and leptin receptor antagonists (SHLA, Lan1 and Lan2) on a–c cell proliferation (BrdU incorporation), d–f cas- pase-3 activity and g–i oestradiol secretion. Each point represents the mean ± SEM from three independent experiments of four repli- cates per treatment group. In a–f, values marked with (p < 0.05) and *(p < 0.01) are significantly different from leptin values. In g–i, val- ues marked with are significantly different from control, while those indicated by #(p < 0.05) are significantly different from leptin Fig. 5 Effect of leptin and leptin receptor antagonists (SHLA, Lan1 and Lan2) on a–c cell proliferation (BrdU incorporation), d–f cas- pase-3 activity and g–i oestradiol secretion. Each point represents the mean ± SEM from three independent experiments of four repli- cates per treatment group. In a–f, values marked with (p < 0.05) and (p < 0.01) are significantly different from leptin values. In g–i, val- ues marked with are significantly different from control, while those indicated by #(p < 0.05) are significantly different from leptin the cell cycle proteins studied. All three blockers decreased the expression of cyclin D1 and cdk2, while Lan2 also reduced cdk4 expression and both Lan1 and Lan2 reduced E2F1 protein. cdk4 but not cyclin D was noted under the influence of Lan1. A marked inhibitory effect on cyclin A and cdk4 protein expression under the influence of Lan2 was noted. None of the antagonists investigated had an effect on E2F1 or E2 (Fig. 6a). In COV434 cells, leptin with the exception of a stimula- tory effect on E2F2 had no effect on the expression of any other investigated cell cycle proteins. Effect on leptin receptor antagonists on selected cell cycle protein expression All three leptin recep- tor blockers decreased cyclin D1 and cdk4 in the following order: SHLA < Lan1 < Lan2. In addition, SHLA decreased cyclin A2 protein expression, Lan1 cyclin A2 and cdk2 expression, and Lan2 cdk2 protein expression. Of the three blockers studied here, only Lan2 had an inhibitory effect on E2F2 expression (Fig. 6b). Discussion As described by us, higher ObR expression and a leptin-stimulatory effect on its own recep- tor in cancer granulosa cells correspond with our previously published data [14] in epithelial ovarian cancer cells, sug- gesting similar effects in both epithelial ovarian cancer and folliculoma. Increased expression of ObR, corresponding to higher risk, has also been described in cases of breast [1] and prostate cancer [25], both hormone-dependent cancer types. In ovarian cancer patients, high leptin levels are associated with poor treatment prognosis [26]. the activation of oestrogen receptor alpha (ERα) through the MAPK pathway in MCF-7 and HeLa cells [31]. In ER+ MCF-7 cells, chronic exposure to leptin has been found to result in a higher ERα/ERβ ratio, enhanced oestrogen tran- scriptional activity, greater cell growth and resistance to the anti-oestrogen compound tamoxifen [32].f It is well known that leptin can directly affect ovarian function by its action on oestradiol secretion [33]. There have been a few studies on blocking leptin activity in the ovary and interactions with oestradiol secretion. Our results show that the leptin receptor antagonists SHLA and Lan1, at concentrations of 100 ng/mL, increased oestradiol (E2) secretion by the HGrC1 cell line, but had no effect on oestra- diol secretion in granulosa cancer cells (except for Lan2, which decreased E2 secretion in KGN cells). The effect on ERα and ERβ expression was variable in this study. In HGrC1 cells, all antagonists decreased ERβ gene expression, but only Lan1 and Lan2 decreased protein levels. The block- ers SHLA and Lan1 decreased ERα in juvenile COV434 cells, while Lan1 and Lan2 had an effect in adult form KGN cells. These results suggest that, independently of the magni- tude of ERα expression, Lan1 was sufficient in both types of cells. Our data are in agreement with Fusco et al. [19], who showed that leptin receptor silencing in MCF-7 breast cancer cells results in decreased ERα expression. The same authors reported that higher leptin levels are more strongly corre- lated with ER+ breast cancer rather than ER−. In another study, Dupuis et al. [11] used the leptin receptor antagonist PEG-SMLA to demonstrate that inhibition of ObR impaired follicle rupture without affecting meiotic maturation of oocytes in ovarian follicles. Cross talk between leptin and oestrogen receptors should also be taken into consideration. It was apparent that, in can- cer cells, elevated ObR expression correlated with elevated ERα expression. Discussion This study clearly demonstrates twofold higher leptin recep- tor gene and protein expression in cancer granulosa cell lines compared to non-cancer cell lines. Additionally, we found that leptin increased its own receptor gene expression only in cancer cell lines. These results on both the short (ObRa) and long (ObRb) forms of the leptin receptor are in agreement with other studies demonstrating both forms of the leptin receptor in In KGN cells, leptin, with the exception of a slight inhibi- tory effect on cdk2, had no effect on the expression any of 1 3 Cancer Chemother Pharmacol (2017) 80:815–827 824 Fig. 6 Expression of cell cycle proteins in HGrC1, COV434 and KGN cell lines under the influence of leptin and leptin receptor antagonists. The representative blots of three experiments are shown in the panels. Cdk2, cyclin A, cdk4, cyclin D and E2F1 densitometry results were normalised to GAPDH loading controls to obtain band ratios. Values are mean ± SEM. All values marked with (p < 0.05), (p < 0.01) are significantly different from untreated control values. All values marked with #(p < 0.05), ##(p < 0.01) are significantly dif- ferent from the values of leptin at 40 ng/mL Fig. 6 Expression of cell cycle proteins in HGrC1, COV434 and KGN cell lines under the influence of leptin and leptin receptor antagonists. The representative blots of three experiments are shown in the panels. Cdk2, cyclin A, cdk4, cyclin D and E2F1 densitometry results were normalised to GAPDH loading controls to obtain band ratios. Values are mean ± SEM. All values marked with (p < 0.05), *(p < 0.01) are significantly different from untreated control values. All values marked with #(p < 0.05), ##(p < 0.01) are significantly dif- ferent from the values of leptin at 40 ng/mL Fig. 6 Expression of cell cycle proteins in HGrC1, COV434 and KGN cell lines under the influence of leptin and leptin receptor antagonists. The representative blots of three experiments are shown in the panels. Cdk2, cyclin A, cdk4, cyclin D and E2F1 densitometry results were normalised to GAPDH loading controls to obtain band ratios. Values are mean ± SEM. All values marked with (p < 0.05), **(p < 0.01) are significantly different from untreated control values. All values marked with #(p < 0.05), ##(p < 0.01) are significantly dif- ferent from the values of leptin at 40 ng/mL granulosa cells [11, 24]. Discussion The direct relationship between ObR and ER is still unresolved; however, the existence of cross talk between these receptors was examined. Fusco et al. [19] described a threefold increase in ERα expression in the pres- ence of 100 ng/mL leptin. Different effects, dependent on the type of ER expression, have been described by Ray et al. [27] and Ozbay et al. [28], who showed that leptin increased the proliferation level to a greater extent in the ER+ breast cancer cell line T47-D than in ER− MDA-MB231 cells. This finding is in line with our results, as all investigated granulosa cell lines demonstrated a higher degree of prolif- eration than in previously examined epithelial ovarian cell lines [14]. Physiologically, the primary sites of oestrogen receptors include the same areas where ObR are located [29]. Leptin can enhance aromatase activity, promoting oestrogen production from androstenedione in adipose tissue and hence stimulate the progression of oestrogen- dependent breast cancer [30]. Furthermore, leptin enhances 1 3 3 Cancer Chemother Pharmacol (2017) 80:815–827 825 Leptin, via direct action through ObR and additionally by enhancing the activation of oestrogen receptor alpha (ERα) through the MAPK pathway, induces cell proliferation. The data presented here show that leptin at a supraphysiological level induced cell proliferation in all investigated cell types, with the greatest effect observed in the normal granulosa cell line HGrC1 where proliferation reached 180% of the control. The mutagenic effect of leptin has been observed in various cell types, including breast cancer [33, 34], endo- metrial cancer [35] and prostate cancer cells [25]. Kato et al. [36] showed that higher leptin levels (above 100 ng/mL) or longer incubation times are required to have an effect on proliferation. However, Fiedor and Gregoraszczuk [14] and Ptak et al. [37], using the same leptin concentration, described a similar effect on the proliferation of epithelial ovarian cancer cells. leptin at 40 ng/mL alone or in combination of SHLA or Lan2 does not affect the activity of caspase-3, -8 or -9 (unpublished data). With regard to the mechanism of action of the ObR antag- onists, we showed that, in non-cancer HGrC1 cells, Lan1 and Lan2 decreased the expression of cyclin A2 and cdk4. In COV434 cells, all tested antagonists decreased cyclin D1 and cdk4 protein expression, while Lan1 also decreased cyc- lin A2 and cdk2, SHLA had an inhibitory effect on cyclin A, and Lan2 inhibited cdk2 expression. Discussion It is generally believed that the critical function of the cyclin A–Cdk2 complex is the phosphorylation of substrates that start DNA replication and co-ordinate the end of S-phase [40]. In KGN cells, only cyclin D1 and cdk2 were decreased by all leptin receptor blockers. Additionally, we observed an inhibitory effect of Lan1 and Lan2 on the expression of the transcription fac- tor E2F1, suggesting that the anti-proliferative effect of this antagonist in ovarian cancer may be mediated, in part, by the down-regulation of E2F1. Our previously published results [14] concerning the action of leptin receptor antagonists on epithelial ovarian cancer cells showed that both antago- nists studied decreased cdk2 and cdk4 protein expression in CaOV-3 and OVCAR-3 cells. Additionally, in CaOV-3 cells, cyclin D1 expression decreased under the influence of SHLA and Lan2. All ObR antagonists used in this study reversed the lep- tin-stimulatory effects on cell proliferation, although with varying degrees of success. In non-cancerous HGrC1 cells, the most potent antagonist was SHLA. In KGN cells, all three antagonists at all concentrations reversed leptin-stim- ulated proliferation. Surprisingly, in the COV434 cell line, the effects of blockers were negligible. The use of leptin receptor antagonists has been well studied in breast cancer. There are reports of the inhibitory action of Aca-1 and Allo- aca on leptin-stimulated proliferation of MCF-7 and MDA- MB231 breast cancer cells [15], and D-Ser and DDD on the proliferation of MCF-7 cells [38]. Catalano et al. [16], using LDFI (leptin binding site I), showed the inhibitory effects on the leptin-induced growth of ERα-positive (MCF-7) and ERα-negative (SKBR3) breast cancer cells. Fusco et al. [19] demonstrated the inhibitory effects of a neutralising mono- clonal antibody (9F8) on cell proliferation in the ER-positive MCF-7 cell line, but not in MDA-MB231 ER-negative cells. We previously described SHLA and Lan2 as a promising treatment for epithelial ovarian cell tumours [14]. To our knowledge, these are the first findings to indicate the pos- sible use of ObR blockers in the treatment of folliculoma cancer. 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Am J Physiol Endocrinol Metab 294:E817–E826 Discussion The ability of these cyclins to activate the cyclin-dependent kinase CDK4 is the most extensively documented mechanism for their oncogenic actions and pro- vides an attractive therapeutic target. In conclusion, taking into consideration that these results are based on experiments performed on cell lines, and did not consider the tumour microenvironment, further studies should be performed using explants of granulosa cancer from patients with folliculoma or the coculture of granulosa cancer cell line with fibroblasts, epithelial cells and other components of the tumour environment. Our results reveal that leptin at 40 ng/mL does not affect caspase-3 activity in non-luteinising cells HGrC1, but could decrease it in COV434 cells and to a small extent in KGN cells. Using granulosa cells, Sirotkin et al. [39] showed a stimulatory effect of leptin at a dose of 100 ng/ mL on Bax protein in human granulosa cells, suggesting that leptin can modulate apoptosis in human ovaries. The discrepancy in the findings may be due to the different concentrations of leptin. All the antagonists investigated here had no effect on caspase-3 activity in HGrC1 or KGN cells. Only Lan1 and Lan2, at the highest concentrations, restored caspase-3 activity close to the control level in COV434 cells. Previous experiments conducted in our laboratory have shown that, in epithelial ovarian tumours, Acknowledgements This study was supported by K/ZDS/006310, K/DSC/003955, DS/MND/WBiNoZ/IZ/15/2016 and Jagiellonian Uni- versity in Kraków, Poland. Compliance with ethical standards Conflict of interest The authors declare that they have no conflicts of interest. 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https://openalex.org/W4308727025	https://www.researchsquare.com/article/rs-2224121/latest.pdf	English	null	The effects of regional quadriceps architecture on angle-specific rapid torque expression	Research Square (Research Square)	2,022	cc-by	7,924	The effects of regional quadriceps architecture on angle-specific rapid torque expression Dustin J. Oranchuk Auckland University of Technology William G. Hopkins Victoria University John B. Cronin Auckland University of Technology Adam G. Storey Auckland University of Technology André R. Nelson (  andre.nelson@vu.edu.au ) Victoria University Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. R d F ll Li License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/19 Page 1/19 Abstract Evaluating anatomical contribution to performance can build an understanding of muscle mechanics and guide physical preparation. While the impact of anatomy on muscular performance is well studied, the effects of regional quadriceps architecture on rapid torque expression are less clear. Regional (proximal, middle, distal) quadriceps (vastus lateralis, rectus femoris, lateral and anterior vastus intermedius) thickness (MT), pennation angle (PA), and fascicle length (FL) of 24 males (48 limbs) were assessed via ultrasonography. Participants performed isometric contractions at 40º, 70º, and 100º of knee flexion to evaluate rate of torque development from 0-200 ms (RTD0 − 200). Measurements were repeated on three separate occasions with the greatest RTD0 − 200 and average muscle architecture measures used for analysis. Linear regression models predicting angle-specific RTD0 − 200 from regional anatomy provided adjusted simple and multiple correlations (√adjR2) with bootstrapped compatibility limits to assess magnitude. Mid-rectus femoris MT (√adjR2 = 0.41–0.51) and mid-vastus lateralis FL (√adjR2 = 0.41–0.45) were the best single predictors of RTD0 − 200, and the only measures to reach acceptable precision with 99%CL. Small simple correlations were found across all regions and joint angles between RTD0 − 200 and vastus lateralis MT (√adjR2 = 0.28 ± 0.13; mean ± SD), vastus lateralis FL (√adjR2 = 0.33 ± 0.10), rectus femoris MT (√adjR2 = 0.38 ± 0.10), and lateral vastus intermedius MT (√adjR2 = 0.24 ± 0.10). Multiple correlations are reported within the article. Researchers should measure mid-region rectus femoris MT and vastus lateralis FL to efficiently and robustly evaluate potential anatomical contributions to changes in rapid knee extension torque expression. Experimental design Using a repeated measures design, we examined measures of regional quadriceps anatomical parameters and RTD at a range of joint angles. Each participant was tested on three separate occasions, separated by 5–8 days. At each session, an ultrasonographic assessment of regional quadriceps MT, PA, and FL, followed by an isometric assessment of RTD at 40º, 70º, and 100º of knee flexion (0º=full extension), was completed. Inter-session reliability was determined for all measures. Average muscle architecture was used for analysis to minimize daily fluctuations in hydration and sonographer error, whereas the greatest RTD from all sessions were used for analysis to minimize issues of motivation and muscular activation. Participants Twenty-four healthy males (mean ± SD: 28.5 ± 4.7 years, 180.1 ± 7.7 cm, 81.6 ± 11.8 kg) volunteered for this study. All participants were required to have at least six months of resistance training experience (6.73 ± 4.83, range: 1–18 years), with at least two weekly sessions of lower-body resistance training (2.54 ± 0.75, range: 2–5 sessions.week− 1), and be free of any musculoskeletal injuries for at least three months before data collection. Participants were instructed to maintain their current level of physical activity throughout the data collection period apart from refraining from strenuous physical activity, alcohol, caffeine, and other ergogenic aids for at least 48 hours before each session. The Auckland University of Technology Research Ethics Committee approved the study (18/232), and all subjects provided written informed consent. All ethical conciderations were performed inline with the World Medical Association and the Declaraton of Helsinki17. Introduction Measures of muscle size (e.g., muscle thickness (MT), cross-sectional area, volume) and architecture (e.g., pennation angle (PA), fascicle length (FL)) have become commonplace when assessing acute or chronic effects of exercise1–3, baseline characteristics of performance, or clinical populations4–6. Several research groups have also aimed to understand the effect of muscle architecture on function and performance5,7−12, often with the intent of guiding exercise prescription relating to contraction type or other variables1,3,13,14. For example, Oranchuk et al.1 compared the acute effects of eccentric and eccentric quasi-isometric resistance exercise on quadriceps muscle architecture and reported differing changes in regional MT and echo intensity, which may relate to long-term adaptations. While the relationships between muscle architecture and maximal force or torque expression are well studied5,7−12, very few studies have utilized rapid force or torque expression as a variable, possibly due to larger measurement variabilities15. One such recent study by Coratella et al.16 examined the relationships between the rate of force development at several epochs and muscle architecture of the vastus lateralis, vastus medialis, rectus femoris, and vastus intermedius. The correlations logically increased with larger epochs, with the greatest correlations between the rate of force development and vastus intermedius MT (r = 0.27–0.69), vastus lateralis FL (r = 0.37–0.63), and vastus intermedius FL (r = 0.34–0.64)16. While Page 2/19 Page 2/19 Coratella and colleagues16 were, to our knowledge, the first to examine the relationship between quadriceps architecture and rapid force expression, only the mid-region of each muscle, and a single joint angle, were examined. Also several findings from Coratella et al. run contrary to a previous study9 that demonstrated smaller correlations between mid-region vastus intermedius MT and maximal isometric torque (√adjR2 = 0.23–0.42). While several researchers have examined the relationships between anatomy and isometric force or torque-time characteristics, none have determined the strength of association between regional architecture and rapid torque expression at multiple joint angles. Based on previous research, we hypothesized that mid and distal vastus lateralis and lateral vastus intermedius MT and distal vastus lateralis FL would be the strongest predictors of rapid torque expression. Additionally, we hypothesized that joint angle would not substantially affect the form-to-function relationship. Testing procedures Ultrasonography Ultrasound settings (frequency, 12 MHz; brightness, maximum; gain, 60 dB; dynamic range, 70; depth, individualized) were recorded and kept consistent across all sessions. All assessments were performed by the same sonographer with ~ 3 years of experience. Upon arrival, participants were positioned in a supine position on a massage table for 15 minutes, to allow for fluid re- distribution18. The participants remained in a supine position with knees and hips fully extended with a foot strap used to prevent excessive external rotation19. During this period, the participants had each thigh measured and marked by an ISAK level-2 anthropometrist. The length of the lateral aspect of the thigh was determined by measuring the distance from the superior border of the greater trochanter to the inferior border of the lateral condyle of the femur. The anterior aspect of the thigh was determined by measuring the distance between the superior border of the patella and the inferior border of the anterior, superior iliac spine20. Thigh lengths were recorded, and markings were made at 30% (proximal), 50% (middle), and 70% (distal) of the lateral and anterior distances, respectively19. The vastus lateralis was marked by determining the most lateral aspect of the thigh. Participants were instructed to briefly tense the quadriceps so that the researcher could mark the rectus femoris. Pilot testing with our resistance- trained male cohort determined that extended field-of-view scans of the vastus intermedius were not consistently clear. Thus, scanning depths were adjusted to only capture the vastus lateralis and rectus femoris muscles. The vastus medialis was excluded as it can be further broken down into the obliquus and longus portions, with deep and superficial fiber bundles21. Furthermore, MT of the vastus medialis oblique and longus have substantially smaller correlations with magnetic resonance imaging derived cross-sectional area when compared to the vastus lateralis or rectus femoris22. MT and PA of the vastus lateralis and lateral vastus intermedius, and the rectus femoris and anterior vastus intermedius were collected in the same snap-shot images, respectively. Typical proximal, middle, and distal lateral and anterior images are provided in Fig. 1. Fascicle length was measured by the extended field-of-view function as extensively detailed previously23,24. The ultrasound probe was moved across a muscle, while a texture mapping algorithm merged the sequence of images into a composite image23,25. Testing procedures Ultrasonography Page 3/19 All ultrasound images were collected using the same transducer and built-in software (45 mm linear array, GE Healthcare, Vivid S5, Chicago, IL). Ultrasound settings (frequency, 12 MHz; brightness, maximum; gain, 60 dB; dynamic range, 70; depth, individualized) were recorded and kept consistent across all sessions. All assessments were performed by the same sonographer with ~ 3 years of experience. Upon arrival, participants were positioned in a supine position on a massage table for 15 minutes, to allow for fluid re- distribution18. The participants remained in a supine position with knees and hips fully extended with a foot strap used to prevent excessive external rotation19. During this period, the participants had each thigh measured and marked by an ISAK level-2 anthropometrist. The length of the lateral aspect of the thigh was determined by measuring the distance from the superior border of the greater trochanter to the inferior border of the lateral condyle of the femur. The anterior aspect of the thigh was determined by measuring the distance between the superior border of the patella and the inferior border of the anterior, superior iliac spine20. Thigh lengths were recorded, and markings were made at 30% (proximal), 50% (middle), and 70% (distal) of the lateral and anterior distances, respectively19. The vastus lateralis was marked by determining the most lateral aspect of the thigh. Participants were instructed to briefly tense the quadriceps so that the researcher could mark the rectus femoris. Pilot testing with our resistance- trained male cohort determined that extended field-of-view scans of the vastus intermedius were not consistently clear. Thus, scanning depths were adjusted to only capture the vastus lateralis and rectus femoris muscles. The vastus medialis was excluded as it can be further broken down into the obliquus and longus portions, with deep and superficial fiber bundles21. Furthermore, MT of the vastus medialis oblique and longus have substantially smaller correlations with magnetic resonance imaging derived cross-sectional area when compared to the vastus lateralis or rectus femoris22. MT and PA of the vastus lateralis and lateral vastus intermedius, and the rectus femoris and anterior vastus intermedius were collected in the same snap-shot images, respectively. Typical proximal, middle, and distal lateral and anterior images are provided in Fig. 1. All ultrasound images were collected using the same transducer and built-in software (45 mm linear array, GE Healthcare, Vivid S5, Chicago, IL). Testing procedures Ultrasonography A water-soluble gel was applied to the scanning head of the ultrasound probe to achieve acoustic coupling, with care taken to avoid tissue deformation23–25. The probe was oriented perpendicular to the skin and parallel to the estimated fascicle direction23–25. Two images were captured for each region or extended field-of-view scan. Two measurements for MT, PA and FL were quantified from each image, and the mean calculated (i.e., image mean). The mean of the two image means was then calculated and used for subsequent analyses24. Isometric dynamometry Following the ultrasonographic assessment, participants warmed up by cycling at low to moderate resistance using a self-selected pace for five minutes15. Subjects were seated on the isokinetic dynamometer (CSMi; Lumex, Ronkonkoma, NY) at a hip angle of 85º, with shoulder, waist, and thigh straps to reduce body movement during contractions15,26. The shin-pad was positioned ~ 5 cm superior Page 4/19 Page 4/19 to the ankle joint malleoli15. Subjects were required to hold handles at the sides of the chair, while the non-working limb was positioned behind a restraining pad15. Knee alignment was determined by visual inspection and unloaded knee extensions. Dynamometer settings were recorded and matched for subsequent sessions. Once fitted to the dynamometer, subjects underwent a series of extensions and flexions of the knee to determine safety stop positions and calibrate gravity correction15. Subjects then completed a standardized warmup of concentric contractions of 30%, 50%, 70%, 85%, and 100% of perceived maximal voluntary contraction15. One minute after the completion of the warmup contractions, the participants' knee was positioned at 40º of flexion, where one familiarization isometric knee extension at 50% of perceived maximum torque was performed 15. Sixty seconds later, two maximal contractions lasting four seconds were completed with 30 seconds separating each contraction15. Participants were instructed to contract "as fast and hard as possible," following a countdown of "3-2-1-go!"15,27. All participants were given strong verbal encouragement along with visual feedback of the torque-time tracing during each trial15,27. Participants were instructed to avoid any pre-tension and countermovement of the knee extensors, while the live torque-time trace was carefully inspected by the examiner leading up to each contraction15,27. The cut-off for pre-tension was set at 10 N15. Any contractions with a countermovement or an unsteady baseline were rejected and repeated15,27. The participants then completed the same series at 70º and 100º of knee flexion. The isometric contractions were always performed in series from 40º to 100º to avoid greater muscle damage and fatigue synonymous with long muscle length contractions20. Ten minutes following the final isometric contraction, the concentric warm-up, and isometric assessments were repeated on the opposite limb. Limb order was randomized throughout the testing sessions and counterbalanced over the participant group. All contractions were collected, without filtering, via a custom-made software (LabVIEW; National Instruments, New Zealand) sampling at 2000 Hz15,27. D t i d l i Ultrasonography Images were stored and analyzed via digitizing software (ImageJ; National Institutes of Health). Muscle thickness (cm) was defined as the perpendicular distance between the deep and superficial aponeurosis, and PA was defined as the angle of the fascicles relative to the deep aponeurosis25. Due to the depth of the muscle, the fascicles on the lateral and anterior vastus intermedius were not consistently visible24. Therefore, PA and FL were not recorded for the lateral and anterior vastus intermedius. As the rectus femoris is complex, in that there are distinctly different directions of pennation20, only 23/48 of the distal rectus femoris FL measurements were able to be determined. Representative vastus lateralis and rectus femoris extended field-of-view images are illustrated in Fig. 2A and B, respectively. Anatomical variables were averaged across all sessions to reduce errors arising from sonography or participant variability. Reliability Measures of RTD, MT, and FL were log-transformed before analysis to reduce non-uniformity of error28,29 and analyzed with an Excel spreadsheet30. To reduce bias in the correlations between variables, only measures with sufficiently high intersession reliability were included in the correlational analysis. Since the mean or maximum of the three sessions was used in the correlation analysis, the intraclass correlation coefficient (ICC) of the mean of the three sessions was used as the measure of interest29. A value of ICC > 0.75 was considered sufficiently reliable31. For comparison with other studies, the between- session typical error of measurement (TEM) was also analyzed and expressed as a percentage for RTD, MT, and FL28,29. TEM is expressed in degrees for PA, as there is an upper limit of 90º, and unlike other measures, the error would not be expected to increase with increasing values28,29. While not a primary aim of the study, the variability of intra-image measures (e.g., the variability of two proximal vastus lateralis FL measures in the same image) was performed to determine variations in quadriceps anatomy across regions and muscles using the same procedure and interpretation as for the intrasession reliability analysis. Intra-image variability statistics were not analyzed for RTD as the greatest value, regardless of contraction or session, was utilized for the correlational analysis. Isometric dynamometry Data were analyzed via a customized MATLAB (MathWorks, Natick, MA) script. As the moment arm substantially affects isometric outputs11,18, all torque data were divided by the length of the dynamometer arm, in meters, to normalize the difference in shank length between participants15. Thus, all torque data are reported in Newtons per second (N·s− 1). Normalized torque over 200 N were identified to signify a full contraction and eliminate false contractions15. The contraction with the highest RTD was analyzed. The maximum values of RTD across the three sessions were utilized to reduce error arising from submaximal voluntary activation. Correlational analysis As the primary goal was to examine the effects of regional quadriceps anatomy on RTD, a measure was included only if it was sufficiently reliable (ICC > 0.75) in all three regions (proximal, middle, distal) and knee joint angles (40º, 70º, 100º). Simple linear regressions were used to predict angle specific RTD with anatomical parameters in isolation (e.g., distal vastus lateralis MT). Multiple linear regressions (explained in detail below) were used to determine if the difference in predictive ability between regions (e.g., distal vastus lateralis FL vs middle vastus lateralis FL) was substantial. All regression analyses were performed with Proc Reg in the Statistical Analysis Software (University Edition of SAS Studio, version 9.4, SAS Institute, Cary NC). Measures of the ability of the regression models to predict RTD were the multiple R (the correlation between observed and predicted values), √R2 (equivalent to the absolute value of Pearson's r for a single predictor), and the adjusted multiple R (the square-root of the R-squared, √adjR2, adjusted for degrees of freedom). The adjustment removes the Page 6/19 Page 6/19 Page 6/19 upward bias in the R-squared that occurs when any predictor is added to a model with a finite sample size. Negative values of adjR2 were expressed as negative correlations by changing the sign before taking the square root. Effects (adjusted multiple R, their means, and their differences) are presented with the qualitative magnitude of their observed (sample) value and interpreted as trivial, small, moderate, large, very large, and extremely large for values < 0.10, ≥ 0.10, ≥ 0.30, ≥ 0.50, ≥ 0.70, and ≥ 0.90 respectively29. Sampling uncertainty in the estimates of effects is presented as 90% compatibility limits (90%CL), which were estimated from percentiles of the effects in 10,000 bootstrapped samples. The bootstrapped effects were estimated initially by performing multiple linear regression with each bootstrapped sample, but the median values of the effects were substantially higher than those in the original sample. Therefore, values in the bootstrapped samples were derived as follows: the regression coefficients in the original sample were used to predict the dependent variable in each bootstrapped sample; the correlation between the predicted and observed values of the dependent variable in each sample was squared and adjusted for degrees of freedom; the square root of this adjusted R-squared then provided values of adjusted multiple R, which showed close agreement between the original and bootstrapped median values. Correlational analysis As a further check on the adequacy of this bootstrap method, the analyses were performed on simulated data with a sample size of 24 and simple correlations between a dependent variable and two predictor variables in two limbs. One hundred analyses each of 10,000 bootstrap samples were performed for correlations ranging from 0.00 to 0.70. Observed and median adjusted multiple R showed a downward bias of 0.05 to 0.10 for true correlations of 0.00-0.30, 0.04 for true correlations of 0.50, and only 0.02 for true correlations of 0.70. Despite the bias, 90%CLs showed less than the 10% expected error rate in their coverage of low values of true correlations and a slightly higher error rate (~ 12%) for true correlations of 0.50 and 0.70. The bootstrapping was therefore judged to be trustworthy. The non-clinical version of magnitude-based decisions with a minimally informative prior was used to interpret the uncertainty in effect magnitudes32. Chances that the true magnitude was a substantial negative value, a trivial value, and a substantially positive value were derived directly from the bootstrapped samples as the proportion of sample values with those magnitudes. Effects were deemed to be clear (to have adequate precision) if the chances of one or other substantial true values were < 5% (i.e., the 90%CLs did not include substantial positive and negative values). Clear effects are reported with a qualitative descriptor for the magnitude with chances > 25% using the following scale: >25%, possibly (); >75%, likely (); >95%, very likely (); and > 99.5%, most likely (**)29. When the chances of a substantial or trivial magnitude were > 95%, the magnitude itself is described as clear. Effects with inadequate precision are described as unclear. Effects with adequate precision defined by 99%CL are highlighted in bold in tables and figures; the overall error rate for coverage of 10 independent true values with such intervals is that of a single effect with a 90%CL (10%), and interpretation of outcomes is focused on these effects. Reliability Intersession, intrasession and intra-image (two fascicles from the same image) reliability of quadriceps architectural parameters have been previously reported in detail9. In brief, all MT and vastus lateralis PA and FL measures were sufficiently reliable (ICC = 0.77–0.98). However, rectus femoris PA and FL were either unreliable, or unable to be consistently evaluated in each participant, and therefore not included in the correlational analysis. RTD0 − 100 was reliable across all limbs at 40º (ICC = 0.85, TEM = 15%), 70º (ICC = 0.79, TEM = 20%), and 100º (ICC = 0.80, TEM = 19%). RTD0 − 200 was slightly more reliable at 40º (ICC = 0.90, TEM = 13%), 70º (ICC = 0.81, TEM = 15%), and 100º (ICC = 0.87, TEM = 12%). Furthermore, correlations between muscle architecture and RTD were always trivially different between 0-100 and 0-200 epochs. Therefore, due to the very large number of correlations, all subsequent results are reported for RTD0 − 200 only. Results Page 7/19 Correlational analysis Mean regional anatomical measures are summarized in Table 1. RTD0 − 200 for the dominant and non- dominant limbs were (mean ± SD) 2510 ± 657 N·s− 1 and 2462 ± 599 N·s− 1 at 40º, 2876 ± 873 N·s− 1 and 2789 ± 725 N·s− 1 at 70º, and 2388 ± 656 N·s− 1 and 2319 ± 701 N·s− 1 at 100º, respectively. All between- limb differences in predictive ability were unclear, or possibly small; therefore, all correlations are presented after pooling dominant and non-dominant limbs. Page 8/19 Page 8/19 Table 1 Regional quadriceps architecture averaged over three sessions. Muscle Measurement Limb Proximal Middle Distal Vastus lateralis MT (cm) Dominant 2.54 ± 0.36 2.81 ± 0.43 2.21 ± 0.34 Non- dominant 2.44 ± 0.33 2.74 ± 0.41 2.20 ± 0.37 PA (°) Dominant 17.1 ± 3.2 20.6 ± 2.6 22.7 ± 2.7 Non- dominant 16.3 ± 2.7 20.2 ± 2.1 22.5 ± 2.8 FL (cm) Dominant 7.73 ± 0.68 8.18 ± 0.83 7.16 ± 0.83 Non- dominant 7.70 ± 0.73 8.16 ± 0.72 7.12 ± 0.90 Rectus femoris MT (cm) Dominant 2.80 ± 0.26 2.69 ± 0.33 1.96 ± 0.30 Non- dominant 2.76 ± 0.33 2.69 ± 0.31 1.99 ± 0.31 Lateral vastus intermedius MT (cm) Dominant 2.02 ± 0.44 2.04 ± 0.54 1.93 ± 0.44 Non- dominant 2.01 ± 0.51 2.03 ± 0.53 1.84 ± 0.42 Anterior vastus intermedius MT (cm) Dominant 2.94 ± 0.46 2.24 ± 0.47 1.88 ± 0.42 Non- dominant 2.96 ± 0.47 2.36 ± 0.49 1.78 ± 0.37 MT = muscle thickness. PA = pennation angle. FL = fascicle length. Data are mean ± SD. Data are across 48 limbs. Table 1 Table 1 Simple linear regressions Correlations and bootstrapped 90%CLs of individual measures of regional muscle anatomy with RTD0 − 200 at 40º, 70º, and 100º of knee flexion are presented in Fig. 3. Regardless of region or joint angle, small correlations were found between RTD0 − 200 and vastus lateralis MT (mean ± SD √adjR2 = 0.28 ± 0.13), vastus lateralis FL (√adjR2 = 0.33 ± 0.10), rectus femoris MT (√adjR2 = 0.38 ± 0.10), and lateral vastus intermedius MT (√adjR2 = 0.24 ± 0.10). Neither vastus lateralis PA (√adjR2 = 0.00 ± 0.18), nor anterior vastus intermedius (√adjR2 = 0.06 ± 0.12) were substantially associated with RTD0 − 200. The largest single correlation was between mid-rectus femoris MT and RTD0 − 200 at 100° (√adjR2 = 0.51, 90%CL: Page 9/19 Page 9/19 0.16–0.73) with mid-rectus femoris MT also having the greatest mean correlation with RTD0 − 200 regardless of joint angle (√adjR2 = 0.47 ± 0.05). This finding was followed by mid-vastus lateralis FL(√adjR2 = 0.43 ± 0.02) and distal rectus femoris MT (√adjR2 = 0.42 ± 0.02) as the only measures consistently correlating with RTD0 − 200 (√adjR2 ≥ 0.40). Additionally, middle rectus femoris MT and middle vastus lateral FL were the only measures to reach acceptable precision with 99%CLs at all three joint angles. Regardless of model, correlations were greatest for RTD0 − 200 at 100º (√adjR2 = 0.24 ± 0.15) and decreased at 70º (√adjR2 = 0.21 ± 0.20) and 40º (√adjR2 = 0.15 ± 19). However, the differences were either unclear, or possibly to likely trivial-small. Multiple linear regressions Between-region differences in simple correlations are presented in Supplementary file 1. Of the 54 between region comparisons, 51 were trivial or small, with 36 decisions being unclear. Distal vastus lateralis MT had likely higher correlations with RTD0 − 200 than proximal vastus lateralis MT at all joint angles (Δ√adjR2 = 0.22–0.27). Likewise, mid vastus lateralis MT was likely better correlated to RTD0 − 200 than proximal vastus lateralis MT at the 70° joint angle (Δ√adjR2 = 0.21). Distal vastus lateralis PA had likely higher correlations with RTD0 − 200 than proximal vastus lateralis PA at all joint angles (Δ√adjR2 = 0.33–0.39). Distal vastus lateralis PA also had a possibly greater correlation to RTD0 − 200 than mid vastus lateralis PA at the 100° joint angle (Δ√adjR2 = 0.27). Middle vastus lateralis FL was likely and possibly better correlated to RTD0 − 200 than distal vastus lateralis FL at 70° (Δ√adjR2 = 0.17) and 100° (Δ√adjR2 = 0.27), respectively. Middle vastus lateralis FL was also possibly better correlated with RTD than proximal vastus lateralis FL at 100° (Δ√adjR2 = 0.17). Middle rectus femoris MT has possibly and likely higher correlations with RTD0 − 200 than proximal rectus femoris MT at 40° (Δ√adjR2 = 0.14) and 100° (Δ√adjR2 = 0.27), respectively. Distal lateral vastus intermedius MT had likely higher correlations with RTD0 − 200 proximal lateral vastus lateralis MT at 70° (Δ√adjR2 = 0.23) and 100° (Δ√adjR2 = 0.26), respectively. Distal lateral vastus lateralis MT was also possibly better correlated to RTD0 − 200 than mid lateral vastus lateralis MT at 70° (Δ√adjR2 = 0.19). Discussion While several studies have examined the effects of quadriceps anatomy on force or torque expression, none have investigated the relationship between regional architecture and rapid torque expression at multiple joint angles. Our primary findings were that mid-region vastus lateralis FL, and mid-region rectus femoris MT were the strongest and most consistent predictors of the rate of torque development. When paired with previous findings, researchers should assess distal or mid vastus lateralis, mid rectus femoris MT and mid vastus lateralis FL to estimate the knee extensors' potential for both maximal and rapid torque expression. Page 10/19 Page 10/19 A key finding of this study was the relatively high and consistent correlations between rectus femoris MT and RTD0 − 200 (√adjR2 = 0.27–0.51). This finding contrasts with previous work demonstrating that rectus femoris MT (√adjR2=-0.01-0.49) was a minor and inconsistent contributor to peak torque when compared to vastus lateralis MT (√adjR2 = 0.18–0.64) or FL (√adjR2 = 0.29–0.60)9. This discrepancy can most likely be explained by several factors. While all quadriceps muscles insert into the patella, the rectus femoris is oriented and attaches directly superior7. Therefore, the rectus femoris has the most direct line- of-pull of the superior quadriceps muscles7, potentially reducing the time required for measurable torque following contraction on-set. However, the rectus femoris is substantially smaller than the vastus lateralis, limiting its contribution to maximal knee extensor strength5,20,33,34. A key finding of this study was the relatively high and consistent correlations between rectus femoris MT and RTD0 − 200 (√adjR2 = 0.27–0.51). This finding contrasts with previous work demonstrating that rectus femoris MT (√adjR2=-0.01-0.49) was a minor and inconsistent contributor to peak torque when compared to vastus lateralis MT (√adjR2 = 0.18–0.64) or FL (√adjR2 = 0.29–0.60)9. This discrepancy can most likely be explained by several factors. While all quadriceps muscles insert into the patella, the rectus femoris is oriented and attaches directly superior7. Therefore, the rectus femoris has the most direct line- of-pull of the superior quadriceps muscles7, potentially reducing the time required for measurable torque following contraction on-set. However, the rectus femoris is substantially smaller than the vastus lateralis, limiting its contribution to maximal knee extensor strength5,20,33,34. Vastus lateralis FL being a top predictor of rapid torque expression is logical given the implication of sarcomeres-in-series to contraction velocity35, or more recently sarcomere resting length theories36. Discussion Though our hypothesis of distal vastus lateralis FL being a top predictor of RTD was not confirmed as mid-region FL had higher correlations at all three joint angles. It is difficult to determine why this is the case, especially as distal vastus lateralis FL was consistently the worst predictor of RTD on the FL measures. However, it is plausible that regional FL is representative of other regions of the same muscle. The mid-region of the vastus lateralis is also thicker than the proximal or distal regions and, therefore, likely has the greatest potential for maximal force and torque expression24. Given that maximal strength is a good predictor of rapid force and torque expression37, our finding is logical. This finding is also interesting as distal vastus lateralis FL had the greatest inter-participant variability. No meaningful relationships between vastus lateralis PA and RTD0 − 200 were found (√adjR2=-0.23–0.32), regardless of region or joint angle. This finding, similar to our previous work examining peak torque9, is contrary to several studies examining maximal or rapid contractile performance8,10,33,38. However, our findings are similar to a recent study also examining knee extension kinetics that found small correlations between vastus lateralis PA and rate of force development over several epochs (r=-0.23– 0.19)16. Though it is difficult to determine the reasons for these conflicting results, it has recently been suggested that assessing PA may lack functional significance due to a variety of factors, including its relationship to MT and FL, changes in PA based on joint position and muscle force, and concepts such as muscle gearing39. Contrary to Coratella et al.,16 this study did not find large or consistent correlations between the RTD measure and vastus intermedius MT. The discrepancy between studies could be due to several factors. Firstly, Coratella et al.,16 utilized a relatively small sample of 17, whereas we examined 48 limbs across 24 individuals. Secondly, and similarly to Ando et al.,12 Coratella et al.16 was only concerned with knee extensor capabilities and muscle architecture on a single occasion, inviting additional uncertainty. Additionally, while our participants were well-trained (≥ 2 lower body resistance-training sessions per week for ≥ 6 months), the participants in the aforementioned studies were 'recreationally trained' and 'healthy', respectively. Therefore, training status may have affected the correlational results as the force or Page 11/19 Page 11/19 torque expression of the well-trained participants is likely more influenced by neural versus anatomical factors40. Discussion These differences in neural control would partially explain the generally weaker correlations in this present study as compared to those of Coratella et al.,16 though this could be due to other factors including the fact that Coratella and colleagues utilized a strain-gauge, whereas an isokinetic dynamometer was used in this study. Finally, while we, and other researchers, have utilized several strategies to maximize reliability, determined good inter-rater variability, it cannot be assumed that sonographers, are without substantive error. Other researchers have determined that cross-sectional areas and muscle volume are stronger predictors of muscle function than MT25. Therefore, it could be recommended that the distal and middle quadriceps cross-sectional area be assessed via panoramic ultrasound to obtain a robust predictor of torque expression in only two scans. Additionally, a third scan examining mid-region vastus lateralis FL could be performed if predicting RTD potential is a priority. Funding Dustin J. Oranchuk was supported by the Auckland University of Technology's Vice-Chancellor's Doctoral Scholarship. There was no other funding provided for this study. Limitations and future research directions While the aims of this study were achieved, there are several limitations to consider. Firstly, the use of surface or needle electromyography to determine individual muscle activation at different joint angles was not utilized; yet similar examinations have reported electromyography to have trivial to moderate correlations to isometric10,38 and cycling performance33, and/or contributed little to multiple regression models10,11,38. Similarly, we could only evaluate our participants' voluntary efforts, not the maximal potential of the quadriceps musculature through peripheral or transcranial stimulation techniques41,42. While the aforementioned strategy to maximize voluntary activation removes many limitations, the lack of joint angle order randomization may have led to the accumulation of non-trivial fatigue levels as contractions progressed from 40º to 100º of flexion. We used the greatest RTD outputs over the three collection sessions to partially address this. Future research could include the knee joint moment arm, which can change through the range of motion10,43. Likewise, compression of the dynamometer padding means that the joint angles reported were likely overestimated and RTD was delayed. Researchers may also wish to scan the quadriceps in the same position as the isometric strength assessment to represent joint angle-specific architecture better. More experienced sonographers or different hardware could have consistently produced extended field-of- view rectus femoris and vastus intermedius FLs, allowing for further elucidation of the relationship between regional quadriceps anatomy and angle-specific torque. Likewise, sonographer error is an ever- present consideration, and this study is no exception. Analyzing the mean fascicle length over multiple regions may also result in interesting findings while diminishing the effect of sonographer error. While several researchers have examined inhomogeneous morphological and architectural adaptations, very few have evaluated more than two regions of an individual muscle44. Therefore, we recommended that future investigations examine three or more regions to illuminate further the effect of training or disuse on region-specific muscle adaptations. Other advanced methods used as muscle volume Page 12/19 assessments and high-density electromyography may unveil additional insights. The bi-articular structure and function of the rectus femoris should be further explored as it is the most commonly injured quadriceps muscle during accelerations and sprinting45, and may play a critical role in clinical populations46. assessments and high-density electromyography may unveil additional insights. Conclusion This study was the first to examine the effect of regional quadriceps architecture on RTD at multiple joint angles. We found that the relative contribution of regional anatomical parameters to RTD does not change at different joint angles. However, middle and distal rectus femoris MT and mid-region vastus lateralis FL were the strongest predictors of RTD, likely due to these respective muscles and structures having the most direct line-of-pull on the musculo-tendonous unit, greatest number serial sarcomeres, respectively. Combined with previous research, ultrasonic evaluations of the quadriceps should focus on mid-region MT and FL of the vastus lateralis and mid-region rectus femoris MT to obtain a thorough yet time-efficient model of quadriceps force expression. Limitations and future research directions The bi-articular structure and function of the rectus femoris should be further explored as it is the most commonly injured quadriceps muscle during accelerations and sprinting45, and may play a critical role in clinical populations46. Conflict of interest The authors have no conflicts of interest to disclose. Author contributions DJO conceived the study. DJO, JBC, AGS and ARN obtained ethical approval. DJO and AGS recruited participants. DJO collected and processed the data. DJO and WGH performed the statistical analysis. DJO, WGH and ARN interpreted the results. DJO created the tables and figures. DJO wrote the manuscript. All authors reviewed and edited, and approved the manuscript before submission. Ethics approval and consent to participate The Auckland University of Technology Research Ethics Committee approved the study (18/232), and all subjects provided written informed consent. Data availability statement Raw data is available upon reasonable request by contacting Dr Dustin J. Oranchuk: dustinoranchuk@gmail.com. Page 13/19 References 1. Oranchuk, D. J., Nelson, A. R., Storey, A. G., Diewald, S. N. & Cronin, J. B. Short-term neuromuscular, morphological, and architectural responses to eccentric quasi-isometric muscle actions. Eur. J. Appl. Physiol. 121, 141–158 (2021). 2. Guex, K., Degache, F., Morisod, C., Sailly, M. & Millet, G. P. Hamstring architectural and functional adaptations following long vs. short muscle length eccentric training. Front. Physiol. 7, 1–9 (2016). 3. McMahon, G. E., Morse, C. I., Burden, A., Winwood, K. & Onambele, G. L. 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Figure 3 Adjusted simple correlations with bootstrapped 90% compatibility limits of normalized maximal voluntary isometric torque at 40º, 70º, and 100º of knee flexion with regional quadriceps architecture. Figures Page 16/19 Figure 1 Representative proximal, middle, and distal B-mode ultrasound images of the lateral (top) and anterior (bottom) quadriceps muscles. MT = muscle thickness. PA = pennation angle. Figure 1 Figure 1 Representative proximal, middle, and distal B-mode ultrasound images of the lateral (top) and anterior (bottom) quadriceps muscles. MT = muscle thickness. PA = pennation angle. Page 17/19 Page 17/19 Page 17/19 Figure 2 Representative extended field-of-view images of the vastus lateralis with proximal, middle, and distal fascicle lengths (A). Typical extended field-of-view image of the rectus femoris (B); note the lack of discernible fascicles towards the distal region. FL = fascicle length. Figure 2 Representative extended field-of-view images of the vastus lateralis with proximal, middle, and distal fascicle lengths (A). Typical extended field-of-view image of the rectus femoris (B); note the lack of discernible fascicles towards the distal region. FL = fascicle length. Figure 2 Representative extended field-of-view images of the vastus lateralis with proximal, middle, and distal fascicle lengths (A). Typical extended field-of-view image of the rectus femoris (B); note the lack of discernible fascicles towards the distal region. FL = fascicle length. Representative extended field-of-view images of the vastus lateralis with proximal, middle, and distal fascicle lengths (A). Typical extended field-of-view image of the rectus femoris (B); note the lack of discernible fascicles towards the distal region. FL = fascicle length. Page 18/19 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. SupTable1.docx Page 19/19
https://openalex.org/W3210472566	https://air.uniud.it/bitstream/11390/1213438/1/A60.%20Attanasio_Preghenella_De%20Toni_Battistella%20-%20Stakeholder%20engagement%20in%20business%20models%20for%20sustainability%20-%20BSE_2021.pdf	English	null	Stakeholder engagement in business models for sustainability: The stakeholder value flow model for sustainable development	Business strategy and the environment	2,021	cc-by	12,068	Abstract Abstract This paper aims to investigate how stakeholder groups engaged by the company con- tribute to the value flow of business models for sustainability. The research aims to expand the knowledge on business models for sustainability by highlighting the most important contributions of stakeholders that are relevant from a value flow and sus- tainable development perspective. The research methodology is a multiple case study in five Italian B corporations. The paper contribution is a stakeholder value flow model of business models for sustainability that categorizes the stakeholders engaged in the specific value flow dimension, namely, value intention, value proposi- tion, value creation, value delivery and value capture. The stakeholder value flow model can facilitate a systematic and deeper analysis of stakeholder contributions to the company business model. Moreover, the stakeholder value flow model can be used to map from the company perspective the most significant relationships and to facilitate the stakeholder engagement towards sustainable development. K E Y W O R D S stakeholder model, stakeholder engagement, sustainable development, value flow, value flow business model for sustainability Giovanna Attanasio1 \| Nadia Preghenella2 \| Alberto Felice De Toni3 \| Cinzia Battistella3 1Department of Economics and Statistics, University of Udine, Udine, Italy 2Department of Technology and Management of Industrial Systems, University of Padova, Padua, Italy 3Polytechnic Department of Engineering and Architecture, University of Udine, Udine, Italy Correspondence Nadia Preghenella, Department of Economics and Statistics, University of Udine, Udine, Italy. Email: nadia.preghenella@phd.unipd.it Funding information University of Udine; University of Padova Bus Strat Env. 2021;1–15. ved: 23 July 2021 Revised: 23 September 2021 Accepted: 8 October 2021 ved: 23 July 2021 Revised: 23 September 2021 Accepted: 8 October 2021 Received: 23 July 2021 Revised: 23 September 2021 Accepted: 8 October 2021 DOI: 10.1002/bse.2922 R E S E A R C H A R T I C L E 2021 The Authors. Business Strategy and The Environment published by ERP Environment and John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. 2.1 \| Stakeholder theory and sustainability The term stakeholder first appeared in 1963 in opposition to the notion that shareholders are the only group that management must refer to (Parmar et al., 2010). A stakeholder can be defined as ‘any group or individual that can influence or be influenced by the achieve- ment of the organization's goals’ (Freeman, 1984, p. 46), while for Dunham, Freeman, and Liedtka (2006, p. 25), it represents ‘a group that the company needs to exist, particularly customers, suppliers, employees, shareholders, and communities’. The concepts of traditional business model and business model for sustainability have been defined and described through different theoretical perspectives, for instance, the activity perspective (Zott & Amit, 2010), the building block perspective (Osterwalder & Pigneur, 2010) or the value flow perspective (Teece, 2010). The value flow in business model for sustainability consists of value intention (Barth et al., 2017), value proposition, value creation, value delivery, and value capture (Bocken et al., 2014; Short et al., 2014), and it should be aimed at multiple stakeholders (Brozovic, 2020). The stake- holder theory sees organizations at the centre of a network of stake- holders that can influence or be influenced by the organization's objectives (Freeman, 2010). Combining stakeholder theory with the business model for sustainability suggests that stakeholders contrib- ute resources and activities to the value flow (Norris et al., 2021). Although the link between stakeholder theory and business model theory is clear, there are extremely few authors who have deepened research in this direction (Freudenreich et al., 2020; Norris et al., 2021; Velter et al., 2021). Stakeholder theory proposes to adopt as a unit of analysis the relationships (unilateral, bilateral or even multiparty) between a com- pany and its stakeholders (Parmar et al., 2010). The stakeholder the- ory permits (1) to resolve the needs of a broad group of stakeholders (Harrison et al., 2010); (2) to manage and shape the relationships to create as much value as possible to be then distributed to all stake- holders (Freeman, 1984); and (3) to assess potential damages and ben- efits to broad groups and individuals (Phillips, 2003; Post et al., 2002; Sisodia et al., 2007). 2 the entire value flow, consisting of value intention, value proposition, value creation, value delivery and value capture (Barth et al., 2017; Bocken et al., 2014; Short et al., 2014). Thus, this work combines the whole value flow of business model for sustainability and the stake- holder theory to address the following research question: How do stakeholders contribute to and benefit from the value flow of the business model for sustainability? business model is focused on the relationship between the company and its customers and takes less into account that all organizations depend on exchanges with other systems to survive (Scott, 1998). The literature on business model is focused on the creation of value for customers in exchange with economic value for company. This tra- ditional view of value creation encourages a separation between stakeholders who receive value and those who contribute to create it. However, sustainable perspective requires removing this distinction and considering value creation as a joint effort between stakeholders and the company (Freudenreich et al., 2020). This paper provides a stakeholder value flow model of business models for sustainability derived from key characteristics of both busi- ness models for sustainability and stakeholder theory. The model depicts the value flows among stakeholders in a business model for sustainability. The interactions of companies with their external environment, including stakeholders, represent a fundamental characteristic of busi- ness models for sustainability (Amankwah-Amoah et al., 2018; Hall & Wagner, 2012; Velter et al., 2020). Particularly, the three fundamental characteristics of a business model for sustainability are reported by Preghenella and Battistella (2021): (1) a long-term business vision; (2) the integration of sustainable value, namely, economic, environ- mental and social, in business the value proposition, delivery and cap- ture; and (3) the stakeholder engagement, integration and management. This paper is structured as follows. Firstly, there is an explanation of the previous literature, highlighting the research gap. Secondly, the conceptual framework coming from previous literature and used for the analysis is described. Then, case studies results are presented and discussed. Finally, the stakeholder value flow model is discussed as main contribution of this research. 2 \| THEORETICAL BACKGROUND Following this perspective, companies need to consider not only the interests of customers but may adopt a multi-stakeholder per- spective at system level (e.g. Evans et al., 2017; Schaltegger, Hansen, & Lüdeke-Freund, 2016) to create sustainable value. Sustainable value is intended as the whole social, environmental and economic benefits that come from a wide range of exchanges in business model (Méndez-Leon et al., 2021; Tao & Yu, 2018). Therefore, value is obtained through the multi-stakeholder collaboration, intended as the participation by diverse people and organizations with several differ- ent competences and resources (Hörisch et al., 2014). 1 \| INTRODUCTION economy, involving entire systems is necessary to implement a signifi- cant change in the scope of the business (Bocken et al., 2014; Boons et al., 2013). Indeed, according to Stubbs (2019), sustainability is increasingly becoming a changing behaviour, and less a technical chal- lenge, because the types of approaches to sustainable innovation are shifting from internally oriented and incremental and focused on effi- ciency to more radical and systemic ones (Adams et al., 2016). Com- pany attention to value is increasing because of its economic and societal implications (Méndez-Leon et al., 2021). Particularly, compa- nies are entities able to produce shared value and long-term prosper- ity (Porter & Kramer, 2011). Growing environmental and social problems combined with popula- tion growth and related consumption of resources led the United Nations in 2018 to issue a plan that represents a call to action and aims to achieve the 17 Sustainable Development Goals (United Nations, 2018). Sustainable development requires the integration of environmental and social issues into the decisions that determine eco- nomic and social development, both by the public and private sectors (WCED, 1987). Companies have a great power on the economy and life in general. Therefore, sustainable development is not possible without sustainable business development (Schaltegger et al., 2012). Since sustainable development requires moving towards a sustainable Since its introduction, the business model has been associated to value with an economic orientation. Generally, research on traditional wileyonlinelibrary.com/journal/bse Bus Strat Env. 2021;1–15. 1 ATTANASIO ET AL. ATTANASIO ET AL. 3 Literature argues that stakeholder theory is useful to address sus- tainability issues. The relations with stakeholders are dynamic and could change depending on the approach to solving sustainability problems (Hall & Wagner, 2012; Mitchell et al., 1997) and allow to share resources and knowledge to solve complex environmental and social problems (Fadeeva, 2005; Gray & Purdy, 2018). As suggested by Vildåsen and Havenvid (2018), the stakeholder relations lead to corporate sustainability through (1) relations on a specific technical project; (2) the achievement and development of mutual sustainability long-term goals; and (3) networking, in which a company systemati- cally relates with the stakeholders in joint sustainability initiatives. Since stakeholder theory is linked to the concept of sustainability (Hörisch et al., 2014), it can be applied to business models for sustain- ability and can add several points to the discussion, as indicated by Freudenreich et al. (2020). On one hand, stakeholder theory recog- nizes that value creation in based on company relationships. Conse- quently, solid relationships with stakeholders make a business model work. On the other hand, the creation of value through the business model takes on multidirectional and multi-stakeholders characteristics oriented towards a common purpose of sustainability. It is established by the literature that stakeholders are a central element in business models for sustainability (e.g. Bocken et al., 2013; Freudenreich et al., 2020; Kujala & Korhonen, 2017; Stubbs & Cocklin, 2008). Furthermore, some scholars have inserted stakeholder relation as part of their business model framework. Although the literature recognizes the importance of stakeholders to implement corporate sustainability, there is a lack of investigation on the contribution of these stakeholders on the value flow of the business model. Traditional and sustainability-oriented business model concepts have been defined in various ways, from various theoretical perspectives, for example, taking a stakeholder, activity, building block or value flow perspective. The relation among company and its stake- holders is even stronger when it goes beyond the practice and becomes the foundation of the organizational sustainability strategy (Fobbe & Hilletofth, 2021) and the business model. Firstly, Stubbs and Cocklin (2008) conceptualized the business model from a systemic perspective, considering all stakeholders, including nature and society. Similarly, Lozano (2018) proposes and defines a framework for business model for sustainability by adopting a holistic and systemic view to integrate organizational approaches, business systems, stakeholders and sustainability dimensions. Starting from this systemic view, some authors have studied the individual dimensions of value flow associated with stakeholders. On one hand, Bocken et al. (2013) focused on the value proposition of the business model by including both an understanding of the differ- ent forms of value and the stakeholders the company must address. On the other hand, Freudenreich et al. (2020) focus on the mul- tidirectional creation of value between the company and its stake- holders around a common purpose through which stakeholders are engaged in the business model. 2.2 \| Business model for sustainability and stakeholders Traditionally, the business model has been viewed as a tool available to companies useful to describe the logic by which an organization creates, delivers and captures value (Osterwalder & Pigneur, 2010). The firm-centric view of the business model has been overcome by Zott and Amit (2010, p. 216), who described the business model ‘as a system of interdependent activities that transcends the focal firm and spans its boundaries’. The system of activities allows the firm, together with its partners, to create value and also to appropriate a share of that value (Zott & Amit, 2010). The same authors call this concept the ‘networked nature of value creation’, claiming that ‘value creation through business models involves a more complex and inter- connected set of relationships and activities among multiple actors’ (Zott & Amit, 2010, p. 1031). These concepts broaden in the case of business models for sustainability. Indeed, business model for sustain- ability goes beyond the economic value and includes a consideration of positive value for a wider set of stakeholders (Bocken et al., 2013). Despite the relevance of the topic, the literature does not specify how stakeholders contribute to all dimensions of the value flow of business models for sustainability. 2.1 \| Stakeholder theory and sustainability The conceptual link of stakeholder theory with sustainability emerges from some shared concepts such as (1) the purpose of busi- ness that must go beyond maximizing short-term shareholder value; (2) the interconnection between ethical and business issues; and (3) the consideration of a long-term perspective that allows for the creation of stakeholder value now, without compromising the ability to create value in the long term (Hörisch et al., 2014). Indeed, a sus- tainability challenge for companies is to relate with stakeholders on a multitude of contemporary social and ecological issues (Hörisch et al., 2014). This is stated also by the United Nations that include multi-stakeholder partnerships as one of the Sustainable Develop- ment Goals (United Nation, 2018). This paper examines the whole business model for sustainability from a stakeholder theory perspective, taking in consideration all the stakeholders that participate in the value flow with their mul- tidirectional influences. Moreover, while current research focuses only on value creation, this paper takes in consideration the perspective of 4.2 2. employees (Bocken et al., 2014; Lozano, 2018); 3. customers (Alberti & Varon Garrido, 2017; Joyce & Paquin, 2016; Wagner & Svensson, 2014); To select information-rich cases, we adopted a targeted sampling strategy (Creswell, 2013; Eisenhardt, 1989). We started identifying potential cases by looking for companies that explicitly set themselves a sustainability mission. We consider a sustainable mission when it includes long-term environmental and social goals. We have also selected business organizations linked to profit and therefore excluded non-profit organizations or social cooperatives. Conse- quently, to guarantee that selected organizations respect all the selec- tion criteria, we chose B Corps as case studies. B corps are profit- driven companies certified by a no-profit organization to satisfy rigor- ous standards regarding environmental and social performance, accountability and transparency (Long et al., 2018). 4. shareholders (Baldassarre et al., 2017; Chang et al., 2017); 5. government (Hart & Milstein, 2003; Yang et al., 2017); 6. society, such as local communities and territory (Geissdoerfer et al., 2016; Joyce & Paquin, 2016), NGOs (Boons & Lüdeke- Freund, 2013), media (Chang et al., 2017; Clarkson, 1995), future generations (Stubbs & Cocklin, 2008; Upward & Jones, 2016); 6. society, such as local communities and territory (Geissdoerfer et al., 2016; Joyce & Paquin, 2016), NGOs (Boons & Lüdeke- Freund, 2013), media (Chang et al., 2017; Clarkson, 1995), future generations (Stubbs & Cocklin, 2008; Upward & Jones, 2016); 7. other organizations/competitors (Melander & Pazirandeh, 2019; Morioka et al., 2017); 7. other organizations/competitors (Melander & Pazirandeh, 2019; Morioka et al., 2017); 8. influence groups, such as corporations, industrial entities, com- mercial entities, lobbies, external agencies, unions and civic insti- tutions (Chang et al., 2017; Stubbs & Cocklin, 2008); 8. influence groups, such as corporations, industrial entities, com- mercial entities, lobbies, external agencies, unions and civic insti- tutions (Chang et al., 2017; Stubbs & Cocklin, 2008); 9. universities/research institutes (Bocken et al., 2013; Parmar et al., 2010); and 9. universities/research institutes (Bocken et al., 2013; Parmar et al., 2010); and According to the literature, B Corps use ‘the power of business to solve social and environmental problems’ (Stubbs, 2017, p. 299). 10. natural environment (Christ et al., 2018; Lozano, 2018). To create a homogeneous sample, we looked at companies of similar size. In terms of geographic location, we have only included companies with headquarter in Italy to ensure that there are no differ- ences in terms of legal and political parameters. 4.1 \| A qualitative research design 5. Value capture. It includes different forms of benefits captured by different key stakeholders (Short et al., 2014). To explore the contribution of stakeholders to the value flow in busi- ness model for sustainability, we chose a qualitative research design (Eisenhardt, 1989; Yin, 2014). The complexity of the business model for sustainability, the in-depth level of understanding we tend to and the relevance of the peculiarities related to the context have further pushed us towards the use of a qualitative research design (Creswell, 2013). We applied a multiple case study approach to achieve the required depth and to be able to compare results (Eisenhardt, 1989; Eisenhardt & Graebner, 2007). Our conceptualiza- tion is based on model descriptions (Cornelissen, 2016), so our induc- tive research design was helpful in bringing theory out of data (Eisenhardt et al., 2016). 4 ATTANASIO ET AL. other organizations/competitors, influence groups, universities/ research institutes and natural environment) are reported. In the col- umns, the described above dimensions of the value flow of a business model for sustainability are reported. The cells of the framework, which connect rows (stakeholder groups) and columns (value flow dimensions), represent stakeholder contribution to the value flow of the business model for sustainability and the benefit that a stake- holder can receive. In particular, in the cells, we described how stake- holders contributed to a specific dimension of the value flow and what they capture in terms of benefits. 1. Value intention. As described by Barth et al. (2017), it is the atti- tude of the entrepreneur to change, innovate towards sustainabil- ity and create sustainable value. 2. Value proposition. It is, as defined by Patala et al. (2016; p. 144), ‘the promise on the economic, environmental and social benefits that a firm's offering delivers to customers and society at large, considering both short-term profits and long-term sustainability’. 3. Value creation. Value creation ‘begins to flesh out the organisation and architecture of the firm. It also specifies and describes the firm's sources of competitive advantage, i.e., its resources and capabilities’ (Richardson, 2008, p. 139). 4. Value delivery. It represents how the value is delivered to different stakeholders. It is the ‘logical next step and is most closely related with the customer. It is focused on customer relationships, cus- tomer segments and channels’ (Bocken et al., 2018, p. 84). 3.1 \| The value flow in business model for sustainability To address the research question, a systematic review of the literature on business models for sustainability was conducted. The literature was furtherly investigated by considering the two main aspects of this discussion: the value flow in a business model for sustainability and the stakeholders. One of the definitions of a business model for sustainability sees it as a simplified representation of the elements, the interrelationship between these elements and the relation with its stakeholders that a company uses to propose, create, deliver and capture sustainable value for, and in collaboration with, a wide range of stakeholders (Geissdoerfer et al., 2016). Furthermore, the notion of business model for sustainability ‘builds on the business model concept and combines it with the important concepts of stakeholder management (Donaldson & Preston, 1995; Freeman, 1984; Post, Preston & Sachs, 2002), sustainable value creation (Short et al., 2012), a long-term per- spective’ (Geissdoerfer et al., 2016, p. 2). The value is no longer a one-way flow between the company and its customers, but it is created by joint actions and formal and informal alliances with stakeholders who are both recipients and creators of value (Beattie & Smith, 2013; Freudenreich et al., 2020). Several authors have depicted the value flow in business models by consider- ing value proposition, value creation and delivery and value capture (Bocken et al., 2014; Short et al., 2014). To these, Barth et al. (2017) added value intention. The dimensions we considered for analysing the value flow are consequently five and they are defined below. 3.2 \| The stakeholders in business model for sustainability Additionally, within the literature on business models for sustainabil- ity, we investigated the stakeholder groups and their categorizations. Following Hart and Milstein (2003), Rezaee (2016), Lozano (2018) and Oskam et al. (2018), we categorized stakeholders as internal and external to the organizations. We also identified the stakeholder groups as follow: 1. entrepreneur (Barth et al., 2017; Dixon & Clifford, 2007); 4.4 Company B was founded in 2012 in Northern Italy and operates in the wholesale/retail industry. The production activity of the com- pany is entirely outsourced. In fact, in addition to the legal and opera- tional headquarters (located in Italy), the company is also in China with a strategic representative office that ensures the production of high-quality garments for women, men and children, final customers of the company. In terms of geographical expansion, the company operates in 30 countries around the world. The export share has reached 50% of sales thanks to a selected network of wholesale stores. The company has 40 employees. Data collection took place both through semi-structured interviews and secondary data (e.g. annual sustainability reports, press releases, archival documents or company websites). The interviews were conducted through online calls. In order to gain multiple perspective and to analyse the whole organization, inter- viewees were selected among the top management or the key roles actively involved in the sustainability processes. Moreover, semi- structured interviews were conducted with two interviewees per company with an average duration of 1 h (details in Table 1). After an initial series of interviews in 2020, we developed a first draft of stake- holder contributions and used external data sources for triangulation. The following year, we held a second series of interviews with the same entrepreneurs and employees to confirm, adjust and complete our initial understanding. Company C was founded in 1989 in the South of Italy, and it operates in the natural cosmetics industry. The company deals with R&D of natural solutions able to re-establish the well-being, balance and health of the skin and to improve the state of mind of the person. The company group consists of three parts: The first deals with marketing, the second with extraction, production and research, and the third with agriculture and hospitality. In this way, the company has established an integrated local supply chain at ‘Km 0’, directly following all the phases of the production cycle: certified organic cul- tivation; extraction of the organic active ingredients; research and innovation and quality control; production of natural and organic cos- metics; and private label customization (ingredients, texture, olfactory note, desired effect, design, organic certification). These choices favour the ‘green quality concept’, now a pillar of the company phi- losophy, which guarantees the highest standards of quality and sus- tainability of both the production process and the finished product. ATTANASIO ET AL. 5 flow of individual companies, we decided to focus on a sample of medium–small and well-rooted companies in the territory. In total, we worked with five business cases. The cases selected and analysed are described in the following section. expanded into new markets thanks to major investments in automatic lathes and improvements in its processes. Each product is tailor-made and manufactured in a constant relationship with the customer. Maxi- mum customization is aimed at optimizing solutions in relation to dif- ferent requirements. The company works in business to business and on order and has 45 employees. 4.3 Company E was founded in 1965 in the north-east of Italy, and it operates luxury furniture industry. Having become much more inter- national, the company now consists of two divisions: mono-brand shop fitting which deals with the furnishing of high-end retail shops; and tailor-made interiors dedicated to special furnishing solutions, aimed in particular at the end customer. It was created through the acquisition of another company specializing in tailor-made furnishings. Thanks to the acquisition of a company in Michigan, Company E also has a strategic production site for both the shop-fitting market and the HORECA sector in North America. The company has 45 employees and operates, as seen, in both business-to-consumer and business-to-business markets. Company A was founded in the 1960s in Northern Italy and operates in the industrial transport industry. It consists of more than 100 com- panies that deal exclusively with transport on behalf of third parties, core business and strategic key of the activity. Thus, company com- bines the advantages of large industrial fleets, such as capillarity, breadth and guaranteed service, with the advantages of small fleets, such as customer care, flexibility and attention to the goods trans- ported. Company A has two logistics companies, two customs compa- nies, one commercial company and one heavy vehicle rental company, all united by a central management. Company's main customers are large companies belonging to different sectors (e.g. food, packaging and processed products). Moreover, the company has 50 employees and 80 affiliated companies. 4.2 From this analysis, we derived and classified the stakeholders that contribute to the value flow in the business model for sustainability, building a conceptual framework organized as follows. In the rows, the internal stakeholders (i.e. entrepreneur and employees) and exter- nal stakeholders (i.e. customers, shareholders, government, society, In addition to our research, we sought further advice from com- munity experts, with a focus on sustainable entrepreneurship. Since our aim was to identify the contributions of stakeholders in the value 4.6 \| Results from with-in case analysis: Value flow perspective and stakeholder contribution Various stakeholders contribute to the value flow of the business model of Case A. Employees, thanks to the awareness of the benefits obtained, contribute to the value proposition by suggesting sustain- ability innovations through a portal made available by the company. The affiliated drivers contribute to the value delivery because they become promoters of the project by participating in various inter- views and encouraging colleagues to adopt a more sustainable trans- port. Other partners are key stakeholders for the value creation: (1) the agro-livestock cooperative, which built a plant that recovers all CO2 in production (the remaining methane is then purified, cooled and used to fuel the truck), and (2) a company which built the first ser- vice station in Lombardy designed for self-service natural gas refuelling for heavy vehicles and open to cars as well. However, the government contributes as a barrier to value proposition and value creation through regulatory changes. In this section, the case studies are described following the two main perspectives of this research: the value flow of the business models for sustainability and the stakeholder contribution on the value flow. 6 The soci- ety, as a stakeholder, benefits from economic sponsorships in favour of local teams and defibrillators available at each company location. The main benefit for the natural environment derives directly from the creation of value and is the reduction in emissions due to the use of alternative fuels. 4.5 \| Data analysis The data analysis was conducted according to the recommendations of Eisenhardt (1989), Yin (2014). All interviews were recorded and fully transcribed and all documents are synthesized. Finally, the inter- viewees have been asked to review and confirm if the data interpreta- tion was correct. The feedback from the interviewees is essential to avoid bias by the observer. We then analysed the collected data. The data analysis was car- ried out in two phases, first by analysing the individual case studies through detailed descriptions (Yin, 2014) and then looking at the cases as a whole through a cross-case analysis, highlighting elements of con- vergence and divergence (Benbasat et al., 1987; Eisenhardt, 1989; Yin, 2014). We interpreted each individual case study as a separate experiment. We first analysed the business model of each company using the business model canvas tool (Osterwalder & Pigneur, 2010) and then systematically mapped the data on the stakeholder contribu- tion to the value flow that characterize the business model for sustainability. 4.4 The company works in business to business market and has 65 employees. The double collection of data through interviews and external sources allowed to validate the results. As a unit of analysis, we focused on each company's individual business model. In the event that the company adopts multiple business models, we have focused on the main business model of the company oriented towards its sus- tainable mission. We developed an interview protocol to act as a guideline for the data collection process and to ensure the consistency of the informa- tion collected (Yin, 2014). We asked the interviewees questions in order to investigate how stakeholders contribute to corporate sustain- ability. In addition, we paid particular attention to the contribution to the value flow within the business models for sustainability. The first questions are related to the company business models with the aim to highlight the main peculiarities. Secondly, the interviewees should answer to the questions on the dimensions of the value flow of the business model. Finally, the questions cover aspects related to the specific stakeholder contribution to the sustainability. Company D was founded in 1957. In 1985, the founder's sons continued with the business activity, that is, production of turned components (metalworker industry) for the automotive, electrome- chanical and automation industry. Over the years, the company has 6 ATTANASIO ET AL. TABLE 1 Information about the interviews and interviewees TABLE 1 Information about the interviews and interviewees TABLE 1 Information about the interviews and interviewees Company Role Interview duration (2020) Interview duration (2021) A CEO 80 min 34 min Environmental specialist 65 min 52 min B Product and sustainability specialist 60 min 36 min Product and sustainability manager 45 min 50 min C CEO 70 min 30 min Sustainability manager 65 min 30 min D CEO 48 min 52 min Sustainability manager 47 min 38 min E OHS and quality manager 62 min 55 min Chief commercial officer 34 min 45 min reduce vehicle emissions. The value created by the company is then delivered to the customer through partnerships. In addition, Case A distributes value through industry-related trade shows or events related to B corporations. The value flow proposed, generated and delivered is then captured not only by the company but also by other stakeholders. For example, employees benefit from a higher insurance policy than the minimum threshold, an extra budget from the welfare project, sustainability-related training and family engagement initia- tives. The drivers that are considered as partners of the company ben- efit from less pollution during the transport, greater involvement of their families and greater safety during work hours, because the com- pany is committed to enforcing work hours and road rules. The soci- ety, as a stakeholder, benefits from economic sponsorships in favour of local teams and defibrillators available at each company location. The main benefit for the natural environment derives directly from the creation of value and is the reduction in emissions due to the use of alternative fuels. reduce vehicle emissions. The value created by the company is then delivered to the customer through partnerships. In addition, Case A distributes value through industry-related trade shows or events related to B corporations. The value flow proposed, generated and delivered is then captured not only by the company but also by other stakeholders. For example, employees benefit from a higher insurance policy than the minimum threshold, an extra budget from the welfare project, sustainability-related training and family engagement initia- tives. The drivers that are considered as partners of the company ben- efit from less pollution during the transport, greater involvement of their families and greater safety during work hours, because the com- pany is committed to enforcing work hours and road rules. Stakeholder contribution on the value flow of 4.8 \| Case A 4.12 \| of Case C Several stakeholders contribute to the value flow of Case C business model. Specifically, several stakeholders contribute to the value crea- tion. For example, the suppliers or partners contribute to the value creation by providing sustainable packaging and resources. Then, the customers contribute to the value creation by requesting and defining product characteristics. Finally, the university tests the product effi- cacy, a key process of the company's activities and the value creation. As regards the other organizations that contribute to the value flow, there are all companies that join the B-corporation network. They contribute to the value delivery by promoting events to spread the culture of sustainability. In the case of C, the natural environment contributes to the value creation by offering quality raw materials. 4.11 Other stakeholder organizations are the members of the B- corporation network and contribute to the value delivery by promot- ing events to spread the culture of sustainability. In Case C, the value intention is dictated by the entrepreneur who wants to enhance the value of his territory by proposing lines of personalized natural cosmetics that respect the environment. The value proposition is then created through the control of the entire supply chain and the use of organic cultivation. Part of the value created comes from the choice of suppliers. Actually, the company turns only to suppliers belonging to the B-corporation network or to companies that have in place a process of change towards sustainability. Some groups of influence contribute to the value creation. In par- ticular, the Italian biogas consortium enables the value creation by all- owing the annual production of 2000 tons of liquefied methane. 4.10 \| of Case B Several stakeholders contribute to the value flow of the Case B busi- ness model. Suppliers contribute to the value creation by selecting, manufacturing and patenting materials that meet the highest quality and environmental standards. Customers contribute to the value delivery by asking for more in-depth product information, for instance, usage, impacts of microfibres and innovative features of collections. Further stakeholders of the company are the media. The media con- tribute to the value creation by discussing with the company both the sustainability trends of the sector and the measurement of environ- mental impacts along the production process. The stakeholders we defined as other organizations contribute to the value delivery through communication activities and strategic brand consulting with a focus on corporate social responsibility strategies and economic, social and environmental sustainability. Other organizations as B cor- porations are stakeholders of Case C and contribute to the value delivery by organizing and promoting events to spread the culture of sustainability. 4.9 \| Value flow perspective in Case B The value intention of Case B is dictated by the entrepreneur who wants to save wildlife and the natural environment by offering a qual- ity product designed with entirely recyclable parts. As regards the value delivery, customers are reached through industry events and demonstrations or events organized by the B- corporation network. In addition to the value captured by the com- pany related to the commissioned product, the company's employees benefit from an extra bonus. The company captures a portion of value related to increased tourism related to the company-owned wellness centre. In addition, the society benefits from sponsorships related to youth entrepreneurship initiatives and donations destined for schools, also with the help of local associations. The natural environment ben- efits from reduced land use and low-impact cultivation. The value creation is possible thanks to the use of materials and components from qualified sources, with low environmental impact and respectful of social and ethical aspects. A key element in the value creation is the choice of suppliers, which are evaluated against social criteria, in addition to meeting economic and quality criteria. Value is delivered to customers through company-owned stores, e-commerce, retailers and pop-ups or through participation in various events orga- nized by the B corporations' movement. Moreover, customers are reached through the main social networks. The value proposed, cre- ated and delivered is not only captured by the company but also by other stakeholders. In particular, employees benefit from better cor- porate welfare; suppliers, located mainly in China, benefit from better working conditions imposed by the company policy. The society, especially disadvantaged communities and countries, benefit from donations made in collaboration with NGOs or other organizations, which include companies belonging to the B-corporation network. The natural environment benefits from reduced pollution and animal protection. 4.12 \| of Case C 7 ATTANASIO ET AL. 4.7 \| Value flow perspective in Case A In Case A, the value intention is dictated by the entrepreneur who wants to pursue common benefit purposes by proposing as customer value a tailor-made service through a sustainable and inclusive logis- tics in the territory. Therefore, the value creation is based on new technologies and advanced biofuels (e.g. LNG, bioLNG and H2) that 4.11 \| Value flow perspective in Case C 4.11 Stakeholder contribution on the value flow B 4.10 \| of Case B ATTANASIO ET AL. 8 project estimate, realization, delivery, installation and aftersales ser- vices of various kinds (maintenance, repair and management of the replaced supply with recycling, reuse and dismantling services). price but is based on the search for high-quality products, technologi- cally advanced and made with respect for man and the environment. The value created is delivered to the customer through the comp- any's website and trade fairs or B-corporation network events. The value proposed, created and delivered is captured not only by the company but also by other stakeholders. For example, employees benefit from continuous training courses with the aim of enhancing the individual within the work group. Training courses are not only related to work activities but also to improving personal health and safety in the workplace. There are awards in cash for achieving goals. In addition, employees are given 1% of the company's shares. Com- pany pays attention to finding solutions that enable employees to make less physical effort and reduce the risk of manual handling of loads. The creation of value for the company is possible thanks to part- ners (who take care of the carpentry and joinery, key processes for the business) that the company has helped to grow and with whom it has a common purpose. With these partners, the company has also shared technological investments and invested in training to increase its know-how on machines and systems. In addition to its partners, the company relies on monopoly suppliers with whom it does not have a high level of trust. Value is delivered through social networks, trade fairs and the company website (which is currently being modified to minimize energy consumption and emissions from browsing). In addition to these, there are B-corporation events. Different stakeholders capture value. Employees benefit from continuing education courses, family audit trails, smart working (even before the pandemic), team building initiatives, a green space built for lunch breaks and various company welfare initiatives. The company also provides concrete help to employees' families by allowing working parents to benefit from reduced fees to enrol their children in a nursery school, of which the company is a founding partner. The society, as a stakeholder, benefits from the development of the regional territory due to the company's choice to select regional suppliers and, where possible, those that respect sustainability princi- ples. 4.14 \| Stakeholder contribution on the value flow of Case D 4.14 \| of Case D Different stakeholders contribute to the value flow of E. Partners con- tribute to the creation of value by committing themselves to the issues of a responsible forest economy, promoting good management and valorization of forests and their products. They also commit to the company's code of conduct. Furthermore, they contribute to the value delivery by promoting partnerships in the communities in which they operate and by actively addressing the social and environmental challenges they face. The costumers contribute to the creation of value by developing the initial design together with the company's designers. Several stakeholders contribute to the value flow of Case D business model. Suppliers contribute to value creation by providing know-how, raw materials and subsidiaries. Customers also contribute to value creation by requesting and defining product characteristics. Universities and other organizations/ competitors contribute to value creation by researching and develop- ing innovative and sustainable solutions. The stakeholder other organizations/competitors, which include the organizations belonging to the B-corporation network, also con- tribute to value delivery by organizing and promoting events together with the company to spread the culture of sustainability. Other stakeholder organizations are members of the B- corporation network and participate in value delivery by promoting events to spread the culture of sustainability or by collaborating with the company on various initiatives. In addition to this, local associations benefit from the company's support and from various support, dialogue and involvement initia- tives. The environment benefits from the preservation of natural resources, reduced environmental impact (also due to 100% green energy supply) and reduced waste. The society benefits from various initiatives in support of the local area and schools in the company's municipality. The environment benefits from the recycling and reuse of processing waste, separate waste collection, efficient energy consumption, the production of energy from renewable sources and the reduction of emissions. 4.16 \| of Case E 4.13 In Case D, the value intention is linked to intrinsic motivations of the entrepreneur to balance all interests and needs of all stakeholders in a responsible and sustainable way. The value proposition, which is not specifically linked to the value intention, is to offer the market turned metal parts and components such as handles, knobs, components and quick-release couplings. The company's value creation is possible thanks both to the use of the latest generation of numerical control machines, which make it possible to achieve minimum precision toler- ances in the complex machining and finishing of products, and to a high level of expertise. Part of the creation of value also comes from the choice of suppliers, which is determined not only by the logic of 8 10 TABLE 2 (Continued) TABLE 2 (Continued) Value intention Value delivery Test products and R&D of innovative and sustainable solutions Offer quality row material the value dimensions of the business model for sustainability. All these dimensions (i.e. value intention, value proposition, value crea- tion, value delivery and value capture) come from the literature review and represent the value flow that starts with the entrepreneur's inten- tion to create sustainable value. Then, this value is proposed as prod- ucts or services, created and delivered. From a sustainable perspective, the value delivered is captured in the form of benefits by the stakeholders. All the dimensions of the value flow are observed in all the cases as shown in Table 2. that contributes to the value proposition are the employees that could suggest innovations to make the product or service offered more sus- tainable. Moreover, the government could contribute to the value proposition by changing the regulations. This is true in Case A because it belongs to the transportation sector which is very sensitive to changes in regulations. Value is created by a range of activities involving a number of stakeholders. The value creation is a multi-stakeholder issue, whose aim is to stimulate the balanced exploitation of natural resources at the local level and to limit the social and environmental impacts. The suppliers and partners strongly contribute to the value creation; they share the know-how and provide sustainable manufacture and resources and are selected through sustainability criteria in almost all the case studies. Moreover, the universities or research centres can play a role in creating sustainable value as they test the sustainability characteristics of the product and research and develop innovative and sustainable solutions (Case D). In the Case C, that is, production to order, customers can contribute to the value creation through requests of sustainable product or service. A final stakeholder that impact on the value creation are the media, especially in the Case B that belongs to the retail industry. The media discuss with the com- pany about the industry sustainability trends and the measurement of environmental impacts along the production process. The rows of Table 2 contain the stakeholders (both internal and external) mentioned in the literature of business models for sustainability. The cells obtained by the intersections between the rows and the first four dimensions of the value (value intention, value proposition, value creation and value delivery) represent the stakeholder(s) contri- bution on the specific value dimension in the business model for sus- tainability. The cells obtained by the intersections between the rows and the last dimension of the value (value capture) represent the ben- efits that stakeholders can receive. 4.17 \| Results from cross-case analysis: Value flow perspective and stakeholder contribution In Company E, the value intention is dictated by the entrepreneur who, considering himself to be the ‘temporary custodian of a common good’, wants the company to continue over time, going beyond the generations. In this section, the results coming from the case studies are crossed following the two main perspectives of this research: the value flow of the business models for sustainability and the stakeholder contribu- tion on the value flow. The results from cross-case analysis are pres- ented in Table 2. In detail, Table 2 is composed of columns containing The value proposition, which again is not specifically linked to the value intention, is to offer the market luxury furnishing parts for shops, restaurants and living spaces, taking care of all the phases: TABLE 2 Results from cross-case analysis Value intention Value proposition Value creation Value delivery Value capture Internal stakeholders Entrepreneur Pursue purposes of common benefit by balancing the interests of all stakeholders (including society, the environment and the local area) to enable the organization to reach beyond generations Employees Suggest sustainability innovations Benefit from corporate welfare, family engagement, training related to ATTANASIO ET AL. 9 ATTANASIO ET AL. 9 TABLE 2 Results from cross-case analysis Value intention Value proposition Value creation Value delivery Value capture Internal stakeholders Entrepreneur Pursue purposes of common benefit by balancing the interests of all stakeholders (including society, the environment and the local area) to enable the organization to reach beyond generations Employees Suggest sustainability innovations Benefit from corporate welfare, family engagement, training related to ATTANASIO ET AL. 9 TABLE 2 Results from cross-case analysis Value creation Value capture Value delivery g p External stakeholders Suppliers/ partners Adopt, manufacture, and patent sustainable materials and practices Share know-how Promote sustainability projects and partnership in the communities in which they operate to address social and environmental challenges Benefit from family engagement, greater safety during work hours, less pollution Customers Request and define product characteristics Ask for in-depth information regarding the sustainability Government Change regulations Change regulations Society Discuss both the sustainability trends of the sector and the measurement of environmental impacts along the production process Benefit from generalized welfare (e.g. donations and sponsorships) and local development Other organizations/ competitors Promote and communicate the culture of sustainability (e.g. 4.17 \| Results from cross-case analysis: Value flow perspective and stakeholder contribution B-corporation network) Influence groups Enabling the production (Continues) External stakeholders Suppliers/ partners Enabling the production (Continues) (Continues) TABLE 2 (Continued) Value intention Value proposition Value creation Value delivery Value capture Universities/ research institutes Test products and R&D of innovative and sustainable solutions Natural environment Offer quality row material Benefit from reduced pollution, animal protection, low- impact cultivation, recycling and reuse of processing waste, separate waste collection, efficient energy consumption, production of energy from renewable sources and the reduction of emissions 10 ATTANASIO ET AL. ATTANASIO ET AL. 5 \| DISCUSSION From the analysis of the case studies, we are able to derive how stakeholders contribute to the sustainability of the business model. This section compares the results summarized in Table 2 with the existing literature of business models for sustainability and stake- holder theory. All the companies analysed are deeply rooted in the territory in which the company operates or in which it has production plants. Therefore, the main objective of the entrepreneur is to be the spokes- person for the interests of all the stakeholders that interface with the company (including society and the environment), pursuing aims of common benefit. Among the companies analysed, Case A, Case C, Case D and Case E have a strong attachment to the territory and the community in which they operate, and the task of the entrepreneur is to strengthen this bond in order to allow his or her company to con- tinue over time and beyond the generations. In the Case B, the entre- preneur is motivated by more general purpose linked to the protection of animals and the environment. We consider stakeholder contribution as the missing piece of the business model for sustainability literature. Therefore, the two per- spectives (value flow and stakeholder contribution) were combined in the discussion. The result is the stakeholder value flow model (Figure 1). The model was defined by generalizing the results. In our research, the business model for sustainability was con- ceived as a value flow that begins with the entrepreneur's intention to create sustainable value. Value intention of the entrepreneur has been recognized by Barth et al. (2017) as the last of the building blocks for business models for sustainability. Entrepreneurs' connection to sus- tainability has been discussed by several authors in the literature. Firstly, Dixon and Clifford (2007) explored how ‘ecopreneurs’ can cre- ate an economically profitable business while maintaining social and environmental values. Then, Schaltegger, Lüdeke-Freund, and Hansen (2016) studied the different approaches of sustainable entre- preneurship for companies that want to contribute to a sustainable development of mass markets. Finally, Vallaster et al. (2019) analysed the motivations and drives of responsible entrepreneurs and the activ- ities and practices they can implement to create value in the society. Employees contribute to the sustainability of the business model by grasping the challenges that the entrepreneur proposes to them and becoming active in suggesting sustainable innovations. ATTANASIO ET AL. 11 regarding the sustainability of product and service. This is true espe- cially in the Case B that belongs to the retail industry and has the abil- ity to come into contact with end users. The media contributes to sustainability by discussing industry sus- tainability trends with the company and measuring environmental impacts throughout the production process (Case B). All case studies show that other organizations/competitors con- tribute to the sustainability of the business model by promoting and communicating the culture of sustainability. This is visible in the cases members of the B-corporation network. The value capture involves other stakeholders, not only the com- pany. The main stakeholders engaged in the value capture are the employees, benefitting from welfare projects, sustainability-related trainings and family engagement initiatives. The society could capture value especially disadvantaged communities and countries through donations by all the case studies. All the companies are committed to have a positive impact on society and the natural environment so that they as stakeholders could partially capture the value deriving from the business activity. In only one case study (Case A), influence groups contribute to sustainability by enabling the production of a key company resource. Universities and research centres in Case C contribute by testing and researching and developing innovative sustainable solutions. The nat- ural environment contributes to sustainability by providing quality raw material to develop their products (Case C). 4.18 \| Value flow perspective The value intention of the entrepreneur is key in the business model for sustainability because he or she wants to pursue purposes of com- mon benefit to defend the environment (Case B and Case C) and resolve common social issues (Case C and Case E). Moreover, the entrepreneur would like to enable the organization to reach beyond generations (Case E). The value delivery mostly concerns the communication and dis- semination activities of the company's sustainability initiatives. There- fore, other organizations as the members of the associations or networks, to which all the cases belong, affect the promotional activi- ties of the product or service that increase consumer awareness towards the company's commitment to sustainable development. Moreover, the suppliers or partners in Case A and Case E have a role in the value delivery, because they promote the sustainability projects of the company. Finally, the customers ask for in-depth information The value proposition is the direct consequence of the value intention that takes the form of a promise to offer value not only to the customer but to a wider group of stakeholders. The stakeholder 5 \| DISCUSSION Moreover, the employees are aware of the benefits that are obtained thanks to the sustainability commitment undertaken by the companies. Suppliers and partners contribute to the sustainability of the busi- ness model by sharing their sustainability know-how, adopting, pro- ducing and patenting sustainable practices and materials to satisfy their customers (all cases). In Case A and Case E, same stakeholders contribute to the sustainability of the client company by promoting sustainability projects and partnerships in the communities where they operate. We consider value intention as the initial and fundamental dimen- sion to reach sustainable impact. From the case studies, it emerges that the sustainable vision together with the entrepreneur's intention- ality is key element to adopt a business model for sustainability. For this reason, in the stakeholder value flow model (Figure 1), we have positioned the value intention as first ideal dimension. The value prop- osition has a greater degree of concreteness than the value intention; therefore, in Figure 1, it has been positioned below. This because value proposition is understood as the product or service that have to generate value for a network of stakeholders (Baldassarre et al., 2017). Customers contribute to the sustainability by exercising their power to choose the company from which to request their sustainable product or service. This is realized in Cases A, C, D and E by defining the characteristics of the product and service that the company will create. In addition to exercising their power of choice, the customers interface directly with the company to request more information about product sustainability (Case B). We learn that the government makes a negative contribution in terms of industry regulations from Case A. The value proposition has been studied by several authors in the literature. For example, Bocken et al. (2013) identified different FIGURE 1 The stakeholder value flow model. Note: Internal stakeholders are marked with black borders [Colour figure can be viewed at wileyonlinelibrary.com] 12 ATTANASIO ATTANASIO ET AL. 12 FIGURE 1 The stakeholder value flow model. Note: Internal stakeholders are marked with black borders [Colour figure can be viewed at wileyonlinelibrary com] FIGURE 1 The stakeholder value flow model. Note: Internal stakeholders are marked with black borders [Colour figure can be viewed at wileyonlinelibrary.com] holder value flow model. Note: Internal stakeholders are marked with black borders [Colour figure can be viewed a types of value to propose to a wide range of stakeholders. 6 \| CONCLUSION Adams, R., Jeanrenaud, S., Bessant, J., Denyer, D., & Overy, P. (2016). Sus- tainability-oriented innovation: A systematic review. International Jour- nal of Management Reviews, 18(2), 180–205. https://doi.org/10.1111/ ijmr.12068 The research aims at contributing to the knowledge on business models for sustainability by looking for relationships with stakeholders that could be replied by other companies. The paper contribution is a stakeholder value flow model of business models for sustainability that categorizes the stakeholders in the specific value flow dimension. The model can facilitate a systematic and deeper analysis of stake- holder contributions, coming largely from outside. Alberti, F. G., & Varon Garrido, M. A. (2017). Can profit and sustainability goals co-exist? New business models for hybrid firms. Journal of Busi- ness Strategy, 38(1), 3–13. https://doi.org/10.1108/JBS-12-2015- 0124 Amankwah-Amoah, J., Danso, A., & Adomako, S. (2018). Entrepreneurial orientation, environmental sustainability and new venture perfor- mance: Does stakeholder integration matter? Business Strategy and the Environment, 28(1), 79–87. https://doi.org/10.1002/bse.2191 Moreover, the stakeholder value flow model can be used to map from the company point of view the most significant relationship and to help companies to inspire and facilitate the stakeholder engage- ment for business models for sustainability in the future. Baldassarre, B., Calabretta, G., Bocken, N. M. P., & Jaskiewicz, T. (2017). Bridging sustainable business model innovation and user-driven inno- vation: A process for sustainable value proposition design. Journal of Cleaner Production, 147, 175–186. https://doi.org/10.1016/j.jclepro. 2017.01.081 Coherently with the objectives of the study, the identification of stakeholder relationships with the business model could support both researchers and enterprises in mapping the value flow. From the aca- demic point of view, the research attempts to reduce the knowledge gap on business models for sustainability and stakeholder theory. From the managerial point of view, the research underlines those entrepreneurs and managers must consider not only the customer but also a wide range of stakeholders. These stakeholders contribute not only to value creation as most of the literature has indicated but also to the other value dimensions of the business model for sustainability. Moreover, the research shows from a practical perspective that busi- ness models are open systems that do not depend exclusively on internal practices, activities and processes or internal stakeholders. For this reason, in addition to the engagement, managers and entre- preneurs must consider the alignment of stakeholders with the organi- zation's objectives. Barth, H., Ulvenblad, P. O., & Ulvenblad, P. (2017). ORCID training and family engagement initiatives. The society benefits from generalized welfare and local development, the suppliers/partners from family engagement, greater safety during work hours and the natural environment benefits from animal protection, low-impact cul- tivation and efficient energy consumption. Giovanna Attanasio https://orcid.org/0000-0002-9091-7300 Nadia Preghenella https://orcid.org/0000-0001-7058-6578 Alberto Felice De Toni https://orcid.org/0000-0001-8961-6378 Cinzia Battistella https://orcid.org/0000-0002-7953-4923 6 \| CONCLUSION Towards a conceptual framework of sustainable business model innovation in the agri-food sector: A systematic literature review. Sustainability (Switzerland), 9(9), 1–15. https://doi.org/10.3390/su9091620 Beattie, V., & Smith, S. J. (2013). Value creation and business models: Refocusing the intellectual capital debate. British Accounting Review, 45(4), 243–254. https://doi.org/10.1016/j.bar.2013.06.001 Benbasat, I., Goldstein, D. K., & Mead, M. (1987). The case research strat- egy in studies of information systems. MIS Quarterly, 11, 368–386. https://doi.org/10.2307/248684 Bocken, N. M. P., Schuit, C. S. C., & Kraaijenhagen, C. (2018). Experimenting with a circular business model: Lessons from eight cases. Environmental Innovation and Societal Transitions, 28, 79–95. https://doi.org/10.1016/j.eist.2018.02.001 Bocken, N. M. P., Short, S., Rana, P., & Evans, S. (2013). A value mapping tool for sustainable business modelling. Corporate Governance, 13, 482–497. https://doi.org/10.1108/CG-06-2013-0078 Bocken, N. M. P., Short, S. W., Rana, P., & Evans, S. (2014). A literature and practice review to develop sustainable business model archetypes. Journal of Cleaner Production, 65(4), 42–56. https://doi.org/10.1016/j. jclepro.2013.11.039 The research proposes a model that could be adopted and implemented in real corporate environments and can be the starting point for future research directions. The relationship between the company and its stakeholders can be explored by adding the time var- iable. Companies at the beginning of their sustainability stage might have different stakeholders with different contributions in value dimensions, compared to a more advanced stage of sustainability. In addition to this, other dimensions of value for business models for sustainability could be identified. Boons, F., & Lüdeke-Freund, F. (2013). Business models for sustainable innovation: State-of-the-art and steps towards a research agenda. Journal of Cleaner Production, 45, 9–19. https://doi.org/10.1016/j. jclepro.2012.07.007 Boons, F., Montalvo, C., Quist, J., & Wagner, M. (2013). Sustainable inno- vation, business models and economic performance: An overview. Journal of Cleaner Production, 45, 1–8. https://doi.org/10.1016/j. jclepro.2012.08.013 Bridoux, F., & Stoelhorst, J. W. (2016). Stakeholder relationships and social welfare: A behavioral theory of contributions to joint value creation. Academy of Management Review, 41(2), 229–251. https://doi.org/10. 5465/amr.2013.0475 5 \| DISCUSSION However, in our research, stakeholders not only receive but are an active part in the various dimensions of value. In fact, the case studies analysis revealed that both the government (external stakeholder) and employees (internal stakeholders) contribute to the value proposi- tion. Stubbs and Cocklin (2008) and Chang et al. (2017) pointed to the government as the stakeholder that plays a significant role in changing the economic and sociopolitical environment of businesses by facilitating the transition to sustainability through, for example, tax incentives. Indeed, government is forcing organizations to address environmental issues through a variety of restrictions. Our research shows that government can contribute negatively by hin- dering the value proposition. Employees who are considered value beneficiaries in the literature (e.g. Bocken et al., 2013) contribute to the value proposition by improving it and making it more sustainable. contributions to joint value creation. Freudenreich et al. (2020) analysed which value is created and exchanged between stakeholders and the organization. Our research specifies which are those contribu- tions and broadens the spectrum, highlighting how external stake- holders contribute to value creation, including customers by requesting and defining sustainable product characteristics, universi- ties and research institutes by testing products and searching for sus- tainable and innovative solutions and society by discussing with the company the impacts along the production processes. At the same level as value creation, there is value delivery. To the best of our knowledge, in the literature, there is no consideration of this dimension of value associated with stakeholder contributions. Therefore, this research identifies the contribution of external stake- holders on value delivery. Customers request more information about sustainability through the company's channels; suppliers/partners and other organizations/competitors act as channels themselves by pro- moting and communicating sustainability initiatives. In the value flow, the value proposition has to be realized through the value creation. Value creation is the focus of many discussions of business models for sustainability and stakeholder theory. Bridoux and Stoelhorst (2016) argue that businesses play a crucial role in pro- moting social welfare through their ability to foster stakeholder Finally, the value flow delivered is captured in the form of bene- fits from internal stakeholders (employees) and external stakeholders (natural environment, suppliers/partners, society). For example, employees benefit from welfare projects, sustainability-related 13 ATTANASIO ET AL. 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https://openalex.org/W2332130592	https://www.per-central.org/items/perc/3686.pdf	English	null	Understanding Student Preparation of Exam Note Sheets	null	2,014	cc-by	3,382	INTRODUCTION Despite the fact that this study yielded rich and interesting data, it was limited in epistemological insight simply because it allowed for only inferences at best. For example, a student who chose to include mostly equations on her note sheet (as was the case frequently) may have seen equations as the only important things in the whole class or the only items worth committing to paper. I also could not say whether students mindlessly commit notes to paper or judiciously choose what to include or leave out. Previous research has used methods such as interviews [1] or multiple-choice surveys [2] to gain insight into student epistemologies (defined here as context-dependent beliefs about the nature of science and learning). This study is an extension of previous work [3] where I tried to uncover student epistemological thinking by studying the note sheets students prepare for their final exams. As indicated in the conclusion of my previous work, I decided to address some of these questions. I incorporated an anonymous survey into my collection of the students’ note sheets with the goal of getting more direct information from students regarding their preparation of these sheets. In that work, I allowed students in two introductory mechanics courses to bring a 4” x 6” notecard for the midterm and one side of an 8.5” x 11” sheet for the final exam. I sought to reduce emphasis on equation memorizing as well as answer the following questions: (1) In what different ways do different students choose to use the allowed note space? (2) Can we find associations between what students choose to include and their exam performance? (3) What can student notecards tell us about what the students see as important in the course and what might be examined? Inspired by feedback I received on my previous work, the questions I asked were: (1) How did you decide what to write on your note sheet? (2) How was either writing or having the note sheet helpful to you, if at all? (3 – for the final only) Was your preparation of notes for this [the final] exam different than it was for the midterm? How? I classified what students included on the sheets with minimal ambiguity. The majority of student exam notes were classified as equations, but other prevalent categories included definitions, units, physical constants, conceptual statements, pictures and diagrams, and guides to symbols. , edited by Engelhardt, Churukian, and Jones; Peer-reviewed, doi:10.1119/perc.2013.pr.050 Published by the American Association of Physics Teachers under a Creative Commons Attribution 3.0 license. Further distribution must maintain attribution to the article’s author, title, proceedings citation, and DOI. 2013 PERC Proceedings Understanding Student Preparation Of Exam Note Sheets Timothy L. McCaskey of Science and Mathematics, Columbia College Chicago, 600 S. Michigan Ave., Chicago IL 60605 Department of Science and Mathematics, Columbia College Chicago, 600 S. Michigan Ave., Ch Abstract: In a previous study, I allowed introductory physics students to create a notecard (or sheet) for their midterm and final exams in an attempt to remove equation memorizing as a focus of the course. I hoped to use the study of these cards as an epistemological lens that would uncover their perceptions and attitudes about the course. Without follow-up questions, though, epistemology remained unclear. I have continued this line of research by adding anonymous survey questions that probe why students chose to include what they did, how (if at all) the notes were helpful, and how their card preparation changed throughout the semester. Through my analysis, I found cases where the survey questions reveal epistemological insight about students the note sheets alone would not. For example, one student with an equation- centered note sheet did not see equations as the most central course component, but he considered himself fluent enough in concepts that he felt he could leave conceptual statements out. I also discuss some of the more thoughtful survey answers I received, some future study possibilities, and efforts to discuss exam preparation in the classroom. Keywords: student notes, epistemology, assessment PACS: 01.40.Fk, 01.40.G- INTRODUCTION Though my data sets were too small for rigorously mathematical conclusions, I found little relationship between the number of notes on a note sheet and the final grade; specifically, two note sheets I analyzed in detail from opposite sides of the grade spectrum were quite similar in raw number of notes but different in organization. As with last year’s data, student note sheets were heavily equation-centered. I will document the survey results and focus on cases where students either had unique takes on the “equation sheet” strategy or explicitly focused on something besides equations. Keywords: student notes, epistemology, assessment PACS: 01.40.Fk, 01.40.G- STUDENT POPULATIONS Columbia College Chicago is one of the largest arts and media colleges in the USA. We have no physics majors, and most students neither major nor minor in 249 retrieved their midterm exam notecards later, but I returned neither the exam nor note sheet from the final. retrieved their midterm exam notecards later, but I returned neither the exam nor note sheet from the final. science. Our two courses focused on mechanics are Physics for Filmmakers [4] (PfF) and Fundamentals of Physics I (FoP). These courses provide the present study’s population. Preparation and Collection The final survey question asked if their note preparation was different between the final exam and the midterm exam. All but two students in the PfF population and Jordan in FoP indicated the process was similar, and three students in each group said the main difference was simply the amount of material to be covered on the exam. In the Filmmakers population, only one student gave a more nuanced answer: “I knew a little better what to expect so I didn’t put ALL the variations of the equations like I did last time. Just the main ones.” The other Filmmakers outlier was the student who barely prepared anything at all. Representing the FoP population, Jordan indicated, “I studied more of the concepts that I didn’t understand more so than I did before.” For both courses, students were informed well in advance that they would be allowed to bring notes to the exam, and they were told they could use the paper space in any way they wished. Students were also informed that the final exam would consist of multiple sections. Both courses’ exams featured two parts: a conceptual section containing multiple choice items, short answer problems, and graphing representations, and a more mathematical section consisting of written problems. The FoP exam was more conceptually and mathematically involved, so those students received a three page guide listing topics and sample problems. Because I was the course instructor, participation in the survey was kept optional and anonymous. All students who consented to my survey questions prepared notes, but not all spent a long time doing so. Students left their names off the note sheets and attached them to their survey responses. Students Observations Regarding Final Exam Survey Questions – Spring 2013 y p p Both courses in this study are algebra-based. PfF uses clips from action and science fiction movies to motivate discussion about kinematics, dynamics, collisions, energy, and other mechanics topics. Labs are often filmed and analyzed using Tracker [5] software. Most of the students in this class come from the Film and Video department. The goal of the course is to help them make more deliberate choices when depicting physics in their artistic work. Students must complete a final project where they demonstrate correct physics in a film they create. The Spring 2013 version of the course started with seventeen students, with fourteen completed the final exam and eleven completing the note sheet with survey questions. As in my previous study, a large portion of the note sheets turned in were devoted to equations. Every note sheet included a number of them with the exception of a single PfF note sheet with only three equations on it. This trend was not a surprise, but I checked to see how many students explicitly mentioned writing down as many formulas as possible as well as why. Eight of the eleven FoP students and seven of ten PfF students prominently mentioned writing out formulas as part of the note-writing process. Students mentioned using other course resources less often when constructing their note sheets. Among the ten students studied in the Spring 2013 Physics for Filmmakers course, two of the students said they referred to old quizzes, four reported using their notes (either online or from their notebooks), and one used his or her textbook as a reference for the note sheet. Among the eleven Spring 2013 FoP students I surveyed, five used their notes (either online or from their notebooks), one mentioned looking through old homework, and one student, who I will codename “Jordan,” explicitly brought up conceptual material. Specifically, Jordan said, “I wanted to get as many concepts as possible with some examples. I followed the concepts / practice problem sheet you gave us as a guideline.” In the next section, I will compare Jordan’s note sheet with those of students who self-reported their preparation as more equation-centered. FoP has more traditional mechanics course coverage and is taken primarily by Acoustics students in our new Bachelor of Science program. The Spring 2013 version of the course had seventeen students. Observations Regarding Final Exam Survey Questions – Spring 2013 Course activities included lectures, labs (some in common with PfF), tutorials, and demo-centered discussions. Ten students completed the survey. FoP included more mathematics, including a small amount of trigonometry which was not a PfF prerequisite. Many FoP students are either enrolled in or are planning to take calculus. There was still noticeable overlap between the final exams for the two courses, especially on conceptual questions. More Reflective Survey Responses From Midterm Exams In addition to Jordan’s response mentioned above, the other two of Jordan’s survey questions were concept-centered, including, “I was able to use a lot of what I wrote to help with concepts on the test,” and “I studied more of the concepts that I didn’t understand.” Not only was Jordan’s note sheet the lengthiest I received this semester, it was most balanced in content and approach. The midterm and final exam surveys occasionally gave different types of student responses. In Fall 2012 and Spring 2013, students seemed more willing to reflect more after the midterm than after the final. I received more thoughtful epistemological responses to my survey items, likely due to the fact that the exam conditions were less stressful. Jordan’s sheet featured over seventy equations, but also at least fifteen detailed conceptual statements, an equal number of diagrams, including a number of translations between position, velocity, and acceleration graphs, ten fully worked examples, and a number of definitions, unit guides, and physical constants. In my past work, I’ve seen sheets as lengthy and well-prepared, but the focus of Jordan’s survey answers is interesting. Without seeing Jordan’s survey, I would not have anticipated such a concept- centered attitude toward the note sheet given how prevalent the equations and mathematics were, despite a larger-than-average focus on concepts. The most recent final exam survey answers in FoP, for example, tended to be terse one or two sentence answers. The lengthiest response not belonging to Jordan said, “I thought the most important thing would be to be able to solve all the equations[;] I also decided to have some SI units and Kepler’s laws.” While some of my midterm responses were that length and depth, other students from the Fall 2012 class were more reflective, such as one who said, “I looked through each homework, lab, and quiz and thought I had a good enough understanding of the concepts that all I needed to know was the formulas. Although after going through the test I realized I didn’t know this stuff well enough conceptually, and so all my equations look meaningless.” Another wrote, “Probably should have included some homework examples, but I didn’t, regretting that now. A Comparison Of Relevant Note Sheets In my previous work, I did a detailed comparison of two note sheets from students who both wrote a large amount of material but performed differently on the 250 definitions from kinematics. In the survey, Dana indicated that this list “was a little helpful. I forgot a few things I should’ve written down.” My best guess is that if Dana had written more, it would have been equations relating to different concepts. Many topics covered on the exam were neglected. Without the survey, I might have assumed Dana was simply focused on equations and poorly-prepared, but his survey response indicated he felt comfortable with the concepts. I could not have known that fact about Dana’s self-efficacy from the note sheet alone. exam. I cannot do an exam comparison in the present study since the surveys were anonymous. However, I will compare the sheets from three Spring 2013 FoP students where the students approached the note sheet task from different epistemological angles. One student I will look at in detail is Jordan, described above. Another is “Georgie,” a student who tersely noted that he “wrote just equations,” and “Having formulas helps a lot.” Finally, I will look at “Dana,” a student who mentioned both concepts and equations in his survey. Dana’s wrote, “I knew concepts really well, so all I really needed was a list of equations I would need.” I will break down the content of these three sheets and discuss their organization. Class Discussions One hope of mine in giving the survey at the midterm was for students to be able to modify or improve their performance on the final. I thought that if given a chance to be reflective on their note-writing process, students would be inspired to discuss that process after the fact and come up with ideas they would use on the final. This past semester, that did not happen much with either class. Though I devoted some time in both classes just before the final to reviewing content and exam composition, the students did not explicitly take it as a chance to improve their note sheets. However, there were examples found in the data where students self-reported insights into their epistemological thinking that would have been difficult to anticipate from the note sheets alone. Jordan’s outlook on the survey questions was centered on concepts, despite the mathematical focus on much of that note sheet. Dana gave the most minimal, equation-centered sheet I received, but this was not due to an equation-centered epistemology necessarily; conceptual self-efficacy was evident. Georgie’s note sheet matched closely with the corresponding survey questions. In summary, epistemological insights I have gained from this study come from directly asking relevant epistemological questions to students. The classification of note sheet contents helped point to pieces of data that would reward deeper study, but it did not serve as an epistemological study in itself. There are a few steps I can take in future semesters to improve this. One is to weave thinking about note sheets into homework. My students are always willing to discuss homework questions at the start of class, so perhaps a writing prompt before or after the midterm would spark a discussion about useful study strategies. A prominent benefit cited by students in making these sheets is that preparing the sheets helps them to study. In fact, I received feedback on an earlier presentation of this work indicating that the students would be better off if their lists of notes were explicitly called “study sheets” rather than “note sheets” or “cheat sheets.” g y The prior semester’s surveys occasionally gave far more reflective survey responses. Ideally, I would like to see similar depth from everyone either in a class discussion or on his or her survey. REFERENCES Another option one could pursue to distinguish between equation-centered students and conceptually- focused ones is to provide a common list of equations for all students and give them the ability to add to that list. Since this method could deprive students of the benefit of writing their own equations, I will not likely pursue this modification in the near future. 1. T. L. McCaskey, Ph.D. dissertation, University of Maryland, College Park, 2009. 1. T. L. McCaskey, Ph.D. dissertation, University of Maryland, College Park, 2009. 2. E. F. Redish, R. N. Steinberg, and J. M. Saul, Am. J. Phys. 66, 212-224 (1998). 2. E. F. Redish, R. N. Steinberg, and J. M. Saul, Am. J. Phys. 66, 212-224 (1998). 3. T. L. McCaskey, “Using Student Notecards As An Epistemological Lens,” AIP Conf. Proc. 1513, 290-293 (2013). 3. T. L. McCaskey, “Using Student Notecards As An Epistemological Lens,” AIP Conf. Proc. 1513, 290-293 (2013). 4. T. L. McCaskey, “Physics for Filmmakers: Goals, Tracker Labs, and Projects,” 2011 AAPT Summer Meeting, Omaha, NE. 4. T. L. McCaskey, “Physics for Filmmakers: Goals, Tracker Labs, and Projects,” 2011 AAPT Summer Meeting, Omaha, NE. Possible Study Extensions It is likely that a semi-structured interview starting with my written survey questions may be the best way to get detailed responses from students. As course instructor, I had ethical concerns interviewing my own students, but I would be in favor of using interviews for a population outside my personal courses. On a written survey, even the most thoughtful students will only write a sentence or two, and even a short interview could indeed prove richer in comparison. Class Discussions Decisions regarding how to get the class to discuss their exam preparation remain; I could either incorporate epistemology- focused prompts into the homework or devote more time to class discussion. Though many students saw the note sheet writing as useful, it is an open question as to what setup of these tasks would help them both understand the material best and sympathize with my main epistemological goal for the courses: that students should see physics understanding as consisting of multiple types of thinking and representation. More Reflective Survey Responses From Midterm Exams I mostly studied by reviewing homework, but I seem to have glossed over some things, thinking I understood them better than I do.” These answers were not only more thoughtfully written out than my final exam surveys in general, they were more reflective about the studying process. Georgie wrote a lengthy and well-organized equation sheet for notes. There were over forty different equations, and five definitions written entirely in equation form. To say that Georgie only wrote equations is not entirely correct, but much of the extra writing was equation-centered also. There were twenty separate symbol guides that explained the equations’ terms. These equations and symbol guides was just about the sum of it; I coded only two statements as conceptual: one stating energy conservation and one explaining the role of the velocity vector in the expression for angular momentum of a free particle. Finally, Georgie included the value of G and the SI unit breakdown of a Newton. Words above groups of equations indicated general subject area, like “Gravity,” “Energy,” or “Centripetal.” One was even more explicitly epistemological. His (or her) notes were centered “mostly around formulas and example problems. I did this not to re-‘replicate’ them, but to have some sort of step-by-step basis and understanding for long answer problems. I knew multiple choice/graphing were to be based on true knowledge.” Having students articulate that more conceptual-styled problems touch on “truer” knowledge is a benefit to this survey methodology one cannot get from a simple analysis of note contents. Dana’s sheet, as indicated, was also extremely equation-centered – in fact, more so than Georgie’s. The sheet included fourteen equations and nothing else. There were no guides to symbols, pictures, examples, conceptual statements, or even equation-like 251 epistemologies from what they choose to include in their allotted space. Previous work including mostly note sheet contents allowed only the most superficial inferences, so the present study added survey questions that asked students to reflect about what they felt was important to include. For the most part, as anticipated, the students felt equations were the most important thing to focus on in preparing the note sheets. FUTURE DIRECTIONS Class Discussions SUMMARY AND CONCLUSIONS I began work on studying student exam note sheets not simply from a desire to review the contents, but to investigate what we can learn about student 5. http://www.cabrillo.edu/~dbrown/tracker/ 5. http://www.cabrillo.edu/~dbrown/tracker/ 252
https://openalex.org/W3017322662	https://europepmc.org/articles/pmc7171129?pdf=render	English	null	Matrix Gla protein is an independent predictor of both intimal and medial vascular calcification in chronic kidney disease	Scientific reports	2,020	cc-by	8,224	Matrix Gla protein is an independent predictor of both intimal and medial vascular calcification in chronic kidney disease PEN Armand M. G. Jaminon1, Lu Dai2, Abdul Rashid Qureshi2, Pieter Evenepoel3, Jonaz Ripsweden4, Magnus Söderberg5, Anna Witasp2, Hannes Olauson2, Leon J. Schurgers1,6 & Peter Stenvinkel2,6* Matrix Gla protein (MGP) is a potent inhibitor of vascular calcification (VC) and requires carboxylation by vitamin K to exert calcification inhibition. Chronic kidney disease (CKD) patients undergo early vascular aging often involving extensive VC. The present cross-sectional study investigated the association between circulating dp-ucMGP levels, MGP expression in vascular tissue and MGP polymorphisms. In 141 CKD stage 5 patients, CAC score was significantly increased in the highest tertile of dp-ucMGP (p = 0.002), and a high medial VC score was associated with elevated dp-ucMGP levels. MGP vascular expression was associated with increased circulating dp-ucMGP and CAC scores. MGP SNP analysis revealed that patients homozygous for the C allele of the rs1800801 variant had a higher CAC score (median 15 [range 0–1312]) compared to patients carrying a T allele (median 0 [range 0–966] AU). These results indicate that plasma levels of dp-ucMGP are an independent predictor of increased VC in CKD5 patients and correlate with both higher CAC scores and degree of medial calcification. Additionally, high vascular expression of MGP was associated with higher CAC scores and plasma dp-ucMGP levels. Taken together, our results support that MGP is involved in the pathogenesis of VC. Matrix Gla protein (MGP) is a vitamin K dependent protein (VKDP) that is involved in the inhibition of vascular calcification (VC). MGP is small secretary protein (14 kD) that is primarily secreted by vascular smooth muscle cells (VSMCs) in the arterial wall1. MGP contains five Glu residues that require carboxylation to become activated and to fulfill its calcification inhibitory function. This carboxylation step cannot take place in the absence of vitamin K, which has an unequivocal role in driving this post-translational step2,3. Vitamin K is a co-factor for the enzyme γ-glutamyl carboxylase that converts glutamic acid (Glu) into γ-carboxyglutamic acid (Gla) residues2. This conversion is critical for the activation of MGP. Additionally, there are three serine residues that need phosphorylation4,5. The exact role of phosphorylation of MGP is still not known, but it is believed to play an important role in the regulation of secretion of the protein3. Upon activation, MGP binds calcium-salts with high affinity, thereby affecting the calcification processes. www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports \| (2020) 10:6586 \| https://doi.org/10.1038/s41598-020-63013-8 Results In a multivariate logistic regression analysis of factors associated with high medial VC (Table 3) (VC score media (0–1) = reference), high medial VC was associated with age (p = 0.006), sex (p = 0.001), dp-ucMGP (p = 0.04) and DM (p = 0.008) after adjusting for hsCRP and CAC score (p = 0.059) (Table 3). High arterial MGP mRNA expression associates with high CAC. We divided patients according to median of MGP mRNA expression. High expression of MGP (n = 41) showed higher CAC score with a median (10th – 90th percentile) of 16 (0–1804), compared to the low MGP expression group with a median (10th – 90th percentile) of 0 (0–255) (p = 0.036) (Fig. 3a). Additionally, high expression of MGP showed higher dp-ucMGP plasma levels, with a median (10th - 90th percentile) of 1459 (711–5313), compared to low MGP expression with a median (10th - 90th percentile) of 1100 (760–1653) (p = 0.034) (Fig. 3b). In a Spearman’s rank (rho) analysis, MGP expression was significantly correlated with CAC score (Rho = 0.39) and medial calcification score (Rho = 0.37). After adjusting for age and sex in a multivariate linear regression analysis, these significant correlations were lost. MGP polymorphisms - relation to circulating dp-ucMGP, arterial MGP mRNA and VC. Distributions of MGP rs4236, rs1800801, rs1800802 polymorphisms in CKD5 patients and controls are presented in Supplemental Table S2. The genotype frequencies of the rs4236 and rs1800801, but not rs1800802, differed significantly between patients and controls: in patients, the rs4236 T/T and rs1800801 C/C genotypes were more common (44% vs. 39% and 53% vs. 44%, respectively), whereas rs4236 C/C and rs1800801 T/T were less frequent (both 6% vs. 15%) compared to controls. A selection of patient characteristics was investigated in relation to MGP genotypes (Supplemental Table S3). None of the MGP SNPs were associated with age, sex, DM or dp-ucMGP levels. Neither the rs1800801 nor the rs1800802 SNPs associated with MGP mRNA levels but when dividing patients according to rs4236 T-homozygotes vs. C-allele carriers, MGP mRNA levels were found to be higher in patients homozygous for the T allele compared to patients with CC and CT genotype (1.3 vs. 0.9 RQ, p = 0.03). Whereas patient’s CAC scores were not associated with MGP genotypes, media VC score differed sig- nificantly according to rs1800801 genotype, with 50% of C homozygotes having a moderate to extensive calcifica- tion (i.e. Results Clinical and biochemical characteristics. Demographic and clinical characteristics are shown in Table 1 and Supplemental Table S1. Patients in the highest tertile of dp-ucMGP (>1491 pM) levels were older and had higher CVD prevalence (25%). Moreover, serum creatinine was higher in the highest tertile of dp-ucMGP com- pared to the other two tertiles (782 μmol/L). Additionally, glu-OC (37.8 ng/mL), mid-OC (90.5 ng/mL) and Osteoprotegerin (7.6 pg/mL) were also elevated in the highest tertile of dp-ucMGP. dp-ucMGP levels were associated with increased CAC and medial calcification score. Patients in the 3rd tertile showed higher CAC score compared to the other two tertiles ranging from a CAC score with a median (10th – 90th percentile) of 0 (0, 236) in the 1st 8 (8, 1392) in the 2nd and 34 (0, 1948) in the 3rd dp-ucMGP tertile (p = 0.002) (Fig. 1a). Medial VC (scored by pathologists, ranging from 0 - no, 1 – mild, 2 – moderate, to 3 - severe calcification) was associated with an increase in dp-ucMGP levels (median 1193 in group 0–1 and 1458 in group 2–3) (p < 0.05) (Fig. 1b). Calcification of the vascular media co-localized with ucMGP. Patients that had a medial VC score of 0 showed no staining for ucMGP (Fig. 2a). On the contrary, patients that had a medial VC score of 3 showed a strong staining of ucMGP around calcified areas (Fig. 2b). Calcified areas are accompanied by a loss of structure and show no cellular components, the arrows indicate calcified areas that stain highly positive for ucMGP (Fig. 2b). Calcification of the vascular media co-localized with ucMGP. Patients that had a medial VC score of 0 showed no staining for ucMGP (Fig. 2a). On the contrary, patients that had a medial VC score of 3 showed a strong staining of ucMGP around calcified areas (Fig. 2b). Calcified areas are accompanied by a loss of structure and show no cellular components, the arrows indicate calcified areas that stain highly positive for ucMGP (Fig. 2b). Multivariate analysis of factors associated with dp-ucMGP. In multivariate linear regression analysis of determinants, bone markers were associated with dp-ucMGP (i.e. 1-SD increase) including ucOC (est 0.48; p = 0.001) and OPG (est 0.28; p = 0.02) after adjusting for 1-SD increase of age and albumin (Table 2). Multivariate analysis of factors associated with medial calcification. www.nature.com/scientificreports/ www.nature.com/scientificreports/ levels associate with VC8,9, cardiovascular morbidity-mortality10 and aortic valve calcification11. However, the connec- tion between local vessel wall expression of MGP (transcription), production (tissue ucMGP) and excretion (plasma dp-ucMGP) is not well explored in CKD. In addition, the vasculature is exposed to a toxic uremic milieu, which has been shown to affect carboxylase activity causing vascular vitamin K deficiency and increased VC12. Additionally, uremia promote VC in a rat model13 and induced bone-specific proteins in cultured VSMCs14.h p pi p The use of dp-ucMGP has clinical potential as a prognostic biomarker of VC15 and might provide complemen- tary information to traditional cardiovascular disease (CVD) risk factors16. Furthermore, carboxylated MGP bind, via the negative charge, to active calcification. The inactive form of MGP (dp-ucMGP) is set free in the circulation because of lack of negative charge to bind to calcium crystals and, thus, might be used as biomarker to identify high-risk CVD patients, allowing early intervention17. Furthermore, although single nucleotide polymorphisms (SNP) of MGP have been associated with outcomes in diabetes and CKD, its association to VC remains obscure. MGP SNP analysis could help us understand the complex nature of MGP expression and regulation. Other VKDPs, such as osteocalcin (OC) share the carboxylation step to become activated. Similar to MGP, uncarboxylated OC (ucOC) is increased in vitamin K deficiency18 and has been reported to have a role in the development of VC19. ( )i y p p We investigated the potential association between MGP and VC in CKD stage 5 patients undergoing liv- ing donor renal transplantation (RTx). We link MGP genetics (SNPs), transcription (mRNA) and protein data (immunohistochemistry and plasma levels) to clinical vascular phenotype (CAC and histology). Matrix Gla protein is an independent predictor of both intimal and medial vascular calcification in chronic kidney disease PEN The importance of MGP in the inhibition of calcification is illustrated by studies of MGP knockout mice, who die within two months after birth due to severe arterial calcification and rupture of the aorta1. ti Chronic kidney disease (CKD) patients have an extremely high risk for developing vascular disease4. VC, manifested both as medial and intimal calcification with distinct pathologies, is a common risk factor in CKD5. Additionally, vitamin K deficiency is frequently encountered in CKD, which is associated with increased plasma levels of dephosphorylated uncarboxylated MGP (dp-ucMGP) plasma levels6,7. Furthermore, increased plasma dp-ucMGP 1Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, the Netherlands. 2Division of Renal Medicine, Department of Clinical Science, Technology and Intervention, Karolinska Institutet, Stockholm, Sweden. 3Department of Immunology and Microbiology, Laboratory of Nephrology, Katholieke Universiteit Leuven, Leuven, Belgium. 4Division of Medical Imaging and Technology, Department of Clinical Science, Intervention and Technology, Karolinska Institutet, Karolinska University Hospital, Huddinge, Stockholm, Sweden. 5Pathology, Clinical Pharmacology and Safety Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden. 6These authors contributed equally: Leon J. Schurgers and Peter Stenvinkel. email: peter.stenvinkel@ki.se Scientific Reports \| (2020) 10:6586 \| https://doi.org/10.1038/s41598-020-63013-8 Results score 2 or 3) compared to 24% and 14%, respectively, in patients with C/T and T/T genotypes (p = 0.01) (Supplemental Table S3). Results When comparing C-allele homozygotes to T-allele carriers in relation to either none/ minimal calcification (score 0–1) or moderate/extensive calcification (score 2–3) the difference became even Scientific Reports \| (2020) 10:6586 \| https://doi.org/10.1038/s41598-020-63013-8 www.nature.com/scientificreports/ All (n = 141) 1st tertile (n = 46) 2nd tertile (n = 47) 3rd tertile (n = 48) p-value Age (years) 47 (24, 63) 43 (22, 63) 49 (23, 62) 50 (32, 68) 0.008 Males, (%) 70 65 68 75 0.57 Diabetes mellitus, (%) 11 7 11 17 0.29 Cardiovascular disease, (%) 16 7 15 25 0.04 BMI, (kg/m2) 24.5 (20.8, 29.9) 24.0 (19.8, 29.1) 23.5 (20.8, 30.4) 25.1 (21.8, 30.2) 0.17 Systolic BP, (mmHg)* 140 (117, 169) 140 (116, 162) 140 (116, 167) 144 (121, 181) 0.29 Diastolic BP, (mmHg)* 84 (69, 96) 82 (68, 92) 82 (72, 100) 86 (67, 99) 0.33 Hemoglobin, (g/L)‡ 114 (99, 132) 111 (98, 129) 118 (99, 135) 113 (99, 132) 0.09 HbA1c, (mmol/mol)† 33 (23, 40) 35 (26, 40) 32 (25, 40) 32 (21, 41) 0.06 Triglycerides, (mmol/L) 1.3 (0.7, 2.4) 1.2 (0.6, 2.2) 1.2 (0.7, 2.6) 1.4 (0.7, 2.6) 0.58 Total cholesterol, (mmol/L) 4.5 (3.1, 6.2) 4.3 (3.0, 6.1) 4.6 (3.2, 6.0) 4.5 (3.0, 6.4) 0.64 HDL cholesterol, (mmol/L) 1.4 (0.9, 2.1) 1.4 (1.0, 2.1) 1.4 (0.9, 2.0) 1.3 (0.8, 2.2) 0.37 Serum Creatinine, (µmol/L) 723 (488, 1022) 622 (388, 985) 743 (517, 1018) 782 (531, 1135) 0.01 Serum Albumin (g/L)* 35 (29, 40) 35 (30, 42) 35 (29, 40) 36 (29, 41) 0.80 Uric Acid, (µmol/L)• 369 (254, 522) 413 (285, 557) 350 (239, 510) 357 (247, 469) 0.02 PEW (SGA > 1), n (%)** 25 31 26 18 0.33 Vascular calcification biomarkers Coronary artery calcium, (AU) ‡‡ 8 (0, 1170) 0 (0, 236) 8 (0, 1392) 34 (0, 1948) 0.002 Calcium, (mmol/L)* 2.3 (2.0, 2.5) 2.3 (2.1, 2.4) 2.3 (2.0, 2.6) 2.3 (1.9, 2.6) 0.67 Phosphate, (mmol/L)* 1.6 (1.0, 2.4) 1.7 (1.0, 2.2) 1.6 (1.1, 2.5) 1.7 (0.9, 2.5) 0.96 Glu-OC, (ng/mL) 14.0 (1.8, 87.6) 7.5 (1.6, 33.7) 15.5 (1.7, 122.6) 37.8 (2.3, 99.2) 0.001 Gla-OC, (ng/mL) 33.2 (11.7, 97.6) 26.7 (11.0, 73.5) 33.2 (11.7, 138.0) 42.9 (9.7, 102.2) 0.23 Mid-OC¸ (ng/mL) ¶¶¶ 59.5 (13.5, 259.5) 37.9 (11.6, 178.6) 61.8 (11.3, 218.6) 90.5 (17.7, 395.8) 0.02 Osteoprotegerin, (pg/ml) ••• 6.1 (3.8, 12.0) 5.6 (2.8, 10.1) 5.9 (3.9, 11.1) 7.6 (4.2, 14.0) 0.002 Table 1. Results Similarly, patients that were homozygous for the rs4236 T-allele had more often moderate-extensive medial VC than patients carrying a C-allele (49% vs. 27%, p = 0.02). i i Results Baseline clinical and biochemical characteristics in 141 CKD 5 patients in relation to tertiles of dp-ucMGP. Data presented as median (10th – 90th percentile) or percentage. Abbreviations: CKD, chronic kidney disease; dp-ucMGP, dephosphorylated uncarboxylated Matrix Gla protein; BMI (body mass index); BP, blood pressure; HbA1c, Hemoglobin A1c; HDL, high-density lipoprotein; PEW, protein energy wasting; SGA, subjective global assessment; AU, Agatson unit; Glu-OC, undercarboxylated osteocalcin; Gla-OC, carboxylated osteocalcin; mid-OC, n-mid osteocalcin. n= 139 n= 137 *n= 109. ‡n= 120 ‡‡n= 110 ‡‡‡n= 103. †n= 12 ††n= 99 †††n= 68. ¶n= 106 ¶¶n= 138 ¶¶¶n= 91. •n= 128 ••n= 140 •••n= 114. Table 1. Baseline clinical and biochemical characteristics in 141 CKD 5 patients in relation to tertiles of dp-ucMGP. Data presented as median (10th – 90th percentile) or percentage. Abbreviations: CKD, chronic kidney disease; dp-ucMGP, dephosphorylated uncarboxylated Matrix Gla protein; BMI (body mass index); BP, blood pressure; HbA1c, Hemoglobin A1c; HDL, high-density lipoprotein; PEW, protein energy wasting; SGA, subjective global assessment; AU, Agatson unit; Glu-OC, undercarboxylated osteocalcin; Gla-OC, carboxylated osteocalcin; mid-OC, n-mid osteocalcin. n= 139 n= 137 n= 109. ‡n= 120 ‡‡n= 110 ‡‡‡n= 103. †n= 121 ††n= 99 †††n= 68. ¶n= 106 ¶¶n= 138 ¶¶¶n= 91. •n= 128 ••n= 140 •••n= 114. idney disease; dp-ucMGP, dephosphorylated uncarboxylated Matrix Gla protein; BMI (body mass index); BP, lood pressure; HbA1c, Hemoglobin A1c; HDL, high-density lipoprotein; PEW, protein energy wasting; SGA, ubjective global assessment; AU, Agatson unit; Glu-OC, undercarboxylated osteocalcin; Gla-OC, carboxylated steocalcin; mid-OC, n-mid osteocalcin. n= 139 n= 137 *n= 109. ‡n= 120 ‡‡n= 110 ‡‡‡n= 103. †n= 121 n= 99 †††n= 68. ¶n= 106 ¶¶n= 138 ¶¶¶n= 91. •n= 128 ••n= 140 •••n= 114. Figure 1. dp-ucMGP levels associate to CAC score and vascular media calcification score. (a) CAC score measured in 110 CKD5 patients in relation to tertiles of dp-ucMGP levels. (b) dp-ucMGP levels of 128 CKD5 patients displayed against low (score 0 + 1) and high (score 2 + 3) media VC. Figure 1. dp-ucMGP levels associate to CAC score and vascular media calcification score. (a) CAC score measured in 110 CKD5 patients in relation to tertiles of dp-ucMGP levels. (b) dp-ucMGP levels of 128 CKD5 patients displayed against low (score 0 + 1) and high (score 2 + 3) media VC. more significant (50% vs. 22%, n = 109; p = 0.005). Table 1. Baseline clinical and biochemical characteristics in 141 CKD 5 patients in relation to tertiles of Table 1. Baseline clinical and biochemical characteristics in 141 CKD 5 patients in relation to tertiles of p-ucMGP. Data presented as median (10th – 90th percentile) or percentage. Abbreviations: CKD, chronic Discussion Model (coefficient + SE, p-value) 1-SD increase of age, years 0.05 + 0.12 (0.69) 1-SD increase of ucOC, ng/ml 0.48 + 0.14 (0.001) 1-SD increase of albumin, g/L 0.13 + 0.12 (0.27) 1-SD increase of Mid-OC, ng/ml −0.21 + 0.15 (0.16) 1-SD increase of osteoprotegerin, pg/ml 0.28 + 0.12 (0.02) 1-SD increase of ucOC, ng/ml 0.48 + 0.14 (0.001) 1-SD increase of albumin, g/L 0.13 + 0.12 (0.27) 1-SD increase of Mid-OC, ng/ml −0.21 + 0.15 (0.16) 1-SD increase of osteoprotegerin, pg/ml 0.28 + 0.12 (0.02) Table 2. Multiple linear regression models for 1-standard deviation increase of plasma dp-ucMGP in 141 CKD 5 patients. Abbreviations; 1-SD, one standard deviation; dp-ucMGP, dephosphorylated uncarboxylated Matrix Gla protein; CKD, chronic kidney disease; SE, standard error; ucOC, undercarboxylated osteocalcin; mid-OC, n-mid osteocalcin. High calcification score media (Q2 + Q3) Odds ratio (95%CI) p-value 1-SD increase of age, years 2.3 (1.3–4.1) 0.006 1-SD increase of dp-ucMGP, pmol/L 1.6 (1.0–2.5) 0.04 Gender (female, male) 7.3 (2.2–25.1) 0.001 1-SD increase of hsCRP, mg/L 1.3 (0.4–1.3) 0.31 Diabetes mellitus (yes, no) 26.0 (2.3–293.2) 0.008 1-SD increase of CAC-score, AU 1.2 (0.5–2.9) 0.59 Table 3. Multinominal logistic regression analysis of factors associated with high calcification score media (2 + 3) (n = 122, pseudo r2 = 0.22). Patients with calcification score media 0 + 1 served as the reference. Abbreviations: 95%CI, 95% confidence interval; 1- SD, one standard deviation; dp-ucMGP, dephosphorylated uncarboxylated Matrix Gla protein; hsCRP, high-sensitivity C-reactive protein; CAC, coronary artery calcium; AU, Agatston unit. Table 2. Multiple linear regression models for 1-standard deviation increase of plasma dp-ucMGP in 141 CKD 5 patients. Abbreviations; 1-SD, one standard deviation; dp-ucMGP, dephosphorylated uncarboxylated Matrix Gla protein; CKD, chronic kidney disease; SE, standard error; ucOC, undercarboxylated osteocalcin; mid-OC, n-mid osteocalcin. Table 2. Multiple linear regression models for 1-standard deviation increase of plasma dp-ucMGP in 141 CKD 5 patients. Abbreviations; 1-SD, one standard deviation; dp-ucMGP, dephosphorylated uncarboxylated Matrix Gla protein; CKD, chronic kidney disease; SE, standard error; ucOC, undercarboxylated osteocalcin; mid-OC, n-mid osteocalcin. Table 2. Multiple linear regression models for 1-standard deviation increase of plasma dp-ucMGP in 141 CKD 5 patients. Abbreviations; 1-SD, one standard deviation; dp-ucMGP, dephosphorylated uncarboxylated Matrix Gla protein; CKD, chronic kidney disease; SE, standard error; ucOC, undercarboxylated osteocalcin; mid-OC, n-mid osteocalcin. p g p p 5 patients. Discussion We examined the role of MGP as a potential biomarker for the extent of VC in CKD5 patients and confirmed that an increase in plasma dp-ucMGP levels was significantly associated with an increase in both CAC score and medial arterial VC. CKD patients are predisposed to VC20, an independent risk factor for CVD morbidity and mortality4,21. CAC score determines the extent of CAC and is correlated to VC at other places in the arterial Scientific Reports \| (2020) 10:6586 \| https://doi.org/10.1038/s41598-020-63013-8 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 2. Immunohistochemical staining of ucMGP in epigastric artery from CKD5 patients. Panel a shows ucMGP staining of epigastric artery of a patient with a medial VC score of 0 (left, 4×; right, 20×). Panel b shows ucMGP staining of an epigastric artery with a medial VC score of 3 (left, 4×; right, 20×). Figure 2. Immunohistochemical staining of ucMGP in epigastric artery from CKD5 patients. Panel a shows ucMGP staining of epigastric artery of a patient with a medial VC score of 0 (left, 4×; right, 20×). Panel b shows ucMGP staining of an epigastric artery with a medial VC score of 3 (left, 4×; right, 20×). Figure 2. Immunohistochemical staining of ucMGP in epigastric artery from CKD5 patients. Panel a shows ucMGP staining of epigastric artery of a patient with a medial VC score of 0 (left, 4×; right, 20×). Panel b shows ucMGP staining of an epigastric artery with a medial VC score of 3 (left, 4×; right, 20×). Model (coefficient + SE, p-value) 1-SD increase of age, years 0.05 + 0.12 (0.69) 1-SD increase of ucOC, ng/ml 0.48 + 0.14 (0.001) 1-SD increase of albumin, g/L 0.13 + 0.12 (0.27) 1-SD increase of Mid-OC, ng/ml −0.21 + 0.15 (0.16) 1-SD increase of osteoprotegerin, pg/ml 0.28 + 0.12 (0.02) Table 2. Multiple linear regression models for 1-standard deviation increase of plasma dp-ucMGP in 141 CKD 5 patients. Abbreviations; 1-SD, one standard deviation; dp-ucMGP, dephosphorylated uncarboxylated Matrix Gla protein; CKD, chronic kidney disease; SE, standard error; ucOC, undercarboxylated osteocalcin; mid-OC, n-mid osteocalcin. Discussion Abbreviations; 1-SD, one standard deviation; dp-ucMGP, dephosphorylated uncarboxylated Matrix Gla protein; CKD, chronic kidney disease; SE, standard error; ucOC, undercarboxylated osteocalcin; mid-OC, n-mid osteocalcin. High calcification score media (Q2 + Q3) Odds ratio (95%CI) p-value 1-SD increase of age, years 2.3 (1.3–4.1) 0.006 1-SD increase of dp-ucMGP, pmol/L 1.6 (1.0–2.5) 0.04 Gender (female, male) 7.3 (2.2–25.1) 0.001 1-SD increase of hsCRP, mg/L 1.3 (0.4–1.3) 0.31 Diabetes mellitus (yes, no) 26.0 (2.3–293.2) 0.008 1-SD increase of CAC-score, AU 1.2 (0.5–2.9) 0.59 Table 3. Multinominal logistic regression analysis of factors associated with high calcification score media (2 + 3) (n = 122, pseudo r2 = 0.22). Patients with calcification score media 0 + 1 served as the reference. Abbreviations: 95%CI, 95% confidence interval; 1- SD, one standard deviation; dp-ucMGP, dephosphorylated uncarboxylated Matrix Gla protein; hsCRP, high-sensitivity C-reactive protein; CAC, coronary artery calcium; AU, Agatston unit. High calcification score media (Q2 + Q3) Odds ratio (95%CI) p-value 1-SD increase of age, years 2.3 (1.3–4.1) 0.006 1-SD increase of dp-ucMGP, pmol/L 1.6 (1.0–2.5) 0.04 Gender (female, male) 7.3 (2.2–25.1) 0.001 1-SD increase of hsCRP, mg/L 1.3 (0.4–1.3) 0.31 Diabetes mellitus (yes, no) 26.0 (2.3–293.2) 0.008 1-SD increase of CAC-score, AU 1.2 (0.5–2.9) 0.59 Table 3. Multinominal logistic regression analysis of factors associated with high calcification score media (2 + 3) (n = 122, pseudo r2 = 0.22). Patients with calcification score media 0 + 1 served as the reference. Abbreviations: 95%CI, 95% confidence interval; 1- SD, one standard deviation; dp-ucMGP, dephosphorylated uncarboxylated Matrix Gla protein; hsCRP, high-sensitivity C-reactive protein; CAC, coronary artery calcium; AU, Agatston unit. High calcification score media (Q2 + Q3) Odds ratio (95%CI) p-value 1-SD increase of age, years 2.3 (1.3–4.1) 0.006 1-SD increase of dp-ucMGP, pmol/L 1.6 (1.0–2.5) 0.04 Gender (female, male) 7.3 (2.2–25.1) 0.001 1-SD increase of hsCRP, mg/L 1.3 (0.4–1.3) 0.31 Diabetes mellitus (yes, no) 26.0 (2.3–293.2) 0.008 1-SD increase of CAC-score, AU 1.2 (0.5–2.9) 0.59 Table 3. Multinominal logistic regression analysis of factors associated with high calcification score media (2 + 3) (n = 122, pseudo r2 = 0.22). Patients with calcification score media 0 + 1 served as the reference. Abbreviations: 95%CI, 95% confidence interval; 1- SD, one standard deviation; dp-ucMGP, dephosphorylated uncarboxylated Matrix Gla protein; hsCRP, high-sensitivity C-reactive protein; CAC, coronary artery calcium; AU, Agatston unit. Table 3. Discussion Multinominal logistic regression analysis of factors associated with high calcification score media (2 + 3) (n = 122, pseudo r2 = 0.22). Patients with calcification score media 0 + 1 served as the reference. Abbreviations: 95%CI, 95% confidence interval; 1- SD, one standard deviation; dp-ucMGP, dephosphorylated uncarboxylated Matrix Gla protein; hsCRP, high-sensitivity C-reactive protein; CAC, coronary artery calcium; AU, Agatston unit. Scientific Reports \| (2020) 10:6586 \| https://doi.org/10.1038/s41598-020-63013-8 www.nature.com/scientificreports/ Figure 3. Association between MGP expression and CAC and dp-ucMGP. (a) MGP expression measured in 41 CKD5 patients in relation to CAC score. (b) MGP expression measured in 41 CKD5D patients in relation to dp- ucMGP plasma levels. Figure 3. Association between MGP expression and CAC and dp-ucMGP. (a) MGP expression measured in 41 CKD5 patients in relation to CAC score. (b) MGP expression measured in 41 CKD5D patients in relation to dp- ucMGP plasma levels. tree22–24. However, as CT of the heart involves radiation and its value to determine microcalcifications is limited, circulating biomarkers that reflect early VC would be cheaper, safer and less invasive. Additionally, to interfere with VC progression, biomarkers that predict initiation phases of VC, such as MGP are of value in prediction and to follow treatment of VC. tree22–24. However, as CT of the heart involves radiation and its value to determine microcalcifications is limited, circulating biomarkers that reflect early VC would be cheaper, safer and less invasive. Additionally, to interfere with VC progression, biomarkers that predict initiation phases of VC, such as MGP are of value in prediction and to follow treatment of VC. MGP is a potent inhibitor of VC produced by VSMCs25, which are pivotal in maintaining MGP homeostasis, as they are involved in both the production and activation of MGP, both of which are important for the subsequent inhibition of VC21,25. Since MGP is an active inhibitor of VC it could be a potential useful biomarker. Indeed, circulating plasma levels of dp-ucMGP correlate with amount of VC22,23 and arterial stiffness26. In accordance, we report that patients with the highest dp-ucMGP levels had a higher CAC score and more extensive medial VC.fi p p g p g CKD patients suffer from vitamin K deficiency and are prone to develop VC24. Since vitamin K is necessary for the activation (carboxylation) of MGP in the vessel wall, MGP will be produced in the inactive form when levels of vitamin K are low25. Discussion We show that next to circulating levels of dp-ucMGP, highly calcified epigastric arteries also show co-localization with ucMGP. This data supports the hypothesis that circulating dp-ucMGP reflects local tissue levels of ucMGP which might be caused by a vascular vitamin K deficiency, a risk factor for VC. Although vitamin K intake or plasma levels were not measured in the present study, our findings indirectly imply that vitamin K status is low since also ucOC was significantly increased27. After correction for age and albumin, dp-ucMGP remained significant for bone markers. Moreover, after correction for CAC score and inflammation, dp-ucMGP remained significant for medial calcification score.i p gii In this study, high MGP expression was associated with CAC score and increased medial calcification (not significant) scored by pathologists. High expression of MGP infers to a high local production of MGP. However, nothing is known about the activation state of this locally produced MGP. As CKD patients are vitamin K defi- cient24, we hypothesize that most of the MGP is present as inactive MGP. This accord with our results showing a strong association between MGP expression and circulating dp-ucMGP. In vitro, low expression of MGP was associated with calcification via upregulation of BMP-228. Additionally, under specific pro-calcific conditions, such as a high calcium concentration, calcification is increased whereas MGP expression is decreased29. This is contradictory to our results but might be explained by the carboxylation efficiency of MGP. Total MGP in the plasma is the sum of both active (cMGP) and inactive (ucMGP) MGP; thus, high levels of dp-ucMGP do not reflect the total level of MGP that is expressed in the tissue. The importance of MGP as inhibitor of VC is demon- strated by MGP ko studies in mice that showed increased soft tissue calcification30. Moreover, MGP-/- mice express higher levels of osteogenic proteins, such as osteopontin, OC and Cbfa131. Due to the complex nature of MGP, further studies need to examine the precise relation between local vasculature MGP expression and circulating MGP. Besides measurement of plasma dp-ucMGP, we genotyped a subgroup of patients for three well-known SNPs (rs4236, rs1800801 and rs1800802) in the MGP gene. In comparison to healthy controls, our subgroup of CKD patients differed in respect to two of the SNPs, rs4236 and rs1800801, with patients displaying clearly lower fre- quencies of the minor alleles C and T, respectively. Patients and study design. Patients and study design. Adult ESRD patients undergoing living donor (LD) kidney transplantation (tx) at the Department of Transplantation Surgery at Karolinska University Hospital between March 2009 and October 2016 were invited to participate in the study. The etiologies of CKD were chronic glomerulonephritis (n = 52), hypertension and renovascular disease (n = 8), diabetic nephropathy (n = 9) and others or unknown causes (n = 72). The cohort included CKD5 non-dialysis (ND) patients (n = 51), prevalent peritoneal dialysis (PD) patients (n = 39) and prevalent hemodialysis (HD) patients (n = 51). PD patients were treated (median vintage time 11.4 months) with different combinations of biocompatible glucose-based or amino acid-based, or, for the long dwell, icodextrin-based solutions. HD-patients were treated by conventional maintenance HD or other dialytic techniques such as hemodiafiltration (median vintage time 14.4 months). Sixteen (11%) out of 141 patients had diabetes. Twenty-three (15%) of the patients had previously been diagnosed with cerebrovascular, cardiovascular, and/or peripheral vascular disease (grouped as CVD). We measured circulating plasma dp-uc- MGP in 141 CKD5 patients including 51 non-dialyzed (CKD5-ND) and 90 CKD5 patients treated by either PD (n = 39) or HD (n = 51). Patient characteristics are shown in Table 1 and a flow chart of patient inclusion is dis- played in Fig. S1. Age ranged from 19 to 75 years and patients were recruited from March 2009 to October 2016. Exclusion criterion was unwillingness to participate. Informed consent was obtained from each patient. The Ethics Committee of the Karolinska Institutet approved study protocols The studies were conducted in adherence Exclusion criterion was unwillingness to participate. Informed consent was obtained from each patient. The Ethics Committee of the Karolinska Institutet approved study protocols. The studies were conducted in adherence to the Declaration of Helsinki.” Biochemical assessments. Prior to the LD-RTx, fasting blood samples were drawn and stored in −80 °C. Biochemical analyses of plasma cholesterol, triglycerides, HDL-cholesterol, hemoglobin, creatinine, calcium, phosphate, albumin (coefficient of variation, CV, 3–4%), were performed at the Clinical Chemical Laboratory of Karolinska University Hospital, Stockholm, Sweden. LDL was calculated using the Friedewald formula: [(total cholesterol) - (high-density lipoprotein cholesterol) – (triglycerides/5)]. Plasma dp-ucMGP. Plasma dp-ucMGP levels were determined using the commercially available IVD CE-marked chemiluminescent InaKtif MGP assay on the IDS-iSYS system (IDS, Boldon, UK). www.nature.com/scientificreports/ and CAC score. An explanation for the conflicting evidence might be the differences in ethnic composition between the study populations and limited power.hh y p p p The present results should be considered in light of some strengths and limitations. The relatively low sam- ple size restricts power of the multivariate analyses and information retrieved from the MGP gene variants. Moreover, the observational nature of the study restricts causal conclusions. Furthermore, results obtained in this study are retrieved from different patient cohorts which means that our study population is heterogenous, for example displaying a population aged from 19–75. Since dp-ucMGP only represents a minor fraction of the total ucMGP content it should be interpreted with care17. CAC determination by cardiac CT cannot distinguish between medial or intimal calcification. However, we did find a strong association between medial VC in the epi- gastric artery and CAC. Finally, expression levels of MGP were only measured in limited number of patients and further studies are needed to examine the role of MGP expression in VC. For the MGP gene variants we need to perform additional in vitro and in vivo experiments to assess its causal role. p p As increased dp-ucMGP levels associate with increased CAC and medial VC our results support that dp-ucMGP is an independent predictor of VC and a risk factor for arterial stiffness and cardiovascular mortal- ity34. Alterations in plasma dp-ucMGP correlate with local tissue expression of ucMGP around areas of VC, high general MGP expression and genomic SNP analysis. Although our data indicate a predictive value of MGP as a biomarker for VC, further studies need to confirm whether these finding translate into CVD morbidity and mor- tality. Additionally, discovering the precise regulation of MGP in VC may provide novel therapeutic approaches with a potential role for vitamin K supplementation. Discussion As none of the SNPs were associated with plasma dp-ucMGP levels, the biological impact is unclear. However, because the MGP rs1800801 T-allele is associated with lower medial VC score, this allele may be protective. The literature is, however, conflicting regarding the potential pro- tective effect of the T-allele on vascular disease. A meta-analysis by Sheng et al.32 showed that the MGP T-allele was associated with a higher risk for VC whereas Taylor et al.33 found no association between the MGP rs1800801 Scientific Reports \| (2020) 10:6586 \| https://doi.org/10.1038/s41598-020-63013-8 www.nature.com/scientificreports/ Patients and study design. In brief, patient samples and internal calibrators were incubated with magnetic particles coated with murine monoclonal antibod- ies against dp-MGP, acridinium-labelled murine monoclonal antibodies against ucMGP, and an assay buffer. The magnetic particles were captured using a magnet and washed to remove any unbound analyte. Trigger reagents were added; the resulting light emitted by the acridinium label was directly proportional to the level of dp-ucMGP in the sample. The within-run and total variations of this assay were 0.8–6.2% and 3.0–8.2%, respectively. The assay measuring range was between 300 and 12,000 pmol/L and was linear up to 11,651 pmol/L. Assays were performed in a single run by Coagulation Profile BV, department of Biochemistry, Maastricht, the Netherlands. Vascular scoring by histology. Inferior epigastric arteries (n = 118) were collected from patients within 20 min from the start of kidney transplantation procedure. After fixation (4% phosphate buffered formalin) and paraffin embedding, 1–2 μm thick tissue sections were stained with hematoxylin, eosin and von Kossa method before evaluation by experienced pathologists. The extent of medial calcification was assessed by a pathologist in vascular biopsies35 and graded as 0 to 3. Patients graded as 0 and 1 represented no/minimal vascular calcification, and those graded 2 and 3 represented moderate/extensive vascular calcification. ucMGP immunohistochemical staining. After deparaffinization and rehydration of inferior epigastric arteries (n = 20), immunostaining for ucMGP was performed as described previously36, using a monoclonal MGP antibody directed against uncarboxylated MGP (1 mg/mL, 1:400 diluted) and a goat anti-mouse HRP (60 minutes at room tem- perature; Dako, Golstrup, Denmark) secondary antibody. Detection was performed using Novared stain (Vector Labs, Burlingame, CA), yielding a red color. The degree of ucMGP staining was measured semi-quantitatively and assessed by independent pathologists and graded as 0 to 3. Patients graded as 0 and 1 represented zero to minimal ucMGP stain- ing, and patients graded 2 and 3 represented moderate to extensive ucMGP positivity. Scientific Reports \| (2020) 10:6586 \| https://doi.org/10.1038/s41598-020-63013-8 www.nature.com/scientificreports/ MGP expression. Inferior epigastric artery samples collected at LD-RTx (n = 41) were incubated overnight in AllProtect Tissue Reagent (Qiagen, Hilden, Germany) and subsequently stored at −70 °C. RNA was isolated from arteries using TRIzol Reagent and MGP mRNA levels were analysed with TaqMan chemistry (Thermo Fisher Scientific, Waltham, MA USA) as previously described37. Genotyping of MGP rs4236, rs1800801 and rs1800802. References 1. Luo, G. et al. Spontaneous calcification of arteries and cartilage in mice lacking matrix GLA protein. Nature 386, 78–81 (1997).i 2. Price, P. A., Faus, S. A. & Williamson, M. K. Warfarin Causes Rapid Calcification of the Elastic Lamellae in Rat Arteries and H Valves. Arteriosclerosis, Thrombosis, and Vascular Biology 18, 1400–1407 (1998). h gy 3. Wajih, N., Borras, T., Xue, W., Hutson, S. M. & Wallin, R. Processing and transport of matrix gamma-carboxyglutamic acid protein and bone morphogenetic protein-2 in cultured human vascular smooth muscle cells: evidence for an uptake mechanism for serum fetuin. J. Biol. Chem. 279, 43052–43060 (2004). 4. O’Shaughnessy, M. M., Liu, S., Montez-Rath, M. E., Lafayette, R. A. & Winkelmayer, W. C. Cause of kidney disease and cardiovascular events in a national cohort of US patients with end-stage renal disease on dialysis: a retrospective analysis. Eur. Heart J. 40, 887–898 (2019). 5. Bashir, A. et al. Coronary Artery Calcium Assessment in CKD: Utility in Cardiovascular Disease Risk Assessment and Treatment? Am. J. Kidney Dis. 65, 937–948 (2015). f ld l ff f l f l d fi h d l d d 6. Westenfeld, R. et al. Effect of vitamin K2 supplementation on functional vitamin K deficiency in hemodialysis patients: a randomized trial. Am. J. Kidney Dis. 59, 186–195 (2012). y 7. Dalmeijer, G. W. et al. Matrix Gla Protein Species and Risk of Cardiovascular Events in Type 2 Diabetic Patients. Diabetes Care 36 3766–3771 (2013). 8. Delanaye, P. et al. Dephosphorylated-uncarboxylated Matrix Gla protein concentration is predictive of vitamin K status and is correlated with vascular calcification in a cohort of hemodialysis patients. BMC Nephrol 15, 145 (2014).hi i 9. Schurgers, L. J. et al. The circulating inactive form of matrix gla protein is a surrogate marker for vascular calcification in chronic kidney disease: a preliminary report. Clin J Am Soc Nephrol 5, 568–575 (2010). y p y p p 0. Ueland, T. et al. Circulating levels of non-phosphorylated undercarboxylated matrix Gla protein are associated with disease severity in patients with chronic heart failure. Clinical Science 121, 119–127 (2011).i p 1. Brandenburg, V. M. et al. Slower Progress of Aortic Valve Calcification With Vitamin K Supplementation: Results From a Prospective Interventional Proof-of-Concept Study. Circulation 135, 2081–2083 (2017). 11. Brandenburg, V. M. et al. Slower Progress of Aortic Valve Calcification With Vitamin K Su Interventional Proof-of-Concept Study. Circulation 135, 2081–2083 (2017). 2. Kaesler, N. Received: 21 October 2019; Accepted: 17 March 2020; Published: xx xx xxxx Received: 21 October 2019; Accepted: 17 March 2020; Published: xx xx xxxx Received: 21 October 2019; Accepted: 17 March 2020; Published: xx xx xxxx Patients and study design. A subset of 117 CKD5 patients with availa- ble peripheral blood samples and 389 controls (anonymous blood donors recruited in Stockholm municipality) underwent genotyping. Genomic DNA was extracted following standard procedures at the Karolinska Biobank. Genotypes for rs4236, rs1800801 and rs1800802 polymorphisms were determined with PCR-based allelic dis- crimination using TaqMan SNP Genotyping Assays, QuantStudio 5 System (Thermo Fisher Scientific, Waltham, MA, USA) and TaqMan Genotyper Software according to manufacturer’s protocols. CAC score. Cardiac computed tomography (CT) scans were performed using a 64-channel detector scanner (Lightspeed VCT; General Electric (GE) Healthcare, Milwaukee, WI). CAC scores were expressed in Agatston units38, the protocol and measurements as described previously in detail38. Total CAC score was calculated as the sum of CAC scores in the left main artery, the left anterior descending artery, the left circumflex artery, and the right coronary artery. Statistical analyses. Continuous data are expressed as median (10th to 90th percentile) and nominal or ordinal data as percentage. Statistical significance was set at the level of P < 0.05. Comparisons between groups were assessed with the non-parametric Wilcoxon test/Kruskal-Wallis ANOVA-test for continuous variables and Chi-square test for nominal variables. Non-parametric Spearman rank correlation analysis was used to determine associations between variables. We performed multiple imputation of missing values for multivariate linear regression and multivariate logistic regression using the function PROC MI, with all variables in the covariate section used to produce the values for imputation. The original n for each variable is given throughout. The results for each imputation were generated using PROC REGRESS and LOGISTIC, and then combined using PROC MIANALYZE. We used 20 imputed datasets for this study to ensure that our effect estimates were not overly inaccurate due to Monte Carlo variability. Multivariate linear regression analyses of dp-ucMGP were used and results were shown as standardized β regression coefficients. We per- formed multinomial logistic regression analysis to examine factors associated for determinants of vascular calcification 0–1 score vs 2–3. Statistical analyses were performed using statistical software SAS version 9.4 (SAS Campus Drive, Cary, NC, USA) and Stata 15.1 (Stata Corporation, College Station, TX, USA)38. Figures were created using GraphPad Prism version 8.3.1 for Windows, GraphPad Software, San Diego, California USA, www.graphpad.com. Author contributions Conceptualization: All authors. Data curation: A.J., L.D., T.Q., P.E., J.R., M.S., A.W., H.O., L.S. and P.S. Formal analysis: A.J., T.Q., A.W. Funding acquisition: L.S., P.E., P.S. Clinical investigation: T.Q. and P.S. Methodology: All authors. Original idea: A.J., L.D., T.Q., L.S. and P.S. Project administration: T.Q., L.S. and P.S. Resources: A.J., T.Q., L.S. and P.S. Software: T.Q. Supervision: L.S. and P.S. Validation: A.J., T.Q. Visualization: T.Q. Writing – original draft: A.J., L.D., T.Q., P.E., J.R., M.S., A.W., H.O., L.S. and P.S. Final manuscript: All authors read and approved the final manuscript. Conceptualization: All authors. Data curation: A.J., L.D., T.Q., P.E., J.R., M.S., A.W., H.O., L.S. and P.S. Formal analysis: A.J., T.Q., A.W. Funding acquisition: L.S., P.E., P.S. Clinical investigation: T.Q. and P.S. Methodology: All authors. Original idea: A.J., L.D., T.Q., L.S. and P.S. Project administration: T.Q., L.S. and P.S. Resources: A.J., T.Q., L.S. and P.S. Software: T.Q. Supervision: L.S. and P.S. Validation: A.J., T.Q. Visualization: T.Q. Writing – original draft: A.J., L.D., T.Q., P.E., J.R., M.S., A.W., H.O., L.S. and P.S. Final manuscript: All authors read and approved the final manuscript. References 89, 1147–1154 (2001).i of smooth muscle lineage markers. Circ. Res. 89, 1147–1154 (200 g 32. Sheng, K. et al. 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Coll. Cardiol. 15, 827–832 (1990). ( ) 38. Agatston, A. S. et al. Quantification of coronary artery calcium using ultrafast computed tomography. J. Am. Coll. Cardio 827–832 (1990). Competing interests p g Dr. Schurgers reports consultancy fee from Immunodiagnostic systems (IDS), outside the submitted work and grants from NattoPharma outside the submitted work; Dr. Evenepoel reports personal fees from Vifor FMC, personal fees from Amgen, personal fees from Medice, grants from Sanofi outside the submitted work. Dr. Stenvinkel reports personal fees from Astellas, grants and personal fees from Astra Zeneca, personal fees from Reata, personal fees from Corvidia, personal fees from Baxter, personal fees from Pfizer, outside the submitted work. All other authors don’t have competing interests. Acknowledgementsh g This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 722609 (INTRICARE). 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Circulating Dephospho-Uncarboxylated Matrix Gla-Protein Is Associated With Kidney Dysfunction and Arterial Stiffness. Am. J. Hypertens. 31, 988–994 (2018). f yp 27. Lian, J. B. & Gundberg, C. M. Osteocalcin. Clinical Orthopaedics and Related Research &NA, 267–291 (1988).ii f yp 27. Lian, J. B. & Gundberg, C. M. Osteocalcin. Clinical Orthopaedic 8. Chiyoya, M. et al. Matrix Gla protein negatively regulates calcification of human aortic valve interstitial cells isolated from calcified aortic valves. J. Pharmacol. Sci. 136, 257–265 (2018). 9. Wang, Q. et al. High Concentration of Calcium Promotes Mineralization in NRK-52E Cells Via Inhibiting the Expression of Matrix Gla Protein. Urology 119, 161.e1–161.e7 (2018).fhlt gy 30. Lanham, S. A., Cagampang, F. R. & Oreffo, R. O. C. The influence of a high fat diet on bone and soft tissue formation in Matrix Gla Protein knockout mice. Sci Rep 8, 3635 (2018).i p 31. Steitz, S. A. et al. Smooth muscle cell phenotypic transition associated with calcification: upregulation of Cbfa1 an of smooth muscle lineage markers. Circ. Res. 89, 1147–1154 (2001). p 31. Steitz, S. A. et al. Smooth muscle cell phenotypic transition associated with calcification: upregulation of Cbfa1 and downregul p eitz, S. A. et al. Smooth muscle cell phenotypic transition associated with calcification: upregulation of Cbfa1 and downregulation smooth muscle lineage markers. Circ. Res. 89, 1147–1154 (2001). 31. Steitz, S. A. et al. Smooth muscle cell phenotypic transition asso 1. Steitz, S. A. et al. Smooth muscle cell phenotypic transition associated with calcification: upregulation of Cbfa1 and downregulation of smooth muscle lineage markers. Circ. Res. 89, 1147–1154 (2001).i , p ypi of smooth muscle lineage markers. Circ. Res. 89, 1147–1154 (2001). yi g of smooth muscle lineage markers. Circ. Res. 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https://openalex.org/W2605007737	https://kclpure.kcl.ac.uk/ws/files/93206614/Cheng_Hui_Cascio_2017.pdf	English	null	Leniency Bias in Performance Ratings: The Big-Five Correlates	Frontiers in psychology	2,017	cc-by	10,555	Citation for published version (APA): Cheng, H. C. K., Hui, C. H., & Cascio, W. F. (2017). Leniency Bias in Performance Ratings: The Big-Five Correlates. Frontiers in Psychology, Article 521. https://doi.org/10.3389%2Ffpsyg.2017.00521 Citation for published version (APA): Cheng, H. C. K., Hui, C. H., & Cascio, W. F. (2017). Leniency Bias in Performance Ratings: The Big-Five Correlates. Frontiers in Psychology, Article 521. https://doi.org/10.3389%2Ffpsyg.2017.00521 Citing this paper Pl h C t g t s pape Please note that where the full-text provided on King's Research Portal is the Author Accepted Manuscript or Post-Print version this may differ from the final Published version. If citing, it is advised that you check and use the publisher's definitive version for pagination, volume/issue, and date of publication details. And where the final published version is provided on the Research Portal, if citing you are again advised to check the publisher's website for any subsequent corrections. Citation for published version (APA): Cheng, H. C. K., Hui, C. H., & Cascio, W. F. (2017). Leniency Bias in Performance Ratings: The Big-Five Correlates. Frontiers in Psychology, Article 521. https://doi.org/10.3389%2Ffpsyg.2017.00521 ORIGINAL RESEARCH published: 10 April 2017 doi: 10.3389/fpsyg.2017.00521 ORIGINAL RESEARCH published: 10 April 2017 doi: 10.3389/fpsyg.2017.00521 Leniency Bias in Performance Ratings: The Big-Five Correlates Kevin H. C. Cheng 1, C. Harry Hui 2 and Wayne F. Cascio 3 1 Innushuk Consult, Sydney, NSW, Australia, 2 Department of Psychology, University of Hong Kong, Hong Kong, Hong Kong, 3 CU Denver Business School, University of Colorado at Denver, Denver, CO, USA Kevin H. C. Cheng 1, C. Harry Hui 2 and Wayne F. Cascio 3 1 Innushuk Consult, Sydney, NSW, Australia, 2 Department of Psychology, University of Hong Kong, Hong Kong, Hong Kong, 3 CU Denver Business School, University of Colorado at Denver, Denver, CO, USA Some researchers assume that employees’ personality characteristics affect leniency in rating others and themselves. However, little research has investigated these two tendencies at the same time. In the present study we developed one index for other-rating leniency and another one for self-rating leniency. Based on a review of the literature, we hypothesized that a generous assessment of peers would more likely be made by those who are extroverted and agreeable than by those who are not. Furthermore, a generous assessment of oneself would more likely be made by people who are conscientious and emotionally stable, than by people who are not. We also investigated if the leniency in rating others and the leniency in rating oneself are part of a more general leniency tendency. Data collected from a sample of real estate dealers provided support for the above hypotheses. Limitations and implications for future research are discussed. Edited by: Con Stough, Swinburne University of Technology, Australia Keywords: rating leniency, work performance, personality, extroversion, Agreeableness, Big Five personality traits, self-rating, others rating Reviewed by: M. Teresa Anguera, University of Barcelona, Spain Riccardo Sartori, University of Verona, Italy INTRODUCTION Correspondence: Kevin H. C. Cheng chenghck@graduate.hku.hk Dispositional Factors Undermining Accuracy of Work Performance Theoretically, a performance rating embodies the following: the ratee’s level of work performance, the rater’s perspective (i.e., self, peer, supervisor, etc.), the rater’s idiosyncrasies, and random error. Using managers’ developmental rating data, Scullen et al. (2000) found that the ratee’s actual performance accounted for only about 20–25% of the variability in performance ratings when averaged across dimensions, perspectives, and instruments. Similar studies agreed that the inﬂuence on performance ratings for all combinations of rater perspectives and performance dimensions originated from rater’s bias (Castilla and Benard, 2010; Ng et al., 2011; Bono et al., 2012). One important component of the bias is a rater’s leniency. Specialty section: This article was submitted to Organizational Psychology, a section of the journal Frontiers in Psychology Received: 27 November 2015 Accepted: 22 March 2017 Published: 10 April 2017 On the basis of reviews on the link between personality and organizational behavior (Barrick and Mount, 1991, 2005; Barrick et al., 2001; Ones et al., 2007; Shaﬀer and Postlethwaite, 2013), we adopt the Big Five model for a better understanding of the personality substratum of rating leniency (Ceschi et al., 2016). The primary premise of this study is that certain personality dimensions of the Big Five are related to leniency in the ratings one assigns to oneself and others. In the following sections we elaborate on what might deﬁne this relationship. Keywords: rating leniency, work performance, personality, extroversion, Agreeableness, Big Five personality traits, self-rating, others rating General rights General rights Copyright and moral rights for the publications made accessible in the Research Portal are retained by the authors and owners and it is a condition of accessing publications that users recognize and abide by the legal requirements associ ral rights for the publications made accessible in the Research Portal are retained by the authors and/or other copyright condition of accessing publications that users recognize and abide by the legal requirements associated with these right •Users may download and print one copy of any publication from the Research Portal for the purpose of private study or research. •You may not further distribute the material or use it for any profit-making activity or commercial gain •You may freely distribute the URL identifying the publication in the Research Portal •Users may download and print one copy of any publication from the Research Portal for the purpose of private study •You may not further distribute the material or use it for any profit-making activity or commercial gain y y p g y •You may freely distribute the URL identifying the publication in the Research Portal Take down policy If you believe that this document breaches copyright please contact librarypure@kcl.ac.uk providing details, and we will remove access to the work immediately and investigate your claim. p y If you believe that this document breaches copyright please contact librarypure@kcl.ac.uk providing details, and we w the work immediately and investigate your claim. Download date: 24. Oct. 2024 Possible Causes of Rating Bias When Rating Others, and the Role of Personality Thus, raters who placed a high value on social interactions and sensitivity to colleagues’ needs were less likely to be unduly harsh, since they would not want to risk their social ties at the workplace (Randall and Sharples, 2012). As Jawahar (2001) suggested, a concern for maintaining social relationships with others may contribute to leniency in rating others. This tendency would be strengthened if there is a concern not to hurt the self-esteem of the person being appraised. This would be found in someone who is socially sensitive and people- oriented. Being friendly, cheerful, and outgoing is a component of the extrovert personality. Based on this conceptualization and empirical ﬁndings, we expected more pronounced leniency bias from employees who have the motivation and energy to befriend others, and among those who are forgiving, understanding, approachable, and socially adept. To the extent that an agreeable and extrovert personality underlies these characteristics, we oﬀer the following hypothesis: Some of these possible reasons for leniency bias can be traced to contextual factors raters face (Tziner et al., 2005). For example, Uber riders reported giving higher ratings to drivers because they know drivers risk being de-registered if their average score drops below 4.6 (anything under the maximum ﬁve stars constitutes a failing grade) (Rosenblat and Stark, 2016). Heslin and VandeWalle (2008) found that a rater’s implicit beliefs about people correlate with rater leniency. In addition to beliefs, at least one study found that cognitive ability is also related to leniency (Truxillo et al., 2008). Raters higher in cognitive ability tended to assign more severe ratings, while those lower in cognitive ability tended to assign more generous ratings. Banks and Murphy (1985) argued assigning ratings was unrelated to cognitive ability. Other sources of rating elevation could be dispositional and demographic characteristics. For example, people who score high in emotional-stability are more likely to provide unbiased ratings across dominant and friendly interpersonal settings, than people low in emotional-stability (Cheng, 2016). Agreeableness, extraversion, and emotional stability were related to performance ratings, and features of the rating context (e.g., study setting, appraisal purpose, accountability) moderated these relationships (Harari et al., 2015). Gender and race/ethnicity are related to magnitude of bias and to changes in bias across time (Tziner et al., 2005; Yun et al., 2005; Rosenman et al., 2011). More recently, a study of Airbnb Inc. Possible Causes of Rating Bias When Rating Others, and the Role of Personality political considerations that often characterize such performance appraisal settings (Gioia and Longenecker, 1994; Latham et al., 2008; Hu and Liden, 2011). Jawahar (2001) found that the rater’s attitude toward providing accurate performance feedback predicted accurate ratings for low self-monitors, but not for high self-monitors. Szarota et al. (2002) reported Dynamism and Excitability elicited higher agreement between self-and peer- ratings than Agreeableness and Intellect, although in case of Conscientiousness judges appeared to be as accurate as in the case of Excitability. Possible Causes of Rating Bias When Rating Others, and the Role of Personality Why would some people rate their subordinates leniently, that is, assign performance ratings that are more generous than might be justiﬁed? More than half a century ago, Thorndike (1949) and Glickman (1955) identiﬁed eight possible explanations. In particular, the rater: (1) may feel that anyone under his or her jurisdiction who is rated unfavorably will reﬂect poorly on the rater’s own worthiness (Paulhus, 1984); (2) may feel that anyone who could have been rated unfavorably had already been discharged from the organization; (3) may feel that a derogatory rating may be revealed to the ratee to the detriment of relations between rater and ratee; (4) may rate leniently in order to win promotions for his or her subordinates and therefore indirectly increase the rater’s future control of those subordinates by earning a reputation as a supervisor with “inﬂuence upstairs;” (5) may be projecting; (6) may feel it necessary always to approve others in order to gain approval for himself or herself (Wayne and Liden, 1995); (7) may be operating on the basis: “Whoever associates with me is meritorious, therefore I am meritorious;” and (8) may rate leniently because there exists in the culture a response set to approve rather than disapprove. Other reasons can be found in Fleenor et al. (2010) and Reich et al. (2007). y Wong and Kwong (2007) found that students who had been primed to maintain group harmony would provide an elevated evaluation of their peers. Bernardin et al. (2009) observed that those high on Agreeableness tended to rate the least eﬀective performers more leniently than did other raters. People who were socially oriented, friendly, conscientious and concerned about other people’s feelings tried not to make the target feel badly, even when a low rating was justiﬁed (Bernardin et al., 2009, 2016). Frontiers in Psychology \| www.frontiersin.org Citation: Cheng KHC, Hui CH and Cascio WF (2017) Leniency Bias in Performance Ratings: The Big-Five Correlates. Front. Psychol. 8:521. doi: 10.3389/fpsyg.2017.00521 April 2017 \| Volume 8 \| Article 521 Frontiers in Psychology \| www.frontiersin.org Leniency Bias in Rating Cheng et al. Possible Causes of Rating Bias When Rating Others, and the Role of Personality found ratings are highly likely to be inﬂuenced by bias on the basis of factors like race or ethnicity. For example, guests with African-American names were about 16% less likely to be accepted as rentees than guests with characteristically white names (Edelman et al., 2016). H1: Leniency in rating others will be associated positively with the raters’ Extroversion and Agreeableness. Possible Causes of Rating Bias When Rating Oneself, and the Role of Personality The psychological literature on leniency in self-rating uses a diﬀerent set of labels, such as better-than-average eﬀect, self- serving bias, self-deception, other-deception, self-enhancement, social desirability, and impression management (Wayne and Liden, 1995). Various explanations have been oﬀered. For example, a cross-cultural perspective in individualism- collectivism (Farh et al., 1991; Xie et al., 2006; Heine and Hamamura, 2007; Cheng and Cascio, 2009; Ng et al., 2011) was found to be relevant in rating biases. Recently, there had been evidence supporting the contrary (Brown, 2010)—that is, while cultural factors may moderate leniency eﬀect, the tendency to self-enhance is universal. Extension of Past Research Our present investigation attempts to extend past leniency research in three ways: the operationalization of the construct; inclusion of peer raters; and a focus on a real-life ﬁeld setting. p p g Warr and Bourne (1999) reported that self-enhancement (i.e., self-ratings being higher than the ratings given by the supervisor) was associated with personality characteristics such as motivation to achieve. Conscientious and hardworking workers were more likely to observe and remember their own desirable work behaviors than those who did not pay much attention to their work. Goﬃn and Anderson (2007) reported similar ﬁndings. That is, inﬂated self-ratings of performance (relative to the ratings assigned by superiors) were associated with high achievement need. Both studies also found that the self-enhancement bias was weaker among people who were worrisome, anxious, and low in self-esteem (Warr and Bourne, 1999; Goﬃn and Anderson, 2007). Our second hypothesis, set in a Big-Five framework, attempts to replicate these ﬁndings: First, we attempt to operationalize leniency more accurately. One of the methods prior research has used is to ask participants to self-report their lenient tendency (e.g., Fried et al., 1999). Another approach we have taken is to treat as lenient those ratings that are distributed in a negatively skewed manner (Taylor and Hastman, 1956), that is, when raters assign a greater proportion of their ratings to the categories above their mean than to those below. However, without a suﬃciently large number of ratings, it is not possible to determine skewness reliably. Even if there is skewness, at least part of it can be attributed to the skewed distribution of workers on their actual behaviors. Hence, unless the ratings were done on a scale anchored with statistically meaningful terms (e.g., a ratio scale based on magnitude estimation), and not with less precise ones (e.g., “very good,” “fairly satisfactory performance,” and so forth), it would not be justiﬁable to regard a high rating as lenient. H2: Leniency in rating oneself will be associated positively with Conscientiousness, and negatively with Neuroticism. Another way to operationalize leniency is to compare the ratings against somebody else’s judgment. This has been a method in many of the studies cited above. For example, in their study of students’ bias in peer ratings, Bernardin et al. (2000) used the professor’s rating as an estimate of a target student’s performance. Possible Causes of Rating Bias When There has been some work on the eﬀects of dispositional characteristics such as gender and self-monitoring. Tziner et al. (2008) reported a strong correlation between self- monitoring and leniency bias. This is understandable given the Robins and Paulhus (2001) postulated that personality traits as grandiosity and dominance served as underlying factors of self-enhancement. Farh and Dobbins (1989) found self-esteem April 2017 \| Volume 8 \| Article 521 Frontiers in Psychology \| www.frontiersin.org 2 Leniency Bias in Rating Cheng et al. higher among those who exhibited a tendency toward self- aggrandizement (which, unfortunately, in their study might have been confounded by true performance). Evidence exists that people who have considered themselves better than average have higher self-esteem and better mental health (see review by Taylor and Brown, 1994). Similar studies on self-enhancement and adjustment were reported by Kwan et al. (2008). consequently, would have some common associations. A positive correlation, therefore, should conﬁrm this speculation and prompt a search for the common dispositional characteristic(s) that may underlie the general leniency bias. For exploratory purposes, we oﬀer the following pair of competing hypotheses: H3a: Other-rating leniency and self-rating leniency will not be associated with each other. At the workplace, Murphy et al. (2004) reported raters purposely manipulated appraisal ratings in pursuit of their own goals. For example, they would over-report others’ behaviors when seeking greater harmony with colleagues, or distort selected behaviors of executives when pursuing their own political goals. Sinha et al. (2012) also found self-raters high on extraversion, dominance, cultural conformity, cynicism, and detail orientation who were somewhat likely to over-rate their performance as compared to peer ratings. H3b: Other-rating leniency and self-rating leniency will be associated with each other, and also with certain common personality factors. Frontiers in Psychology \| www.frontiersin.org Extension of Past Research It should be noted, however, that such a measure inevitably confounds leniency with the target’s actual performance, as well as with the professor’s rating bias. In the present investigation, we derived a more objective measure of work performance without relying too much on the information provided by a single rater, who might have been equally biased. In doing so, the “leniency index” can be used to represent the psychological construct that we are interested in. Is There a General Factor for Rating Bias? In addition to evaluating the two hypotheses, we explored the relationship between self-rating leniency and other-rating leniency. Very few researchers have examined in the same study elevation bias in performance appraisal as well as self- enhancement and grandiosity. Besides the lack of a common metric for measuring the two types of leniency, another probable reason that such a study had previously not been carried out is that the research community does not see much conceptual linkage or psychological commonality between self- rating leniency and other-rating leniency. If there indeed were no substantively meaningful connections between the two rating types, we would observe a null correlation. This would imply that the elevation biases in self-ratings and other-ratings are driven by two diﬀerent psychological processes. Second, we directed our attention to the self as raters. Most early work on rater accuracy used the immediate supervisor as the only rater. With the proliferation of 360◦feedback, however, ratees as well as non-supervisors served as raters. This multi- rater procedure currently used in performance management and employee development rests on a rather strong assumption that ratings of behavior obtained from diverse sources are equally reliable and valid. Indeed, factorial structures of ratings scales have shown to be largely invariant across rater perspectives such as peer and self (Scullen et al., 2003; Hoﬀman and Woehr, 2009; Monahan et al., 2013). Nevertheless, researchers have also found discrepancies between self-ratings and ratings received This need not be true, however. Consider that both the tasks of rating oneself and rating others on a series of behavioral items require prior storage of behavioral episodes, retrieval of information from memory, and organization of the information for reporting. It is possible that some heuristics are used. Participants and Procedures This function was emphasized when the participants were instructed to choose as their raters those people who had had opportunities to observe them in action, and in whom they could trust for feedback. With this arrangement, all participants served both as raters and ratees. Two of the 74 participants were dropped from our sample because of incomplete data. The total number of observations in the study was 216. Each ratee had an average of 3.24 peer raters. The present study meets the above criteria in four ways. First, the study sampled professional real estate agents in a developmental workshop. The second and third ways were that they were sampled based on service climate and personality, respectively. Fourth, during the workshop, the real estate agents completed a personality scale as well as a task to provide performance appraisal ratings to self and to their colleagues. Many have argued that ratings of performance pose threats to employees when done for promotional or remunerative purposes (Iqbal et al., 2015). The workshop hosts stressed that the ratings were for developmental purposes and that the data collected was not fed back to their employers. One reason for the relative scarcity of published ﬁeld studies in this area was that supervisors were dispersed in diﬀerent work units, and consequently they rated their own staﬀ, not others. Any observed diﬀerence between average ratings provided by two supervisors may suggest a diﬀerence in leniency, a genuine inter- unit diﬀerence in work performance, or both. While it is good research design to have two or more supervisors rating the same employee, often this is not feasible in ﬁeld settings. Moreover, ﬁeld settings often present diﬃculties for researchers who want to implement completely crossed experimental designs, in which each participant serves both as a rater as well as a ratee. In the present investigation, we extended previous laboratory studies by working with a sample that provided us with data that allowed us to compute leniency indices largely not confounded Materials: Personality Measures Personality was assessed on a Chinese translation of the 60- item NEO-FFI (Yik and Bond, 1993), with 12 items for each of the ﬁve dimensions. Cronbach’s α (for the English version of the instrument) was 0.86 for the Neuroticism scale, 0.77 for Extroversion, 0.73 for Openness to Experience, 0.68 for Agreeableness, and 0.81 for Conscientiousness (Costa and McCrae, 1992). Participants and Procedures In the ﬁeld study, we collected data during a personal- development workshop for employees of an established real estate agency in Hong Kong. The attendees included 52 and 22 personnel in operational and managerial positions, respectively. For the operational staﬀ, their average age was 33 years old. Those in managerial posts averaged 51 years old. The workshop discussed themes on personality at work, organizational climate, 360◦rating systems and customer service quality. On completion of the workshop, the author and one master trainer consultant supervised an exercise designed to put participants’ understanding of the above themes into practice. This included completing various scales related to the above themes. Role-plays on how quality service should be delivered also took place at this point. Third, the current study is a ﬁeld study. A ﬁeld study collects data outside a laboratory. Usually, it involves a range of well-deﬁned variables in participants’ natural environment (Cronbach, 1982). The strength of a ﬁeld study is that it has ecological validity and demand characteristics are generally low. Field research is deﬁned by its degree of naturalism (Reis and Gosling, 2010). Cronbach (1982) suggested when assessing the degree of naturalism of a study, one should refer to the units, treatments, setting and observations. As Paluck and Cialdini (2014, p. 82) illustrated, when studying the eﬀects of interpersonal empathy, an eﬀective way is to ask adults (not college undergraduates) to watch television episodes (not written instructions or simulated pictorials) with varying degree of emotionally arousing scenes while coding their facial reactions (instead of a written measure). Fifty-six male and eighteen female real estate agents rated themselves (i.e., self-rating) on a performance rating scale (to be described in criterion measures). Each agent was allowed up to ﬁve participants (including those in either operational or managerial posts) to provide ratings of their customer service performance (i.e., peer rating). Prior to the workshop the consultant speciﬁcally made arrangements with the agency’s human resource personnel so that all participants were acquainted with at least three people. To ease possible concerns stemming from the evaluations, the hosts reiterated issues on anonymity and the University’s policy on data privacy of participants. Participants were reassured the rating practice carried no administrative/remunerative consequences. Further, the ratings were to be aggregated and delivered anonymously and conﬁdentially to the ratees from the hosts for purposes of personal development. Extension of Past Research If all leniencies are primarily a response set that people employ to do the rating task with minimal cognitive eﬀort, self-rating leniency and other-rating leniency might utilize the same heuristics, and, April 2017 \| Volume 8 \| Article 521 3 Cheng et al. Leniency Bias in Rating from peers. For instance, self-appraisals have tended to be more lenient, less variable, more biased, and shown less agreement with the judgments made by others (Fox and Dinur, 1988; Diefendorﬀ et al., 2005; Woehr et al., 2005; Hannum, 2007). by participants’ positions and roles. The sample is not large by conventional standards, but it allowed us to execute a meticulous procedure for data collection. Despite the conceptual similarity between leniency in rating oneself and leniency in rating others, there is very little overlap between the two bodies of literature. For example, previous research has not compared whether one is more lenient with the self or with others. There is also no work to understand if the same set of psychological mechanisms operates upon the two leniencies. This is probably due to a lack of a common metric that researchers can use to index a person’s leniency. To extend our knowledge, the present investigation examined how self-rating leniency and other-rating leniency might be associated with each other, and if leniency in these two sources could be ascribed to the same set of personality antecedents. Frontiers in Psychology \| www.frontiersin.org RESULTS Table 1 displays reliability estimates, means, and standard deviations of each of the personality scales, other major variables, as well as bivariate correlations among them. While the ratees’ ﬁve personality scores did not correlate signiﬁcantly with performance rating received from other raters, three (Neuroticism, Agreeableness, and Conscientiousness) correlated signiﬁcantly with the ratees’ self-rating. This strongly suggests common source problem and the possible impact of personality on the process of rating oneself. Table 1 displays reliability estimates, means, and standard deviations of each of the personality scales, other major variables, as well as bivariate correlations among them. While the ratees’ ﬁve personality scores did not correlate signiﬁcantly with performance rating received from other raters, three (Neuroticism, Agreeableness, and Conscientiousness) correlated signiﬁcantly with the ratees’ self-rating. This strongly suggests common source problem and the possible impact of personality on the process of rating oneself. To recapitulate, in the present study three diﬀerent sets of data were collected to investigate rater leniency. These included: measures of each participant’s personality, his or her self-rating, and his or her ratings of several other people in the sample. From these data, we derived the following three measurement constructs: In our sample, the diﬀerence between the rating assigned to oneself and the average rating assigned to others ranged from −0.85 to 1.17, with a median of 0.06. For 58% of the participants, the ratings they gave to themselves were higher than the average ratings they had given to others. Self-serving bias (i.e., rating oneself more favorably than rating others) existed in some, but not the majority of participants. Ratings assigned to self (M = 4.09, SD = 0.42, median = 4.06) were not diﬀerent from ratings assigned to others (M = 4.02, SD = 0.46, median = 4.00). Nevertheless, these two ratings were highly correlated (r = 0.66, p < 0.001, n = 72, 95% C.I. = 0.52–0.79), suggesting individual diﬀerences in response sets such as leniency and extreme response style. 1. One’s actual performance, as averaged over all peer ratings assigned to that person. Understandably, ratings from diﬀerent peer raters may not have coincided exactly with each other. The peers had seen the person in action at diﬀerent times, and in diﬀerent situations. In addition, each peer may have had his or her own bias. Furthermore, since the ratings came from people nominated by the target persons, errors of measurement would not be completely random. Participants and Procedures The Cronbach’s α scores for the Chinese version on the same ﬁve scales were somewhat lower: 0.79, 0.65, 0.56, 0.49, and 0.75, respectively. As we did not develop a hypothesis on Openness to Experience, its reliability is not an issue here. April 2017 \| Volume 8 \| Article 521 4 Leniency Bias in Rating Cheng et al. Nonetheless, interpretation of any null eﬀects for Agreeableness should be made with caution. Nonetheless, interpretation of any null eﬀects for Agreeableness should be made with caution. randomly selected colleagues, whose variation in knowledge about the target persons may have introduced even more noise to the data and undercut external validity of any ﬁndings. 2. 2. Other-rating leniency (ORL): this was the elevation of the ratings one assigned to a target person relative to the average ratings on the same target person by other peers, averaged across all targets that the rater has rated. This may be expressed algebraically as: Criterion Measures + Rp p Nd (1) (1) Nd dP j = 1 nP i = 1 Rk = ratings re ORL = other-rating leniency; dP j = 1 nP i = 1 Rk = ratings where ORL = other-rating leniency; dP j = 1 nP i = 1 Rk = ratings assigned to target persons i = 1 through n across dimensions j = 1 through d by rater k; R1, R2... Rp = ratings assigned by raters 1, 2 through p of the same target person; p = the number of raters of each target person excluding rater k; N = the total number of target persons rated by rater k. Psychologically, this indicated how much more favorably this person rated others when compared to his or her fellow-raters. 3. Self-rating leniency (SRL): the elevation of ratings one assigned to oneself relative to the average ratings assigned by others to him or her. Algebraically: SRLj = dP j = 1 Rk − R1 + R2 + ... + Rp p d (2) (2) where all terms were as deﬁned above. where all terms were as deﬁned above. Frontiers in Psychology \| www.frontiersin.org Criterion Measures The real estate agent’s job is primarily to facilitate transactions between a buyer and a seller, or between a landlord and a tenant. To devise an industry-appropriate measure of sales- oriented customer-service behavior, we asked a manager and ﬁve property dealers in the company to describe independently the daily routine of an agent, and what they considered to be the most important and desirable activities that a real estate agent should exhibit. Critical incidents were also solicited from the managers by three researchers, with education and training in industrial-organizational psychology. The interviews resulted in a summary of 16 behavioral statements agreed by the three researchers to reﬂect on contextual performance (for details of the item constructions, refer to Hui et al., 2003, 2007). As customer-service quality can be represented by how frequently these behaviors are displayed, we reformatted each item so that the frequency of the behaviors could be rated on a 1–5 Likert scale (1 = never, 3 = occasionally, 5 = very often). The Cronbach’s α was 0.82 for self-rating, and 0.87 for rating of others. Sample statements were: “provides services not available from competitors,” “is able to negotiate a good price on customer’s behalf,” and “makes customers feel that he/she is reliable and trustworthy.” For target persons (n = 15) rated by three peer raters, ICC was 0.03. For target persons rated by four and ﬁve peer raters, ICCs were 0.40 (n = 16) and 0.70 (n = 5), respectively. For target persons rated by six and seven peer raters, ICCs were 0.75 (n = 5) and 0.84 (n = 2), respectively. The low agreement among raters could be attributed to diﬀerences in contexts in which the target had been observed, but also to rater idiosyncrasies in leniency, which is the subject of this research. In the present study, we used as the true-score estimate the average of the peer ratings. We operationalized leniency as the elevation of the rating from the average of the ratings all others assigned to that same person. Since leniency for a given individual was deﬁned relative to other people, the expected value of the overall mean of the leniency index was zero. ORLij = dP j = 1 nP i = 1 Rk − R1 + R2 + ... RESULTS Nevertheless, the average peer rating appeared to be the best estimate of the target person’s “actual” performance. It is at least better than the alternative, namely, ratings obtained from a set of April 2017 \| Volume 8 \| Article 521 Frontiers in Psychology \| www.frontiersin.org 5 Cheng et al. Leniency Bias in Rating Analyses on the Leniency Indices SRL ranged from −1.06 to 1.03 (n = 71; SD = 0.49), and was more variable than ORL, which ranged from −0.74 to 0.89 (n = 72; SD = 0.34). The diﬀerence was statistically signiﬁcant by Levene’s test (F = 9.00, df = 1,141, p < 0.005). The two leniency indices were highly correlated (r = 0.55, p < 0.001, n = 69, 95% C.I. = 0.37–0.70), despite the fact that the average peer rating used for computing ORL was diﬀerent from the one used to assess each participant’s SRL. Partial correlations between the two indices with one of the Big Five traits controlled for each time ranged from 0.52 to 0.56 (p < 0.001, df = 65–66). TABLE 1 \| Descriptive statistics of demographics, personality, and rating measures. Reliability Mean SD Gender Rank N E O A C RAO RAS RRO ORL SRL Gender (1 = male, 2 = female) 1.24 0.43 Rank (1 = non-manager, 2 = manager) 1.28 0.45 −0.01 Neuroticism (N) 0.79a 29.10 7.07 0.23 −0.15 Extroversion (E) 0.65a 34.90 5.37 0.05 0.19 −0.42** Openness to experience (O) 0.56a 30.96 5.09 0.03 0.14 −0.09 0.10 Agreeableness (A) 0.49a 28.92 4.96 0.05 −0.11 −0.26* 0.20 −0.05 Conscientiousness (C) 0.75a 39.66 5.34 0.02 0.33 −0.34 0.22 0.06 0.34 Rating assigned to others (RAO) 0.40–0.70b 4.02 0.46 0.07 0.41* −0.29* 0.40* 0.13 0.18 0.34 Rating assigned to self (RAS) 0.82a 4.07 0.42 0.09 0.35 −0.30 0.20 0.18 0.25* 0.33 0.66* Rating received from others (RRO) 0.40–0.70b 4.05 0.35 0.10 0.62* 0.09 0.16 0.05 −0.11 0.04 0.30 0.19 Other-Rating Leniency (ORL) – −0.01 0.34 0.10 −0.03 −0.22 0.31** 0.13 0.24* 0.24* 0.82* 0.55* −0.11 Self-Rating Leniency (SRL) – 0.02 0.49 0.01 −0.15 −0.32 0.06 0.12 0.30 0.26* 0.37 0.72* −0.55* 0.55* General Leniency Index – 0.01 0.72 0.05 −0.11 −0.31 0.19 0.14 0.31 0.28* 0.63* 0.74* −0.42* 0.83* 0.92*** ontiers in Psychology \| www.frontiersin.org g p f Our main hypothesis is that personality is related to leniency. RESULTS Results indicated that ORL correlated with three of the Big Five personality traits: Extroversion (r = 0.31, p < 0.01, 95% C.I. = 0.08–0.51), Agreeableness (r = 0.24, p < 0.05, 95% C.I. = 0.01–0.45), and Conscientiousness (r = 0.24, p < 0.05, 95% C.I. = 0.01–0.45). SRL correlated negatively with Neuroticism (r = −0.32, p < 0.01, 95% C.I. = −0.52 to −0.09) and positively with Conscientiousness (r = 0.26, p < 0.05, 95% C.I. = 0.03– 0.47) and Agreeableness (r = 0.30, p < 0.05, 95% C.I. = 0.07– 0.50). We regressed ORL and SRL on the Big Five personality dimensions, after controlling for two demographic variables, gender and position (supervisory or otherwise). Results showed that the demographic variables did not have any eﬀects on ORL, but neither did the personality factors. As for SRL, Neuroticism was a valid predictor (β = −0.30, p < 0.05). Since there was a strong correlation between SRL and ORL, we aggregated ORL and SRL to form a general leniency index (GLI). We found that this new index correlated with Neuroticism (r = −0.31, p < 0.01, 95% C.I. = −0.51–0.08), Agreeableness (r = 0.31, p < 0.01, 95% C.I. = 0.08–0.51), and Conscientiousness (r = 0.28, p < 0.05, 95% C.I. = 0.05–0.49). In a hierarchical regression, after controlling for the eﬀects of demographic variables, we identiﬁed two personality predictors: Neuroticism (R2 change = 0.13, F = 9.96, df = 1,66, p < 0.005, β = −0.29) and Conscientiousness (R2 change = 0.06, F = 4.51, df = 1,65, p < 0.05, β = 0.26). Emotionally stable and conscientious people were more generous in assigning their ratings. All of these ﬁndings were consistent with Hypothesis 3b, that there was a general leniency index. We also found in the same analysis that supervisors were more stringent than frontline staﬀ(β = −0.25, p < 0.05)1. 1The above observation is partially supported by two discriminant analyses. In the ﬁrst, we formed four groups by median-splitting the sample according to ORL and SRL. Extroversion discriminated the two harsh-on-others groups from the two lenient-on-others groups (Wilks’ Lambda = 0.85, F = 3.74, df = 3.64, p < 0.05). The more sociable one is, the more likely that she or he will assign lenient ratings to herself or himself and to others. Thirty-ﬁve percent of the cases were classiﬁed correctly. In a second discriminant analysis, we formed three groups (those lenient on both self and others, those harsh on both self and others, and the rest). Neuroticism loaded on the discriminant function (Wilks’ Lambda = 0.91, F = 3.41, df = 2.65, p < 0.05), and 48% of the cases were classiﬁed correctly. Regression Analyses In response to concerns raised about use of certain diﬀerence scores (Edwards, 1995), and to be conservative, we performed a parallel set of regression analyses without deriving any scores April 2017 \| Volume 8 \| Article 521 6 Cheng et al. Leniency Bias in Rating TABLE 2 \| Hierarchical regression analysis predicting peers’ ratings assigned to target persons. Coefﬁcients Standard Error p Level Rater Gender 0.10 0.05 ns Seniority −0.02 0.05 ns Neuroticism 0.01 0.00 ns Extroversion 0.01 0.00 0.001 Openness to Experience −0.00 0.01 cns Agreeableness 0.00 0.01 ns Conscientiousness 0.01 0.01 0.04 Ratee Intercept −0.70 0.30 0.02 Actual performance 0.89 0.04 0.0005 TABLE 2 \| Hierarchical regression analysis predicting peers’ ratings assigned to target persons. by subtracting one value from another. The criterion variables in these supplementary analyses were the ratings participants assigned, and not the leniency scores. We conducted the ﬁrst analysis, on ratings assigned to peers, using hierarchical linear modeling. This analytic technique allowed us to make simultaneous inferences on the eﬀects of variations in the raters’ personalities (at the individual, i.e., rater, level) and in the ratees’ true performance (at the nested, i.e., ratee, level)2, which was statistically controlled. Table 2 contains the regression coeﬃcients of the hierarchical regression analyses. At the ratee level, a substantial amount of variance in the ratings could be attributed to the true performance score. At the rater level, the model depicts the impact of two of the Big-5 personality dimensions. The raters’ extroversion and conscientiousness contributed statistically signiﬁcant variance to the criterion variable. This partially replicated results from analyses on ORL, thus lending additional support to Hypothesis 1. Not everyone was generous in rating himself or herself, and at the same time harsh in rating others. This is consistent with the meta-analytic ﬁnding that the self-enhancement eﬀect among Asians is not as strong as that among North Americans (Heine and Hamamura, 2007). There is, in fact, considerable inter-rater variability. We conducted the second series of hierarchical regression analyses to predict the ratings participants gave themselves. In the ﬁrst step we entered the demographic variables as predictors, in the second step we entered the participants’ true-scores, and in the third step we entered personality scores. Results showed that while demographic variables accounted for 12.9% of the variance in the criterion, adding the true score to the model did not improve the prediction of a person’s self-rating. 2In HLM, the group-level variable (i.e., the true performance of the ratee) must, by deﬁnition, be a constant within each cluster of respondents reporting on the same ratee. Therefore, for this analysis we estimated “actual” performance by aggregating ratings received from all peer raters. Because of this, we expected a slight overestimation of the eﬀect of this variable in the model. Personality Correlates of ORL Agreeableness and extroversion are the personality antecedents responsible for rating other people leniently. We can now speculate on the psychological mechanism that links these two traits to ORL. According to Murphy and Cleveland (1995), a rater may consider four possible goals: task performance (e.g., to raise or maintain target’s task performance), interpersonal (e.g., to raise or maintain interpersonal relations with target), strategic (e.g., to increase target’s chance of organizational advancement), and internalized (e.g., to rate according to some values and beliefs). Regression Analyses However, adding the participants’ personality scores explained a signiﬁcant amount of variance. The 1R2 for Neuroticism was.08 [F(1, 65) = 6.38, p < 0.05], for Agreeableness it was 0.08 [F(1, 66) = 6.92, p < 0.05], and for Conscientiousness it was.05 [F(1, 66) = 3.93, p = 0.052]. Adding an interaction term between personality and true score did not account for additional variability. This pattern is consistent with the ﬁndings for SRL, as reported in the previous subsection, and it conﬁrms Hypothesis 2, that even after demographic variables and a person’s actual performance have been controlled statistically, personality still has a substantial impact on one’s self-rating. Results supported our general hypothesis that leniency can be traced, at least in part, to the rater’s personality. It conﬁrmed Hypothesis 1, that people who are outgoing, conscientious, and agreeable assign more generous ratings to others. People who are amiable tend to perceive a greater frequency of desirable behaviors in their peers’ daily work. Furthermore, the study conﬁrmed Hypothesis 2, that people who are emotionally stable and conscientious assign more generous ratings to themselves. Moreover, ORL and SRL are somewhat related to each other. However, controlling for personality did not attenuate the correlation between the two leniency indices. This suggests that any individual diﬀerence in general leniency in rating self and others could be due to characteristics beyond the Big Five. April 2017 \| Volume 8 \| Article 521 Personality Correlate of SRL Our ﬁndings are consistent with previous research on the eﬀect of self-esteem on self-rating. SRL was associated with low Neuroticism. People who are emotionally less stable (hence more likely to be low on self-esteem as well) were harsher when evaluating themselves than is warranted, while those who were emotionally stable tended to be more self-aggrandizing. It is possible that self-esteem and performance level were reciprocally related. People who were more conﬁdent, self-eﬃcacious, and energetic had better work performance. Those who had a track record of high performance had built up positive self-esteem in various aspects of their lives, while people who had not achieved a high level of success may, as a result, feel less personal worth. Their low performance level was observable to raters, whose input we used to compute the average peer rating. The fact remains, however, that persons who had low self-esteem tended to assess their own performance even lower than their peers actually saw them. Conversely, the emotionally stable employees rated themselves higher than others rated them. Incidentally, it is worth noting that elevated ratings might still be psychologically useful. First, ratings in our study were assigned for developmental purposes only, while in organizational settings ratings have been usually assigned for administrative decision-making as well as for developmental purposes (Mount et al., 1998). Can ﬁndings from ratings assigned for developmental purposes be generalizable to those assigned for administrative purposes? Kraiger and Ford (1985) investigated a similar question in their meta-analysis and noted that the eﬀects they found were not moderated by the administrative or developmental purpose of the ratings. In light of their ﬁndings, the generalizability concern might be relatively minor. A second potential limitation is that while in real life supervisors provide most ratings, peers provided the ratings in the present study. A third potential limitation is that we assured participants of complete anonymity and conﬁdentiality between the peer raters and the target persons. In performance appraisals other than 360◦feedback programs, however, the boss (rater) usually has the responsibility to deliver the results to the target person face-to-face. Faced with such a situational imperative without the protection of anonymity, the boss may be under pressure to distort his or her ratings for a variety of political or motivational reasons (e.g., Longenecker et al., 1987; Gioia and Longenecker, 1994), none of which we have considered in this paper. Personality Correlate of SRL These limitations may restrict the generalizability of our ﬁndings to peer, or 360◦ratings, more so than to supervisory ratings. We therefore welcome more research in this direction. The unspoken norm in most organizational settings is to manage others’ (especially supervisors’) impressions (e.g., Wayne and Liden, 1995). This involves, among other things, providing socially desirable responses in a self-appraisal. Lenient self- appraisals were used for impression management (to gain scarce rewards such as a promotion or an increase in salary), and to establish and maintain self-worth (see Paulhus, 1984). However, in the present research, where ratings were assigned conﬁdentially and for a developmental purpose, and were not seen by company managers, SRL could be attributed only to the latter of these two motives. People generous in their self- reports thought highly of themselves (which is a sign of emotional stability), and people who thought highly of themselves tended to rate themselves more generously. A fourth limitation is that there is the usual concern whether ﬁndings from data collected in one part of the world is generalizable elsewhere. To the extent that the Big Five model and measurement of personality are applicable in both the Chinese sample and other world populations, and that the ﬁve scales correlated in a pattern similar to that found in previous American and European studies, the personality-leniency correlation may be generalizable beyond our present sample. Although, we consider it a strength that our data came from employees in a single organization (and therefore participants experienced a similar organizational culture), replications in other industries and countries are essential to establishing the robustness of this relationship. Could this correlation be spurious, with high SRL individuals who had inﬂated their own scores on desirable personality dimensions? It is not surprising that people with a positive view of themselves recalled more of their own positive work- related behaviors while completing a self-appraisal form, as well as recalled behaviors that were related to desirable traits while completing a personality inventory. The supplementary regression analysis also revealed the disturbing fact that one’s self-rating on performance was not predicted by evaluations that others provided, but by one’s own personality. Future research on this topic should therefore use alternative personality measures that do not rely upon self-reports, or at least instruments that are less susceptible to distortion motivated by needs for social and self-approval. DISCUSSION This is the ﬁrst study that used a common metric to assess employees’ tendency to assign lenient ratings to self and to colleagues. Our basic premise was that raters’ personalities might be related to leniency when they report on their own and others’ work performance. We also considered the possibility that other-rating leniency and self-rating leniency might be aﬀected by the same personality characteristic. Unlike most previous studies that investigated how the target person’s self-rating diﬀers from another person’s (usually the supervisor’s) rating, our study focused on the leniency operationalized by comparing the employee’s judgment against an aggregation of ratings by several individuals, each of whom is reasonably knowledgeable about the target person. In the present investigation, where raters were not necessarily the targets’ managers (they were mostly peers) and the purpose of rating was non-administrative, the ﬁrst and third goals were not salient. As for the second, interpersonal goal, it is quite possible that the raters provided elevated ratings out of concerns about maintaining good relationships with the ratees. Given that our participants had been assured that their reports would not be seen by the target persons, there was minimal motivation for impression management. Other-rating leniency, reﬂecting a reluctance to deliver unfavorable observations to the recipients, could therefore be attributed to the person’s genuine or habitual April 2017 \| Volume 8 \| Article 521 April 2017 \| Volume 8 \| Article 521 Frontiers in Psychology \| www.frontiersin.org 7 Leniency Bias in Rating Cheng et al. concern for others. Their internalized value to be friendly and caring manifested itself through their agreeable personalities, and resulted in favorable reports about their peers. improving a rater’s ability to judge performance. Our study echoes this by identifying an element in the rating process, namely the rater’s personality characteristics, which may have nothing to do with ability. That one’s self-ratings could not be predicted by the aggregated ratings received from peers, but could be predicted by one’s own personality, adds to the ongoing controversy surrounding the accuracy and usefulness of this measurement (e.g., Atwater and Yammarino, 1997; Jones and Fletcher, 2002). A few limitations should be mentioned. Personality Correlate of SRL A ﬁfth and ﬁnal potential limitation is the use of averaged peer ratings for computing the leniency indices. This procedure might possibly result in the loss of information about rater (dis)agreement (Sulsky and Balzer, 1988). However, because no objective measures of performance were available for many types of jobs, averaging is by far the best method to remove at least some measurement errors and systematic, rater-dependent biases. On the positive side, since the same contextual factors likely to promote or reduce leniency for a given rater also REFERENCES recommendations for research and practice. J. Bus. 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Innushuk consult is the fund provider for the publication fee. Implications and Limitations With regard to the implementation of performance-management systems, Banks and Murphy (1985) questioned the eﬃcacy of April 2017 \| Volume 8 \| Article 521 Frontiers in Psychology \| www.frontiersin.org 8 Cheng et al. Leniency Bias in Rating to the use of their data, the data will be destroyed after suitable feedbacks are provided to them. All participants consented and signed the consent form. applied to other raters, any diﬀerence between the ratings assigned by that person and the ratings averaged from other people would primarily reﬂect dispositional factors and their interaction with situational factors, and not the situational factors alone. ETHICS STATEMENT KC, CH, and WC are the sole authors of this research paper. Each author contributed equally to the work of this manuscript in all aspects. The University of Hong Kong Research Committee. 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https://openalex.org/W4386686743	https://www.nature.com/articles/s41598-023-42367-9.pdf	English	null	Monitoring the impacts of crop residue cover on agricultural productivity and soil chemical and physical characteristics	Scientific reports	2,023	cc-by	10,805	Mohammad Kazemi Garajeh 1, Keyvan Hassangholizadeh 2, Amir Reza Bakhshi Lomer 3, Amin Ranjbari 2, Ladan Ebadi 4 & Mostafa Sadeghnejad 5 Mohammad Kazemi Garajeh 1, Keyvan Hassangholizadeh 2, Amir Reza Bakhshi Lomer 3, Amin Ranjbari 2, Ladan Ebadi 4 & Mostafa Sadeghnejad 5 To the best of our knowledge, the impacts of crop residue cover (CRC) on agricultural productivity and soil fertility have not been studied by previous researchers. In this regard, this study aims to apply an integrated approach of remote sensing and geospatial analysis to detect CRC and monitor the effects of CRC on agricultural productivity, as well as soil chemical and physical characteristics. To achieve this, a series of Landsat images and 275 ground control points (GCPs) collected from the study areas for the years 2013, 2015, and 2021 were used. A convolutional neural network (CNN), a class of artificial neural network has commonly applied to analyze visual imagery, was employed in this study for CRC detection in two classes (Not-CRC and CRC) for the years 2013, 2015, and 2021. To assess the effects of CRC, the Normalized Difference Vegetation Index (NDVI) was applied to Landsat image series for the years 2015 (22 images), 2019 (20 images), and 2022 (23 images). Furthermore, this study evaluates the impacts of CRC on soil fertility based on collected field observation data. The results show a high performance (Accuracy of > 0.95) of the CNN for CRC detection and mapping. The findings also reveal positive effects of CRC on agricultural productivity, indicating an increase in vegetation density by about 0.1909 and 0.1377 for study areas 1 and 2, respectively, from 2015 to 2022. The results also indicate an increase in soil chemical and physical characteristics, including EC, PH, Na, Mg, HCO3, K, silt, sand, and clay from 2015 to 2022, based on physical examination. In general, the findings underscore that the value of an integrated approach of remote sensing and geospatial analysis for detecting CRC and monitoring its impacts on agricultural productivity and soil fertility. This research can offer valuable insight to researchers and decision-makers in the field of soil science, land management and agriculture. Crop residue cover (CRC) refers to the deceased crop biomass that remains in fields after crop harvesting1,2. CRC is critical for preventing soil erosion and enhancing soil fertility due to its rich nitrogen, potassium, and phosphorus contents3,4. CRC has a significant impact on agriculture production and can influence biological production and biodiversity by increasing vegetation density and soil fertility5,6. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports/ Previous researchers have predominantly applied various indexes (e.g., CAI) and machine learning techniques (e.g., Random Forest) for CRC detection and mapping48,49. The present research, however, intends to accomplish three primary objectives: (1) map and monitor the effects of CRC on agricultural productivity using NDVI from 2015 to 2022, (2) model and monitor the impacts of CRC on soil chemical and physical characteristics from 2015 to 2022, and (3) examine the efficiency of a convolutional neu- ral network (CNN) in detecting and mapping CRC over the study areas. Landsat series images hold substantial potential for monitoring long-term changes in various features50,51. In this regard, Landsat series images used to detect CRC and evaluate its impacts on agricultural productivity and soil fertility in Maragheh and Heris, East Azerbaijan Province, Iran, spanning from 2013 to 2022. i Remote sensing also provides various spectral indexes for monitoring and mapping the structure (i.e., density and complexity) and distribution of green areas44. The Normalized Difference Vegetation Index (NDVI) is one of the vegetation indexes that has frequently used to assess the long-term trends of vegetation on a large scale. NDVI proves to be an effective method for monitoring plant productivity over time45, investigating the relation- ship between productivity and biodiversity, and determining vegetation composition and landscape structure46,47.f According to the literature, few studies have explored the effects of CRC on agricultural productivity and soil chemical and physical characteristics. Previous researchers have predominantly applied various indexes (e.g., CAI) and machine learning techniques (e.g., Random Forest) for CRC detection and mapping48,49. The present research, however, intends to accomplish three primary objectives: (1) map and monitor the effects of CRC on agricultural productivity using NDVI from 2015 to 2022, (2) model and monitor the impacts of CRC on soil chemical and physical characteristics from 2015 to 2022, and (3) examine the efficiency of a convolutional neu- ral network (CNN) in detecting and mapping CRC over the study areas. Landsat series images hold substantial potential for monitoring long-term changes in various features50,51. In this regard, Landsat series images used to detect CRC and evaluate its impacts on agricultural productivity and soil fertility in Maragheh and Heris, East Azerbaijan Province, Iran, spanning from 2013 to 2022. www.nature.com/scientificreports/ affected the quality and accuracy of the data17,18. Additionally, field-based approaches cannot provide timely information on CRC and its impacts over a wide region19. Therefore, it is necessary to employ a timely, cost- effective, and accurate approach to map and monitor CRC and its effects on agricultural production. ff Remote sensing provides multi-spectral and hyper-spectral datasets for mapping CRC and monitoring vegeta- tion density across large areas20. Within the realm of CRC mapping, the primary challenge lies in distinguishing CRC from bare soil21,22. This issue has been addressed by identifying specific bands near 2100 nm, leading to the development of several indexes, such as the Cellulose Absorption Index (CAI)23, Lignin Cellulose Absorption (LCA)24, Shortwave Infrared Normalized Difference Residue Index (SINDRI), Normalized Difference Index (NDI), and Dead Fuel Index (DFI)25–27. These indexes, on the other hand, are susceptible to atmosphere and ground features since they are based on spectral reflectance28,29. Additionally, the spectral reflectance for CRC indexes can be influenced by the type and shape of crop residues30. Many CRC indexes also exhibit reduced sen- sitivity in areas densely covered by CRC and can saturate to various extents31. Soil Adjusted Corn Residue Index (SACRI) and Modified Soil Adjusted Corn Residue Index (MSACRI), for instance, are both sensitive to CRC and become saturated at CRC values up to 0.2532. Therefore, learning-based approaches, such as machine and deep learning have been employed to retrieve bio-geophysical variables due to their computational efficiency32–39. These approaches excel in capturing the non-linear relationships between input variables and desired outputs40. According to the literature review, few studies have investigated the efficiency of deep learning convolutional neural networks for CRC monitoring and mapping. However, they have applied for landuse/cover mapping41,42 and crop type identification43. p ypi Remote sensing also provides various spectral indexes for monitoring and mapping the structure (i.e., density and complexity) and distribution of green areas44. The Normalized Difference Vegetation Index (NDVI) is one of the vegetation indexes that has frequently used to assess the long-term trends of vegetation on a large scale. NDVI proves to be an effective method for monitoring plant productivity over time45, investigating the relation- ship between productivity and biodiversity, and determining vegetation composition and landscape structure46,47. According to the literature, few studies have explored the effects of CRC on agricultural productivity and soil chemical and physical characteristics. Materials and methodology Materials. To evaluate the impacts of CRC on agricultural productivity and soil chemical and physical char- acteristics, Landsat images with a spatial resolution of 30 m were utilized for the years 2013/09/28, 2015/08/17, 2019, 2021/08/17, and 2022 (Table 1). A total of 275 ground control points (GCPs) were collected from the study areas using the global positioning system (GPS) and Google Earth, which were then used to construct CNN models. Of these GCPs, 70% were allocated for training the CRC models, while the remaining 30% were employed to validate the accuracy of the CNNs. p y y Various variables, including precipitation, soil temperature, and soil moisture, were generated from the Mod- erate Resolution Imaging Spectroradiometer (MODIS) and employed in this study to properly monitor the impacts of CRC on agricultural productivity and soil chemical and physical characteristics. Monthly and annual data of precipitation rate data were obtained from the General Department of Meteorology of East Azarbaijan Province (www.wamo.ir). We also accessed monthly and annual soil temperature and soil moisture data from https://giovanni.gsfc.nasa.gov. Soil sample points collection and laboratory measurement. To assess the effects of CRC on soil chemical and physical characteristics, soil samples were collected from the study areas at the end of June, prior to any plowing of agricultural land, ensuring the pristine condition of the topsoil texture. A total of 74 and 62 GCPs were collected from study areas 1 and 2, respectively. These GCPs were selected based on real field observations, existing digital soil type information, and landuse/cover maps spanning from 2015 to 2022. At each sampling point, four topsoil samples were collected and combined using a soil drill. To record the geographical position of soil sampling points, a portable GPS (UniStrong G120 with a positioning accuracy of 0.5 m) was used. Following collection, all soil samples were properly sealed, labelled, and moved to the laboratory for the measurement of key physicochemical soil attributes. To facilitate accurate analysis, soil samples were fully air-dried and sieved Table 1. More details regarding the employed Landsat images to monitor the effects of CRC on agricultural productivity and soil chemical and physical characteristics. Study areas CRC NDVI 1 2013/09/28 2015/08/17 2021/08/17 2015/05/13 2019/05/24 2022/05/23 2 2013/09/28 2015/08/17 2021/08/17 2015/05/13 2019/05/24 2022/05/23 Table 1. More details regarding the employed Landsat images to monitor the effects of CRC on agricultural productivity and soil chemical and physical characteristics. Mohammad Kazemi Garajeh 1, Keyvan Hassangholizadeh 2, Amir Reza Bakhshi Lomer 3, Amin Ranjbari 2, Ladan Ebadi 4 & Mostafa Sadeghnejad 5 It can also enhance the speed of enzymatic activities, decrease water consumption, lower soil temperature, and reduce air pollution and harmful gas emissions7–9. In summary, CRC can contribute to the sustainability of agriculture10,11.i g y y g Having accurate spatially and temporally resolved information about field-scale CRC and its impacts on vegetation and soil fertility will assist decision-makers in assessing the effectiveness of government conserva- tion programs and voluntary ecosystem service markets. This information will also facilitate the quantification of cropland biogeochemical processes through agro-ecosystem modeling12,13. The most conventional method for obtaining information about CRC and agricultural production is through field-based data collection, which involves farmers participating in commercial and governmental programs and annual residue surveys14–16. These techniques, however, are time-consuming and labor-intensive. Furthermore, several factors, including the per- sonal judgment of surveyors, the high cost of field surveys, and farmers’ concerns regarding data privacy, have 1Department of Civil, Constructional and Environmental Engineering, Sapienza University of Rome, 00138 Rome, Italy. 2Department of Remote Sensing and GIS, University of Tabriz, Tabriz, Iran. 3Department of Geography, Birkbeck, University of London, London WC1E 7HX, UK. 4Department of Surveying Engineering, Faculty of Engineering, Golestan University, Aliabad Katoul, Iran. 5Department of Geography and Geospatial Sciences, Kansas State University, 920 N17th Street, Manhattan, KS, USA. email: kazemi20.0432@gmail.com \| https://doi.org/10.1038/s41598-023-42367-9 Scientific Reports \| (2023) 13:15054 www.nature.com/scientificreports/ Methodology Th d l d This study applied an integrated approach of remote sensing and deep learning data-driven for assessing the impacts of CRC on agricultural productivity and soil chemical and physical characteristics. The methodology consisted of several phase. In the first phase, Convolutional Neural Network (CNN) was utilized to detect all instances of CRC within the study areas. Moving to the second phase, a series of Landsat 8 images were employed to estimate the Normalized Difference Vegetation Index (NDVI), which served as a means to monitor the den- sity of vegetation in the study areas between 2015 and 2022. Additionally, changes in soil chemical and physical characteristics across the study areas from 2015 to 2022 were assessed. Finally, various quantitative approaches were applied to validate the accuracy of the obtained results. Figure 1 reveals an overview of the methodology employed in this study for assessing the effects of CRC on agricultural productivity and soil chemical and physi- cal characteristics. Convolutional neural network for CRC mapping. CNN is a neural network architecture inspired by neuroscience findings52,53. It has been widely applied in the field of remote sensing for modeling and map- ping various Earth features, including landforms54, forest fires55, soil salinity distribution56, landslide57, landuse/ cover41 and more. It is made of layers of artificial neurons called nodes. Each node calculates a weighted sum of inputs and returns an activation map53,58. Each node in a layer is defined by its weight values59. In the context of images, when a layer receives input, such as an image, it extracts visual properties. Initially, the CNN finds edges in the image. This partial definition of the image is then passed to the next layer, which begins to identify features like corners and color groups. This refined image definition is further processed in subsequent layers until the network makes prediction60,61. This research utilized GCPs in conjunction with Landsat 8 imagery, featuring a 30-m spatial resolution, to facilitate the training of CRC models. A comprehensive set of 275 GCPs was used for the model training process. Subsequently, all generated CRC models were categorized into two classes: Not-CRC and CRC. In the realm of input images, elevating the model’s input resolution yields enhanced performance. The rationale behind this phenomenon is that the inclusion of finer details (specifically, information) results in improved overall performance. Materials and methodology Study areas CRC NDVI 1 2013/09/28 2015/08/17 2021/08/17 2015/05/13 2019/05/24 2022/05/23 2 2013/09/28 2015/08/17 2021/08/17 2015/05/13 2019/05/24 2022/05/23 Table 1. More details regarding the employed Landsat images to monitor the effects of CRC on agricultural productivity and soil chemical and physical characteristics. Table 1. More details regarding the employed Landsat images to monitor the effects of CRC on agricultura productivity and soil chemical and physical characteristics. https://doi.org/10.1038/s41598-023-42367-9 Scientific Reports \| (2023) 13:15054 \| www.nature.com/scientificreports/ through a 2 mm sieve to eliminate extraneous materials. Using a digital multi-parameter measuring apparatus (Multi 3420 Set B, WTWGmbH, Germany) at a room temperature of 25 °C, soil electrical conductivity and soil water ratio were determined based on the leachate prepared at a soil-to-water ration of 1:2.5. through a 2 mm sieve to eliminate extraneous materials. Using a digital multi-parameter measuring apparatus (Multi 3420 Set B, WTWGmbH, Germany) at a room temperature of 25 °C, soil electrical conductivity and soil water ratio were determined based on the leachate prepared at a soil-to-water ration of 1:2.5. Methodology Th d l d , k (1) (2) f (x) = tanh (x) = ex −e−x ex + e−x (2) where f denotes the non-linear function, ∗ represents the represented convolutional operator, k denotes convo- lutional kernels, wj represents the weights, and bj denotes the bias. where f denotes the non-linear function, ∗ represents the represented convolutional operator, k denotes convo- lutional kernels, wj represents the weights, and bj denotes the bias. (3) aj = max N×1 an×1 j µ(n, 1) (3) where µ(n, 1) is the window function to patch the earlier layer and aj is the maximum in the patch. h h d l l d b dd h l l l Th h j In the next step, a weighted sum was calculated by adding up the convolutional layers. This sum was then passed through an activation function to generate output. The Rectified Linear Unit (ReLu) function employed in this study to construct the CRC model. Equation (4) is defined the ReLu70. (4) f (x) = 0, x < 0 x, x ≥0 (4) Finally, all parameters in the CNN were optimized using the back-propagation algorithm. The parameter optimization is applied to reduce the loss function value, which is expressed by Eq. (5). The results obtained from the CRC-CNNs were categorized into two classes: not-CRC and CRC (See Fig. 4). (5) L y, ˆy = −1 N N i=1 yi log ˆyi + 1 −yi log 1 −ˆyi (5) where N is the total number of the data samples, yi is the actual result of sample i (0 or 1), ˆyi is the predicted kelihood of sample i having the output 1, and y and yi are the vectors of real outputs and predicted probabilities Normalized difference vegetation (NDVI) for extracting vegetation density. In numerous stud- ies, satellite-derived vegetation indexes are frequently employed to monitor vegetation status, with the Normal- ized Difference Vegetation Index (NDVI) being a commonly utilized choice. NDVI serves to gauge the spa- tial distribution and relative abundance of vegetation71–73. These remote sensing-based spectral indexes utilize reflectance measurements gathered from satellites and aircraft to evaluate vegetation status71. NDVI is com- monly used to measure vegetation cover and is closely associated with chlorophyll content, energy absorption, and photosynthetic capacity74. This study applied the NDVI indicator for assessing the impacts of CRC on agri- cultural productivity from 2015 to 2022, as defined by Eq. (6). Methodology Th d l d The underlying hypothesis posits that the augmentation of performance with increased image size is solely attributed to the infusion of more intricate details62. Table 2 represents further details regarding the applied parameters in CNN. g g In CNN, like in other neural networks, there are three layers, namely an input layer, a hidden layer (can be more than one), and an output layer63. An input layer is comprised of a m × n matrix with a respective feature value at each node. A convolutional layer that is immediately adjacent to the input layer is sometimes referred to as a feature extractor. The reason for this is that it is used to extract the features of an image. A back-propagation algorithm optimizes several convolutional kernels in the convolutional layer64,65. The output of the convolutional layer will be the input for the next layer66. A convolutional layer consists of a pooling layer, multiple weights, and Figure 1. An overview of the employed methodology to evaluate the effects of CRC on agricultural productivity and soil fertility. Figure 1. An overview of the employed methodology to evaluate the effects of CRC on agricultural productivity and soil fertility. https://doi.org/10.1038/s41598-023-42367-9 Scientific Reports \| (2023) 13:15054 \| www.nature.com/scientificreports/ Table 2. Characteristics of employed CNN models for detecting and mapping CRC. Year Activation function Loss function Optimizer 2013 Case study areas 1 and 2 ReLu Cross-Entropy ADAM 2015 ReLu Cross-Entropy ADAM 2021 ReLu Cross-Entropy ADAM Table 2. Characteristics of employed CNN models for detecting and mapping CRC. an activation function67. Max-pooling is similar to the convolutional layer but instead of taking a dot product between the input and the kernel, it takes the max of the region from the input overlapped by the kernel. In this study, a maximum operator was used to downsample the feature maps in the encoder. Maximum pooling is required to split the feature maps into several rectangular regions for generating maximum values in each layer68. The fully connected layer is used to reduce the loss function and subsequently outputs the classification result69. Equations (1)–(3) are defined convolutional manipulation and max pooling, respectively. (1) Cj = N i f w∗ j vi + bj , j = 1, 2, . . . , k (1) Cj = N i f w∗ j vi + bj , j = 1, 2, . . . Study area Study a ea Case study area 1. The case study area is located at 46° 27ʹ 21ʺ E, 37° 15ʹ 44ʺ N in Maragheh, East Azerbai- jan Province, Iran (Fig. 2a, b and e). Clay loam is the main soil texture in the study area. Wheat and pea are the major crops, sometimes in rotation with forage crops. Figure 2c reveals the observed CRC in the case study area 1. Since tillage and planting were mostly conducted under conservation tillage and reduced tillage procedures, different percentages of CRC were readily available. Based on our observation, this area has a regular rotation of products. This convinces us to focus on this area for assessing the impacts of CRC on agricultural productivity and soil chemical and physical characteristics. The annual precipitation rate and temperature are 330 ml and 12.5 °C, respectively for the study area76,77. Case study area 2. Figure 2a, b and f shows the case study area 2, located in Heris, East Azerbaijan Prov- ince, Iran. Wheat is the predominant crop grown in this area. Figure 2d reveals the observed CRC over the case study area 2. From a soil perspective, clay is the dominant soil type in this region. Additionally, this area is characterized as a semi-arid region with hot summers and cold winters. The annual precipitation rate and tem- perature for this study area are approximately 315 mm and 8.5 °C, respectively. Methodology Th d l d The NDVI uses the Red (620–670 nm) and Near Infra-Red (NIR) (841–876 nm) spectrums to estimate vegetation density. (6) NDVI = (PNIR −PRed) (PNIR + PRed) (6) where PNIR and PRed are top of atmosphere reflectance (TOA) in the NIR and Red bands, respectively. NDVI values range from − 1 to + 1. Since chlorophyll absorbs light, the red spectrum reflection is always lower than the NIR band spectrum reflectance for green vegetation. NDVI values in vegetation regions cannot be less than 0 and below or equal to 0.1 represent water bodies or bare ground due to low reflectance recorded in NIR. The values 0.2–0.5 represent sparse vegetation, while values near 1 represent dense vegetation. Thus this study employed a series of Landsat 8 images (A total number of 65 images) from 2015 to 2022, which were individually converted to NDVI using Eq. (6) for each year. https://doi.org/10.1038/s41598-023-42367-9 Scientific Reports \| (2023) 13:15054 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ Accuracy assessment. Accuracy assessment is a crucial step in the domain of image classification75. In this regard, a set of quantitative approaches, including Intersection Over Union (IOU) values, Recall (RC), Precision (PC), Specificity (SP), F-measure (FM), Accuracy (ACC), and Kappa (KP) applied to assess the accuracy of the classification. Equations (7)–(11) describe the employed approaches. The results of the CNN for detecting and monitoring CRC changes also are presented in Table 3. According to the information presented in Table 3, the CNN performs well with the ACC of > 0.95 for CRC mapping. (7) IOU = AO ∩EO AO ∪EO = TP TP + FP + FN (8) Recall = TP TP + FN (9) Speciﬁcity = TN TN + FP (10) Accuracy = TP + TN TP + TN + FN + FP (11) Kappa = TP + TN −TPexpected −TNexpected TP + TN + FN + FP −TPexpected −TNexpected (7) IOU = AO ∩EO AO ∪EO = TP TP + FP + FN (7) (8) (9) (10) (11) where AO is actual output; EO is on behalf of expected result; TP , FP , FN , and TN are true positive, false positive, false negative, and true negative, respectively. Scientific Reports \| (2023) 13:15054 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ According to Fig. 4, the highest NDVI values were 0.5945 and 0.6288 for the year 2015 in case study areas 1 and Figure 2. Location of study areas, generated in the ArcGIS 10.6 software (www. esri.com): (a) in the world, (b) in the north-west of Iran, (c) and (d) examples of observed CRC over the study areas, (e) location of study area in Maragheh city and (f) location of study area in Heris city. Figure 2. Location of study areas, generated in the ArcGIS 10.6 software (www. esri.com): (a) in t in the north-west of Iran, (c) and (d) examples of observed CRC over the study areas, (e) location o in Maragheh city and (f) location of study area in Heris city. Figure 2. Location of study areas, generated in the ArcGIS 10.6 software (www. esri.com): (a) in the world, (b) in the north-west of Iran, (c) and (d) examples of observed CRC over the study areas, (e) location of study area in Maragheh city and (f) location of study area in Heris city. Figure 2. Location of study areas, generated in the ArcGIS 10.6 software (www. esri.com): (a) in the world, (b) in the north-west of Iran, (c) and (d) examples of observed CRC over the study areas, (e) location of study area in Maragheh city and (f) location of study area in Heris city. Figure 2. Location of study areas, generated in the ArcGIS 10.6 software (www. esri.com): (a) in the world, (b) in the north-west of Iran, (c) and (d) examples of observed CRC over the study areas, (e) location of study area in Maragheh city and (f) location of study area in Heris city. According to Fig. 4, the highest NDVI values were 0.5945 and 0.6288 for the year 2015 in case study areas 1 and 2, respectively. However, the NDVI values increased by about 0.1909 and 0.1377 in case study areas 1 and 2, respectively for the year 2022 (as shown in Fig. 4). This means that the impacts of CRC on agricultural productiv- ity may be positive. For being sure about the positive effects of CRC on agricultural productivity, monthly and annual changes in certain predisposing variables, including precipitation, soil moisture, and soil temperature, were evaluated. The results indicate no significant changes in precipitation, soil moisture, and soil temperature. Resultsh This study applied an integrated approach of remote sensing and geospatial analysis for assessing the effects of CRC on agricultural productivity and soil chemical and physical characteristics. To this end, an automated CNN employed to detect CRC in two classes: not-CRC and CRC, for the years 2013, 2015, and 2021. A total of 275 GCPs were incorporated into the CRC models, with 70% for training and 30% for testing the results of CRC net- works. The results show the high efficiency of CNN for detecting and mapping CRC. According to Table 3, CNN performed well in mapping CRC with an ACC of > 0.95 for the years 2013, 2015, and 2021. Figure 3 provides the results of applied CNN for detecting and monitoring CRC for the years 2013, 2015, and 2021. pp g g y A series of Landsat images was then used to evaluate vegetation status using NDVI for the years 2015, 2019, and 2022. Figure 4 reveals the results of NDVI in the study areas. This study also considered the effects of other predisposing variables, including precipitation, soil moisture, and soil temperature, on the changes in vegetation density for the years 2015, 2019, and 2022. The results reveal an increase in NDVI values from 2015 to 2022. Table 3. The results of CNN for CRC detecting and mapping. Stud area Years IOU RC PC KP ACC 1 2013 0.8699 0.8845 0.9641 0.9154 0.9685 2015 0.8700 0.8701 0.9581 0.9014 0.9780 2021 0.8612 0.8835 0.9600 0.9123 0.9614 2 2013 0.8534 0.8778 0.9514 0.9124 0.9612 2015 0.8614 0.8701 0.9435 0.99012 0.9564 2021 0.8542 0.8714 0.9524 0.9089 0.9587 Table 3. The results of CNN for CRC detecting and mapping. Stud area Years IOU RC PC KP ACC 1 2013 0.8699 0.8845 0.9641 0.9154 0.9685 2015 0.8700 0.8701 0.9581 0.9014 0.9780 2021 0.8612 0.8835 0.9600 0.9123 0.9614 2 2013 0.8534 0.8778 0.9514 0.9124 0.9612 2015 0.8614 0.8701 0.9435 0.99012 0.9564 2021 0.8542 0.8714 0.9524 0.9089 0.9587 Table 3. The results of CNN for CRC detecting and mapping. https://doi.org/10.1038/s41598-023-42367-9 Scientific Reports \| (2023) 13:15054 \| www.nature.com/scientificreports/ As seen in Table 4, several points were created over the study areas, and the Zonal Statistics as Table tool in the Arc GIS 10.8 environment was used to assess the changes in precipitation, soil moisture, and soil temperature for the years 2015, 2019, and 2022. According to Table 4, the mean precipitation values were 331, 329, and 328 for study area 1 for the years 2015, 2019, and 2022. Similarly, the mean precipitation values for study area 2 were estimated at 252, 250, and 249 for the years 2015, 2019, and 2022 (Table 4). Regarding soil moisture, the mean values for study area 1 were 0.3804, 0.3919, and 0.3878 for the years 2015, 2019, and 2022, respectively. The mean soil moisture values were estimated at 0.4247, 0.4122, and 0.4214 for the same years, as shown in Table 4. Lastly, the mean soil temperature values in Kelvin (K) were 283.52 K, 282.61 K, and 283.21 K for study area 1 for the years 2015, 2019, and 2022, respectively. Similarly, for study area 2, the mean soil temperature values were 282.85 K, 281.86 K, and 281.98 K for the same years, as presented in Table 4. https://doi.org/10.1038/s41598-023-42367-9 Scientific Reports \| (2023) 13:15054 \| www.nature.com/scientificreports/ Figure 3. Generated CRC maps using an automated CNN approach in two classes in the ArcGIS 10.6 software (www. esri.com), namely not-CRC and CRC: (a and b) for the year 2013 for the case study areas 1 and 2, respectively, (c and d) for the year 2015 for the case study areas 1 and 2, respectively, and e and f) for the year 2021 for the case study areas 1 and 2, respectively. Figure 3. Generated CRC maps using an automated CNN approach in two classes in the ArcGIS 10.6 software (www. esri.com), namely not-CRC and CRC: (a and b) for the year 2013 for the case study areas 1 and 2, respectively, (c and d) for the year 2015 for the case study areas 1 and 2, respectively, and e and f) for the year 2021 for the case study areas 1 and 2, respectively. Discussion l di General discussion. CRC improves soil structure, increases organic matter content, enhances vegetation density, reduces evaporation, and aids in carbon dioxide fixation. Managing residues on agricultural land has a variety of positive effects on soil quality. Moreover, crop residues can be used for the production of biofuels35,78. Previous studies have not explored the effects of CRC on agricultural productivity and the chemical and physical characteristics of the soil. They have applied several spectral indexes, including LCA, SINDRI, NDI, and DFI, for detecting and mapping CRC. Moisture tends to darken soils and residue more equally in the visible bands, unless the residue is very fresh79. When attempting to use spectral differences (multi-band indices) for residue characterization, the influence of moisture on this process becomes more intricate. Under dry conditions, it is possible to distinguish the adsorption characteristics of crop residue from soil80. Well-calibrated broadband spectral indexes like Normalized Difference Tillage Index (NDTI) can accurately map CRC during such condi- tions. However, in wet conditions, differences between Landsat SWIR bands become less noticeable, causing these indexes to lose effectiveness. This is because moisture presence can both consistently lower reflectance across the SWIR spectrum and variably impact the reflectance (due to moisture absorption features) of both soil and residue, which may dry or wet at varying rates. Addressing both these challenges requires applying a moisture correction to NDTI to generate reliable percent residue assessments under high moisture conditions81. Recent studies have also applied learning-based approaches such as machine learning algorithms32–34 for detect- ing and mapping CRC. This study, on the other hand, employed an integrated approach of remote sensing and https://doi.org/10.1038/s41598-023-42367-9 Scientific Reports \| (2023) 13:15054 \| www.nature.com/scientificreports/ Figure 4. Generated vegetation cover maps using NDVI in the ArcGIS 10.6 software (www. esri.com): (a and b) for the year 2015 for the case study areas 1 and 2, respectively, (c and d) for the year 2019 for the case study areas 1 and 2, respectively, and (e and f) for the year 2022 for the case study areas 1 and 2, respectively. Figure 4. Generated vegetation cover maps using NDVI in the ArcGIS 10.6 software (www. Table 4. Annual precipitation (ml), soil moisture, and soil temperature (K) changes for the years 2015, 201 and 2022. Discussion l di Results of the Zonal Statistics as Table tool in the Arc GIS 10.8 environment for assessing the change in vegetation status for the years 2015, 2019, and 2022 for the study area 1. Table 5. Results of the Zonal Statistics as Table tool in the Arc GIS 10.8 environment for assessing the change in vegetation status for the years 2015, 2019, and 2022 for the study area 1. Table 6. Results of the Zonal Statistics as Table tool in the Arc GIS 10.8 environment for assessing the change in vegetation status for the years 2015, 2019, and 2022 for the study area 2. Point 2015 2019 2022 1 0.6149 0.6095 0.6704 2 0.6055 0.6305 0.6726 3 0.5961 0.5396 0.5904 4 0.6006 0.5951 0.7055 5 0.5951 0.6478 0.7543 6 0.6164 0.6518 0.7571 7 0.5888 0.5151 0.6749 8 0.6021 0.6164 0.7394 Table 6. Results of the Zonal Statistics as Table tool in the Arc GIS 10.8 environment for assessing the change in vegetation status for the years 2015, 2019, and 2022 for the study area 2. Point 2015 2019 2022 1 0.6149 0.6095 0.6704 2 0.6055 0.6305 0.6726 3 0.5961 0.5396 0.5904 4 0.6006 0.5951 0.7055 5 0.5951 0.6478 0.7543 6 0.6164 0.6518 0.7571 7 0.5888 0.5151 0.6749 8 0.6021 0.6164 0.7394 Table 6. Results of the Zonal Statistics as Table tool in the Arc GIS 10.8 environment for assessing the change in vegetation status for the years 2015, 2019, and 2022 for the study area 2. Table 6. Results of the Zonal Statistics as Table tool in the Arc GIS 10.8 environment for assessing the change in vegetation status for the years 2015, 2019, and 2022 for the study area 2. geospatial analysis to map and monitor the CRC and assess its impacts on agricultural productivity and soil chemical and physical characteristics. The findings highlight the high ability of automated CNN in detecting and mapping CRC. The outcomes of this study demonstrate the CNN’s capacity for mapping CRC using Landsat remote sensing images. In comparison to traditional machine learning methods like support vector machines and neural networks, CNN mitigates the loss of edge information during the extraction of CRC from remote sensing images. Traditional machine learning techniques primarily rely on the accuracy of the extracted features, encompassing factors like pixel values, shapes, textures, and positions. Discussion l di esri.com): (a and b) for the year 2015 for the case study areas 1 and 2, respectively, (c and d) for the year 2019 for the case study areas 1 and 2, respectively, and (e and f) for the year 2022 for the case study areas 1 and 2, respectively. Table 4. Annual precipitation (ml), soil moisture, and soil temperature (K) changes for the years 2015, 2019, and 2022. Variable Study area 2015 2019 2022 Precipitation 1 331 329 328 2 252 250 249 Soil moisture 1 0.3804 0.3919 0.3878 2 0.4247 0.4122 0.4214 Soil temperature 1 283.52 282.61 283.21 2 282.85 281.86 281.98 Table 4. Annual precipitation (ml), soil moisture, and soil temperature (K) changes for the years 2015, 2019, and 2022. Variable Study area 2015 2019 2022 Precipitation 1 331 329 328 2 252 250 249 Soil moisture 1 0.3804 0.3919 0.3878 2 0.4247 0.4122 0.4214 Soil temperature 1 283.52 282.61 283.21 2 282.85 281.86 281.98 Table 4. Annual precipitation (ml), soil moisture, and soil temperature (K) changes for the years 2015, 2019, and 2022. Table 4. Annual precipitation (ml), soil moisture, and soil temperature (K) changes for the years 2015, 2019, and 2022. https://doi.org/10.1038/s41598-023-42367-9 Scientific Reports \| (2023) 13:15054 \| www.nature.com/scientificreports/ Table 5. Results of the Zonal Statistics as Table tool in the Arc GIS 10.8 environment for assessing the change in vegetation status for the years 2015, 2019, and 2022 for the study area 1. Point 2015 2019 2022 1 0.5204 0.6089 0.7243 2 0.5090 0.5790 0.6601 3 0.5080 0.6186 0.7045 4 0.4069 0.6704 0.6980 5 0.4585 0.5972 0.6469 6 0.3562 0.5798 0.6173 7 0.5371 0.6987 0.7154 8 0.4436 0.5824 0.6334 9 0.3986 0.6368 0.6562 10 0.5285 0.6936 0.7250 Table 5. Results of the Zonal Statistics as Table tool in the Arc GIS 10.8 environment for assessing the change in vegetation status for the years 2015, 2019, and 2022 for the study area 1. Point 2015 2019 2022 1 0.5204 0.6089 0.7243 2 0.5090 0.5790 0.6601 3 0.5080 0.6186 0.7045 4 0.4069 0.6704 0.6980 5 0.4585 0.5972 0.6469 6 0.3562 0.5798 0.6173 7 0.5371 0.6987 0.7154 8 0.4436 0.5824 0.6334 9 0.3986 0.6368 0.6562 10 0.5285 0.6936 0.7250 Table 5. Results of the Zonal Statistics as Table tool in the Arc GIS 10.8 environment for assessing the change in vegetation status for the years 2015, 2019, and 2022 for the study area 1. Table 5. Discussion l di Soil properties Case study area 1 Case study area 2 2015 2019 2022 2015 2019 2022 Sand 25.14 25.19 25.31 30.48 30.89 31.02 Silt 32.85 32.99 33.01 67.25 67.87 68.12 Clay 8.44 8.64 8.87 7.89 7.90 7.90 Soil properties Case study area 1 Case study area 2 2015 2019 2022 2015 2019 2022 Sand 25.14 25.19 25.31 30.48 30.89 31.02 Silt 32.85 32.99 33.01 67.25 67.87 68.12 Clay 8.44 8.64 8.87 7.89 7.90 7.90 Table 8. Results of the collected GCPs from the study areas for assessing the effects of CRC on soil physical haracteristics from 2015 to 2022. strategically positioned within the ArcGIS 10.8 environment. In order to accurately quantify the effects of CRC on agricultural productivity, we utilized the Zonal Statistics as Table tool within the ArcGIS 10.8 environment. Tables 5 and 6 show the results of applied Zonal Statistics as Table tool over the study areas 1 and 2, respectively. strategically positioned within the ArcGIS 10.8 environment. In order to accurately quantify the effects of CRC on agricultural productivity, we utilized the Zonal Statistics as Table tool within the ArcGIS 10.8 environment. Tables 5 and 6 show the results of applied Zonal Statistics as Table tool over the study areas 1 and 2, respectively. y y According to Table 5, for point 1, the NDVI mean value increased by about 0.2039 from 2015 to 2022 in study area 1. Similarly, the NDVI mean value for point 2 increased by about 0.1511 from 2015 to 2022, as shown in Table 5. As we can see in Table 5, for point 3, the NDVI mean value increased by about 0.1965 from 2015 to 2022. Point 4 also exhibited an increase in the NDVI mean value of about 0.2911 from 2015 to 2022, as indicated in Table 5. According to Table 5, for point 5, the NDVI mean value increased by about 0.1884 from 2015 to 2022. Point 6 depicted a similar trend with an increase of about 0.2611 in the NDVI mean value from 2015 to 2022. As we can see in Table 5, for point 7, the NDVI mean value increased by about 0.1783 from 2015 to 2022. The NDVI mean value also increased by about 0.1898 from 2015 to 2022 for point 8, as shown in Table 5. According to Table 5, for point 9, the NDVI mean value increased by about 0.2576 from 2015 to 2022. Discussion l di Similarly, the NDVI mean value for point 10 increased by about 0.1965 from 2015 to 2022, as shown in Table 5. p y According to Table 6, for point 1, the NDVI mean value increased by about 0.0555 from 2015 to 2022 in study area 2. Similarly, the NDVI mean value for point 2 increased by about 0.0671 from 2015 to 2022, as shown in Table 6. As we can see in Table 6, for point 3, the NDVI mean value increased by about 0.0057 from 2015 to 2022. Point 4 also saw an increase in the NDVI mean value by about 0.1049 from 2015 to 2022, as indicated in Table 6. According to Table 6, for point 5, the NDVI mean value increased by about 0.1592 from 2015 to 2022 in study area 2. Point 6 exhibited a similar trend with an increase of about 0.1407 in the NDVI mean value from 2015 to 2022. As we can see in Table 6, for point 7, the NDVI mean value increased by about 0.0861 from 2015 to 2022. Lastly, point 8 indicated an increase in the NDVI mean value of about 0.1373 from 2015 to 2022, as shown in Table 6. The impacts of CRC on soil chemical and physical characteristics. Tables 7 and 8 show the results of CRC’s effects on soil chemical and physical characteristics. As we can see in Table 7, the EC level increased by about 0.04 and 0.02 from 2015 to 2022 in case study areas 1 and 2, respectively. According to Table 7, while the PH decreased by about 0.02 for study area 1 from 2015 to 2022, it increased by about 0.03 for study area 2. The Na values also exhibited an increase of about 0.02 and 0.01 for study areas 1 and 2, respectively, during the same period, as shown in Table 7. As we can see in Table 7, the Mg level increased by about 0.08 and 0.06 from 2015 to 2022 in case study areas 1 and 2, respectively. The HCO3 values also shown an increase of about 0.07 and 0.22 for study areas 1 and 2, respectively, from 2015–2022, as shown in Table 7. According to Table 7, while the K level decreased by about 0.01 for study area 1 from 2015 to 2022, it increased by about 0.01 for study area 2. Discussion l di Conversely, deep learning through con- volutional neural networks can autonomously acquire pertinent high-level features directly from remote sensing images for CRC classification. This streamlines the process, lessening the need to design custom feature extrac- tors for every classification task. The results of this study also reveal the positive impacts of CRC on agricultural productivity and soil fertility (See Fig. 4 and Tables 7 and 8). The impacts of CRC on vegetation density. As mentioned in previous sections, a primary objective of this study is to assess the effects of CRC on agricultural productivity. In this regard, the NDVI index applied to Landsat series images captured in the years 2015, 2019, and 2022 to map the greenness of the study areas. Subsequently, we established a total of 10 points for study area 1 and 8 points for study area 2. These points were Table 7. Results of the collected GCPs from the study areas for assessing the effects of CRC on soil chemical characteristics from 2015 to 2022. Soil properties Case study area 1 Case study area 2 2015 2019 2022 2015 2019 2022 EC 0.59 0.61 0.63 0.49 0.50 0.51 PH 7.11 7.09 7.09 6.78 6.80 6.81 Na 0.56 0.57 0.58 0.53 0.53 0.54 Mg 1.68 1.75 1.76 1.33 1.35 1.39 HCO3 3.73 3.78 3.80 2.35 2.58 2.57 K 0.13 0.12 0.12 0.11 0.12 0.12 Table 7. Results of the collected GCPs from the study areas for assessing the effects of CRC on soil chemical characteristics from 2015 to 2022. Soil properties Case study area 1 Case study area 2 2015 2019 2022 2015 2019 2022 EC 0.59 0.61 0.63 0.49 0.50 0.51 PH 7.11 7.09 7.09 6.78 6.80 6.81 Na 0.56 0.57 0.58 0.53 0.53 0.54 Mg 1.68 1.75 1.76 1.33 1.35 1.39 HCO3 3.73 3.78 3.80 2.35 2.58 2.57 K 0.13 0.12 0.12 0.11 0.12 0.12 Table 7. Results of the collected GCPs from the study areas for assessing the effects of CRC on soil chemical characteristics from 2015 to 2022. Table 7. Results of the collected GCPs from the study areas for assessing the effects of CRC on soil chemical characteristics from 2015 to 2022. https://doi.org/10.1038/s41598-023-42367-9 Scientific Reports \| (2023) 13:15054 \| www.nature.com/scientificreports/ Table 8. Results of the collected GCPs from the study areas for assessing the effects of CRC on soil physical characteristics from 2015 to 2022. Conclusionh This study introduced an integrated approach combining remote sensing and geospatial analysis to effectively identify and map CRC while also evaluating its impact on vegetation density, as well as soil chemical and physical attributes. The results exhibit the remarkable efficiency of an automated CNN data-driven approach (> 0.96) in detecting and mapping CRC. Furthermore, the outcomes underscore the positive influence of CRC on vegetation density and soil chemical and physical characteristics.hi y y The findings of this study demonstrate that the integration of remote sensing and geospatial analysis is adept at accurately detecting and mapping CRC while simultaneously assessing its effects on vegetation status and soil fertility. The efficiency of Landsat series images in mapping and monitoring dynamic features like vegetation and soil is also emphasized in this research.hfii The study underscores the efficacy of learning-based methods in proficiently classifying Earth’s features. When paired with satellite-based datasets and Geographic Information Systems (GIS), this computer-based approach proves to be notably efficient. Importantly, the research reveals that crop residue cover contributes to heightened soil productivity in terms of chemical and physical attributes. Moreover, it serves as means to maintain soil moisture levels, a critical factor for crop survival, particularly in semi-arid and arid regions.hi The insights from this study hold significant value for researchers engaged in soil science and agriculture. Additionally, the findings provide essential information for decision-makers and planners involved in land man- agement and soil erosion control. Ultimately, our research contributes to enhancing the understanding of the multifaceted impacts of CRC, benefiting both scientific advancements and practical land management strategies. Discussion l di As seen in Table 8, the amount of sand increased by about 0.21 and 0.54 in case study areas 1 and 2, respec- tively, from 2015 to 2022. Similarly, the amount of silt also increased by about 0.16 and 0.87 in the study areas 1 and 2, respectively during the same period, as shown in Table 8. According to Table 8, there was an increase of 0.43 and 0.01 in clay content for study areas 1 and 2, respectively, between 2015 and 2022. Limitation of this study. This study developed an integrated approach of remote sensing and geospatial analysis for monitoring and mapping the effects of CRC on agricultural productivity and soil chemical and phys- ical characteristics. Despite the satisfying results obtained from this work, there exist two primary limitations that future research in the domain of CRC and its impacts on vegetation density and soil fertility could address. Firstly, the current study relied on Landsat series images with a spatial resolution of 30 m. In future endeavors, the availability of Sentinel-2 images could be exploited as a potential alternative for a more extended time period, aiding in the detection and mapping of CRC. Secondly, a recommended avenue for forthcoming research is the adoption of an integrated methodology that combines object-based image analysis and Convolutional Neural Networks (CNNs) to monitor CRC changes. This amalgamation is noteworthy because object-based image anal- ysis harnesses numerous features, such as brightness, to detect specific features. This comprehensive approach enhances the precision and accuracy of the outcomes obtained. In conclusion, this study’s innovative approach has provided valuable insights into the effects of CRC on agricultural systems. 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https://openalex.org/W4223954210	https://www.researchsquare.com/article/rs-1540013/latest.pdf	English	null	Finite element simulation of treatment with locking platefor distal fibula fractures	Research Square (Research Square)	2,022	cc-by	4,128	Finite element simulation of treatment with locking platefor distal fibula fractures Yafeng Li (  liyf@tiangong.edu.cn ) Yafeng Li (  liyf@tiangong.edu.cn ) Tiangong University Wei Yang Tiangong University Xi Zhang Tianjin Hospital Cheng Yu Tiangong University Zhengzhao Ji Tiangong University Jie Sun Tianjin Hospital Jing Zhang Indiana University - Purdue University Research Article Keywords: distal fibula fracture, fibula model, finite element analysis, stress masking effect. Posted Date: April 14th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-1540013/v1 DOI: https://doi.org/10.21203/rs.3.rs-1540013/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/15 Page 1/15 Abstract In this study, an improved finite element model is applied to compare the biomechanical stability of plates with three different options in the treatment of distal fibula fractures. A CT scan of the knee to ankle segment of a volunteer was performed. A three-dimensional finite element model of the fibula was reconstructed based on the CT data. Three different loads (uni-pedal standing, torsion, and twisting) were applied, the same as in the experiments in the literature. The stresses and strains of the three options were compared under the same loads, using a 4-hole locking plate (Option A), a 5-hole locking plate (Option B), and a 6-hole locking plate (Option C) in a standard plate for lateral internal fixation. The simulation results show that all three options showed a stress masking effect. Option C had the best overall biomechanical performance and could effectively distribute the transferred weight. This is because option C has greater torsional stiffness and better biomechanical stability than options A and B, and therefore, option C is the recommended internal fixation method for distal fibula fractures. The finite element analysis method developed in this work is applicable to the stress analysis of fracture treatment options in other body parts. 1 Introduction Distal fibular fractures account for approximately 3.92% of all fractures in the human body(Ju et al. 2019). Most distal fibula fractures require internal fixation, with plate internal fixation techniques being the most common. In the previous study (Fan et al. 2019), the use of a 4-hole angle stabilization plate and screws, in the clinical treatment of distal fibular fractures, improved the time of bone healing for eight weeks. In another study (Rozbruch et al. 1998), the 4-hole locking plate, 5-hole locking plate, and 6-hole locking plate internal fixation protocols were chosen. Another study (Fang et al. 2018) showed that the use of two to three screws would be sufficient for each major fracture block. In parallel to experiments, the finite element model has been used to stimulate the bone healing process, with different screw layouts of the joint plate (Hess and Sommer 2011) (Cao et al. 2019). The relationship between the effective length of the plate on the stability of the tibiofibular was studied. Although the above efforts, there is still lacking how the stress and deformation occur in a realistic shaped distal fibula fracture. In this study, an improved finite element model is proposed, which uses the reconstructed CT images. The developed model is firstly validated with experimental data in the literature. Then, in order to compare the biomechanical effectiveness and stability of different protocols in the treatment, three options with 4- hole, 5-hole, or 6-hole lateral internal fixation plates under different external loads (uni-axial compression, bending, and torsion) are considered. Their stress and displacement distributions are simulated. These results will be used to select the best treatment protocol for distal fibula fractures and provide theoretical understanding for clinical treatment. 2 Methods Page 2/15 Page 2/15 2.1 CT scan data and geometry reconstruction CT scan was conducted on the lower limb of a male volunteer patient (height 175 cm, weight 75 kg, age 29 years, and healthy lower limb bones). The 64-row spiral CT uses a scanning layer thickness of 0.5 mm. The CT data were converted to DICOM format and then imported into Mimics 20.0 software (Materialise, Belgium) to reconstruct the skin and cancellous bone into a 3D geometrical model in STL format. The fibula surface was processed by constructing grids and fitting surfaces in Geomagic Studio 13.0 software (Raindrop, USA), and then the solid model was created and exported into STEP format file. 2.2 Finite element modeling of internal fixation The STEP format file was imported into Solidworks 20.0 (Dassault Systemes S.A, USA) for the assembly of the skin and cancellous bone. The models were imported into Anasys workbench 19.0 (ANSYS, USA) software for the finite element analysis. Following the AO (Arbeitsgemeinschaft für Osteosynthesefragen) or the Association of the Study of Internal Fixation (ASIF) standard, the clavicle plate was assembled with the fibula fracture model according to the simulated surgery, thus completing the lateral internal fixation options of the distal fibula fracture. The 4-hole lateral internal fixation plate model was defined as "Option A", the 5-hole lateral internal fixation plate model was defined as "Option B", and the 6-hole lateral internal fixation plate model was defined as "Option C ". The 4-hole, 5-hole, 6-hole locking plates, and screws were created. The threads of the screws were ignored in the model to simplify the computational time. As an example, the components and assembly of Option A are shown in Fig. 1a, which resemble the experimental setup in the literature (Knutsen et al. 2016). The screw and plate dimensions of the model are given in Table 1. Table 1 Dimensions of three options of plates and screw Diameter (mm) Length (mm) Width (mm) Height (mm) (OptionA) 4-hole with bone plate 0 48 10 3 (Option B) 5-hole with bone plate 0 60 10 3 (Option C) 6-hole with bone plate 0 72 10 3 Screw 3.5 15 0 0 2 3 Material model 2.4 Mechanical load boundary conditions 2.4 Mechanical load boundary conditions In this study, three different mechanical boundary conditions are applied similar to the reference (Knutsen et al. 2016). Three types of loads were applied to the fibula model: (1) normal uni-pedal standing posture: a uniaxial compressive force of F = 200 N was applied to the fibular tuberosity with the full constraint of the external fibular ankle (Fig. 2 − 1). (2) Simulated bending motion: a moment of M = 1 N-m was applied at the fibular ankle, with the fibular head fully restrained (Fig. <link rid="fig2">2</link>–2). (3) Simulated torsional motion: a torsion moment of T = 1 N-m at the fibular tuberosity was used, and full constraint at the fibular external ankle (Fig. 2–3). The contact of steel plate, screw, skin, and cancellous bone were set as bound; the fracture surface was set as frictional contact with a friction coefficient of 0.2 (Galloway et al. 2013). It is noted that the fibula fracture has an exponential decrease in fibula model stiffness during prior simulation analyses (Cao et al. 2019), therefore, in this work no artificial cut was made at the outer ankle of the fibula to simulate a distal fibula fracture. 2.5 Validation test To validate our model, the structural stiffnesses of options A, B, and C under torsional loading are calculated compared against the experimental test in the literature (Knutsen et al. 2016). 2.3 Material model 2.3 Material model The cortical bone at the proximal and distal ends of the fibula is very thin, ~ 2 mm. Therefore, the material properties of the outermost layer of the model were set to the cortical bone in the model. The material properties of the remaining parts were set to the cancellous bone, screw, and locking Page 3/15 plates, which are summarized in Table 2(Yang et al. 2021) (Shuangpeng et al. 2014). plates, which are summarized in Table 2(Yang et al. 2021) (Shuangpeng et al. 2014). Table 2 Material properties of the components in the finite element model (Yang et al. 2021) (Shuangpeng et al. 2014) Material Elastic modulus (MPa) Poisson’s ratio Bone density (g/cm3) Yield strength (MPa) Tensile strength (MPa) Cortical bone 17,000 0.36 2.1 142 71 Cancellous bone 300 0.30 1.5 50 3.5 Locking plates 113,000 0.33 4.5 850 900 Screw 113,000 0.33 4.5 850 900 2 4 M h i l l d b d diti 3.1 Validation test results The comparison of this model with the experimental result (Knutsen et al. 2016) is shown in Fig. 3. The comparisons suggest that the predictions are in reasonably good agreement with the experimental data. The structural stiffness of option A is 102% of that of the fibula assembled 5-hole pressurized plate, the Page 4/15 Page 4/15 structural stiffness of option B is 116% of that of the fibula assembled 5-hole locking plate, and the structural stiffness of option C is 195% of that of the fibula assembled 6-hole tabbed plate. structural stiffness of option B is 116% of that of the fibula assembled 5-hole locking plate, and the structural stiffness of option C is 195% of that of the fibula assembled 6-hole tabbed plate. 3.2 Stress distributions The peak stresses for three different options for various loads (Fig. 2) are analyzed. The summarized stress contours are given in Table 3. Table 3: Simulated stress contours of fibula under different loads, with the maximum stress, labeled Table 3: Simulated stress contours of fibula under different loads, with the maximum stress, labeled Page 5/15 In the fibula fracture model under uni-axial load, the maximum stresses for options A, B, a occurred at the plate nail connection at the fractured joint, with 96.6 MPa, 98.4 MPa, and respectively. These peak stresses are much below the allowable failure strength. The max on the fibula were 50.0 MPa, 51.7 MPa, and 54.1 MPa for the three options. Again all of t than the compressive strength of the cortical bone. In the fibula fracture model under uni-axial load, the maximum stresses for options A, B, and C all occurred at the plate nail connection at the fractured joint, with 96.6 MPa, 98.4 MPa, and 91.2 MPa, respectively. These peak stresses are much below the allowable failure strength. The maximum stresses on the fibula were 50.0 MPa, 51.7 MPa, and 54.1 MPa for the three options. Again all of them were less than the compressive strength of the cortical bone. In the fibula fracture model under uni-axial load, the maximum stresses for options A, B, and C all occurred at the plate nail connection at the fractured joint, with 96.6 MPa, 98.4 MPa, and 91.2 MPa, respectively. These peak stresses are much below the allowable failure strength. The maximum stresses on the fibula were 50.0 MPa, 51.7 MPa, and 54.1 MPa for the three options. Again all of them were less than the compressive strength of the cortical bone. Page 6/15 Page 6/15 In the fibula fracture model under torque loading, the maximum displacements of options A and B occurred at the uppermost plate nail connection, which was 99.5 MPa and 97.3 MPa, respectively. In comparison, the maximum displacement of option C occurred at the plate connection at the bone joint, which was 71.4 MPa. The maximum stresses on the fibula were 17.3 MPa, 17.1 MPa, and 13.0 MPa, respectively. During the stress analysis, the stresses on the clavicle plate, screws, skin, and cancellous bone did not exceed the permissible strengths of their respective materials. In the fibula fracture model under torque loading, the maximum displacements of options A and B occurred at the uppermost plate nail connection, which was 99.5 MPa and 97.3 MPa, respectively. In comparison, the maximum displacement of option C occurred at the plate connection at the bone joint, which was 71.4 MPa. The maximum stresses on the fibula were 17.3 MPa, 17.1 MPa, and 13.0 MPa, respectively. During the stress analysis, the stresses on the clavicle plate, screws, skin, and cancellous bone did not exceed the permissible strengths of their respective materials. In the fibula fracture model under moment loading, the maximum displacements of options A, B, and C all occurred at the plate nail connection at the fractured joint, which was 49.5 MPa, 56.1 MPa, and 50.0 MPa, respectively. The maximum stresses on the fibula were 16.3 MPa, 15.3 MPa, and 14.6 MPa, respectively. Throughout the stress analysis, the stresses on the clavicle plate and screw did not exceed their strengths 2.3 Comparison of the distribution of peak displacements for the three options The displacement contours are summarized in Table 4. For the fibula fracture model under axial load, the maximum displacements of options A, B, and C all appeared at the fibular tuberosity, 8.79 mm, 8.40 mm, and 7.62 mm, respectively. Under the torque load, the maximum displacements of options A, B, and C appeared at the fibular tuberosity, which was 0.99 mm, 0.91 mm, and 0.88 mm, respectively. Under a bending moment load, the maximum displacements of options A, B, and C all occurred in the external ankle, which was 3.39 mm, 3.30 mm, and 3.10 mm, respectively. Table 4: Displacement contours of the fibula under different loads, with maximum displacement values Page 7/15 Page 7/15 4 Discussion Page 8/15 Peak stress and displacement values of fibula and plate under different options are summarized in Fig. 4. In this study, common fibula fractures and typical motions were analyzed using finite elements and other means. The results showed that the overall structural stiffness of Option C was the highest, and its resistance to bending and deformation was the strongest. In the surgical model of fibula fracture under axial load, the maximum stresses all appeared at the plate and nail connection near the fracture joint, Page 8/15 which is likely that the elastic modulus of the plate and screw is much larger than that of the fibula, and the stress masking phenomenon occurs near the fracture line. which is likely that the elastic modulus of the plate and screw is much larger than that of the fibula, and the stress masking phenomenon occurs near the fracture line. For the approximate beam structure like the plate, the stresses are mainly concentrated near the fracture line under the action of gravity. Option C has smaller peak stresses and displacements compared with options A and B, indicating that the overall structural stiffness of the fibula is increasing with the increase in plate length and screws, and the use of relatively longer plates is beneficial to the recovery of the fibula, reducing the occurrence of delayed healing or nonhealing of the fibula after surgery and improving the structural stability of the distal fibular fracture. This is consistent with several clinical studies that have concluded that the use of relatively long plates is an important factor in reducing the risk of surgery (Ricci et al. 2014; Weber and Krause 2005). In the surgical model of fibula fracture under torque loading, the peak stresses in the plates of options A and B appear in the uppermost screw. This may be caused by the stress concentration phenomenon between the plate and the fibula projection, which can be avoided by optimizing the shape of the plate. Therefore, movements such as flexion should be avoided during the early rehabilitation of fibular fractures, in order to avoid excessive stresses on the fibula and delay bone healing. 4 Discussion The maximum stresses on the fibula in options A, B, and C were all less than 20 MPa and the maximum displacements were all less than 3.40 mm in the surgical model of fibula fracture under bending moment loading, which was much less than the yield strengths of the plate and screw(Stoffel et al. 2003). Since the distal lateral fibula plate is on the extruded side, it is subjected to relatively small stresses and displacements. The results suggest that the choice of distal lateral fibula implantation plate is correct. The stress contours of the fibula are shown in Fig. 5. It is noted that there is a stress masking phenomenon that occurs in all fractures under different loads (SL et al. 2012), which is due to a large difference between the elastic modulus of the titanium joint plate and that of the fibula. In this study, the stress masking phenomenon in option A at different loads is shown in Fig. 5. The stress values at the bone joints were lower than the stress stimulus required for normal bone growth, which is not helpful to bone healing. Declarations Ethics approval and consent to participate: The participant has the consent to participate in the study. The need for approval was waived. Ethics approval and consent to participate: The participant has the consent to participate in the study. The need for approval was waived. Consent for publication: Not applicable. Availability of data and materials: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests: The authors declare that they have no competing interests Funding: Tianjin Education Scientific Research Project (2018KJ204) and China Scholarship Council (CSC) Scholarship. Funding: Tianjin Education Scientific Research Project (2018KJ204) and China Scholarship Council (CSC) Scholarship. Acknowledgments: Not applicable. Acknowledgments: Not applicable. 5 Conclusions In this study, an improved finite element modeling approach, based on reconstructed CT images, has been successfully developed. The model can simulate the physiological loading of the fibula after surgery, therefore exploring the best surgical solution for distal fibular fractures. The conclusions of the study according to the distal fibula fracture options are as follows: 1. The use of a relatively long plate (option C) will increase the overall structural stiffness of its surgical options, and improve its resistance to torsion and deformation. 1. The use of a relatively long plate (option C) will increase the overall structural stiffness of its surgical options, and improve its resistance to torsion and deformation. 2. With the use of metal plates, stress masking is inevitable at the fracture site. For non-load-bearing bones like the fibula, materials with elastic modulus close to that of the fibula should be considered. 2. With the use of metal plates, stress masking is inevitable at the fracture site. For non-load-bearing bones like the fibula, materials with elastic modulus close to that of the fibula should be considered. Page 9/15 This will reduce the stress masking effect, and the healing of the fibula can be facilitated. This will reduce the stress masking effect, and the healing of the fibula can be facilitated. References 1. Cao Y, Zhang Y, Huang L, Huang X (2019) The impact of plate length, fibula integrity and plate placement on tibial shaft fixation stability: a finite element study J Journal of orthopaedic surgery research 14:1–7 2. Fan Z, Peng J, Zhou L, Hai-tao S, Huang Y, Wu J (2019) Meta-analysis of whether open reduction and internal fixation combined with thread anchors in the treatment of ankle fracture complicated with deltoid ligament injury J Chinese Tissue Engineering Study 23:1307–1312 2. Fan Z, Peng J, Zhou L, Hai-tao S, Huang Y, Wu J (2019) Meta-analysis of whether open reduction and internal fixation combined with thread anchors in the treatment of ankle fracture complicated with deltoid ligament injury J Chinese Tissue Engineering Study 23:1307–1312 3. Fang R, Ji A, Sheng W, Long D, Chen C (2018) Finite Element Analysis on Bone Healing Under Different Screw Configurations Journal of Medical Biomechanics 33:435–441 3. Fang R, Ji A, Sheng W, Long D, Chen C (2018) Finite Element Analysis on Bone Healing Under Different Screw Configurations Journal of Medical Biomechanics 33:435–441 4. Galloway F, Kahnt M, Ramm H, Worsley P, Zachow S, Nair P, Taylor M (2013) A large scale finite element study of a cementless osseointegrated tibial tray J Journal of biomechanics 46:1900–1906 4. Galloway F, Kahnt M, Ramm H, Worsley P, Zachow S, Nair P, Taylor M (2013) A large scale finite element study of a cementless osseointegrated tibial tray J Journal of biomechanics 46:1900–1906 5. Hess F, Sommer C (2011) Minimally invasive plate osteosynthesis of the distal fibula with the locking compression plate: first experience of 20 cases J Journal of orthopaedic trauma 25:110–115 5. Hess F, Sommer C (2011) Minimally invasive plate osteosynthesis of the distal fibula with the locking compression plate: first experience of 20 cases J Journal of orthopaedic trauma 25:110–115 6. Ju DG, Debbi EM, Neustein AZ, Moon CN (2019) Fibular Lengthening Osteotomy With Revision Syndesmotic Repair for Ankle Fracture Malunion J Journal of orthopaedic trauma 33:S38-S39 6. Ju DG, Debbi EM, Neustein AZ, Moon CN (2019) Fibular Lengthening Osteotomy With Revision Syndesmotic Repair for Ankle Fracture Malunion J Journal of orthopaedic trauma 33:S38-S39 7. Knutsen AR et al. (2016) Distal fibula fracture fixation: biomechanical evaluation of three different fixation implants J Foot Ankle Surgery 22:278–285 7. Knutsen AR et al. References (2016) Distal fibula fracture fixation: biomechanical evaluation of three different fixation implants J Foot Ankle Surgery 22:278–285 8. Ricci WM, Streubel PN, Morshed S, Collinge CA, Nork SE, Gardner MJ (2014) Risk factors for failure of locked plate fixation of distal femur fractures: an analysis of 335 cases J Journal of orthopaedic trauma 28:83–89 8. Ricci WM, Streubel PN, Morshed S, Collinge CA, Nork SE, Gardner MJ (2014) Risk factors for failure of locked plate fixation of distal femur fractures: an analysis of 335 cases J Journal of orthopaedic trauma 28:83–89 Page 10/15 9. Rozbruch RS, Müller U, Gautier E, Ganz R (1998) The evolution of femoral shaft plating technique Clinical orthopaedics and related research 354:195–208 10. Shuangpeng D, wang C-t, JiBaoFen, Shu Z, JiaoYongZhe, lu4 Z, Chen C-s (2014) Parametric study on the influence of plant material selection and pretightening force on humerus J Chinese Journal of Bone and Joint Surgery 7:35–39 11. SL L, G T, DM W, SP D, CT W (2012) Finite element analysis in femoral fixation with medical 316L stainless steel plate Beijing Biomedical Engineering 31:445–449 11. SL L, G T, DM W, SP D, CT W (2012) Finite element analysis in femoral fixation with medical 316L stainless steel plate Beijing Biomedical Engineering 31:445–449 12. Stoffel K, Dieter U, Stachowiak G, Gächter A, Kuster MS (2003) Biomechanical testing of the LCP– how can stability in locked internal fixators be controlled? Injury 34:B11-19 12. Stoffel K, Dieter U, Stachowiak G, Gächter A, Kuster MS (2003) Biomechanical testing of the LCP– how can stability in locked internal fixators be controlled? Injury 34:B11-19 13. Weber M, Krause F (2005) Peroneal tendon lesions caused by antiglide plates used for fixation of lateral malleolar fractures: the effect of plate and screw position J Foot ankle international 26:281– 285 14. Yang JC-S, Lin K-Y, Lin H-H, Lee OK (2021) Biomechanical evaluation of high tibial osteotomy plate with internal support block using finite element analysis J Plos one 16:e0247412 14. Yang JC-S, Lin K-Y, Lin H-H, Lee OK (2021) Biomechanical evaluation of high tibial osteotomy plate with internal support block using finite element analysis J Plos one 16:e0247412 Figures Page 11/15 Figure 1 Figure 1 Page 11/15 Figure 1 Page 11/15 (a) Finite element assembly of the fibula fracture model (Option A) in this work, (b) experimental assembly (Knutsen et al. 2016). (a) Finite element assembly of the fibula fracture model (Option A) in this work, (b) experimental assembly (Knutsen et al. 2016). (a) Finite element assembly of the fibula fracture model (Option A) in this work, (b) experimental assembly (Knutsen et al. 2016). Figure 2 Schematic mechanical boundary conditions applied on the distal fibula fracture model (1) uni-axial compression force F, (2) bending moment M, and (3) torsion T. Figure 2 Figure 2 Schematic mechanical boundary conditions applied on the distal fibula fracture model (1) uni-axial compression force F, (2) bending moment M, and (3) torsion T. Page 12/15 Page 12/15 Figure 3 Structure stiffness comparison between the simulation in this study and experimental literature data (Knutsen et al. 2016) Figure 3 Figure 3 Structure stiffness comparison between the simulation in this study and experimental literature data (Knutsen et al. 2016) Page 13/15 Page 13/15 ure 4 k stress and displacement values of fibula and plate under different options. (a) peak stress for fi ding, (b) peak displacement for fibula loading, (c) peak stress for plate loading, and (d) peak placement for plate loading Figure 4 Peak stress and displacement values of fibula and plate under different options. (a) peak stress for fibula loading, (b) peak displacement for fibula loading, (c) peak stress for plate loading, and (d) peak displacement for plate loading Peak stress and displacement values of fibula and plate under different options. (a) peak stress for fibula loading, (b) peak displacement for fibula loading, (c) peak stress for plate loading, and (d) peak displacement for plate loading Page 14/15 Figure 5 Stress contours of the fibula. (a) under uni-pedal stance, (b) torque loading, and (c) bending moment loading Figure 5 Stress contours of the fibula. (a) under uni-pedal stance, (b) torque loading, and (c) bending moment loading Stress contours of the fibula. (a) under uni-pedal stance, (b) torque loading, and (c) bending moment loading Page 15/15
https://openalex.org/W2888212900	https://europepmc.org/articles/pmc6113202?pdf=render	English	null	A novel molecular rotor facilitates detection of p53-DNA interactions using the Fluorescent Intercalator Displacement Assay	Scientific reports	2,018	cc-by	10,810	A novel molecular rotor facilitates detection of p53-DNA interactions using the Fluorescent Intercalator Displacement Assay Walter L. Goh2, Min Yen Lee1, Ting Xiang Lim2, Joy S. Chua2, Sydney Brenner1, Farid J. Ghadessy2 & Yin Nah Teo1 Received: 14 March 2018 Accepted: 13 August 2018 Published: xx xx xxxx Received: 14 March 2018 Accepted: 13 August 2018 Published: xx xx xxxx We have investigated the use of fluorescent molecular rotors as probes for detection of p53 binding to DNA. These are a class of fluorophores that undergo twisted intramolecular charge transfer (TICT). They are non-fluorescent in a freely rotating conformation and experience a fluorescence increase when restricted in the planar conformation. We hypothesized that intercalation of a molecular rotor between DNA base pairs would result in a fluorescence turn-on signal. Upon displacement by a DNA binding protein, measurable loss of signal would facilitate use of the molecular rotor in the fluorescent intercalator displacement (FID) assay. A panel of probes was interrogated using the well-established p53 model system across various DNA response elements. A novel, readily synthesizable molecular rotor incorporating an acridine orange DNA intercalating group (AO-R) outperformed other conventional dyes in the FID assay. It enabled relative measurement of p53 sequence-specific DNA interactions and study of the dominant-negative effects of cancer-associated p53 mutants. In a further application, AO-R also proved useful for staining apoptotic cells in live zebrafish embryos. Interactions between proteins and DNA are essential cellular processes. Examples of DNA transacting pro- teins include transcription factors, polymerases, telomerases as well as factors involved in DNA repair path- ways. Compromised protein-DNA interactions can give rise to severe disease phenotypes, notably cancer1. There exists therefore, a requirement for robust assays enabling fundamental understanding of molecular interactions, high-throughput identification of compounds that restore correct DNA-binding in compromised cellular targets, and assessment of DNA binding proteins in clinical diagnostics2. The p53 protein is a tumor suppressor crucial in preventing cancer through the maintenance of cellular homeostasis and genomic integrity3. It senses intracellu- lar disturbances, particularly those that promote tumorigenesis (radiation damage, hypoxia, glucose starvation, oncogene activation), and functions to limit damage by augmenting sophisticated cellular responses that include cell-cycle arrest, DNA repair, apoptosis and cellular senescence3. As a transcription factor, p53 recognizes and binds cognate DNA elements to transcriptionally regulate a plethora of gene targets. Mutations in p53 have been found in more than half of human cancers4 and typically result in the translation of full-length mutant proteins defective in DNA binding.t g Traditional methods for determining protein-DNA binding include the electrophoretic mobility shift assay (EMSA)5 and DNA footprinting6. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports 1Molecular Engineering Lab, Biomedical Sciences Institutes, ASTAR, 61 Biopolis Drive, Singapore, 138673, Singapore. 2p53 Laboratory, ASTAR, 8A Biomedical Grove, #06-06 Immunos, Singapore, 138648, Singapore. Walter L. Goh and Min Yen Lee contributed equally. Correspondence and requests for materials should be addressed to F.J.G. (email: fghadessy@p53Lab.a-star.edu.sg) or Y.N.T. (email: ynteo1@illumina.com) Received: 14 March 2018 Accepted: 13 August 2018 Published: xx xx xxxx www.nature.com/scientificreports/ undoubtedly facile, it requires defined labeling of each target DNA, potentially limiting its use in higher through- put screening campaigns interrogating protein binding to larger DNA libraries.hli undoubtedly facile, it requires defined labeling of each target DNA, potentially limiting its use in higher through- put screening campaigns interrogating protein binding to larger DNA libraries.hli p g p g g g p g g The availability of a homogenous, low volume, non-radioactive assay with fluorescent readout will benefit both academia and industry. The fluorescent intercalation displacement (FID) assay meets many of these cri- teria in addition to being cost-effective, facile and robust. FID measures binding of ligands to DNA through the displacement of a fluorogenic compound (e.g. ethidium bromide and thiazole orange) pre-bound to DNA, resulting in a decrease in fluorescence intensity16. These features have led to development of high-throughput applications in combination with cognate site identification (CSI-FID) in a microarray platform to examine netropsin-DNA interaction17. Whilst the FID assay has been used to evaluate binding of numerous compounds, there have only been few reports of its use with proteins17–21. Herein, we explore the utility of a novel class of fluorescent compounds known as molecular rotors for detection of p53 binding to DNA in assays modeled after the FID phenomenon.l p Molecular rotors are a collective group of fluorescent compounds that possess the ability to undergo twisted intramolecular charge transfer (TICT)22. They typically consist of three parts: an electron-donating unit, an electron-accepting unit and a π-conjugated linking moiety which allows electron transfer to occur in the pla- nar conformation. Upon irradiation, electrostatic forces result in the molecule adopting a twisted conformation around the σ-bond in the linker region. This non-planar, twisted conformation has a lower excited state energy, and is thus associated with either a red-shifted fluorescence emission or a non-radiative torsional relaxation path- way, depending on the molecular structure of the rotor. The TICT properties of molecular rotors are highly sen- sitive to their micro-environment. Hindrances in intramolecular rotation, through higher viscosity for instance, prevent the non-radiative pathway and results in fluorescence restoration as the molecule adopts a planar configu- ration. The inherent sensitivity of molecular rotors to the polarity and viscosity of their environment, allowing for simultaneous fluorescence readouts, makes them highly attractive as fluorescent probes. The julolidine malononi- triles have been used as viscosity sensor probes in lipid membranes23. Results Ch i Chemical design and fluorescent properties of molecular rotors. A panel of fluorescent compounds was synthesized and characterized against commonly used fluorescent dyes. 9-(2-carboxy-2-cyano)vinyl-juloli- dine (CCVJ, Jul-R, Fig. 1) is a previously described molecular rotor which displayed strong TICT in detecting protein-protein interaction31. In addition, two new molecular rotors were designed to incorporate a DNA inter- calating unit as the electron donor moiety in place of the non-planar tertiary amine of the julolidine motif. The two units chosen, pyrene (Py-R) and acridine orange (AO-R), have flat aromatic structures likely to facilitate intercalation into the DNA helix (Fig. 1, Scheme 1). Utility of these rotors was compared against common probes used in FID assays, ethidium bromide (EtBr) and thiazole orange (TO-C), as well as commercial acridine orange (AO-C). Thiazole orange was also further modified to include a primary amine handle (TO) that TO-C lacks. Furthermore, we studied the use of two minor groove binders with the carbazole motif (Carb-2 and Carb-3) for use in FID (Fig. 1).hli g The fluorescence intensity of each compound was first examined in solutions with varying viscosities. An increase in viscosity impedes rotation around the bond connecting the two pi-systems, leading to higher struc- tural rigidity and a consequent increase in the fluorescence signal. The extent of fluorescence signal increase relative to the increase in viscosity is an indication of its sensitivity as a molecular sensor. Experiments were per- formed using ethylene glycol in glycerol where the amount of glycerol was adjusted to vary viscosity. All the rotors synthesized showed an increase in fluorescence upon viscosity increase (Supplementary Fig. 1), demonstrating rotor functionality. Optimal excitation and emission wavelengths recorded were used for further experiments (Table 1). Characterization of DNA-dependent fluorescence turn-on. Increasing concentrations of 30-bp double-stranded oligonucleotides were next added to each compound and induced fluorescence upon DNA binding measured. All synthesized compounds showed variable increases in fluorescence signal in the presence of DNA (Fig. 2). Compounds TO and TO-C showed very similar fluorescence profiles. Notably, AO-R was dis- tinctly more sensitive to DNA-dependent fluorescence turn-on than AO-C, displaying similar levels of signal enhancement when in the presence of 50-fold less DNA (Fig. 2C). AO-R also showed lower background signal and displayed a stronger turn-on signal over AO-C (82% and 52%, respectively, in 5 μM DNA). www.nature.com/scientificreports/ Other motifs such as thiophene-containing rotors have also been developed as viscosity sensors24. Besides fluorescence emission, the fluorescence lifespan of benzylidene malononitriles have also been used as a quantitative parameter for ratiometric measurements of vis- cosity in live cells25,26. Furthermore, acridizinium molecular rotors have been shown to be sensitive to DNA bind- ing through intercalation27,28. Upon intercalating to DNA, the intramolecular rotation of the molecule around the N-phenyl bond is restricted, leading to an increase in fluorescence signal. N-(halogenphenyl)-9-acridizinium ions in the presence of an anionic surfactant are also capable of binding to albumin proteins, giving a 20-fold increase in fluorescence29,30. To date, molecular rotors have demonstrated great value as fluorescent probes, granting quan- titative readouts upon viscosity changes as well as molecular interactions within biological systems. An earlier study by our group showed that specific interactions between proteins, as well as their antagonism, can be probed by molecular rotors31. Here, we report the design of several novel fluorogenic compounds, and explore their util- ity in FID assays through assessment of their DNA-binding fluorogenic properties. In particular, a new molecular rotor incorporating an acridine orange DNA intercalating group (AO-R) displayed superior performance over conventional dyes in the FID assay, allowing sensitive measurement of p53 sequence-specific DNA interaction. A novel molecular rotor facilitates detection of p53-DNA interactions using the Fluorescent Intercalator Displacement Assay Walter L. Goh2, Min Yen Lee1, Ting Xiang Lim2, Joy S. Chua2, Sydney Brenner1, Farid J. Ghadessy2 & Yin Nah Teo1 These methods are technically demanding, have limited sensitivity and throughput, and often require the use of radioisotopes. A safer, more facile alternative has been developed based on the ELISA format7, and more recent variations include using flow analysis on fluorescently-labelled micro- spheres8, and bead-based microscopy imaging9. Other techniques involving surface plasmon resonance (SPR)10, protein induced fluorescence enhancement (PIFE)11, and quantitative-PCR12 are powerful but require expensive instrumentation or synthetic labels, and may not be suited for high-throughput applications. Fluorescence ani- sotropy measurement is a widely adopted method for fast, accurate analysis of DNA-protein binding13–15. Whilst 1Molecular Engineering Lab, Biomedical Sciences Institutes, ASTAR, 61 Biopolis Drive, Singapore, 138673, Singapore. 2p53 Laboratory, ASTAR, 8A Biomedical Grove, #06-06 Immunos, Singapore, 138648, Singapore. Walter L. Goh and Min Yen Lee contributed equally. Correspondence and requests for materials should be addressed to F.J.G. (email: fghadessy@p53Lab.a-star.edu.sg) or Y.N.T. (email: ynteo1@illumina.com) ScIEntIfIc ReporTs \| (2018) 8:12946 \| DOI:10.1038/s41598-018-31197-9 1 www.nature.com/scientificreports/ Results Ch i Compounds TO, TO-C, and AO-R showed fluorescence gains at the lowest concentration of duplex DNA tested (10 nM), equiva- lent to that observed for the commonly used FID dye ethidium bromide (Fig. 2G). Both minor groove binders, Carb-2 and Carb-3, displayed a gradual but consistent turn-on activity that appeared to saturate in the presence of ScIEntIfIc ReporTs \| (2018) 8:12946 \| DOI:10.1038/s41598-018-31197-9 2 www.nature.com/scientificreports/ Compound ID Compound Excitation wavelength (nm) Emission wavelength (nm) I Jul-R 433 506 II Py-R 342, 376 527 III AO-R 480 526 IV TO 510 552 V Carb-2 458 572 VI Carb-3 456 582 Table 1. Excitation and emission properties of compounds tested. Table 1. Excitation and emission properties of compounds tested. Table 1. Excitation and emission properties of compounds tested. a relatively lower concentration of duplex DNA (100nM), possibly indicating stronger binding but comparativ Figure 1. Chemical structures of the molecular rotors used. Shown below are synthetic routes for compound II, III and IV. Figure 1. Chemical structures of the molecular rotors used. Shown below are synthetic routes for compounds II, III and IV. Figure 1. Chemical structures of the molecular rotors used. Shown below are synthetic routes for compounds II, III and IV. a relatively lower concentration of duplex DNA (100 nM), possibly indicating stronger binding but comparatively weaker signal activation (Fig. 2E and F). For Jul-R (Fig. 2A) and Py-R (Fig. 2B, Supplementary Fig. 2), notable fluorescence was only apparent at the highest concentration of duplex DNA used (5 µM) (Fig. 2A,B). This may be attributed to topological exclusion arising from the non-planar Jul-R and bulky Py-R, rotor units. The two com- pounds were deemed unsuitable and excluded from further experiments.hi a relatively lower concentration of duplex DNA (100 nM), possibly indicating stronger binding but comparatively weaker signal activation (Fig. 2E and F). For Jul-R (Fig. 2A) and Py-R (Fig. 2B, Supplementary Fig. 2), notable fluorescence was only apparent at the highest concentration of duplex DNA used (5 µM) (Fig. 2A,B). This may be attributed to topological exclusion arising from the non-planar Jul-R and bulky Py-R, rotor units. The two com- pounds were deemed unsuitable and excluded from further experiments.hi p p The turn-on signal of each dye was next examined in a reverse configuration by increasing dye concentra- tions against a fixed amount of DNA (Fig. 2H). Results Ch i DNA-dependent fluorescence enhancement was determined ScIEntIfIc ReporTs \| (2018) 8:12946 \| DOI:10.1038/s41598-018-31197-9 3 entificreports/ Figure 2. Fluorescence response of molecular rotors in the presence of DNA. (A–G) Fluorescence signal of rotor molecules at a fixed concentration (100 nM) titrated against increasing concentrations of double-stranded DNA fragments. Black-bar insets show fluorescence signal at maximum measurement configuration and highest DNA concentration. Hashed-lines depict levels of dye-only background fluorescence. (H) Levels of turn-on fluorescence from increasing concentrations of each compound in the presence of a fixed concentration of DNA (200 nM). All data shows mean ± S.D. of 3 individual experiments. For Py-R with dual excitation wavelengths, results for 376 nM wavelength shown. Similar results were observed upon excitation at 342 nM (Supplementary Fig. 2). www.nature.com/scientificreports/ Figure 2. Fluorescence response of molecular rotors in the presence of DNA. (A–G) Fluorescence signal of Figure 2. Fluorescence response of molecular rotors in the presence of DNA. (A–G) Fluorescence signal of rotor molecules at a fixed concentration (100 nM) titrated against increasing concentrations of double-stranded DNA fragments. Black-bar insets show fluorescence signal at maximum measurement configuration and highest DNA concentration. Hashed-lines depict levels of dye-only background fluorescence. (H) Levels of turn-on fluorescence from increasing concentrations of each compound in the presence of a fixed concentration of DNA (200 nM). All data shows mean ± S.D. of 3 individual experiments. For Py-R with dual excitation wavelengths, results for 376 nM wavelength shown. Similar results were observed upon excitation at 342 nM (Supplementary Fig. 2). by subtracting dye-only background from total signal in the presence of 200 nM duplex DNA (Supplementary Fig. 3). The EtBr control displayed strong gain, approaching saturation at ~40-fold molar excess over duplex DNA molecules, closely correlating to the expected occupancy of a strong binding intercalator. Both carbazole compounds displayed stronger DNA-induced signal gain than previously observed, which increased with increas- ing compound concentrations (Supplementary Fig. 3), suggesting optimal activity when present at ~10–15-fold molar excess over DNA molecules. AO-R displayed markedly brighter turn-on signal from DNA binding com- pared to all dyes, most notably its parental compound, AO-C. In addition, it showed no decline in signal gain at the highest concentration tested, unlike AO-C which displayed diminishing turn-on activity above 3.2 µM dye (16-fold molar excess over duplex DNA molecules). This suggests that fluorescence activation from AO-R is largely DNA-dependent with lesser background signal. Results Ch i Color spectrum depicts absolute amount of fluorescence gained for each dye in the presence of DNA (color scale bar: violet = self-referenced dye-only signal, red = highest observed Δa.u. across all dyes, set at 10 000 a.u.). Each row depicts fluorescence signals from individual compounds with lower limit (violet) benchmarked to respective “dye only” background signals. The p53 tumor suppressor acts as a transcription regulator in cells and mediates gene expression by binding DNA response elements (DNA-RE) carrying the canonical sequence RRRC(A/T) (T/A)GYYY (where R = purine and Y = pyrimidine). Each p53 dimer makes contact with a decamer half-site to form a DNA-tetramer com- plex32,33. Binding of two p53 tetramers to one DNA-RE has also been observed34. Further understanding of p53 biochemistry has revealed a high level of degeneracy in DNA recognition35, likely evolved from requirements in functional plasticity under varying cellular conditions. In addition to the several hundreds of experimentally verified p53 response elements, thousands more putative sites have been identified in whole genome studies36–38. As such, an ideal FID probe for studying highly promiscuous DNA-binding proteins like p53 should possess a strong turn-on signal to afford discernment in DNA selectivity, and also bind DNA with minimal sequence bias to give similar fluorescence baselines across diverse sequences.l gl q To assess the correlation between DNA sequence and fluorescence intensity, the compounds were tested against a panel of unique DNA sequences comprised of a non-binding scrambled DNA control (Scram) and p53-REs of varying affinities (Supplementary Table 1). A dye concentration of 15-fold molar excess over duplex DNA was used for the purpose of comparative assessment, as it gave the best signal-to-noise ratio for all dyes while being below concentrations that could contribute to aberrant signal readouts observed earlier for certain compounds (Fig. 2H). DNA-induced signals were contrasted across all dyes using a colored heat map to repre- sent fluorescence gain. An ideal rotor would show both a high fluorescence turn-on signal (warm colors in the presence of DNA) and a uniform fluorescence response across different REs. Carbazole compounds displayed the highest degree of signal disparity across DNA sequences. As before, Carb-2 appeared brighter than Carb-3 when DNA-bound, and additionally possessed the potential to be “hyper”-activated in the presence of particular DNA sequences (pDINP1 and IGF-BP3), to which both compounds appear to have an affinity towards (Fig. 3). Results Ch i Auto-fluorescence was also observed for TO-C, Carb-2 and Carb-3 when used at higher concentrations (Supplementary Fig. 3). In most cases, the phenomenon of back- ground (dye only) signal eclipsing DNA-induced fluorescence was observed, and became increasingly obvious at higher dye concentrations, likely due to off-target interaction or auto-fluorescence from poor solubility. by subtracting dye-only background from total signal in the presence of 200 nM duplex DNA (Supplementary Fig. 3). The EtBr control displayed strong gain, approaching saturation at ~40-fold molar excess over duplex DNA molecules, closely correlating to the expected occupancy of a strong binding intercalator. Both carbazole compounds displayed stronger DNA-induced signal gain than previously observed, which increased with increas- ing compound concentrations (Supplementary Fig. 3), suggesting optimal activity when present at ~10–15-fold molar excess over DNA molecules. AO-R displayed markedly brighter turn-on signal from DNA binding com- pared to all dyes, most notably its parental compound, AO-C. In addition, it showed no decline in signal gain at the highest concentration tested, unlike AO-C which displayed diminishing turn-on activity above 3.2 µM dye (16-fold molar excess over duplex DNA molecules). This suggests that fluorescence activation from AO-R is largely DNA-dependent with lesser background signal. Auto-fluorescence was also observed for TO-C, Carb-2 and Carb-3 when used at higher concentrations (Supplementary Fig. 3). In most cases, the phenomenon of back- ground (dye only) signal eclipsing DNA-induced fluorescence was observed, and became increasingly obvious at higher dye concentrations, likely due to off-target interaction or auto-fluorescence from poor solubility. ScIEntIfIc ReporTs \| (2018) 8:12946 \| DOI:10.1038/s41598-018-31197-9 4 www.nature.com/scientificreports/ l Figure 3. Signal intensity heat map of DNA-induced and sequence-dependent fluorescence intensity across rotor compounds. Color representative heat map showing rotor-specific fluorescence response to a panel of DNA oligonucleotide sequences containing p53 response elements and control sequence (scram). Color spectrum depicts absolute amount of fluorescence gained for each dye in the presence of DNA (color scale bar: violet = self-referenced dye-only signal, red = highest observed Δa.u. across all dyes, set at 10 000 a.u.). Each row depicts fluorescence signals from individual compounds with lower limit (violet) benchmarked to respective “dye only” background signals. Figure 3. Signal intensity heat map of DNA-induced and sequence-dependent fluorescence intensity across rotor compounds. Color representative heat map showing rotor-specific fluorescence response to a panel of DNA oligonucleotide sequences containing p53 response elements and control sequence (scram). Results Ch i Both arms of Carb-2 rotor could simultaneously bind within minor grooves of DNA, leading to a brighter response over the 3-arm carbazole, which can still experience significant TICT through a sterically excluded unbound third arm. In contrast, EtBr, TO and TO-C showed more uniform fluorescence across diverse DNA sequences, but only a moderate turn-on fluorescence compared to AO-R, which displayed the highest degree of fluorescence activa- tion and was also sequence-agnostic, especially when contrasted against its commercial variant, AO-C (Fig. 3). Molecular rotors as probes for protein-DNA interactions. We next investigated the suitability of each compound in an FID assay to measure p53-DNA binding. Purified wildtype p53 protein (p53-WT) was added to specific DNA-REs pre-incubated with rotor compounds. A p53 non-binding sequence (scram) was included as control DNA for non-specific fluorescence attenuation, and together with selected REs known either to bind ScIEntIfIc ReporTs \| (2018) 8:12946 \| DOI:10.1038/s41598-018-31197-9 5 www.nature.com/scientificreports/ Figure 4. Fluorescent intercalator displacement assay with rotor compounds. Percentage change in fluorescence intensity when purified wildtype-p53 protein is added to mixtures containing rotor pre-complexed with different DNA fragments. Black-hash line depicts 100% reference value (rotor and DNA only), below which shows a displacement event. Grey-hash line shows percentage-change in signal for scrambled-DNA sequence. Data shows mean ± S.D. for 2 biological replicates. Figure 4. Fluorescent intercalator displacement assay with rotor compounds. Percentage change in fluorescence intensity when purified wildtype-p53 protein is added to mixtures containing rotor pre-complexed with different DNA fragments. Black-hash line depicts 100% reference value (rotor and DNA only), below which shows a displacement event. Grey-hash line shows percentage-change in signal for scrambled-DNA sequence. Data shows mean ± S.D. for 2 biological replicates. p53 moderately (Bax, pDINP1) or tightly (conA, p21, RGC), formed the representative panel of DNA response elements. The resultant signal, expressed as a percentage of the reference signal (rotors in the presence of DNA), gives an indication of p53-dependent changes in fluorescence. A displacement event occurs when fluorescence falls below the reference signal in the presence of p53 (black-hashed line, Fig. 4), suggesting liberation of the bulky rotor unit from the intercalating site due to p53-DNA binding, while the scram signal gives a further indication of sequence-specificity. q pi y Compounds AO-C and TO-C displayed sequence-nonspecific decreases in fluorescence whilst TO and Carb-2 showed signal increase (Fig. 4), as seen in the respective signal changes of scram control in the presence of p53. Results Ch i The indiscriminate behavior of these dyes could arise from off-target interaction between dye and p53 molecules, the inability of p53 to recognize or approach DNA motifs due to steric distortions on DNA molecules caused by rotor interaction, or from the sequence-biased turn-on activity discussed earlier. Such factors may be further compounded by solubility issues in aqueous buffers, as noted earlier for several compounds when present at higher concentrations (Fig. 2H) and in thiazole orange compounds (Supplementary Figs 3 and 4). Fluorescence intensity from DNA-RE complexed with Etbr, AO-R and Carb-3 fell below signals from both reference value and scrambled-DNA in the presence of p53-WT, suggesting sequence-specific DNA recognition and displacement by p53. Notably, the pattern of RE-binding across the panel was identical between AO-R and EtBr, and similar in Carb-3 (with the exception of pDINP1 and p21, likely associated to the preferential binding observed earlier in Fig. 3) (Fig. 4). Critically, the magnitude of displacement for each RE also correlated with their respective binding affinities towards p53 (Supplementary Table 1), in the order Con-A < p21, RGC < Bax, pDINP1< scram. AO-R, however, provided the best resolution between response elements, showing almost no displacement activity on the scrambled-DNA control sequence (Fig. 4), hence affording a high level of binding discernment. q gf g g g Direct fluorescence titration of AO-R and AO-C against p21 RE yielded Kds per bp of 4.8 ± 0.8 and 12.5 ± 1.0 μM respectively (Supplementary Fig. 5). The determined Kd for AO-C is comparable with the ~36 μM value obtained using spectroscopic and isothermal calorimetry methods39. Furthermore, the Kd of AO-R is highly comparable to the range of values reported for the common FID dyes ethidium bromide and thiazole orange (1.1–15 and 0.3–20 μM respectively)16,39,40. In-vitro applications of AO-R molecular rotor for studying p53-DNA binding. Rotor AO-R was selected for further characterization due to its favorable FID properties. The p21 and RGC sequence motifs repre- sent two of the most well-characterized and physiologically relevant p53-binding DNA elements that govern cell fate41,42. FID experiments using compound AO-R showed a p53 concentration-dependent binding to both p21 and RGC response elements, but not scrambled DNA control (Fig. 5a). Additionally, DNA-binding was markedly ScIEntIfIc ReporTs \| (2018) 8:12946 \| DOI:10.1038/s41598-018-31197-9 6 www.nature.com/scientificreports/ Figure 5. Acridine orange rotor (AO-R) as a fluorescence probe for sequence-specific p53-DNA binding. Results Ch i (a) FID assay using purified wildtype p53 protein at indicated concentrations on response elements RGC, p21, and scrambled control DNA (black bars) pre-complexed with AO-R. Data normalized to scram control showing FID effects through sequence-specific DNA binding. (b) FID assay using either wildtype p53, p53 G245S mutant or p53 R2733H mutant (10 µM) on DNA response elements pre-mixed with AO-R. (c) FID assay using AO-R to examine dominant effects of mutant p53 proteins, which were first prepared to contain different proportions of wildtype and mutant p53 molecules (10 µM final). Left: FID response of AO-R pre-complexed RGC-RE exposed to p53 mixtures containing wildtype and G245S mutant proteins. Right: FID response of AO-R pre-complexed p21-RE exposed to p53 mixtures containing wildtype and R273H mutant proteins. Black bar on right shows RE-AO-R complex reference signal at 100% (black hash line). All data shows mean ± S.D. of 3 individual FID experiments. Figure 5. Acridine orange rotor (AO-R) as a fluorescence probe for sequence-specific p53-DNA binding. (a) FID assay using purified wildtype p53 protein at indicated concentrations on response elements RGC, p21, and scrambled control DNA (black bars) pre-complexed with AO-R. Data normalized to scram control showing FID effects through sequence-specific DNA binding. (b) FID assay using either wildtype p53, p53 G245S mutant or p53 R2733H mutant (10 µM) on DNA response elements pre-mixed with AO-R. (c) FID assay using AO-R to examine dominant effects of mutant p53 proteins, which were first prepared to contain different proportions of wildtype and mutant p53 molecules (10 µM final). Left: FID response of AO-R pre-complexed RGC-RE exposed to p53 mixtures containing wildtype and G245S mutant proteins. Right: FID response of AO-R pre-complexed p21-RE exposed to p53 mixtures containing wildtype and R273H mutant proteins. Black bar on right shows RE-AO-R complex reference signal at 100% (black hash line). All data shows mean ± S.D. of 3 individual FID experiments. Figure 5. Acridine orange rotor (AO-R) as a fluorescence probe for sequence-specific p53-DNA binding. (a) FID i p ifi d ildt p p53 p t i t i di t d t ti p l t RGC p21 d Figure 5. Acridine orange rotor (AO-R) as a fluorescence probe for sequence-specific p53-DNA binding. (a) FID assay using purified wildtype p53 protein at indicated concentrations on response elements RGC, p21, and scrambled control DNA (black bars) pre-complexed with AO-R. Results Ch i Data normalized to scram control showing FID effects through sequence-specific DNA binding. (b) FID assay using either wildtype p53, p53 G245S mutant or p53 R2733H mutant (10 µM) on DNA response elements pre-mixed with AO-R. (c) FID assay using AO-R to examine dominant effects of mutant p53 proteins, which were first prepared to contain different proportions of wildtype and mutant p53 molecules (10 µM final). Left: FID response of AO-R pre-complexed RGC-RE exposed to p53 mixtures containing wildtype and G245S mutant proteins. Right: FID response of AO-R pre-complexed p21-RE exposed to p53 mixtures containing wildtype and R273H mutant proteins. Black bar on right shows RE-AO-R complex reference signal at 100% (black hash line). All data shows mean ± S.D. of 3 individual FID experiments. reduced when two cancer-associated inactive p53 mutants (R273H or G245S mutation)43 were assayed (Fig. 5b). p53 binds DNA as a homotetramer with momomeric subunits associating through the tetramerization domain. Cells heterozygous (p53mutant/+) for the mutant allele (germline or somatic mutated) are predisposed to cellular transformation due in part to diminished p53 activity from dominant-negative effects exerted through the for- mation of mutant-wildtype hetero-tetramers44. The often ensuing loss-of-heterozygosity (LOH) in the remaining functional wildtype allele during cancer development, frequently recapitulated in animal studies45,46, alludes to pro-survival advantages provided by p53 mutants. In addition, p53mutant/+ cancers exhibit unique tumor spec- tra that are allele-specific and metastasize more frequently47,48, strongly suggesting mutant-dependent dominant gain-of-functions when in the presence of wildtype p53. We investigated mutant and gene-specific dominant effects by studying changes in canonical DNA binding using mutant and wildtype p53 protein mixtures. The p53-G245S mutant protein displayed a strong dominant-negative effect on wildtype p53′s ability to bind the RGC response element (Fig. 5c), where a notable reduction in DNA binding activity was observed in the presence of low mutant concentrations (20% mutant), and completely ablated with 80% mutant protein. Contrastingly, wildtype p53 binding of p21-RE was more recalcitrant to the dominant negative effects of p53-R273H mutant, as binding remained unchanged even in the presence of 40% mutant protein, and was retained considerably even at the highest proportion of mutant protein tested (Fig. 5c). reduced when two cancer-associated inactive p53 mutants (R273H or G245S mutation)43 were assayed (Fig. 5b). p53 binds DNA as a homotetramer with momomeric subunits associating through the tetramerization domain. Results Ch i Cells heterozygous (p53mutant/+) for the mutant allele (germline or somatic mutated) are predisposed to cellular transformation due in part to diminished p53 activity from dominant-negative effects exerted through the for- mation of mutant-wildtype hetero-tetramers44. The often ensuing loss-of-heterozygosity (LOH) in the remaining functional wildtype allele during cancer development, frequently recapitulated in animal studies45,46, alludes to pro-survival advantages provided by p53 mutants. In addition, p53mutant/+ cancers exhibit unique tumor spec- tra that are allele-specific and metastasize more frequently47,48, strongly suggesting mutant-dependent dominant gain-of-functions when in the presence of wildtype p53. We investigated mutant and gene-specific dominant effects by studying changes in canonical DNA binding using mutant and wildtype p53 protein mixtures. The p53-G245S mutant protein displayed a strong dominant-negative effect on wildtype p53′s ability to bind the RGC response element (Fig. 5c), where a notable reduction in DNA binding activity was observed in the presence of low mutant concentrations (20% mutant), and completely ablated with 80% mutant protein. Contrastingly, wildtype p53 binding of p21-RE was more recalcitrant to the dominant negative effects of p53-R273H mutant, as binding remained unchanged even in the presence of 40% mutant protein, and was retained considerably even at the highest proportion of mutant protein tested (Fig. 5c). Use of AO-R molecular rotor for studying apoptosis in live organisms. Acridine orange is widely used as a highly selective dye in live embryos of zebrafish (danio rerio)49 and drosophila melanogaster50, where it strongly labels both the cytoplasm and nucleus of apoptotic cells. This phenomenon, which isn’t observed in live ScIEntIfIc ReporTs \| (2018) 8:12946 \| DOI:10.1038/s41598-018-31197-9 7 www.nature.com/scientificreports/ Figure 6. In-vivo staining of live zebrafish embryos using compound AO-R. (a) Live imaging of either wildtype (top panels), or Mdm−/− (bottom panels) 1 day post fertilization zebrafish embryos stained using either AO-C (left panels) or AO-R (right panels) dyes. Scale bars measure 1 mm. (b) Fluorescence intensity of individually measured whole, wildtype or Mdm2 knock-out, zebrafish embryos stained with either AO-C or AO-R dyes. (N = 22; P < 0.0001, unpaired, two-tailed student t test). Figure 6. In-vivo staining of live zebrafish embryos using compound AO-R. (a) Live imaging of either wildtype (top panels), or Mdm−/− (bottom panels) 1 day post fertilization zebrafish embryos stained using either AO-C (left panels) or AO-R (right panels) dyes. Scale bars measure 1 mm. Results Ch i (b) Fluorescence intensity of individually measured whole, wildtype or Mdm2 knock-out, zebrafish embryos stained with either AO-C or AO-R dyes. (N = 22; P < 0.0001, unpaired, two-tailed student t test). or necrotic cells, is attributed to specific apoptosis-related cellular changes that promote nucleic acid intercalation and staining of condensed apoptotic nuclei51,52. Cellular levels of pro-apoptotic p53 are stringently controlled by the E3 ligase Mdm2, deletion of which results in an embryonic lethal phenotype53. We therefore compared the fluorescence intensity of AO-C and AO-R on either wildtype or Mdm2−/− zebrafish embryos (generated through incrossing of Mdm2+/− fish), previously reported to stain strongly with acridine orange due to the large number of apoptotic cells that are otherwise absent in wildtype fish54. The results, similar to the response seen with DNA intercalation, showed considerably lower levels of background (wildtype embryos) with comparable levels of fluorescence activation (mutant embryos) when using AO-R over AO-C (Fig. 6a). Fluorescence signal of the embryos individually quantified showed ~3-fold reduction in background signal and a 32% increase in turn-on fluorescence (2.5-fold compared to 1.9-fold) for AO-R compared to AO-C. Further experiments are required to understand the improved performance of AO-R in this assay, in particular the signal contribution arising from DNA-binding. Discussion We tested Jul-R, 2 new fluorescent molecular rotors and 3 minor groove binders that possess an intercalating electron-donor moiety in their ability to detect DNA and p53-DNA interactions. The rotors displayed an expected enhancement in fluorescence intensity both in an environment of increasing viscosity, and in the presence of double-stranded DNA. The titration of increasing compound concentration measuring DNA-induced turn-on fluorescence showed that the synthesized rotors were comparable or brighter than several commercial dyes. A screen of the compounds against a panel of DNA response elements suggest that several displayed preferential activities in response to certain DNA sequences, rendering them less suitable for FID assays on proteins with the propensity to target extensive arrays of DNA motifs and sequences (eg. master transcription regulators like p53), but possibly useful for other downstream applications involving decorating or targeting specific DNA motifs. Compounds EtBr, AO-R, and to a lesser extent, Carb-3, were more suitable for the FID assay, and displayed rela- tive p53-dependent displacement activity consistent with published p53-RE binding affinities. The DNA binding affinity of AO-R was also comparable to ethidium bromide and thiazole orange, the most commonly used FID dyes. Along with its relatively lower sequence discrimination, AO-R may find use in screening campaigns inter- rogating large numbers of variant DNAs. Furthermore, in certain cases the spectral properties of AO-R (Table 1) may be more compatible with assay development, for example when potentially auto-fluorescent small molecules are being evaluated for modulation of p53 activity (e.g. reactivation of non DNA-binding mutant p53). ScIEntIfIc ReporTs \| (2018) 8:12946 \| DOI:10.1038/s41598-018-31197-9 8 www.nature.com/scientificreports/ Modifications of the molecular structure have been carried out to tune the electronic properties of the molec- ular rotors. Previous studies have shown that replacement of the amino electron donor group with a weaker elec- tron donor results in a blue shift in both the excitation and emission maxima22. The longer the conjugation length of the linker moiety, the longer the emission wavelength and the larger the Stokes shift. In addition, replacement of the nitrile electron acceptor moiety with methyl ester or phenyl sulfonyl results in an increase in the emission signals due to the stabilization of the excited state brought about by the change in dipole moment. Molecular rotors based on the aminobenzylidene–cyanoacetamide moiety have also been directly incorporated into DNA using modified nucleosides, with subsequent protein binding resulting in fluorescence turn-on55. Materials and Methods Molecular rotor synthesis. Syntheses of Compounds I, V and VI were carried out as previously reported31,58. Molecular rotor synthesis. Syntheses of Compounds I, V and VI were carried out as previously reported31,58. Preparation of Py-R (II). Pyrenecarboxaldehyde and cyanoacetic acid was weighed into a 25 mL round-bottom flask flushed with argon. Triethylamine was then added to the reaction mixture after solvating in anhydrous THF. The reaction mixture was then heated to 55 °C overnight, then evaporated to dryness and puri- fied via column chromatography to yield pale orange solids. i g p y y p g 1H NMR (DMSO, 400 MHz) δ8.14 (t, J = 7.6 Hz, 1H), 8.23 (d, 1H, J = 8.9 Hz), 8.30 (d, 1H, J = 8.7 Hz), 8.34 (d, 1H, J = 7.5 Hz), 8.39 (m, 3H), 8.57 (d, 1H, J = 8.1 Hz), 9.00 (s, 1H). 13C NMR (100 MHz, DMSO) δ 162.8, 145.8, 132.2, 130.7, 130.1, 129.1, 128.8, 127.9, 127.1, 126.7, 126.3, 126.1, 125.8, 124.9, 123.8, 123.6, 122.6, 120.4, 118.9. Preparation of AO-R (III). Acridine Orange aldehyde (0.12 g, 0.42 mmol, 1 eq.) and cyanoacetic acid (0.053 g, 0.63 mmol, 1.5 eq.) was weighed into a 25 mL round-bottom flask flushed with argon. Triethylamine (0.29 mL, 2.09 mmol) was then added to the reaction mixture after solvating in anhydrous DMF. The reaction mixture was heated to 55 °C overnight, then evaporated to dryness and purified via column chromatography to yield orange solids (1.69 mg, 1%). y g ( g ) 1H NMR (CDCl3, 400 MHz) δ 1.26 (s, 3H), 3.05 (s, 3H), 3.24 (s, 6H), 7.06 (dd, 1H, J = 2.4, 9.3 Hz), 7.31 (s, 1H), 7.47 (s, 1H), 7.60 (d, 1H, J = 9.0 Hz), 7.69 (d, 1H, J = 9.4 Hz), 8.36 (s, 1H). 13C NMR (100 MHz, CDCl3) δ 154.8, 154.6, 143.7, 142.8, 141.5, 130.4, 130.3, 117.8, 117.2, 115.2, 95.4, 93.8, 40.6, 30.4, 29.9. y g g 1H NMR (CDCl3, 400 MHz) δ 1.26 (s, 3H), 3.05 (s, 3H), 3.24 (s, 6H), 7.06 (dd, 1H, J = 2.4, 9.3 Hz), 7.31 (s, 1H), 7.47 (s, 1H), 7.60 (d, 1H, J = 9.0 Hz), 7.69 (d, 1H, J = 9.4 Hz), 8.36 (s, 1H). 13C NMR (100 MHz, CDCl3) δ 154.8, 154.6, 143.7, 142.8, 141.5, 130.4, 130.3, 117.8, 117.2, 115.2, 95.4, 93.8, 40.6, 30.4, 29.9. Preparation of 3-methyl-2-(methylthio)benzo[d]thiazol-3-ium 4-methylbenzenesulfonate (12). Discussion In our study, AO-R gave the largest signal increase upon DNA binding and provided a high level of sensitivity and precision to p53-DNA interaction in a sequence-dependent manner. The difference between AO-R and commercial acri- dine orange is the addition of a (2-carboxy-2-cyano) vinyl moiety in AO-R. This endows the molecule its twisted intercalating charge transfer properties. The AO-R rotor also has a lower background than commercial acridine orange because of an additional non-radiative relaxation pathway when in free solution. Excited state energy can be dissipated through twisted intramolecular charge transfer via the rotation of this vinyl bond. This also contributes to its increased sensitivity to binding interactions and greater increase in fluorescence compared to acridine orange. g AO-R enabled discrimination between wild type and mutant p53 DNA binding in the facile and homogenous FID assay. The assay measured gradated inhibition of wt-p53 in the presence of increasing amounts of two com- mon mutant p53 variants, suggesting utility in small-molecule mutant p53 reactivation studies/screening cam- paigns, where a reciprocal phenotype is expected. The assay will be useful for higher throughput combinatorial sampling of the dominant-negative activity of cancer-associated p53 mutants across diverse panels of response elements, and to investigate how interactions of wild-type/mutant p53 with other p53 family members (p63 and p73)56 impacts DNA binding. Furthermore, it may fuel identification of novel therapeutically relevant genes or protein targets, especially when used in combination with fluorescence correlation spectroscopy (FCS) that allows simultaneous inspection of p53 oligomerization and DNA-binding kinetics57. p p g g Further studies to both delineate the precise binding mode(s) of AO-R with DNA and assess its applicability to other DNA binding proteins are required to fully benchmark this new probe against the robust and commonly used FID dyes EtBr and thiazole orange. Materials and Methods 2-methylthiobenzothiazole (5.00 g, 27.62 mmol, 1 eq.) was weighed into a 25 mL round bottom flask under argon. Methyl p-toluenesulfonate (4.58 mL, 30.39 mmol, 1.1 eq.) was then added and the reaction mixture was allowed to stir at 130 °C for 1 hour. Acetone was then added after cooling to 70 °C until white precipitate appeared. The mixture was then refluxed for another 30 minutes before cooling to room temperature. Precipitate was collected by filtration and dried to yield pale yellow solids (10.02 g, 99%). yi y p y ( g ) 1H NMR (CH3OD, 400 MHz) δ 2.35 (s, 3H), 3.12 (s, 3H), 4.15 (s, 3H), 7.19 (d, 2H, J = 4.0 Hz), 7.67 (d, 2H, J = 8.0 Hz), 7.73 (t, 1H, J = 8.0 Hz), 7.85 (t, 1H, J = 8.0 Hz), 8.07 (d, 1H, J = 8.0 Hz), 8.22 (d, 1H, J = 8.0 Hz). 13C NMR (CH3OD, 100 MHz) δ 183.3, 144.2, 143.7, 141.6, 130.8, 130.0, 129.8, 128.6, 127.0, 124.7, 116.5, 36.9, 21.3, 18.5. Preparation of 1-(3-aminopropyl)-4-methylquinolin-1-ium bromide (13). 3-bromopropylamine hydrobromide (6.06 g, 27.68 mmol, 1.46 eq.) was weighed into an argon-flushed 25 mL round-bottom flask. Ethanol (5 mL) was then added to dissolve. Upon addition of Lepidine (2.5 mL, 18.91 mmol, 1 eq.), the reaction mixture was heated to 40 °C overnight. Pale pink precipitate (1.67 g, 32%) formed was filtered, washed and dried. 1H NMR (D2O, 400 MHz) δ 2.55 (quin, 2H, J = 8.0 Hz), 3.10 (s, 3H), 3.29 (t, 2H, J = 8.0 Hz), 5.16 (t, 2H, J = 8.0 Hz), 7.98 (d, 1H, J = 4.0 Hz), 8.11 (t, 1H, J = 4.0 Hz), 8.32 (t, 1H, J = 8.0 Hz), 8.46 (d, 1H, J = 8.0 Hz), 8.60 (d, 1H, J = 8.0 Hz). 13C NMR (100 MHz, D2O) δ 160.6, 147.3, 137.2, 135.6, 129.8, 127.3, 122.6, 118.2, 54.3, 36.5, 27.0 19.6. Preparation of 1-(3-aminopropyl)-4-methylquinolin-1-ium bromide (13). 3-bromopropylamine hydrobromide (6.06 g, 27.68 mmol, 1.46 eq.) was weighed into an argon-flushed 25 mL round-bottom flask. Ethanol (5 mL) was then added to dissolve. Upon addition of Lepidine (2.5 mL, 18.91 mmol, 1 eq.), the reaction mixture was heated to 40 °C overnight. Pale pink precipitate (1.67 g, 32%) formed was filtered, washed and dried. Materials and Methods pi 1H NMR (CH3OD, 400 MHz) δ 1.21 (t, 1H, J = 8.0 Hz), 2.31 (td, 2H, J = Hz), 3.00 (s, 3H), 3.18 (m, 3H), 3.88 (s, 3H), 6.70 (t, 1H, J = 8.0 Hz), 6.74 (s, 1H), 6.77 (d, 1H, J = 8.0 Hz), 6.90 (td, 1H, J = 4.0, 8.0 Hz), 7.14 (d, 1H, J = 8.0 Hz), 7.23 (dd, 1H, J = 4.0, 8.0 Hz), 7.32 (t, 1H, J = 8.0 Hz), 7.50–7.71 (m, 4H), 7.88 (t, 1H, J = 8.0 Hz), 8.08 (d, 1H, J = 8.0 Hz), 8.29 (d, 1H, J = 8.0 Hz), 8.54 (d, 1H, J = 8.0 Hz). 13C NMR (100 MHz, CH3OD) δ 162.1, 158.4, 150.8, 145.3, 142.2, 141.9, 139.1, 134.3, 129.4, 128.1, 127.5, 126.5, 123.7, 123.3, 122.1, 119.4, 113.6, 110.0, 109.5, 89.1, 55.2, 52.3, 34.0, 30.5, 30.2. Viscosity experiments. The rotors were dissolved in spectroscopy-grade DMSO to obtain a concentrated stock solution of 100 mM. The solution was vortexed to ensure complete dissolution. For each rotor, 6 µL of the 100 mM stock was dissolved in 1200 µL of ethylene glycol in each 1.5 mL tube. 3 mL of glycerol was then heated in a boiling bath to reduce viscosity and improve pipetting. In 5 separate tubes, add 200 µL of stained ethylene glycol (i.e. with respective rotor added) to 800 µL of unstained ethylene glycol; 200 µL of stained ethylene glycol, 600 µL of unstained ethylene glycol to 200 µL glycerol; 200 µL of stained ethylene glycol, 400 µL of unstained ethylene glycol to 400 µL of glycerol; 200 µL of stained ethylene glycol, 200 µL of unstained ethylene glycol to 600 µL glyc- erol; and 200 µL of stained ethylene glycol to 800 µL glycerol. The fluids will then have a glycerol content of 0%, 20%, 40%, 60%, 80% respectively. The five tubes were then placed on an inverting mixer and allowed to mix for at least 2 hours while the temperature equilibrate to room temperature. Measurements were taken using 96-well Corning Flat-bottom Black plate and Tecan Infinite M-1000 Spectrophotometer. All samples have a final rotor concentration of 100 µM. Experimental for characterization of rotor-DNA binding. Materials and Methods Mixtures containing specified DNA and rotor concentrations were set-up in 50 μL reaction volumes consisting of 5% (v/v) DMSO, 80% (v/v) p53 binding buffer (25 mM NaPi, pH 7.2, 150 mM KCl, 4 mM DTT), 15% (v/v) DNA buffer (10 mM Tris-HCl, pH 8.0, 50 mM NaCl). Rotor-DNA mixtures were constituted in 96-well plates (round-bottom black plate, Corning), and incu- bated in the dark at room temperature for 20 mins before fluorescence intensity was measured using the EnVision Multilabel Plate Reader (PerkinElmer) following Ex/Em wavelengths specified in Table 1. Scrambled-DNA (scram) double-stranded oligonucleotide molecules are used in experiments for evaluating compound-DNA binding. Measurement parameters used were: excitation light, 100%; detector gain, 500; no. flashes, 200. Figures are plotted using raw fluorescence signals from triplicate experimental repeats where no data manipulation/ transformation was performed (error = ±standard deviation). To generate plot for DNA-dependent turn-on fluorescence (Fig. 2H), ‘dye-only’ background signal were subtracted from total fluorescence signal, raw data prior to background subtraction are shown in Supplementary Fig. 3. For Jul-R and Py-R readings were also taken at maximum measurement configuration (excitation light, 100%, detector gain, 1000, no. flashes, 5000). For consistency, compounds were always pre-diluted in 100% DMSO solution. Rotor-DNA binding heat map was generated by measuring fluorescence signal from 4 μM of each fluorogen alone, or in the presence of 250 nM of dsDNA oligonucleotide from a panel of unique sequences. For each series (specific compounds), fluorescence turn-on is shown on a color-represented scale where the lower limit (violet) is self-referenced to respective back- ground signals (no DNA) and the upper limit (red) to the maximum gain observed in the entire experiment (10 000 a.u.), allowing a standardized frame of comparison for turn-on fluorescence across different compounds. Double-stranded DNA response elements (DNA-RE) were made by mixing equi-molar concentrations of com- plimentary ssDNA oligomers (IDT, Singapore) in DNA buffer (10 mM Tris-Cl pH 7.5, 50 mM NaCl) and heated at 95 °C for 5 minutes before slowly cooling to room temperature. DNA oligonucleotide sequences can be found in Supplementary Figures, Table 1. Direct fluorescence titration of AO-R and AO-C versus a hairpin p21 RE (5′-GAAGAAGACTGGGCATGTCTAAAAAAGACATGCCCAGTCTTCTTC-3′) was carried out in 1 mL cuvettes comprising 80% (v/v) p53 binding buffer (25 mM NaPi, pH 7.2, 150 mM KCl and 4 mM DTT), 15% (v/v) DNA buffer (10 mM Tris-HCl, pH 8.0, 50 mM NaCl) and AO-R/AO-C (100 nM). Materials and Methods 1H NMR (D2O, 400 MHz) δ 2.55 (quin, 2H, J = 8.0 Hz), 3.10 (s, 3H), 3.29 (t, 2H, J = 8.0 Hz), 5.16 (t, 2H, J = 8.0 Hz), 7.98 (d, 1H, J = 4.0 Hz), 8.11 (t, 1H, J = 4.0 Hz), 8.32 (t, 1H, J = 8.0 Hz), 8.46 (d, 1H, J = 8.0 Hz), 8.60 (d, 1H, J = 8.0 Hz). 13C NMR (100 MHz, D2O) δ 160.6, 147.3, 137.2, 135.6, 129.8, 127.3, 122.6, 118.2, 54.3, 36.5, 27.0 19.6. ScIEntIfIc ReporTs \| (2018) 8:12946 \| DOI:10.1038/s41598-018-31197-9 9 www.nature.com/scientificreports/ Preparation of TO (IV). 3-methyl-2-(methylthio)benzo[d]thiazol-3-ium 4-methylbenzenesulfonate (1.05 g, 2.86 mmol, 1 eq.) and 1-(3-aminopropyl)-4-methylquinolin-1-ium bromide (0.8 g, 2.86 mmol, 1 eq.) was weighed into 25 mL round-bottom flask under argon. 30 mL of ethanol was added to dissolve. Triethylamine (0.8 mL) was then added and the reaction was stirred at room temperature for 1 hour. Red precipitate (0.16 g, 11%) formed upon addition of ether was filtered, washed and dried. pi , 1H NMR (CH3OD, 400 MHz) δ 1.21 (t, 1H, J = 8.0 Hz), 2.31 (td, 2H, J = Hz), 3.00 (s, 3H), 3.18 (m, 3H), 3.88 (s, 3H), 6.70 (t, 1H, J = 8.0 Hz), 6.74 (s, 1H), 6.77 (d, 1H, J = 8.0 Hz), 6.90 (td, 1H, J = 4.0, 8.0 Hz), 7.14 (d, 1H, J = 8.0 Hz), 7.23 (dd, 1H, J = 4.0, 8.0 Hz), 7.32 (t, 1H, J = 8.0 Hz), 7.50–7.71 (m, 4H), 7.88 (t, 1H, J = 8.0 Hz), 8.08 (d, 1H, J = 8.0 Hz), 8.29 (d, 1H, J = 8.0 Hz), 8.54 (d, 1H, J = 8.0 Hz). 13C NMR (100 MHz, CH3OD) δ 162.1, 158.4, 150.8, 145.3, 142.2, 141.9, 139.1, 134.3, 129.4, 128.1, 127.5, 126.5, 123.7, 123.3, 122.1, 119.4, 113.6, 110.0, 109.5, 89.1, 55.2, 52.3, 34.0, 30.5, 30.2. Materials and Methods p21-RE (50 μM) was added in 2 μL aliquots and fluorescence measured after 5 min incubation at room temperature. Addition of DNA contin- ued until system reached saturation and fluorescence remained constant. Titrations were carried out in dupli- cate with measurement parameters set to: excitation light, 100%; detector gain, 500; no. flashes, 200. The molar fluorescence of free dye was determined by measuring the fluorescence of increasing concentrations of each dye (in the absence of DNA) and using linear regression to find the gradient. This value was used as described59 for determination of approximate Kds derived by least squares fitting of data using using GraphPad Prism version 7.03 (GraphPad Software, La, Jolla California USA). Expression and purification of p53 protein. Recombinant p53 proteins (residues 94–360, carry- ing N-terminal hexa-histidine tag) were expressed in Rosetta (DE3)-T1R cells from the pNIC28-Bsa4 vector. Expression cultures supplemented with 100 μM ZnSO4 were induced with 0.5 mM IPTG (OD600) and grown overnight at 18 °C, before cells are collected and resuspended in lysis buffer (100 mM HEPES pH 8.0, 500 mM NaCl, 0.5 mM TCEP, 10 mM Imidazole, 10% (v/v) glycerol, with Benzonase endonuclease (Merck) and Protease Inhibitor Cocktail Set III, EDTA free (Calbiochem)) the following day. Cells were lysed on ice by sonication and supernatants were first filtered (1.2 μm, Sartorius) and affinity purified through a Ni-NTA column (AKTA Express, GE Healthcare). Columns were washed with 20-column volumes of wash buffer 1 (20 mM HEPES pH 7.5, 500 mM NaCl, 0.5 mM TCEP, 10 mM Imidazole, 10% (v/v) glycerol), 20-column volumes of wash buffer 2 (20 mM HEPES pH 7.5, 500 mM NaCl, 0.5 mM TCEP, 25 mM imidazole, 10% (v/v) glycerol) before proteins were ScIEntIfIc ReporTs \| (2018) 8:12946 \| DOI:10.1038/s41598-018-31197-9 10 www.nature.com/scientificreports/ eluted with 20 mM HEPES pH 7.5, 500 mM NaCl, 0.5 mM TCEP, 500 mM Imidazole, 10% (v/v) glycerol, and further passed through a gel filtration column (HiLoad 16/60 Superdex 200 prep, GE healthcare). Eluted fractions were SDS-PAGE analysed and further concentrated (10 kDa Amicon ultra-15 centrifugal filter units, Millipore) before buffer exchange into p53 binding buffer 25 mM NaPi, pH 7.2, 150 mM NaCl and 4 mM DTT (Zeba desalt- ing spin columns, Thermo Fisher Scientific). Protein identity was additionally mass spectrometry verified. Experimental for p53-DNA Nbinding assay. p53-DNA binding reaction mixtures were prepared by first mixing DNA-REs with fluorogenic compounds for 20 minutes before p53 protein is added. References References 1. Lee, T. I. & Young, R. A. Transcriptional regulation and its misregulation in disease. Cell 152, 1237–1251 (2013). e e e ces 1. Lee, T. I. & Young, R. A. Transcriptional regulation and its misregulation in disease. Cell 152, 1237–1251 (2013). g g g 2. Brown, C. J., Cheok, C. F., Verma, C. S. & Lane, D. P. Reactivation of p53: from peptides to small molecules. Trends Pharmacol S 53–62 (2011). 53 62 (2011). 3. Bieging, K. T., Mello, S. S. & Attardi, L. D. Unravelling mechanisms of p53-mediated tumour suppression. Nat Rev Cancer 14, 359–370 (2014). 3. Bieging, K. T., Mello, S. S. & Attardi, L. D. Unravelling mechanisms of p53-mediated tumour suppression. Nat Rev Cancer 14, 359–370 (2014).fi 359 370 (2014). 4. Khoo, K. H., Verma, C. S. & Lane, D. P. 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R., Rotkiewicz, K. & Rettig, W. Structural changes accompanying intramolecular electron transfer: focus on twisted intramolecular charge-transfer states and structures. Chem. Rev 103, 3899–4032 (2003). g R , ( ) 23. Nipper, M. E. ScIEntIfIc ReporTs \| (2018) 8:12946 \| DOI:10.1038/s41598-018-31197-9 Materials and Methods After an additional 20 minutes, fluorescence intensity was measured on the EnVision Multilabel Plate Reader (PerkinElmer). To account for slight signal variabilities between wells, fluorescence turn-on signal from DNA-rotor interaction was first quantified, before the addition of proteins, and used for signal normalization within each experimental set. Each FID assay data point was obtained by measuring in replicates, differences in fluorescence intensity between samples with or without p53 proteins. Binding reactions were prepared to contain 1μM DNA response element and 10 μM rotor compounds in 50 μL reaction volumes with or without p53 proteins. Final reaction mixtures consist of 5% (v/v) DMSO, 80% (v/v) p53 binding buffer (25 mM NaPi, pH 7.2, 150 mM KCl and 4 mM DTT) and 15% (v/v) DNA buffer (10 mM Tris-HCl, pH 8.0, 50 mM NaCl). Staining and imaging of zebrMafish embryos. Zebrafish embryos collected at 1-day post fertilisation were incubated with 0.1% acridine orange solution in egg water (0.3% w/v sea-salt solution) for 30 minutes at room temperature before washing in fresh egg water 5 times for 10 minutes each. The embryos were imaged under a green fluorescent filter on the Leica M165FC fluorescence stereomicroscope (Leica, Solms, Germany). Individual embryos were then transferred onto a 96-well plate and quantified using the EnVision Multilabel Plate Reader (PerkinElmer). Data shown are background subtracted using egg water-only values from respective sam- ples. All experiments were conducted in accordance to the ethical guidelines and approved by the Institutional Animal Care and Use Committee, Biomedical Research Council. Data availability. The datasets generated during and/or analysed during the current study are available from the corresponding authors on reasonable request. Data availability. The datasets generated during and/or analysed during the current study are available from the corresponding authors on reasonable request. www.nature.com/scientificreports/ Mutant p53 gain of function in two mouse models of Li-Fraumeni syndrome. Cell 119, 847–860 (2004).i 49. Hammerschmidt, M. et al. dino and mercedes, two genes regulating dorsal development in the zebrafish embryo. Developmen 95–102 (1996). 50. Denton, D. & Kumar, S. Using the vital dye acridine orange to detect dying cells in Drosophila. Cold Spring Harb Protoc 2015, 572–575 (2015). ( ) 1. Abrams, J. M., White, K., Fessler, L. I. & Steller, H. Programmed cell death during Drosophila embryogenesis. Development 117 29–43 (1993). ( ) 52. Delic, J., Coppey, J., Magdelenat, H. & Coppey-Moisan, M. Impossibility of acridine orange intercalation in nuclear DNA of the living cell. Exp Cell Res 194, 147–153 (1991).i g p 3. Montes de Oca Luna, R., Wagner, D. S. & Lozano, G. Rescue of early embryonic lethality in mdm2-deficient mice by deletion of p53 Nature 378, 203–206 (1995).ii ( ) 4. Chua, J. S., Liew, H. P., Guo, L. & Lane, D. P. Tumor-specific signaling to p53 is mimicked by Mdm2 inactivation in zebrafish: insight from mdm2 and mdm4 mutant zebrafish. Oncogene 34, 5933–5941 (2015).l i 55. Dziuba, D., Pohl, R. & Hocek, M. Polymerase synthesis of DNA labelled with benzylidene cyanoacetamide-based fluorescent molecular rotors: fluorescent light-up probes for DNA-binding proteins. Chem Commun (Camb) 51, 4880–4882 (2015). l g p p g p 56. Stindt, M. H. et al. Functional interplay between MDM2, p63/p73 and mutant p53. Oncogene 34, 4300–4310 (2015).l 57. Kanno, D. M. & Levitus, M. Protein oligomerization equilibria and kinetics investigated by fluorescence correlation spectroscopy: a mathematical treatment. J Phys Chem B 118, 12404–12415 (2014).l y 58. Dumat, B. et al. N-phenyl-carbazole-based two-photon fluorescent probes: strong sequence dependence of the duplex vs quadruplex selectivity. Biochimie 93, 1209–1218 (2011).l y 9. “GraphPad - FAQ 1725 - Fitting binding of fluorescent ligands”. Accessed 7th March 2018. https://www.graphpad.com/support/faq fitting-binding-of-fluorescent-ligands/ (2011). www.nature.com/scientificreports/ 5. Levitt, J. A. et al. Membrane-Bound Molecular Rotors Measure Viscosity in Live Cells via Fluorescence Lifetime Imaging. J. Phys Chem. C 113, 11634–11642 (2009).l 6. Kuimova, M. K., Yahioglu, G., Levitt, J. A. & Suhling, K. Molecular rotor measures viscosity of live cells via fluorescence lifetime imaging. J Am Chem Soc 130, 6672–6673 (2008). g g 7. Ihmels, H. et al. Acridizinium salts as a novel class of DNA-binding and site-selective DNA-photodamaging chromophores Photochem Photobiol 74, 505–511 (2001). 8. Ihmels, H., Engels, B., Faulhaber, K. & Lennartz, C. New dyes based on amino-substituted acridizinium salts-synthesis and exceptional photochemical properties. Chem-Eur J. 6, 2854–2864 (2000).l p p p p 29. Granzhan, A., Ihmels, H. & Viola, G. 9-donor-substituted acridizinium salts: versatile environment-sensitive fluorophores fo detection of biomacromolecules J Am Chem Soc 129 1254 1267 (2007) p p p p 29. Granzhan, A., Ihmels, H. & Viola, G. 9-donor-substituted acridizinium salts: versatile environment-sensitive fluorophores for the detection of biomacromolecules. J Am Chem Soc 129, 1254–1267 (2007).l detection of biomacromolecules. J Am Chem Soc 129, 1254–1267 (2 30. Granzhan, A. & Ihmels, H. N-aryl-9-amino-substituted acridizinium derivatives as fluorescent “light-up” probes for DNA protein detection. Org Lett 7, 5119–5122 (2005).l protein detection. Org Lett 7, 5119–5122 (2005). 1. Goh, W. L. et al. Molecular rotors as conditionally fluorescent labels for rapid detection of biomolecular interactions. J Am Chem So p g 1. Goh, W. L. et al. Molecular rotors as conditionally fluorescent labels for rapid detection of biomolecular interactions. J Am Chem Soc 136, 6159–6162 (2014).i 32. el-Deiry, W. S., Kern, S. E., Pietenpol, J. A., Kinzler, K. W. & Vogelstein, B. Definition of a consensus binding site for p53. Nat Genet 1, 45–49 (1992). ( ) 33. Funk, W. D., Pak, D. T., Karas, R. H., Wright, W. E. & Shay, J. W. A transcriptionally active DNA-binding site for human p53 protein complexes. Mol Cell Biol 12, 2866–2871 (1992). p 4. Kearns, S., Lurz, R., Orlova, E. V. & Okorokov, A. L. Two p53 tetramers bind one consensus DNA response element. Nucleic Acid Res 44, 6185–6199 (2016).i ( ) 35. Wang, B., Xiao, Z. & Ren, E. C. Redefining the p53 response element. Proc Natl Acad Sci USA 106, 14373–14378 (2009). 35. Wang, B., Xiao, Z. & Ren, E. C. Redefining the p53 response e 6. Wang, L. et al. www.nature.com/scientificreports/ Analyses of p53 target genes in the human genome by bioinformatic and microarray approaches. J Biol Chem 276 43604–43610 (2001). 7. Li, M. et al. Distinct regulatory mechanisms and functions for p53-activated and p53-repressed DNA damage response genes in embryonic stem cells. Mol Cell 46, 30–42 (2012). 38. Tebaldi, T. et al. Whole-genome cartography of p53 response elements ranked on transactivation potential. BMC Genomics 16, 464 (2015).i 9. Shohreh Nafisi, A. A., Saboury, N., Keramat, J.-F., Neault & Tajmir-Riahi, H.-A. Stability and structural features of DNA intercalation with ethidium bromide, acridine orange and methylene blue. Journal of Molecular Structure 827, 35–43 (2007). k b Th b h fl d h l d l 0. Nygren, J., Svanvik, N. & Kubista, M. The interactions between the fluorescent dye thiazole orange and DNA. Biopolymers 46, 39–51 (1998). 1. Jordan, J. J. et al. Low-level p53 expression changes transactivation rules and reveals superactivating sequences. Proc Natl Acad Sc USA 109, 14387–14392 (2012). 42. Balagurumoorthy, P. et al. Four p53 DNA-binding domain peptides bind natural p53-response elements and bend the DNA. Natl Acad Sci USA 92, 8591–8595 (1995). ( ) 43. Bullock, A. N. & Fersht, A. R. Rescuing the function of mutant p53. Nat Rev Cancer 1, 68–76 (2001 43. Bullock, A. N. & Fersht, A. R. Rescuing the function of mutant p53. Nat Rev Cancer 1, 68–76 (2001). 4. Rivlin, N., Brosh, R., Oren, M. & Rotter, V. Mutations in the p53 Tumor Suppressor Gene: Important Milestones at the Various Step of Tumorigenesis. Genes Cancer 2, 466–474 (2011). g 5. van Boxtel, R. et al. Homozygous and heterozygous p53 knockout rats develop metastasizing sarcomas with high frequency. Am Pathol 179, 1616–1622 (2011). 6. Alexandrova, E. M. et al. p53 loss-of-heterozygosity is a necessary prerequisite for mutant p53 stabilization and gain-of-function in vivo. Cell Death Dis 8, e2661 (2017). 46. Alexandrova, E. M. et al. p53 loss of heterozygosity is a necessary prerequisite for mutant p53 stabilization and gain of function in vivo. Cell Death Dis 8, e2661 (2017). 47 Liu G et al High metastatic potential in mice inheriting a targeted p53 missense mutation Proc Natl Acad Sci USA 97 4174 4179 47. Liu, G. et al. High metastatic potential in mice inheriting a targeted p53 missense mutation. Proc Natl Acad Sci USA 97, 4174– (2000). ( ) 48. Olive, K. P. et al. Author Contributions W.G., M.L., T.L. and J.C. carried out experiments. W.G., S.B., F.G. and Y.T. devised experiments. W.G., F.G. and Y.T. wrote manuscript. All authors analysed data. W.G., M.L., T.L. and J.C. carried out experiments. W.G., S.B., F.G. and Y.T. devised experiments. W.G., F.G. and Y.T. wrote manuscript. All authors analysed data. References et al. Characterization of changes in the viscosity of lipid membranes with the molecular rotor FCVJ. Biochim Biophys Acta 1778, 1148–1153 (2008). 24. Sutharsan, J. et al. Molecular rotors: Synthesis and evaluation as viscosity sensors. Tetrahedron 66, 2582–2588 (2010). ScIEntIfIc ReporTs \| (2018) 8:12946 \| DOI:10.1038/s41598-018-31197-9 11 www.nature.com/scientificreports/ ScIEntIfIc ReporTs \| (2018) 8:12946 \| DOI:10.1038/s41598-018-31197-9 Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-31197-9 Competing Interests: The authors declare no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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https://openalex.org/W2988816548	https://www.nature.com/articles/s42004-019-0229-x.pdf	English	null	Amination of β-hydroxyl acid esters via cooperative catalysis enables access to bio-based β-amino acid esters	Communications chemistry	2,019	cc-by	6,592	1 Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 4, Groningen 9747 AG, The Netherlands. email: k.barta@rug.nl COMMUNICATIONS CHEMISTRY \| (2019) 2:127 \| https://doi.org/10.1038/s42004-019-0229-x \| www.nature.com/commschem MMUNICATIONS CHEMISTRY \| (2019) 2:127 \| https://doi.org/10.1038/s42004-019-0229-x \| www.nature.com/commschem Amination of β-hydroxyl acid esters via cooperative catalysis enables access to bio-based β-amino acid esters https://doi.org/10.1038/s42004-019-0229-x OPEN Anastasiia Afanasenko 1, Tao Yan 1 & Katalin Barta 1 β-amino acid esters are important scaffolds in medicinal chemistry and valuable building blocks for materials synthesis. Surprisingly, the waste-free construction of such moieties from readily available or renewable starting materials has not yet been addressed. Here we report on a robust and versatile method for obtaining β-amino acid esters by direct amination of β- hydroxyl acid esters via the borrowing hydrogen methodology using a cooperative catalytic system that comprises a homogeneous ruthenium catalyst and an appropriate Brønsted acid additive. This method allows for the direct amination of esters of 3-hydroxypropionic acid, a top value-added bio-based platform chemical, opening a simple route to access β-amino acid esters from a range of renewable polyols including sugars and glycerol. 1 Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 4, Groningen 9747 AG, The Netherlands. *ema COMMUNICATIONS CHEMISTRY \| (2019) 2:127 \| https://doi.org/10.1038/s42004-019-0229-x \| www.nature.com/commschem 1 COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-019-0229-x COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-019-0229-x Fig. 2 Catalytic amination of the β-hydroxyl acid esters via the hydrogen borrowing strategy. a Proposed mechanism; b hydrogenation of 3”aa in the presence of Shvo’s catalyst (Cat) and/or diphenyl phosphate additive (A1). Reaction conditions: atm. H2, 90 °C, 15 min Fig. 2 Catalytic amination of the β-hydroxyl acid esters via the hydrogen borrowing strategy. a Proposed mechanism; b hydrogenation of 3”aa in the presence of Shvo’s catalyst (Cat) and/or diphenyl phosphate additive (A1). Reaction conditions: atm. H2, 90 °C, 15 min Table 1 Reaction condition optimization for the β-amino acid esters synthesis Entry Cat [mol%] Additive [mol%] Temp. [°C] Solvent Conv. [%] Sel. 3aa [%] 1 1 – 120 Toluene 17 7 2 1 A1 (5) 120 Toluene >99 >99(87) 3 1 A1 (5) 100 Toluene 21 21 4 1 A1 (5) 110 Toluene 47 47 5 0.5 A1 (5) 120 Toluene 51 51 6 – – 120 Toluene 0 0 7 – A1 (5) 120 Toluene 0 0 8 1 A1 (5) 120 CPME 67 67 9 1 A1 (5) 120 1,4-Dioxane 25 25 10 1 A1 (5) 120 CH3CN 0 0 11 1 A1 (5) 120 THF 19 19 12a 1 A1 (5) 120 Toluene >99 85 13b 1 A1 (5) 120 Toluene 76 63 General reaction conditions: General Procedure (see Supplementary information, page 1-3), 1 mmol of 1a, 0.5 mmol of 2a, 0.5-1 mol% Shvo’s complex (Cat), 5 mol% additive (A1), 2 mL of solvent, 18 h, 100–120 °C, under argon, isolated yields in parentheses. Conversion and selectivity were determined by GC-FID. a1.5 equiv. of 1a was used. b1 equiv. of 1a was used Table 1 Reaction condition optimization for the β-amino acid esters synthesis Entry Cat [mol%] Additive [mol%] Temp. [°C] Solvent Conv. [%] Sel. COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-019-0229-x 3aa [%] 1 1 – 120 Toluene 17 7 2 1 A1 (5) 120 Toluene >99 >99(87) 3 1 A1 (5) 100 Toluene 21 21 4 1 A1 (5) 110 Toluene 47 47 5 0.5 A1 (5) 120 Toluene 51 51 6 – – 120 Toluene 0 0 7 – A1 (5) 120 Toluene 0 0 8 1 A1 (5) 120 CPME 67 67 9 1 A1 (5) 120 1,4-Dioxane 25 25 10 1 A1 (5) 120 CH3CN 0 0 11 1 A1 (5) 120 THF 19 19 12a 1 A1 (5) 120 Toluene >99 85 13b 1 A1 (5) 120 Toluene 76 63 General reaction conditions: General Procedure (see Supplementary information, page 1-3), 1 mmol of 1a, 0.5 mmol of 2a, 0.5-1 mol% Shvo’s complex (Cat), 5 mol% additive (A1), 2 mL of solvent, 18 h, 100–120 °C, under argon, isolated yields in parentheses. Conversion and selectivity were determined by GC-FID. a1.5 equiv. of 1a was used. b1 equiv. of 1a was used Table 1 Reaction condition optimization for the β-amino acid esters synthesis agricultural or forestry waste materials18, as well as glycerol, the major byproduct of biodiesel production19. esters to chemical intermediates11–14, including acrylonitrile15. Interestingly, among these (de)functionalization pathways, the direct and selective amination of the (3-HP) alcohol moiety has not been recognized or achieved yet. In recent years, much attention has been devoted to the development of industrially relevant, scalable methods for the production of 3-HP and its ethyl ester from renewable polyols (Fig. 1c)12,15–17 Thus realizing the above mentioned one-step catalytic amination would create access to valuable synthetic β-amino acid esters from diverse renewable sugar feedstocks, including non-edible lignocellulosic esters to chemical intermediates11–14, including acrylonitrile15. Interestingly, among these (de)functionalization pathways, the direct and selective amination of the (3-HP) alcohol moiety has not been recognized or achieved yet. In recent years, much attention has been devoted to the development of industrially relevant, scalable methods for the production of 3-HP and its ethyl ester from renewable polyols (Fig. 1c)12,15–17 Thus realizing the above mentioned one-step catalytic amination would create access to valuable synthetic β-amino acid esters from diverse renewable sugar feedstocks, including non-edible lignocellulosic An attractive method for carrying out the desired catalytic C–N bond formation is the direct amination of alcohols via the bor- rowing hydrogen approach (Fig. 2a)20–23. ARTICLE ARTICLE COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-019-0229-x β w p a β -Amino acid esters are privileged structural motifs in a wide variety of biologically active compounds1 and indis- pensable building blocks for the synthesis of β-peptides2,3 and β-lactam antibiotics4,5. While β-amino acid moieties can be readily constructed by classical stoichiometric methods, these approaches frequently involve the use of toxic reagents and generate signiﬁcant amounts of waste (Fig. 1a)6–9. Surprisingly, no waste-free catalytic methods, capable of creating β-amino acid scaffolds via direct coupling of β-hydroxyl acids or esters with amines, have been reported to date. Nonetheless, for targeting pharmaceutical compounds as well as functional materials and polymers, a clean synthetic approach would be certainly preferred (Fig. 1b)10. Moreover, such atom-economic method would enable the unprecedented, direct catalytic amination of important bio- based β-hydroxyl acid ester building blocks. β y y g 3-Hydroxypropionic acid (3-HP) has been identiﬁed as one of the top twelve value-added renewable platform chemicals11–13, hence there is a clear demand for its diversiﬁcation beyond already existing targets11–14. Several chemo- and biocatalytic routes have been proposed for the conversion of 3-HP and its Fig. 1 Strategies to access β-amino acid esters. a Classical, stoichiometric pathways; b novel catalytic method for N-alkylation of β-amino acid esters via th hydrogen borrowing strategy established here; c new route to bio-based β-amino acid esters from renewable polyols and subsequent transformation t valuable bio-based building blocks Fig. 1 Strategies to access β-amino acid esters. a Classical, stoichiometric pathways; b novel catalytic method for N-alkylation of β-amino acid esters via the hydrogen borrowing strategy established here; c new route to bio-based β-amino acid esters from renewable polyols and subsequent transformation to valuable bio-based building blocks COMMUNICATIONS CHEMISTRY \| (2019) 2:127 \| https://doi.org/10.1038/s42004-019-0229-x \| www.nature.com/commschem 2 ARTICLE COMMUNICATIONS CHEMISTRY \| (2019) 2:127 \| https://doi.org/10.1038/s42004-019-0229-x \| www.nature.com/commschem Thus, inspired by the remarkable achievements in cooperative transition-metal and Brønsted acid catalysis34–42 we have applied g Here we set to realize the catalytic amination of β-hydroxyl acid esters, including esters of the bio-based 3-hydroxypropionic acid. Fig. 3 a An imine-enamine equilibrium. Herein 1H NMR spectrum of the 3-(4-methoxyphenylamino)-but-2-enoic acid ethyl ester (3”aa) in presence of diphenyl phosphate additive (A1) is displayed. b Proposed adducts involved in cooperative catalysis. Details of the 31P NMR investigation are reported in Supplementary Fig. 12, Supplementary Note 1 biomass-derived α-hydroxyl acids with ammonia, using hetero- geneous Ru-based catalysts28. This work pioneered sustainable pathways from sugars to α-amino acids by a tandem biocatalysis/ heterogeneous catalysis approach. Earlier, Beller described the ﬁrst example of catalytic amination of α-hydroxyl amides with amines using [Ru3(CO)12]/DCPE29. This study also included methyl 2-hydroxypropanoate as substrate, but only the corre- sponding α-amino amide was formed, indicating low ester functional group tolerance under the reported conditions. developed the ﬁrst N-alkylation of unprotected α-amino acids with alcohols using the Ru-based Shvo’s catalyst33. This robust and base-free catalytic system appeared as excellent starting point for the synthesis of β-amino acid esters from β-hydroxyl acid esters and various amines (Fig. 1b). We started our investigation using ethyl 3-hydroxybutanoate and p-anisidine, with the Ru- based Shvo’s catalyst. Very poor substrate conversion was seen even at 120 °C, and the desired product was observed only in traces beside a small amount of imine (Table 1, entry 1). biomass-derived α-hydroxyl acids with ammonia, using hetero- geneous Ru-based catalysts28. This work pioneered sustainable pathways from sugars to α-amino acids by a tandem biocatalysis/ heterogeneous catalysis approach. Earlier, Beller described the ﬁrst example of catalytic amination of α-hydroxyl amides with amines using [Ru3(CO)12]/DCPE29. This study also included methyl 2-hydroxypropanoate as substrate, but only the corre- sponding α-amino amide was formed, indicating low ester functional group tolerance under the reported conditions. y In view of the possible side reactions and with the aim to keep low catalyst loading and mild reaction conditions, we explored alternative ways of enhancing reactivity. Achiral and chiral Brønsted acids have emerged as powerful tools in a wide variety of transformations34–42. In particular, the use of Brønsted acids in combination with transition metal catalysts have shown beneﬁcial in hydrogenation reactions, such as Ru34, Ir35 and Fe-catalyzed hydrogenation of imines36, as well as reductive amination37. Interestingly, recently Zhao has demonstrated the enantioselec- tive amination of alcohols by a cooperative catalytic system comprising an iridium complex and an appropriate chiral phosphoric acid, via the borrowing hydrogen methodology38. COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-019-0229-x COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-019-0229-x COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-019-0229-x A1 Toluene 3’aa E-isomer of 3’’aa Z-isomer of 3’’aa A B Fig. 3 a An imine-enamine equilibrium. Herein 1H NMR spectrum of the 3-(4-methoxyphenylamino)-but-2-enoic acid ethyl ester (3”aa) in presence of diphenyl phosphate additive (A1) is displayed. b Proposed adducts involved in cooperative catalysis. Details of the 31P NMR investigation are reported in Supplementary Fig. 12, Supplementary Note 1 A1 Toluene 3’aa E-isomer of 3’’aa Z-isomer of 3’’aa A A1 Toluene 3’aa E-isomer of 3’’aa Z-isomer of 3’’aa A A B Fig. 3 a An imine-enamine equilibrium. Herein 1H NMR spectrum of the 3-(4-methoxyphenylamino)-but-2-enoic acid ethyl ester (3”aa) in presence of diphenyl phosphate additive (A1) is displayed. b Proposed adducts involved in cooperative catalysis. Details of the 31P NMR investigation are reported in Supplementary Fig. 12, Supplementary Note 1 B Fig. 3 a An imine-enamine equilibrium. Herein 1H NMR spectrum of the 3-(4-methoxyphenylamino)-but-2-enoic acid ethyl ester (3”aa) in presence of diphenyl phosphate additive (A1) is displayed. b Proposed adducts involved in cooperative catalysis. Details of the 31P NMR investigation are reported in Supplementary Fig. 12, Supplementary Note 1 B B COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-019-0229-x Despite tremendous progress24–27, methodology development in the ﬁeld has gen- erally overlooked the use of potentially strongly coordinating substrates and no examples on β-hydroxyl acids or derivatives have been reported. In the recent pioneering work, Yan and co- workers have reported the ﬁrst example of catalytic amination of 3 ARTICLE COMMUNICATIONS CHEMISTRY \| (2019) 2:127 \| https://doi.org/10.1038/s42004-019-0229-x \| www.nature.com/commschem COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-019-0229-x COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-019-0229-x Fig. 4 Scope with variation of the amine substrate. General reaction conditions: General Procedure (see Supplementary information, page 1-3), 1 mmol of 1a, 0.5 mmol of 2a-r, 1 mol% Shvo complex (Cat), 5 mol% additive (A1), 2 mL toluene, 18 h, 120 °C, under argon, full conversion unless otherwise indicated, isolated yields are presented. a 95% conversion. b 94% conversion. See also Supplementary Table 1 Fig. 4 Scope with variation of the amine substrate. General reaction conditions: General Procedure (see Supplementary information, page 1-3), 1 mmol of 1a, 0.5 mmol of 2a-r, 1 mol% Shvo complex (Cat), 5 mol% additive (A1), 2 mL toluene, 18 h, 120 °C, under argon, full conversion unless otherwise indicated, isolated yields are presented. a 95% conversion. b 94% conversion. See also Supplementary Table 1 diphenyl phosphate (A1) as a Brønsted acid additive, assuming that it may facilitate imine reduction by bifunctional catalysis, and in addition potentially enhance imine formation, both steps involved in the borrowing hydrogen cycle (Fig. 2a). proposed borrowing hydrogen mechanism Fig. 2a. Deuterium incorporation experiments using the separately prepared, selec- tively D-labeled key substrate ethyl 3-hydroxyhexanoate-3-d (1b- d1) and applying the simpler substrate, benzyl alcohol-α,α-d227 (see Supplementary Note 2) showed deuterium transfer from the substrate to the amine product in accordance with a borrowing hydrogen mechanism. Furthermore amination of chiral alcohols (see Supplementary Note 3), namely ethyl (S)-3-hydroxybutyrate ((S)-1a) and ethyl (R)-3-hydroxybutyrate ((R)-1a) with p-anisidine (2a) lead to racemic amine products, as further evidence for the existence of the borrowing hydrogen pathway over an ionic mechanism43. The former pathway proceeds through a loss of the chirality of the substrate alcohol by its dehydrogenation to furnish the corresponding achiral carbonyl compound. Indeed, perfect (>99%) conversion and selectivity (>99%) were achieved using diphenyl phosphate (A1) and Cat at 120 °C (Table 1, entry 2). The high level of product selectivity shows that under these carefully selected conditions, the tendency for β- elimination is overcome in favor of dehydrogenation and imine formation. Further lowering the reaction temperature or catalyst amount have not proven beneﬁcial (Table 1, entries 3–5). A blank reaction in the absence of catalyst and additive, or just with A1, gave no detectable conversion (Table 1, entries 6–7). Solvent screening showed moderate success (Table 1, entries 8–11). Results Establishment of the reaction conditions. This transformation is expected to be challenging because of side reactions such as intermolecular transesteriﬁcation, partial ester hydrolysis or β- amino acid amide formation. Moreover, the β-hydroxyl acids or corresponding β-ketoacid/β-iminoacid intermediates (Fig. 2a) may form chelating complexes with the homogeneous catalyst, blocking coordination sites necessary for efﬁcient catalysis30–32. Therefore, the desired transformation requires a robust catalytic system with great functional group tolerance. Very recently, we OMMUNICATIONS CHEMISTRY \| (2019) 2:127 \| https://doi.org/10.1038/s42004-019-0229-x \| www.nature.com/commschem 4 ARTICLE COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-019-0229-x Conducting this reaction step separately also shows the advantage of the full borrowing hydrogen cycle that starts from the alcohol directly and results in the stable amine product. Advantageously, in this case the ketone and apparently labile imine intermediates are kept at low concentration thereby minimizing the possibility for side reactions. Next, we examined the hydrogenation of the enamine (3”aa), which was obtained via synthetic procedure44, in the presence of 1 mol% Shvo’s catalyst (Cat) with/without acid co- catalyst (A1) (Fig. 2b). The excellent, 99% 3aa yield in the presence of A1 compared to the lower 61% 3aa yield obtained in the absence of A1 underscores its beneﬁcial effect on the rate of imine hydrogenation. Scope of the methodology. Next, the scope and limitation of the newly established method were explored. A wide range of anilines were effectively coupled with ethyl 3-hydroxybutanoate (Fig. 4, Supplementary Table 1). With anilines bearing electron-donating substituents (2a-f), including those with bulky groups (2e, 2 f), 48–87% isolated product yields were achieved. Anilines with electron-withdrawing substituents (2g-l) also showed generally high reactivity affording products 3ag-al in 47–87% isolated yield. Functional groups such as –NO2, –CN, –CH3COOCH3 were well-tolerated under the reaction conditions. Notably, also when (2p) and (2r) containing heterocycles were examined, the alky- lated β-amino acid esters (3ap, 3ar) were obtained in 78% and 44% isolated yield, respectively. y p y Furthermore, we examined different β-hydroxyl acid esters as coupling partners to p-anisidine (2a)/p-bromoaniline (2i) (Fig. 5, Supplementary Table 2). Employing β-hydroxyl acid esters with bulky aliphatic substituents at β-position (1b, 1c) delivered the desired β-amino acid esters (3ba, 3bi, 3ca and 3ci) in good yields (79%, 68%, 78%, and 54%, respectively) while with ethyl 3- hydroxy-3-phenylpropanoate (1d) generally lower isolated yields were obtained (3da-3di, 43–48%). Excellent results (81–96%) were obtained with ethyl 3-hydroxy-2,2-dimethylpropanoate (1e) comprising two methyl substituents in the α-position (3ea-3ei, 81–96%). In comparison, 1f bearing an α-phenyl substituent displayed moderate results (3fa-3ﬁ, 33-59%). y g To further understand how this rate enhancement occurs, and to gain more insight into a possible cooperative catalysis by Cat- A1, in situ 31P NMR spectroscopic investigations using toluene- d8 as solvent at 60 °C were conducted (Supplementary Fig. 12, Supplementary Note 1). These experiments have provided support for the formation of adducts between Shvo’s complex (Cat) and diphenyl phosphate (A1) (Fig. COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-019-0229-x Decreasing the amount of alcohol to 1.5 and 1 equivalents (Table 1, entries 12–13) gradually declined conversion therefore for future study an alcohol: amine ratio of 2:1 was kept. Role of the Brønsted acid additive. Gratifyingly, additional 1H NMR experiments also revealed the existence of an imine (3’aa) - enamine (3”aa) equilibrium and the shift of this equilibrium in the presence of additive A1 toward the more reactive imine 3’aa form (Fig. 3a). Additional in situ 1D and 2D 1H NMR (Supplementary Figs. 1– 8) and GC-FID and GC-MS studies (Supplementary Figs. 9–10) of the amination of ethyl 3-hydroxybutanoate (1a) and ethyl 3- hydroxy-2,2-dimethylpropanoate (shown later, 1e) with p-anisi- dine (2a) in presence of Cat with/without diphenyl phosphate additive (A1) were performed. All key intermediates (Supplemen- tary Figs. 1–4, 7–10), such as the corresponding imine (3’ea), enamine (3”aa) and ketone (1’a), were detected that afﬁrmed the More experiments were conducted to further elaborate on the role of the acid additive in the crucial imine formation and imine hydrogenation steps of the hydrogen borrowing cycle. Reactions between ketone (1’a) and p-anisidine (2a) with and without COMMUNICATIONS CHEMISTRY \| (2019) 2:127 \| https://doi.org/10.1038/s42004-019-0229-x \| www.nature.com/commschem 5 ARTICLE COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-019-0229-x additive A1 were conducted, showing a beneﬁcial effect of the additive on the imine formation step, as expected: full conversion and >99% selectivity were achieved with A1 while 64% conversion and 22% selectivity were seen without A1. Conducting this reaction step separately also shows the advantage of the full borrowing hydrogen cycle that starts from the alcohol directly and results in the stable amine product. Advantageously, in this case the ketone and apparently labile imine intermediates are kept at low concentration thereby minimizing the possibility for side reactions. Next, we examined the hydrogenation of the enamine (3”aa), which was obtained via synthetic procedure44, in the presence of 1 mol% Shvo’s catalyst (Cat) with/without acid co- catalyst (A1) (Fig. 2b). The excellent, 99% 3aa yield in the presence of A1 compared to the lower 61% 3aa yield obtained in the absence of A1 underscores its beneﬁcial effect on the rate of imine hydrogenation. additive A1 were conducted, showing a beneﬁcial effect of the additive on the imine formation step, as expected: full conversion and >99% selectivity were achieved with A1 while 64% conversion and 22% selectivity were seen without A1. COMMUNICATIONS CHEMISTRY \| (2019) 2:127 \| https://doi.org/10.1038/s42004-019-0229-x \| www.nature.com/commschem COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-019-0229-x 3b, Complex 1) and between the imine 3’aa, Shvo’s complex (Cat) and diphenyl phosphate (A1) (Fig. 3b, Complex 2) desired in cooperative catalysis36. The interaction between enamine 3”aa and A1 was also conﬁrmed (Fig. 3b, Adduct 3). We assume that in the absence of A1, tautomerization of the imine 3’aa (formed during the borrowing hydrogen cycle) to the corresponding enamine 3”aa would take place, while in the presence of Cat and A1, 3’aa is rapidly reduced to the desired β-amino acid ester (3) via the ruthenium-amine complex (Fig. 3b, Complex 2). Having a highly selective method in hand for obtaining 3ei, the power of our developed catalytic method was demonstrated in the two-step, gram-scale synthesis of a β-lactam (4ei, Fig. 6). A 12- fold upscale of the amination of 1e with p-bromoaniline 2i (Fig. 5) furnished the desired β-amino acid ester (3ei) with excellent isolated yield (86%), which was subsequently cyclized following a known literature procedure (Fig. 6)45. Fig. 5 Scope with variation of the β-hydroxyl acid ester substrate. General reaction conditions: General Procedure (see Supplementary information, page 1-3), 1 mmol of 1a-f, 0.5 mmol of 2a or 2i, 1 mol% Shvo’s complex (Cat), 5 mol% additive (A1), 2 mL toluene, 18 h, 120 °C, under argon, full conversion unless otherwise indicated, isolated yields are presented. a 48 h. b 12 mmol of 1e, 6 mmol of 2i, 1 mol% Shvo’s complex (Cat), 5 mol% additive (A1), 5 mL toluene, 18 h, 120 °C, under argon. c 79% conversion. d 83% conversion. See also Supplementary Table 2 Fig. 5 Scope with variation of the β-hydroxyl acid ester substrate. General reaction conditions: General Procedure (see Supplementary information, page 1-3), 1 mmol of 1a-f, 0.5 mmol of 2a or 2i, 1 mol% Shvo’s complex (Cat), 5 mol% additive (A1), 2 mL toluene, 18 h, 120 °C, under argon, full conversion unless otherwise indicated, isolated yields are presented. a 48 h. b 12 mmol of 1e, 6 mmol of 2i, 1 mol% Shvo’s complex (Cat), 5 mol% additive (A1), 5 mL toluene, 18 h, 120 °C, under argon. c 79% conversion. d 83% conversion. See also Supplementary Table 2 6 ARTICLE COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-019-0229-x Bio-based β-amino acid esters from 3-hydroxypropionates. COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-019-0229-x Finally, to demonstrate the feasibility of this method for obtaining renewable β-amino acid esters in a remarkably simple manner, we turned our attention to the direct catalytic amination of esters of bio-based 3-HP, identiﬁed as one of the Top 12 value-added platform chemicals11,13. It is important to mention that the ethyl ester of 3-HP can be directly obtained from renewable resources, similarly to the acid 3-HP itself15,16. Herein we have investigated the use of commercially available tert-butyl 3-hydroxypropionate (1i) as well as ethyl 3-hydroxypropanoate (1j) as substrates. Gratifyingly, both (1i) as well as (1j) were smoothly aminated with 2a-o using the methodology developed herein (Fig. 7, Sup- plementary Table 3). Notably, the reaction conversion was sig- niﬁcantly decreased in the absence of the additive A1 (Supplementary Table 3, entry 7), conﬁrming the necessity of the catalytic system designed above. Interestingly, selective double N- alkylation of 2a with 1i-j was easily achieved by doubling the catalyst amount to 2 mol%, showing modularity of the method. The isolated yields of products obtained from the 3-HP esters, were somewhat lower compared to previously tested substrates (especially 3-hydroxy-2,2-dimethylpropanoate (1e)), thus the possibility of side reactions cannot be ruled out, although no side products (e.g. amides) were detectable by GC-MS or GC-FID methods. Hydrolysis of the 3-HP esters or the product β-amino acid esters to the corresponding carboxylic acids would be a possible pathway. Interestingly, with substrate 1e, minimal amount of side products attributable to intermolecular transesteriﬁcation processes (Supplementary Figs. 9–10) were seen. Similar reactivity may also be expected starting from the bio-based 3-HP esters 1i or 1j albeit presumably toward higher molecular weight analogs due to the decreased steric hindrance of the primary alcohol moiety. Discussion In summary, we have achieved the ﬁrst direct catalytic coupling of β-hydroxyl acid esters with amines to construct β-amino acid esters by cooperative catalysis using the combination of the Shvo’s catalyst and a Brønsted acid additive. The methodology is highly atom-economic, demonstrates a broad scope, excellent functional-group tolerance and potential application for the synthesis of β-lactams. Notably, the method allows for catalytic amination of a commercially available ester of 3- hydroxypropionic acid, an important bio-based platform chemi- cal, opening an entirely new possibility to access valuable β- amino acid scaffolds from several classes of abundant renewable resources. The obtained β-amino acid esters can be applied as value-added building blocks or further transformed to a variety of bio-based amines, diamines, amino-alcohols usable in the ﬁne chemical, materials or polymer chemistry sectors. The novel cooperative catalytic system presented should be broadly applied, in the future, for the waste-free amination of other highly oxy- genated renewable building blocks. Fig. 6 Application of the developed method in a β-lactam (4ei) synthesis General procedure for in situ 31P NMR study. An oven-dried 20 mL Schlenk tube, equipped with a stirring bar, was charged (depending on the experiment) h Sh ’ l ( l ) d h l h h ( l 16. Kumar, V., Ashok, S. & Park, S. Recent advances in biological production of 3- hydroxypropionic acid. Biotechnol. Adv. 31, 945–961 (2013). General procedure for in situ 31P NMR study. An oven-dried 20 mL Schlenk tube, equipped with a stirring bar, was charged (depending on the experiment) with Shvo’s catalyst (0.02 mmol, 1 equiv.), diphenyl phosphate (0.02 mmol, 1 equiv.) and/or 3-(4-methoxyphenylamino)-but-2-enoic acid ethyl ester (3”aa, 0.02 mmol, 1 equiv.) and toluene-d8 (as a solvent, 1 mL). Solid materials were weighed into the Schlenk tube under air. Then the Schlenk tube was subsequently connected to an argon line and vacuum-argon exchange was performed three times. Liquid starting materials and solvent were charged under an argon stream. The Schlenk tube was capped and the mixture was rapidly stirred at room temperature for 1 min, then was placed into a pre-heated oil bath (60°C) and stirred for 15 min. Then, the reaction mixture was cooled down to room temperature. Preparing the sample, 0.6 mL of the reaction mixture was placed to a J-Young NMR tube under argon. All spectra were recorded using Bruker Avance NEO 600 machine. 17. Matsakas, L., Topakas, E. & Christakopoulos, P. New trends in microbial production of 3-hydroxypropionic acid. Curr. Biochem. Eng. 1, 141–154 (2014). 18. Sheldon, R. A., Poliakoff, M., Perez, E., Tuck, C. O. & Horvath, I. T. Valorization of biomass: deriving more value from waste. Science 337, 695–699 (2012). 19. Len, C. & Luque, R. Continuous ﬂow transformations of glycerol to valuable products: an overview. Sustain. Chem. Process. 2, 1–10 (2014). 20. Bähn, S. et al. The catalytic amination of alcohols. ChemCatChem 3, 1853–1864 (2011). 21. Corma, A., Navas, J. & Sabater, M. J. Advances in one-pot synthesis through borrowing hydrogen catalysis. Chem. Rev. 118, 1410–1459 (2018). General procedure for the hydrogenation of N-aryl enamine. An oven-dried 20 mL Schlenk tube, equipped with a stirring bar, was charged with Shvo’s catalyst (0.002 mmol, 1 equiv.), diphenyl phosphate (0.01 mmol, 1 equiv.) or 3-(4-meth- oxyphenylamino)-but-2-enoic acid ethyl ester (3”aa, 0.2 mmol, 1 equiv.) and toluene (as a solvent, 2 mL). Solid materials were weighed into the Schlenk tube under air. Then the Schlenk tube was subsequently connected to an argon line and vacuum-argon exchange was performed three times. Liquid starting materials and solvent were charged under an argon stream. The Schlenk tube was capped and the mixture was rapidly stirred at room temperature for 1 min. General procedure for in situ 31P NMR study. An oven-dried 20 mL Schlenk tube, equipped with a stirring bar, was charged (depending on the experiment) h Sh ’ l ( l ) d h l h h ( l At the same time the pre-dried autoclave, equipped with the stirring bar, was purged three times with hydrogen. Under the stream of hydrogen, the reaction mixture was transferred from the Schlenk tube to the autoclave and heated at 90 °C for 15 min. The autoclave was then cooled to RT and the reaction mixture was transferred to a ﬂask. The reaction mixture was analyzed by GS–MS and GC-FID to determine conversion. 22. Reed-Berendt, B. G., Polidano, K. & Morrill, L. C. Biomolecular Chemistry hydrogen catalysis using earth-abundant ﬁrst row transition metals. Org. Biomol. Chem. 17, 1595–1607 (2019). 23. Wei, D. & Darcel, C. Iron catalysis in reduction and hydrometalation reactions. Chem. Rev. 119, 2550–2610 (2019). 24. Yan, T., Feringa, B. L. & Barta, K. Iron catalysed direct alkylation of amines with alcohols. Nat. Commun. 5, 5602 (2014). 25. Zhang, G., Yin, Z. & Zheng, S. Cobalt-catalyzed N‑alkylation of amines with alcohols. Org. Lett. 18, 300–303 (2016). g 26. Elangovan, S. et al. Efﬁcient and selective N-alkylation of amines with alcohols catalysed by manganese pincer complexes. Nat. Commun. 7, 12641 (2016). 27. Vellakkaran, M., Singh, K. & Banerjee, D. An Efﬁcient and selective nickel- catalyzed direct N-alkylation of anilines with alcohols. ACS Catal. 7, 8152–8158 (2017). 28. Deng, W. et al. Catalytic amino acid production from biomass-derived intermediates. Proc. Natl Acad. Sci. USA 115, 5093–5098 (2018). Data availability 29. Zhang, M., Imm, S., Bähn, S., Neumann, H. & Beller, M. Synthesis of α-amino acid amides: Ruthenium-catalyzed amination of α-hydroxy amides. Angew. Chem. Int. Ed. 50, 11197–11201 (2011). The authors declare that all other data supporting the ﬁndings of this study are available within the article and Supplementary Information ﬁles, and also are available from the corresponding author upon reasonable request. 30. Yang, W., Fu, H., Song, Q., Zhang, M. & Ding, Y. Amidate iridium(III) bis(2- pyridyl)phenyl complexes: application examples of amidate ancillary ligands in iridium(III)-cyclometalated complexes. Organometallics 30, 77–83 (2011). Received: 4 June 2019; Accepted: 18 October 2019; Received: 4 June 2019; Accepted: 18 October 2019; 31. Zhang, Z., Leitch, D. C., Lu, M., Patrick, B. O. & Schafer, L. L. An Easy-to-use, regioselective, and robust bis(amidate) titanium hydroamination precatalyst: Mechanistic and synthetic investigations toward the preparation of tetrahydroisoquinolines and benzoquinolizine alkaloids. Chem. A Eur. J. 13, 2012–2022 (2007). 32. Haas, K. & Beck, W. Formation of peptides at half-sandwich complexes using β- and γ-amino acid esters and a facile dipeptide synthesis from an N,O- glycinato half sandwich complex [RuCl(NH2CH2CO2)(η6-C6Me6)]. Eur. J. Inorg. Chem. 2001, 2485–2488 (2001). References 1. Kudo, F., Miyanaga, A. & Eguchi, T. Biosynthesis of natural products containing β-amino acids. Nat. Prod. Rep. 31, 1056–1073 (2014). 1. Kudo, F., Miyanaga, A. & Eguchi, T. Biosynthesis of natural products containing β-amino acids. Nat. Prod. Rep. 31, 1056–1073 (2014). 33. Yan, T., Feringa, B. L. & Barta, K. Direct N-alkylation of unprotected amino acids with alcohols. Sci. Adv. 3, eaao6494 (2017). 2. Lelais, G. & Seebach, D. β2-Amino acids - syntheses, occurrence in natural products, and components of β-peptides1,2. Biopolymer 76, 206–243 (2004). Wiley Periodicals, Inc. 2. Lelais, G. & Seebach, D. β2-Amino acids - syntheses, occurrence in natural products, and components of β-peptides1,2. Biopolymer 76, 206–243 (2004). Wiley Periodicals, Inc. 34. Ding, Z., Chen, F., Qin, J., He, Y. & Fan, Q. Asymmetric hydrogenation of 2,4- disubstituted 1,5-benzodiazepines using cationic ruthenium diamine catalysts: An Unusual achiral counteranion induced reversal of enantioselectivity. Angew. Chem. Int. Ed. 51, 5706–5710 (2012). y 3. Cheng, R. P., Gellman, S. H. & DeGrado, W. F. β-peptides: from structure to function. Chem. Rev. 101, 3219–3232 (2001). 3. Cheng, R. P., Gellman, S. H. & DeGrado, W. F. β-peptides: from structure to function. Chem. Rev. 101, 3219–3232 (2001). 4. Alcaide, B., Almendros, P. & Aragoncillo, C. β-lactams: Versatile building blocks for the stereoselective synthesis of non-β-lactam products. Chem. Rev. 107, 4437–4492 (2007). 4. Alcaide, B., Almendros, P. & Aragoncillo, C. β-lactams: Versatile building blocks for the stereoselective synthesis of non-β-lactam products. Chem. Rev. 107, 4437–4492 (2007). 35. Li, C., Wang, C., Villa-Marcos, B. & Xiao, J. Chiral counteranion-aided asymmetric hydrogenation of acyclic imines. J. Am. Chem. Soc. 130, 14450–14451 (2008). 5. Hughes, A. B. Amino Acids, Peptides and Proteins in Organic Chemistry. Building (Wiley-VCH Verlag GmbH & Co. KGaA, 2011). 36. Zhou, S., Fleischer, S., Junge, K. & Beller, M. Cooperative transition-metal and chiral Brønsted acid catalysis: enantioselective hydrogenation of imines to form amines. Angew. Chem. Int. Ed. 50, 5120–5124 (2011). 6. Pollack, M. A. Growth effects of α-methyl homologs of pantothenic acid and β-alanine. J. Am. Chem. Soc. 65, 1335–1339 (1943). 6. Pollack, M. A. Growth effects of α-methyl homologs of pantothenic acid and β-alanine. J. Am. Chem. Soc. 65, 1335–1339 (1943). 37. Li, C., Villa-Marcos, B. & Xiao, J. Metal - Brønsted acid cooperative catalysis for asymmetric reductive amination. J. Am. Chem. Soc. 131, 6967–6969 (2009). β 7. Gardner, P. & Brandon, R. Preparation and reactions of some arylalkyl cyanoacetic esters. Methods Synthesis and characterization. For general information about used chemicals, analytical methods, synthetic procedures, please see Supplementary Methods. 1H, 13C, 31P NMR spectra, GC-FID, GC-MS chromatograms related to the mechanism of the observed catalytic reaction are available in Supplementary Figs. 1–16 and Supplementary Notes 1–3. Full procedures for synthetic transformations to com- pounds 3aa-3jo, 4ei are available in Supplementary Tables 1–3. 1H, 13C NMR spectra of puriﬁed compounds are available in Supplementary Figs. 17–58. Fig. 6 Application of the developed method in a β-lactam (4ei) synthesis Fig. 7 Novel route to bio-based β-amino acid esters via direct catalytic amination of 3-HP esters. General reaction conditions: General Procedure (see Supplementary information, page 1-3), 1 mmol of 1i-j, 0.5 mmol of 2a-o, 1 mol% Shvo’s complex (Cat), 5 mol% additive (A1), 2 mL toluene, 18 h, 120 °C, under argon, isolated yields are presented. a 48 h. b 1 mmol of 1i-j, 0.5 mmol of 2a, 2 mol% Shvo’s complex (Cat), 5 mol% additive (A1), 2 mL toluene, 48 h, 120 °C, under argon Fig. 7 Novel route to bio-based β-amino acid esters via direct catalytic amination of 3-HP esters. General reaction conditions: General Procedure (see Supplementary information, page 1-3), 1 mmol of 1i-j, 0.5 mmol of 2a-o, 1 mol% Shvo’s complex (Cat), 5 mol% additive (A1), 2 mL toluene, 18 h, 120 °C, under argon, isolated yields are presented. a 48 h. b 1 mmol of 1i-j, 0.5 mmol of 2a, 2 mol% Shvo’s complex (Cat), 5 mol% additive (A1), 2 mL toluene, 48 h, 120 °C, under argon COMMUNICATIONS CHEMISTRY \| (2019) 2:127 \| https://doi.org/10.1038/s42004-019-0229-x \| www.nature.com/commschem COMMUNICATIONS CHEMISTRY \| (2019) 2:127 \| https://doi.org/10.1038/s42004-019-0229-x \| www.nature.com/commschem 7 ARTICLE ARTICLE COMMUNICATIONS CHEMISTRY \| https://doi.org/10.1038/s42004-019-0229-x General procedure for the preparation of β-amino acid esters. An oven-dried 20 mL Schlenk tube, equipped with a stirring bar, was charged with amine (0.5 mmol, 1 equiv.), β-hydroxyl acid ester (1 mmol, 2 equiv.), Shvo’s catalyst (0.005 mmol, 1 mol%), diphenyl phosphate (0.025 mmol, 5 mol%) and toluene (as a solvent, 2 mL). Solid materials were weighed into the Schlenk tube under air. Then the Schlenk tube was subsequently connected to an argon line and vacuum-argon exchange was performed three times. Liquid starting materials and solvent were charged under an argon stream. The Schlenk tube was capped and the mixture was rapidly stirred at room temperature for 1 min, then was placed into a pre-heated oil bath at 120 °C and stirred for a given time (typically, 18 h). Then, the reaction mixture was cooled down to room temperature. After taking a sample (app. 0.5 mL) for GC analysis, the crude mixture was ﬁltered through silica gel, eluted with ethyl-acetate, and concentrated in vacuo. The residue was puriﬁed by ﬂash column chromatography to provide the pure β-amino acid ester. 10. Dunn, P. J., Wells, A. S. & Williams, M. T. Green Chemistry in the Pharmaceutical Industry (Wiley-VCH Verlag GmbH & Co. KGaA, 20 10. Dunn, P. J., Wells, A. S. & Williams, M. T. Green Chemistry in the Ph ti l I d t (Wil VCH V l G bH & C KG A 2 Pharmaceutical Industry (Wiley-VCH Verlag GmbH & Co 11. Mika, L. T., Cséfalvay, E. & Németh, Á. Catalytic conversion of carbohydrates to initial platform chemicals: chemistry and sustainability. Chem. Rev. 118, 505–613 (2018). 12. Brar, S. K., Sarma, S. J. & Pakshirajan, K. 3-Hydroxypropionic Acid In Platform Chemical Bioreﬁnery: Future Green Industry (Elsevier: Amsterdam, 2016). 13. Werpy, T. & Petersen, G. Top value added chemicals from biomass: Volume I - results of screening for potential candidates from sugar and synthesis gas. in Report no. DOE/GO-102004-1992 (National Renewable Energy Lab., Golden, CO, 2004). 14. Della Pina, C., Falletta, E. & Rossi, M. A green approach to chemical building blocks. case 3-hydroxypropanoic acid. Green. Chem. 13, 1624–1632 (2011). 15. Karp, E. M. et al. Renewable acrylonitrile production. Science 358, 1307–1310 (2017). General procedure for in situ 31P NMR study. An oven-dried 20 mL Schlenk tube, equipped with a stirring bar, was charged (depending on the experiment) Acknowledgements K.B. thanks the European Research Council, ERC Starting Grant 2015 (CatASus) 638076. This work is part of the research programme Talent Scheme (Vidi) with project number 723.015.005 (for K.B.), which is partly ﬁnanced by the Netherlands Organization for Scientiﬁc Research (NWO). A.A. thanks Bálint Fridrich for help with hydrogenation experiments. Reprints and permission information is available at http://www.nature.com/reprints 43. Banerjee, D., Junge, K. & Beller, M. Cooperative catalysis by palladium and a chiral phosphoric acid: enantioselective amination of racemic allylic alcohols. Angew. Chem. Int. Ed. 53, 13049–13053 (2014). Reprints and permission information is available at http://www.nature.com/reprints Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. 44. Thomas, K. D., Adhikari, A. V. & Shetty, N. S. Design, synthesis and antimicrobial activities of some new quinoline derivatives carrying 1,2,3- triazole moiety. Eur. J. Med. Chem. 45, 3803–3810 (2010). 45. Wang, H. et al. A facile synthesis of 4-gem-diﬂuoromethylene β-lactam and its derivatives from BrCF2CF2Br. J. Fluor. Chem. 127, 1195–1203 (2006). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. References J. Org. Chem. 22, 1704–1705 (1957). 7. Gardner, P. & Brandon, R. Preparation and reactions of some arylalkyl cyanoacetic esters. J. Org. 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Chiral Brønsted acids for asymmetric organocatalysis. in Asymmetric Organocatalysis. Topics in Current Chemistry (ed. List, B.) 291, 395–456 (Springer, Berlin, Heidelberg, 2010). Correspondence and requests for materials should be addressed to K.B. Correspondence and requests for materials should be addressed to K.B. Competing interests p g The authors declare no competing interests. Author contributions A.A. conducted and designed the experiments, collected and analyzed the data and wrote the manuscript draft. T.Y. conceived the catalyst system, performed exploratory studies © The Author(s) 2019 COMMUNICATIONS CHEMISTRY \| (2019) 2:127 \| https://doi.org/10.1038/s42004-019-0229-x \| www.nature.com/commschem 9
https://openalex.org/W2800246013	https://www.frontiersin.org/articles/10.3389/fmars.2018.00130/pdf	English	null	ALOHA From the Edge: Reconciling Three Decades of in Situ Eulerian Observations and Geographic Variability in the North Pacific Subtropical Gyre	Frontiers in marine science	2,018	cc-by	12,270	ALOHA From the Edge: Reconciling Three Decades of in Situ Eulerian Observations and Geographic Variability in the North Paciﬁc Subtropical Gyre Maria T. Kavanaugh 1, Matthew J. Church 2, Curtiss O. Davis 1, David M. Karl 3, Ricardo M. Letelier 1 and Scott C. Doney 4 Maria T. Kavanaugh 1, Matthew J. Church 2, Curtiss O. Davis 1, David M. Karl 3, Ricardo M. Letelier 1 and Scott C. Doney 4 1 College of Earth, Ocean and Atmospheric Sciences, Oregon State University, Corvallis, OR, United States, 2 Flathead Lake Biological Station, University of Montana, Polson, MT, United States, 3 Daniel K. Inouye Center for Microbial Oceanography: Research and Education (C-MORE), University of Hawaii at Manoa, Honolulu, HI, United States, 4 Department of Environmental Sciences, University of Virginia, Charlottesville, VA, United States Global analyses of satellite and modeled data suggest decreased phytoplankton abundance and primary productivity in oligotrophic gyres as they expand in response to increased surface temperatures, shoaling of surface mixed layers, and decreased supply of subsurface macronutrients. However, analogous changes in the phytoplankton have not been evident in situ at Hawaii Ocean Time-series (HOT) Station ALOHA (22◦45′N, 158◦00′W), suggesting that physiological or structural reorganization not observed from space, uncorrected sensor drift, or uncharacterized geographic variability may be responsible for the apparent discrepancy. To address the latter, we compared interannual patterns of in situ phytoplankton dynamics and mixed layer properties to gyre extent and boundary location based on multiple deﬁnitions including dynamic topography, a threshold of satellite surface chlorophyll (chl a) ≤0.07 mg m−3, and multivariate biophysical seascapes using modeled or satellite data. Secular increases in gyre extent were apparent, although the rate of expansion was much slower than previously reported, whereas strong interannual oscillations were evident for all deﬁnitions of the gyre. Modeled and satellite-based multivariate seascapes agreed well in terms of expansion (surface area of seascapes) and isolation of Station ALOHA (distance to seascape boundary) resulting in a combined data record of nearly three decades. Isolation was associated positively with the North Paciﬁc Gyre Oscillation (NPGO), and negatively with Multivariate ENSO Index (MEI), and Paciﬁc Decadal Oscillation (PDO). The converse was true for the gyre’s expansion. Expansion followed a shoaling and freshening of the surface mixed layer and declines of in situ net primary production (PP) suggesting that Station ALOHA may serve as an early indicator of gyre biogeographic patterns. ORIGINAL RESEARCH published: 20 April 2018 doi: 10.3389/fmars.2018.00130 ALOHA From the Edge: Reconciling Three Decades of in Situ Eulerian Observations and Geographic Variability in the North Paciﬁc Subtropical Gyre Lags between geographic indicators and in situ conditions appear to partially explain past observed discrepancies between patterns from satellite remote sensing and those from in situ conditions at Station ALOHA. Keywords: climate oscillations, North Paciﬁc Subtropical Gyre, dynamic geography, seascape ecology, Station ALOHA Edited by: Alison Buchan, University of Tennessee, Knoxville, United States Reviewed by: Jeffrey Polovina, National Oceanic and Atmospheric Administration (NOAA), United States Andrew Irwin, Mount Allison University, Canada Reviewed by: Jeffrey Polovina, National Oceanic and Atmospheric Administration (NOAA), United States Andrew Irwin, Mount Allison University, Canada *Correspondence: Maria T. Kavanaugh mkavanau@ceoas.oregonstate.edu Specialty section: This article was submitted to Aquatic Microbiology, a section of the journal Frontiers in Marine Science Received: 06 December 2017 Accepted: 28 March 2018 Published: 20 April 2018 INTRODUCTION ) Contrary ﬁndings between gyre-scale and in situ analyses, attribution of change to diﬀering climate modes, and the variations in the shape and extent of the gyre suggest that regional oceanographic context needs to be considered when examining the role of interannual variability and secular trends on large scale shifts in the subtropical gyres (e.g., Hammond et al., 2017). One such regional approach considers the temporal patterns of features as a composite of biophysical interactions (Oliver and Irwin, 2008; Kavanaugh et al., 2014b, 2016). Seasonally evolving satellite-derived seascapes describe regional variability in the North Paciﬁc with diﬀerences in biogeochemical interactions (Kavanaugh et al., 2014b), and community structure (Kavanaugh et al., 2014a). Here, we use interannual varying seascapes to further our understanding of subtropical gyre dynamics and compare seascape variability relative to circulation-based or low- level chl a-based deﬁnitions of the gyre. We hypothesized that year-to-year variability in the size of the subtropical gyre and location of the gyre boundary is reﬂected in the patterns of phytoplankton abundance and productivity at Station ALOHA. The location of Station ALOHA is highlighted, with trends and oscillations of seascape area and isolation compared across deﬁnitions and analyzed in concert with in situ patterns. Speciﬁcally, we address the following questions: y gy y Because of their immense size and age, oligotrophic gyres have been considered historically to support pelagic ecosystems in a climax state relatively resulting from stable environmental forcing in space and time; however, they are now recognized to display substantial spatial, seasonal, and interannual variability (Venrick, 1995; Karl, 2010; Karl and Church, 2017). While dominated by small phytoplankton and regenerated productivity (Letelier et al., 1996; Li et al., 2011), relatively short-lived perturbations in the NPSG contribute not only to momentary increases in net primary productivity (NPP) but also to the decoupling of autotrophic and heterotrophic processes and subsequent export of particulate carbon. These include deep vertical mixing events followed by water column restratiﬁcation (DiTullio and Laws, 1991) associated with the passage of cyclonic eddies (Letelier et al., 2000; Wilson and Adamec, 2001), Rossby waves (Sakamoto et al., 2004), and the breaking of internal waves. INTRODUCTION index, the chl a trend was largely driven by changes in the subtropical gyres. Both Polovina et al. (2008) and Irwin and Oliver (2009) noted ENSO or PDO-associated increases in the areal extent of the most oligotrophic water in subtropical gyres. Finally, recent analyses of ocean color data from 1997 to 2010 and 1997 to 2013 suggest gradual, yet signiﬁcant, decreases in surface chl a in subjectively deﬁned polygons that encompassed the oligotrophic regions of the North Paciﬁc as well as other basins (Signorini and McClain, 2012; Signorini et al., 2015). Conversely, Siegel et al. (2013) argue that the tropical/sub-tropical negative correlation between satellite chl a and sea surface temperature (SST) is primarily a physiological signal of variable chl a to carbon (C) ratio rather than a phytoplankton biomass signal, likely associated with photoacclimation (Letelier et al., 1993, 2017; Winn et al., 1993). While some analyses of long-term in situ data support the trend toward oligotrophy found in early large- scale studies (Boyce et al., 2010, 2011, 2014), the majority of in situ time series do not (Chavez et al., 2011; McQuatters-Gollop et al., 2011). Covering over 20 million square kilometers, the North Paciﬁc Subtropical Gyre (NPSG) is the largest ecosystem on the planet surface (Sverdrup et al., 1942; Karl, 2010). The permanently stratiﬁed surface layer in this system restricts the vertical ﬂuxes of inorganic nutrients into the well-lit surface region, resulting in low primary production (PP) and export of carbon to the deep ocean. However, because of their size, open ocean regions like the NPSG are responsible for substantial oceanic primary and export production (Martin et al., 1987; Emerson et al., 1997). The debate is ongoing, however, as to how these processes are changing in response to longer term shifts in environmental forcing such as changes in ocean chemistry (Dore et al., 2009) and temperature (Karl et al., 2001; Corno et al., 2007; Saba et al., 2010). Accurate estimation of global ocean production and export, therefore, will require not only reliable estimation of NPSG ecosystem processes (Karl, 2010) but also a detailed understanding of the oceanographic context in which these processes are occurring, including an accurate characterization of gyre geography and the dynamics of gyre areal extent and boundary location. Citation: Kavanaugh MT, Church MJ, Davis CO, Karl DM, Letelier RM and Doney SC (2018) ALOHA From the Edge: Reconciling Three Decades of in Situ Eulerian Observations and Geographic Variability in the North Paciﬁc Subtropical Gyre. Front. Mar. Sci. 5:130. doi: 10.3389/fmars.2018.00130 April 2018 \| Volume 5 \| Article 130 Frontiers in Marine Science \| www.frontiersin.org 1 Aloha From the Edge Kavanaugh et al. INTRODUCTION On longer time scales, in situ PP and phytoplankton biomass (as determined by chlorophyll chl a) in the NPSG, measured during various programs such as CLIMAX and the Hawaii Ocean Time-series (HOT) have increased over the past four decades (Venrick et al., 1987; Karl et al., 2001; Corno et al., 2007). These increases have been linked to a shift in the phase of the Paciﬁc Decadal Oscillation (PDO, Karl et al., 2001) or El Niño Southern Oscillation (ENSO)/PDO interactions (Corno et al., 2007) and resulting changes in stratiﬁcation. 1. What are the interannual trends across diﬀerent metrics of gyre expansion? 2. To what degree are variations in subtropical seascape geography related to interannual to decadal climate modes? g g Station ALOHA (22.75◦N, 158◦W: A Long-term Oligotrophic Habitat Assessment) is a benchmark monitoring site in the NPSG where in situ sampling of ocean physical, biological, and chemical parameters has occurred since 1988 (Karl, 2010). However, the patterns of increased biomass and primary productivity found in situ are contrary to synoptic patterns observed through analyses of derived satellite products. Behrenfeld et al. (2006) observed that global ocean net PP and phytoplankton biomass (as deﬁned by chl a) decreased from 1999 to 2006 in response to increased water column stratiﬁcation. Hypothesized as an ecological response to an increase in the multivariate ENSO 3. Finally, how do variations in seascape geography relate to the interannual patterns of phytoplankton abundance and primary productivity observed at Station ALOHA? Frontiers in Marine Science \| www.frontiersin.org METHODS Oligotrophic Chl a Gyre Extent Oligotrophic Chl a Gyre Extent Monthly means of each ﬁeld were calculated. Because the determination of extent of features estimated from geostrophic ﬂow lines can be confounded by the eﬀect of steric sea level rise on ADT (Gille, 2014), we downloaded steric sea level anomalies (Levitus et al., 2012) for the upper 2,000 m from NOAA’s National Center for Environmental Information (https://www.nodc.noaa.gov/OC5/ 3M_HEAT_CONTENT/index.html), and calculated the trend in steric sea-level rise from 1993 to 2015 on a per pixel basis. Dynamic height was adjusted by subtracting the interpolated steric sea-level trend from the monthly satellite dynamic height ﬁeld. The gyre was then classiﬁed as the region contained within closing contours of original ADT and adjusted ADT (ADT_adj) at 1.05 m, the minimum height at which the contour lines recirculated (closed) completely to the western boundary (Figure 1B). Oligotrophic Chl a Gyre Extent Area of the extent of highly oligotrophic gyre (a threshold deﬁned as chl a ≤0.07 mg m−3; Polovina et al., 2008, hereafter oligotrophic threshold chl a) was calculated from monthly April 2018 \| Volume 5 \| Article 130 2 Aloha From the Edge Kavanaugh et al. SeaWiFS (R2017, completed January 2018) and Aqua-MODIS (R2017, completed December 2017) satellite ocean color derived chl a (Figure 1A). The SeaWiFS data record extends from autumn of 1997–2010 albeit with episodic gaps during 2008–2010 due to periodic sensor malfunction. Reprocessing (Franz et al., 2007; Franz, 2009) has removed much of the drift in the SeaWiFS and Aqua-MODIS instruments. Data were merged by comparing the per pixel seasonal cycle of SeaWiFS to that of MODIS-Aqua from 2003 to 2007, and then applying a seasonal correction to the historical SeaWiFS record. Merged data include seasonally adjusted SeaWiFS data until July of 2002, after which MODIS- Aqua data were used to December 2016. We also compared the trends between the OCI algorithm (Hu et al., 2012) to that of the previously standard OC3/OC4 (OCX) algorithms for SeaWiFS and MODIS-Aqua. The OCI algorithm is more accurate for [chl a] <0.2 mg m−3, converges with the OCX values above 0.2 mg m−3, and has been the standard NASA chl a algorithm since 2014. SeaWiFS (R2017, completed January 2018) and Aqua-MODIS (R2017, completed December 2017) satellite ocean color derived chl a (Figure 1A). The SeaWiFS data record extends from autumn of 1997–2010 albeit with episodic gaps during 2008–2010 due to periodic sensor malfunction. Reprocessing (Franz et al., 2007; Franz, 2009) has removed much of the drift in the SeaWiFS and Aqua-MODIS instruments. Data were merged by comparing the per pixel seasonal cycle of SeaWiFS to that of MODIS-Aqua from 2003 to 2007, and then applying a seasonal correction to the historical SeaWiFS record. Merged data include seasonally adjusted SeaWiFS data until July of 2002, after which MODIS- Aqua data were used to December 2016. We also compared the trends between the OCI algorithm (Hu et al., 2012) to that of the previously standard OC3/OC4 (OCX) algorithms for SeaWiFS and MODIS-Aqua. The OCI algorithm is more accurate for [chl a] <0.2 mg m−3, converges with the OCX values above 0.2 mg m−3, and has been the standard NASA chl a algorithm since 2014. where u and v are the zonal and meridional velocities calculated from sea surface height anomalies. Seascape Classiﬁcation While this does not isolate the eﬀect of photoacclimation, it assures that modeled chl a and satellite chl a are responding similarly to physical forcing, reﬂecting the phytoplankton physiological responses observed at Station ALOHA. All data sets were standardized prior to classiﬁcation. Satellite and model seascapes were classiﬁed separately (Figures 1C,D). in the Community Earth System Model (CESM-BEC, Doney et al., 2009; Moore et al., 2013). Surface chl a concentration is sensitive to mixed layer depth in the subtropics (e.g., Letelier et al., 1996; Siegel et al., 2013); thus we chose the CESM-BEC because of its inclusion of photoacclimation. While this does not isolate the eﬀect of photoacclimation, it assures that modeled chl a and satellite chl a are responding similarly to physical forcing, reﬂecting the phytoplankton physiological responses observed at Station ALOHA. All data sets were standardized prior to classiﬁcation. Satellite and model seascapes were classiﬁed separately (Figures 1C,D). sum of the area of all pixels within the appropriate contour or identiﬁed seascape in the North Paciﬁc. Surface area of each pixel was calculated by correcting for the spheroid-eﬀect on distance between lines of longitude. For each time step, the distance from Station ALOHA to the subtropical seascape boundary was also calculated using the mean of the 5th quantile of all boundary distances in a quadrant 45◦on either side of due NE from Station ALOHA Figure 1D); encroachment of less oligotrophic waters from the NE was most common. When the oligotrophic boundary encroached beyond (south and or east of) Station ALOHA, the distance values were negative. All area-based analyses were truncated at 10◦N to minimize the eﬀect of the equatorial upwelling region. y Climatological seasonal means of PAR, SST, and chl a were deﬁned for both data sets from 2003 to 2007. We used a probabilistic self-organizing mapping algorithm (PrSOM, Anouar et al., 1998) to reduce the 3-variable (SSTx,y,m, PARx,y, chl a x,y,m) spatiotemporal data set onto a 15 × 15 neuronal map resulting in 225 classes, each with its own 3-D weight based on the maximum likelihood estimation (3-D MLEs). The neural net size (15 × 15 nodes) was chosen to maximize sensitivity to mesoscale processes while preventing underpopulated nodes (deﬁned as <500 pixels). The 3-D MLEs were then further reduced using a hierarchical agglomerative clustering (HAC) with Ward linkages (Ward, 1963). Station ALOHA Data In situ NPP, Total chl a (Tchl a, the sum of divinyl and monovinyl chl a), and in situ physical patterns in the subtropical North Paciﬁc were assessed using archived data from Station ALOHA (http://hahana.soest.hawaii.edu/hot/hot-dogs/interface. html). Net PP was determined by daytime incubation in bottles spiked with 14C bicarbonate incubated in situ to maintain natural light and temperature (Letelier et al., 1996). Chl a and photosynthetic accessory pigments were measured by high performance liquid chromatography (HPLC) according to Wright et al. (1991). Mean pigment concentrations and NPP patterns were quantiﬁed separately for the light-saturated surface (0–45 m) and light-limited region of the euphotic zone that contained the deep chlorophyll maximum (DCM, 75–150 m). Potential temperature and bottle salinity were also recorded for the upper 45 m. Mixed layer depth was calculated using Seascape Classiﬁcation This linkage method uses combinatorial, Euclidian distances that conserve the original data space with sequential linkages (McCune et al., 2002). Euclidian distances here are equivalent to within-group and total sum of squares. To understand the role of mesoscale variability on basin- scale geography, we examined the relationship between mean EKE and the diﬀerence in extent between the gyre deﬁned by circulation and that deﬁned by oligotrophy (see above). Pixels were categorized as belonging in (1) both the oligotrophic and physical gyres, (2) only in the physical gyre, or (3) only in the oligotrophic gyre. The mean EKE was calculated over the pixels for each of the three aforementioned groups. Climatic Indices To infer the eﬀect of climatic forcing on the interannual dynamics of gyre geography, three diﬀerent indices were used. The multivariate El Niño Southern Oscillation Index (MEI) is computed from a principal component analysis (PCA) of six variables including sea level pressure, zonal and meridional wind components, cloudiness, and sea surface and air temperatures (Wolter and Timlin, 1993). These data are available from NOAA (http://www.esrl.noaa.gov/psd/enso/mei/). The North Paciﬁc Gyre Oscillation (NPGO, Di Lorenzo et al., 2008; http:// www.o3d.org/npgo/) is the second dominant mode calculated from North Paciﬁc sea surface height anomalies and is associated with accelerated North Paciﬁc, Alaska, and California Currents (Chavez et al., 2011). The Paciﬁc Decadal Oscillation (PDO, Mantua et al., 1997; Zhang et al., 1997) is associated with the dominant mode of North Paciﬁc SST anomalies and the data are available from the University of Washington (http://jisao. washington.edu/pdo/). Previously, we found that eight seascapes classiﬁed from seasonal climatologies of satellite-derived SST, chl a, and PAR represented seasonal shifts in biogeochemical patterns and planktonic assemblages (Kavanaugh et al., 2014a,b). Here, we followed the methodology of Kavanaugh et al. (2014b), with three exceptions. First, the seasonal variability of PAR was removed on a per-pixel basis to minimize discontinuities associated with the spring and fall transitions, with interannual and spatial variability remaining. Second, stepwise agglomerations were conducted using the Ward method until four seascapes for each of the model and satellite data sets were deﬁned: a subtropical seascape, oligotrophic transition, a mesotrophic transition, and a subarctic seascape. These four regions bear the spatial signature of analogous seasonally evolving regions classiﬁed previously (Kavanaugh et al., 2014b) and represent a trade-oﬀbetween variance explained in both the satellite and model records, and spatial match-up between satellite and modeled seascapes (Figure S1). Finally, once the seasonal and spatial vectors were classiﬁed, the means, variances and covariances within seascapes informed a multivariate Gaussian mixture model (GMM). Class assignments of individual months were then determined by their maximum posterior probabilities. New classes were then predicted from the CESM and satellite GMMs using the monthly means of chl a, SST, and PAR of the respective model and satellite time series. Seascape Classiﬁcation Seascapes were classiﬁed from Level 3 9-km merged chl a (described above), PAR, and SST from the Advanced Very High Resolution Radiometer (AVHRR) and MODIS-Aqua. Similar to chl a, monthly averages of PAR and SST were computed from the combined SeaWiFS and MODIS product; all three variables were binned to 1 degree to match model output. Model output of SST, PAR, and surface chl a from 1985 to 2005 were taken from the Biogeochemical Element Cycling Model embedded Near daily, gridded sea level anomalies (SLA) and absolute dynamic topography (ADT) ﬁelds derived from satellite altimetry were downloaded from AVISO (https://www.aviso.altimetry. fr/en/data/products.html). Eddy kinetic energy (EKE) was calculated from the near daily ﬁelds: EKE (m2 s−2) = 0.5 × [u2 + v2] (1) (1) FIGURE 1 \| Extent of the North Paciﬁc Subtropical Gyre (NPSG) for June of 2003, showing (A) oligotrophic threshold (≤0.07 mg m−3) satellite ocean color derived surface chlorophyll, chl a; (B) geostrophic recirculation deﬁned by the 1.05 m absolute dynamic height relative to the geoid; and multivariate seascapes classiﬁed from (C) CESM-BEC model output and (D) satellite data. Dark blue, subtropical seascape; light blue, oligotrophic boundary seascape; green, transition seascape; yellow, subarctic seascape. Arrow in (D) shows distance between Station ALOHA (white circle) and the boundary of the subtropical seascape. FIGURE 1 \| Extent of the North Paciﬁc Subtropical Gyre (NPSG) for June of 2003, showing (A) oligotrophic threshold (≤0.07 mg m−3) satellite ocean color derived surface chlorophyll, chl a; (B) geostrophic recirculation deﬁned by the 1.05 m absolute dynamic height relative to the geoid; and multivariate seascapes classiﬁed from (C) CESM-BEC model output and (D) satellite data. Dark blue, subtropical seascape; light blue, oligotrophic boundary seascape; green, transition seascape; yellow, subarctic seascape. Arrow in (D) shows distance between Station ALOHA (white circle) and the boundary of the subtropical seascape. Frontiers in Marine Science \| www.frontiersin.org April 2018 \| Volume 5 \| Article 130 3 Aloha From the Edge Kavanaugh et al. in the Community Earth System Model (CESM-BEC, Doney et al., 2009; Moore et al., 2013). Surface chl a concentration is sensitive to mixed layer depth in the subtropics (e.g., Letelier et al., 1996; Siegel et al., 2013); thus we chose the CESM-BEC because of its inclusion of photoacclimation. Statistical Analysis Cross correlation analyses were conducted to determine the correlations between climate and geographic metrics, the relationship between seascape dynamics and in situ conditions at Station ALOHA, and the time scales or lags at which the correlation was strongest. Cross correlation analysis was also conducted to determine the eﬀect of EKE on spatial mismatch between geographic metrics. Prior to analysis, monthly climatological means were calculated by removing outliers (exceeding ± 3 standard deviations), then missing in situ data were interpolated using a 3-month LOESS ﬁlter to minimize the eﬀect of within-year data density on interannual trends. Then, anomalies for each time series were calculated by subtracting the monthly climatological mean, calculated from the smoothed data, and then smoothing with a 12- month LOESS ﬁlter prior to correlation analysis. Statistical signiﬁcance of correlation coeﬃcients were determined using a critical value which adjusted degrees of freedom to account for autocorrelation in covariance (Glover et al., 2011). Correlations that exceeded this value are considered signiﬁcant (p < 0.05). p y Gyre extent, deﬁned by oligotrophic threshold chl a, was positively correlated to the MEI, with positive MEI preceding expansion of low-level chl a area by 6–12 months (Figure 4A). The areal extent of recirculation was positively correlated with the NPGO and strongly negatively correlated with the PDO and MEI (Figure 4B). Similar to the area of low-level chl a, the subtropical seascape was strongly correlated to the MEI and was also related to the PDO (Figure 4C), whereas the isolation of Station ALOHA within the subtropical gyre was positively correlated with the NPGO and negatively correlated with the MEI and PDO (Figure 4D). While covering a much larger area, areal extent of multivariate seascapes was weakly correlated to that of threshold chl a patterns, (Figure S3A: r = 0.24, p < 0.05); however, patterns preceded the chl-only index by approximately 7 months. Multivariate seascape expansion was anti-correlated to both the dynamic topography extent described above (r = −0.48, p < 0.05), and the distance of Station ALOHA to the edge of the subtropical seascape (Figure S3B: r = −0.36, p < 0.05). Isolation distance was positively correlated to gyre extent as measured by geostrophy (r = 0.33, p < 0.05), with isolation shifts preceding that of gyre extent also by 7 months. Isolation was not correlated to the low-level chl a metric. Statistical Analysis Trends over time were adjusted to account for the eﬀect of climate oscillations. We conducted a PCA across the NPGO, PDO, and MEI indices; multiple linear regression was conducted using the PCA scores as predictors and individual seasonally- detrended anomalies as responses. Trends were then a result of a simple linear ﬁt to the residuals of the multiple linear regression model. Geographic Metrics We focus on the dynamics of the subtropical seascape and the boundary between the subtropical and oligotrophic transition seascape. Total area of oligotrophy, recirculation, and multivariate subtropical seascape was assigned based on the April 2018 \| Volume 5 \| Article 130 Frontiers in Marine Science \| www.frontiersin.org 4 Aloha From the Edge Kavanaugh et al. (Table 1). Seasonal and interannual oscillations as well as weak increases (0.5% per year) in the physical extent of the gyre were evident from the altimetry-based time series (Figure 3B, Table 1). Trends were weaker in data adjusted for steric sea- level changes than those unadjusted for absolute dynamic height (ADT_adj and ADT, respectively, Table S1). As with the extent of oligotrophy, presence or magnitude of a trend depended on the length of the time series, with no trend evident from 1998 to 2006 (Figure 3B, Table S1), despite the strong ENSO signal during that span. Because modeled and satellite seascapes were highly correlated and in phase for both indices (Figures S1B,C; expansion: r = 0.64, p < 0.05; isolation r = 0.76, p < 0.05), the data records were combined and a single index of expansion or isolation was used that spanned the entire data record (1985–2016). There was evidence of slight expansion of the subtropical seascape from 1985 to 2016 (Table 1). Isolation distance also varied from year to year, although aseasonal, within-year variability was higher relative to the other metrics (Figure 3D). A linear trend was evident from 1985 to 2016 (but not over a shorter duration), with isolation distance increasing by 0.9% per year (Table 1). a potential density gradient threshold of 0.005 kg m−4 and a potential density oﬀset of 0.125 kg m−3 from the surface (Karl and Lukas, 1996). Climate Variability All series were smoothed using a 12-month LOESS ﬁlter. FIGURE 3 \| Temporal trends of North Paciﬁc subtropical gyre dynamic geography indices showing (A) areal extent calculated from oligotrophic threshold chl a across different satellite ocean color algorithms; (B) areal extent (expansion/contraction) calculated from satellite altimetry derived dynamic topography with and without adjustment for steric sea-level rise; (C) merged model and satellite seascape extent; (D) distance of Station ALOHA to the edge of the merged modeled and satellite subtropical seascape (isolation). All series were smoothed using a 12-month LOESS ﬁlter. Trends calculated over various durations are reported in Table 1 and Table S1. where instabilities associated with the Kuroshio extension appeared to blend the physical and chl a-based boundaries. On th t rn nd th rn d th p ti l mi m t h In Situ Trends Local SST averaged over the upper 45 m at Station ALOHA hibit d t ill ti i t h i li t FIGURE 2 \| Time series of climate indices including (a) the North Paciﬁc Gyre Oscillation, NPGO; (b) the Multivariate ENSO Index, MEI; and (c) the Paciﬁc Decadal Oscillation, PDO. Data sources are included in Methods. All series were smoothed using a 12-month LOESS ﬁlter. FIGURE 2 \| Time series of climate indices including (a) the North Paciﬁc Gyre Oscillation, NPGO; (b) the Multivariate ENSO Index, MEI; and (c) the Paciﬁc Decadal Oscillation, PDO. Data sources are included in Methods. All series were smoothed using a 12-month LOESS ﬁlter. FIGURE 2 \| Time series of climate indices including (a) the North Paciﬁc Gyre Oscillation, NPGO; (b) the Multivariate ENSO Index, MEI; and (c) the Paciﬁc Decadal Oscillation, PDO. Data sources are included in Methods. All series were smoothed using a 12-month LOESS ﬁlter. FIGURE 3 \| Temporal trends of North Paciﬁc subtropical gyre dynamic geography indices showing (A) areal extent calculated from oligotrophic threshold chl a across different satellite ocean color algorithms; (B) areal extent (expansion/contraction) calculated from satellite altimetry derived dynamic topography with and without adjustment for steric sea-level rise; (C) merged model and satellite seascape extent; (D) distance of Station ALOHA to the edge of the merged modeled and satellite subtropical seascape (isolation). All series were smoothed using a 12-month LOESS ﬁlter. Trends calculated over various durations are reported in Table 1 and Table S1. Climate Variability FIGURE 3 \| Temporal trends of North Paciﬁc subtropical gyre dynamic geography indices showing (A) areal extent calculated from oligotrophic threshold chl a across different satellite ocean color algorithms; (B) areal extent (expansion/contraction) calculated from satellite altimetry derived dynamic topography with and without adjustment for steric sea-level rise; (C) merged model and satellite seascape extent; (D) distance of Station ALOHA to the edge of the merged modeled and satellite subtropical seascape (isolation). All series were smoothed using a 12-month LOESS ﬁlter. Trends calculated over various durations are reported in Table 1 and Table S1. Climate Variability From 1985 to 2016, three to four cycles of the NPGO, MEI, and PDO were evident (Figure 2). In the early part of the record (1985–1998), the PDO and MEI were out of phase but moved into phase following the 1997–1998 El Niño and subsequent La Niña, remaining in phase for the rest of the data record (r = 0.6, p < 0.001). Throughout our study period, the NPGO was anticorrelated (r = −0.5, p < 0.001) with the other two indices, but also led the PDO and MEI by 5 and 8 months, respectively (Figure S2). Thus, any secular trend reported is that which remains after accounting for the eﬀect of oscillations in the sign and magnitude of the climate indicators. The NPSG is most contracted in winter and at its greatest extent in September for both deﬁnitions of the gyre (Figures 5A,B). The northern winter boundary of the physical gyre exceeds that of low-level chl a, and the summer eastern boundary extends less than the low-level chl-a. However, on interannual scales, the physical extent and low-level chl a-based extent were anti-correlated, due to diﬀerent climate forcing. The physical extent is strongly positively correlated with the NPGO, whereas the extent of low-level chl a is positively correlated with the PDO and MEI. Increased EKE resulted in expansion of the areal extent of oligotrophic- threshold chl a. The result is that oﬀof the Kuroshio, the spatial mismatch between the two deﬁnitions is minimized with increased EKE between their boundaries (Figure 5C), Expansion and Isolation Trends and Drivers Patterns and trends among the diﬀerent gyre geographic metrics varied and were associated with diﬀerent climate forcing. The area of low-level chl a was dependent on algorithm, with the lower sensitivity of the OCX resulting in a larger expanse of oligotrophy (Figure 3A, Table 1). Large interannual oscillations were evident, with any linear trend being highly dependent on record length (Table S1). From 1998 to 2016, expansion of the area of low-level chl a occurred at a rate of 0.36% year−1 April 2018 \| Volume 5 \| Article 130 Frontiers in Marine Science \| www.frontiersin.org 5 Aloha From the Edge Kavanaugh et al. FIGURE 2 \| Time series of climate indices including (a) the North Paciﬁc Gyre Oscillation, NPGO; (b) the Multivariate ENSO Index, MEI; and (c) the Paciﬁc Decadal Oscillation, PDO. Data sources are included in Methods. In Situ Trends where instabilities associated with the Kuroshio extension appeared to blend the physical and chl a-based boundaries. On the eastern and southern edge, the spatial mismatch increases with increased EKE (Figure 5D), where expanded oligotrophy is associated with greater EKE, particularly in summer. Local SST averaged over the upper 45 m at Station ALOHA exhibited strong oscillations in response to changing climate forcing over the data record, with warmer temperatures associated with positive ENSO and PDO and negative NPGO (Figure 6A, Figure S4). While there was a signiﬁcant linear April 2018 \| Volume 5 \| Article 130 Frontiers in Marine Science \| www.frontiersin.org 6 Aloha From the Edge Kavanaugh et al. TABLE 1 \| Secular trends across geographic metrics, chl a algorithms, and in situ characteristics at Station ALOHA. Algorithm Span Span mean Trend (year−1) Trend (SE) % change year−1 R2 p-Value AREA METRICS (km2) ocx: chl a ≤0.07 mg m−3 1998–2016 2.23E+007 4.87E+04 1.39E+04 0.220 0.05 <0.001 oci: chl a ≤0.07 mg m−3 1998–2016 1.95E+007 7.00E+04 1.36E+04 0.360 0.11 <0.001 ADT_adj 1993–2016 1.76E+007 8.66E+04 1.11E+04 0.491 0.42 <0.001 ADT_adj 1998–2016 1.76E+007 8.48E+04 1.08E+04 0.481 0.27 <0.001 Subtropical Seascape 1985–2016 2.32E+007 1.64+04 6.03E+03 0.071 0.02 <0.01 DISTANCE METRICS (km) Station ALOHA to SS edge 1985–2016 3.28E+02 3.20E+00 8.90E-01 0.970 0.04 <0.001 STATION ALOHA SST (◦C) 1989–2016 24.8 0.013 0.004 0.052 0.04 <0.01 SSS (psu) 1989–2016 35.1 0.008 0.001 0.023 0.14 <0.001 MLD (surface offset, m) 1989–2016 58.7 0.29 0.13 0.494 0.02 0.02 MLD (gradient threshold, m) 1989–2016 32.5 0.14 0.11 0.431 0 0.2 [chl a]: 0–45 m (mg chl a m−3) 1989–2016 0.09 0 0 0.000 0 0.72 [chl a]: 75–150 m (mg chl a m −3) 1989–2016 0.17 0.001 0.0002 0.588 0.06 <0.001 NPP: 0–45 m (mg C m−3 d−1) 1989–2016 6.07 0.053 0.01 0.873 0.08 <0.001 NPP: 75–150 m (mg C m−3 d−1) 1989–2016 1.13 0.045 0.004 3.982 0.34 <0.001 NPP: Int to150 m (mg C m−2 d−1) 1989–2016 513 5.63 0.85 1.097 0.18 <0.001 Italicized values denote no signiﬁcant trend over the duration measured. Signatures of the NPGO, PDO, and MEI have been removed from each time series prior to analysis (see section Methods). Italicized values denote no signiﬁcant trend over the duration measured. Signatures of the NPGO, PDO, and MEI have been removed from each time series prior to analysis (see section Methods). In Situ Trends FIGURE 4 \| Lagged correlations between climate indices and dynamic geography indices of the North Paciﬁc Subtropical Gyre. Where lag is negative, climate index leads geographic metric; the converse is true for positive lags. Correlations are signiﬁcant when they exceed the critical value (dotted red lines). (A) Gyre area deﬁned by chl a ≤0.07 mg m−3. (B) Gyre area deﬁned by closing contour of dynamic height (1.05 m). (C) Gyre area deﬁned by the extent of the multivariate subtropical seascape. (D) Distance from Station ALOHA to the edge of the subtropical seascape. FIGURE 4 \| Lagged correlations between climate indices and dynamic geography indices of the North Paciﬁc Subtropical Gyre. Where lag is negative, climate index leads geographic metric; the converse is true for positive lags. Correlations are signiﬁcant when they exceed the critical value (dotted red lines). (A) Gyre area deﬁned by chl a ≤0.07 mg m−3. (B) Gyre area deﬁned by closing contour of dynamic height (1.05 m). (C) Gyre area deﬁned by the extent of the multivariate subtropical seascape. (D) Distance from Station ALOHA to the edge of the subtropical seascape. April 2018 \| Volume 5 \| Article 130 Frontiers in Marine Science \| www.frontiersin.org 7 Aloha From the Edge Kavanaugh et al. FIGURE 5 \| Drivers of spatiotemporal differences in gyre extent based on dynamic height and oligotrophic threshold chl a. (A) Extent of gyre in February 2003 showing topographic (red) and pigment (blue)- based extents and region of overlap (green). (B) Same as (A), but for September 2003. (C) Cross correlation of mean eddy kinetic energy (EKE) in region represented solely by the physical deﬁnition with areal extent of that region. (D) Same as (C), but in extents solely represented by oligotrophic threshold chl a. FIGURE 5 \| Drivers of spatiotemporal differences in gyre extent based on dynamic height and oligotrophic threshold chl a. (A) Extent of gyre in February 2003 showing topographic (red) and pigment (blue)- based extents and region of overlap (green). (B) Same as (A), but for September 2003. (C) Cross correlation of mean eddy kinetic energy (EKE) in region represented solely by the physical deﬁnition with areal extent of that region. (D) Same as (C), but in extents solely represented by oligotrophic threshold chl a. p < 0.001, R2 = 0.34). In Situ Trends Together, these trends contributed to a relatively strong linear increase in NPP integrated over 150 m (Figure 7C, 5.63 ± 0.85 mg C m−2 d−1year−1, p < 0.001, R2 = 0.18). Oscillations were also evident in both chl a and NPP time series. trend from 1989 to 2016 (Table 1: 0.010 ± 0.004◦C year−1, p < 0.01), it was very weak (R2 = 0.04). Sea surface salinity exhibited large oscillations in the opposite direction of SST and also a relatively strong increasing trend over the data record (0.008 ± 0.001 psu year−1, p < 0.001, R2 = 0.18; Figure 6B). Mixed layer depth had large interannual variability over the data record, but also oscillated in response to changing surface salinity and temperature. A weak linear increase was apparent over the data record for mixed layer calculated via the surface oﬀset method (0.29 ± 0.13 m year−1, p = 0.02, R2 = 0.02; Figure 6D), although none was apparent with the mixed layer depth calculated via the gradient method (Figure 6C, Table 1). Correlation of Seascape Geography With in Situ Physics and Biology y gy Increased mixed layer depth followed isolation of Station ALOHA (Figure 8A, r = 0.32, p < 0.05) and preceded a contracted subtropical seascape (Figure 8B, r = −0.32, p < 0.05) by ∼6 months. Salinity was positively associated with isolation but at longer lags (r = 0.28–0.32, lags > 20 months) and negatively associated with, but preceding expansion of the subtropical seascape (r = −0.35, p < 0.05, ∼1year). SST was positively associated with subtropical seascape expansion (Figure 8B), but preceded expansion by ∼1 year (r = 0.3, p < 0.05). These patterns generally followed what would be expected by the climate forcing of geography (Figure 4, Figure S4) with SST at Station ALOHA positively correlated with the PDO and MEI (and negatively with the NPGO), and surface salinity and mixed layer depth positively correlated with the NPGO (and negatively correlated with the PDO and MEI) (Figure S4). Time lags and wavelengths were much longer and broader when considering climate indices directly, rather than their geographic signatures. With the exception of isolated events (e.g., early 2001), seasonal patterns of T-chl a in the surface and at depth were out of phase (Figure 7A). Concentrations in the light-limited euphotic zone were approximately double that of the surface, and also increased over the data record (Table 1: 0.001 ± 0.0002 mg chl a m−3 year−1, p < 0.001, R2 = 0.08). No analogous trend was evident in surface chlorophyll. NPP increased over the data record in both the surface and at depth (Figure 7B, Table 1). Mean rates in the surface were ∼4x higher than that in the 75–150 m depth bin (Table 1: 5–7 mg C m−3 d−1 vs. 1–2 mg C m−3 d−1). However, the relative rate of increase in NPP over time was stronger in the light limited euphotic zone vs. the surface (Table 1: surface ∼1% year−1, trend = 0.053 ± 0.010 mg C m−3 d−1year−1, p < 0.001, R2 = 0.09; At depth ∼3% year−1, trend = 0.045 ± 0.004 mg C m−3 d−1year−1, Station ALOHA T chl a and NPP were also correlated to gyre geography following local physical forcing. Isolation was Frontiers in Marine Science \| www.frontiersin.org April 2018 \| Volume 5 \| Article 130 Frontiers in Marine Science \| www.frontiersin.org 8 Aloha From the Edge Kavanaugh et al. Correlation of Seascape Geography With in Situ Physics and Biology FIGURE 7 \| Phytoplankton dynamics at Station ALOHA showing anomalies for (A) T chl a in the surface and deep chlorophyll maximum, DCM, (B) Net primary productivity in the surface (0–45 m) and depth (75–150 m) as calculated from daytime incubations, and (C) vertically integrated NPP from 0 to 150 m. FIGURE 6 \| In situ surface conditions at Station ALOHA showing anomalies for (A) sea surface temperature (SST); (B) sea surface salinity; (C) mixed layer depth as deﬁned by a vertical gradient threshold of 0.005 kg m−3 m−1 for potential density and (D) mixed layer depth as deﬁned by a density surface offset of 0.125 kg m−3. FIGURE 7 \| Phytoplankton dynamics at Station ALOHA showing anomalies for (A) T chl a in the surface and deep chlorophyll maximum, DCM, (B) Net primary productivity in the surface (0–45 m) and depth (75–150 m) as calculated from daytime incubations, and (C) vertically integrated NPP from 0 to 150 m. FIGURE 6 \| In situ surface conditions at Station ALOHA showing anomalies for (A) sea surface temperature (SST); (B) sea surface salinity; (C) mixed layer depth as deﬁned by a vertical gradient threshold of 0.005 kg m−3 m−1 for potential density and (D) mixed layer depth as deﬁned by a density surface offset of 0.125 kg m−3. gyre were evident, they were dominated by large oscillations, correlated with changes in ENSO and PDO conditions as well as the NPGO. With oscillations, we observe that in situ biophysical conditions at Station ALOHA are congruent to what would be predicted during periods of expanded oligotrophy via an expanded subtropical seascape including warmer SST and declines in T chl a and NPP. We also observe that conditions at Station ALOHA shift prior to multivariate seascape expansion, suggesting that Station ALOHA may serve as a leading indicator for changes throughout the North Paciﬁc subtropical region. associated with increased chl a in the surface (Figure 8C, r = 0.32, p < 0.05), and increased surface and integrated NPP (Figure 8E, r = 0.32 to 0.4, p < 0.05) followed isolation by ∼5 months. Decreased Tchl a at depth was strongly associated with expansion of the subtropical seascape (Figure 8D: r = −0.41, p < 0.05) as was decreased NPP throughout the water column (Figure 8F: r = −0.28 to −0.39, p < 0.05). Frontiers in Marine Science \| www.frontiersin.org DISCUSSION The location of Station ALOHA at the edge of the subtropics (this study) and other pivot points of climate forcing (Chavez et al., 2011; Messié and Chavez, 2011) may result in diﬀerent climate eﬀects depending on the interaction of multiple climate modes. During positive ENSO phases, water column stabilization restricts upward mixing of new nitrate and selects for organisms that can ﬁx atmospheric nitrogen (Karl et al., 1995, 2001; Campbell et al., 1997). During positive PDO, the Aleutian Low is further south and leads to a southern anomaly in the transition zone chlorophyll front and potentially higher than average primary productivity in the subtropics (Chai et al., 2003). Positive NPGO periods are associated with more northward Aleutian Lows (Chavez et al., 2011) along with stronger North Paciﬁc and California currents, higher pressure and deeper thermocline depths at the center of the subtropics (Di Lorenzo et al., 2008, 2009), potentially leading to decreased access to Where satellite metrics have suggested that oligotrophic systems such as the NPSG will expand in response to warming oceans (Behrenfeld et al., 2006; Polovina et al., 2008; Irwin and Oliver, 2009), analogous changes have not been evident at Station ALOHA (Corno et al., 2007) or the Bermuda Atlantic Time-series Station BATS in the subtropical North Atlantic (Lomas et al., 2010). This suggests that there may be physiological or structural reorganization not observed from space, uncharacterized spatiotemporal variability, or satellite sensor drift. We examined the geographic dynamics of the NPSG, the role of climate forcing on those dynamics, and the relationships between shifting gyre geography and local in situ conditions at the long-term time-series station, Station ALOHA over a 31-year data record. While weak secular trends in the expansion of diﬀerent metrics of the subtropical April 2018 \| Volume 5 \| Article 130 9 Aloha From the Edge Kavanaugh et al. FIGURE 8 \| Correlation between in situ biophysical variables at Station ALOHA and isolation of Station ALOHA (DIST) and expansion of subtropical seascape (AREA). (A,B) SST, mixed layer depth, and salinity; (C,D) Tchl a in the surface and DCM; (E,F) NPP in surface, DCM, and integrated over the upper 150 m. Geographic metrics lead biophysical variables when lag is negative. FIGURE 8 \| Correlation between in situ biophysical variables at Station ALOHA and isolation of Station ALOHA (DIST) and expansion of subtropical seascape (AREA). Frontiers in Marine Science \| www.frontiersin.org DISCUSSION Concomitant to geographic and physical trends, we also conﬁrm the increase of primary production, as well as phytoplankton standing stock derived from chl a in the euphotic zone, throughout the data record at Station ALOHA, as previously reported by Corno et al. (2007) and Karl and Church (2017). It is unclear whether long-term trends in T chl a and NPP are associated with changes in nutrient inputs (Luo et al., 2012; Letelier et al., in preparation), community structure, and or increased eﬃciency of photosynthesis, particularly in the DCM (Letelier et al., 2017). While lags in how climate oscillations aﬀect in situ conditions at Station ALOHA relative to the rest of the gyre may suggest better agreement with respect to expanding oligotrophy, the divergence of secular trends measured by satellite-based geography and in situ conditions at Station ALOHA point toward an alternative hypothesis. In situ PP in the surface appears to be balanced by loss on diurnal time scales (White et al., 2017), allowing no accumulation of biomass through time. In our study, we found that secular trends of phytoplankton appear to be stronger in the deeper light-limited region of the euphotic zone, below the optical depth that satellites can see. Increased NPP in the DCM is correlated with greater abundances of nanophytoplankton (White et al., 2015), which represent over 40% of the particulate carbon (Barone et al., 2015). However, the debate about what exactly 14 C incubations measure (NPP, gross PP or something in between, e.g., Marra, 2009) is exacerbated when considering shifts in community structure, particularly toward increased dominance by nano- and picophytoplankton. Species with relatively high ratios of respiration to photosynthesis may tend to substantially overestimate net primary production, perhaps because a substantial percentage of the carbon respired by such species is old carbon (Pei and Laws, 2013). 14C also overestimates NPP at low growth rates, although the discrepancy is less in 12-h than in 24-h incubations (Pei and Laws, 2014). Certainly, careful intercomparison of incubation and bottle-free Secular increases in the gyre geographical extent are weak, but evident within all four geographic indices. Limited by the satellite record at the time of publication, Polovina et al. (2008) reported increases in the areal extent of oligotrophic threshold chl a in the North Paciﬁc at a rate of 2.2% per year from 1998 to 2007. DISCUSSION Finally, we see no secular trend, but strong oscillations and correlation with both ENSO and PDO were apparent with the extent of the multivariate subtropical seascape (Figures 3C, 4C), where chl a dynamics may have been balanced in the classiﬁcation by SST and spatial light variability. The latter result is in agreement with Behrenfeld et al. (2016), who found large oscillations in surface chl a concentrations in the subtropical gyres after accounting for the eﬀect of photoacclimation. over the duration of the study. During positive NPGO conditions, transport through the North Paciﬁc and California currents is stronger (Di Lorenzo et al., 2008), and meanders of the southern edge of the North Paciﬁc Current-California current bifurcation may be reduced leading to the associated increase in isolation of Station ALOHA within the northeast corner of the subtropical seascape. Eddy kinetic energy appears to expand the geographic signature of oligotrophy, resulting in a temporal mismatch between the gyre deﬁned by low-level chl a and dynamic topography. While this suggests that the net eﬀect of mesoscale processes on chl a (extent of oligotrophy) is negative (expansive), the observed eﬀect of eddies on phytoplankton biomass and NPP near Station ALOHA are mixed. Upwelling within cyclonic eddies have also been purported to change community structure (Bidigare et al., 2003), primary and new production (Vaillancourt et al., 2003). Anticyclonic eddies can result in increased nitrogen ﬁxation (Fong et al., 2008; Church et al., 2009) which is responsible for over 40% of the new/export production at HOT. Finally, regions between anticyclonic and cyclonic eddies have also been shown to have increased PP and particulate organic carbon ﬂux (Guidi et al., 2012). Sea surface height variation in general, has been found to aﬀect the location and species composition in the DCM, with a shallower DCM with higher abundance of larger eukaryotic phytoplankton occurring with lower SSH (Barone et al., in review). Future studies could use existing databases to investigate the relative frequency of eddies (e.g., Chelton et al., 2011) in the NPSG during diﬀerent climate modes, and determine whether changes in frequency are accompanied by changes in nitrogen ﬁxation, NPP, and export at Station ALOHA. After accounting for climate oscillations in the in situ data record at Station ALOHA, we observed that SST, salinity, and mixed layer depth (but only as deﬁned by the surface oﬀset method) were weakly increasing over 1989–2016. DISCUSSION (A,B) SST, mixed layer depth, and salinity; (C,D) Tchl a in the surface and DCM; (E,F) NPP in surface, DCM, and integrated over the upper 150 m. Geographic metrics lead biophysical variables when lag is negative. Station ALOHA tend to precede expansion by a few months. Thus, despite its location on the northeastern edge of the NPSG, or perhaps because of it, the time-series station provides an early indication of biogeographic conditions of the gyre as a whole. deep nutrients in the interior of the gyre. However we found that positive phases of the NPGO were associated with increased mixed layer depth, salinity, and isolation of Station ALOHA which preceded increased T chl a and NPP. Conversely, the subtropical seascape (as well as the extent of oligotrophy deﬁned solely by the threshold chl a) were associated with positive phases of the MEI and PDO, a shoaling and freshening mixed layer, and declines in T chl a and NPP at Station ALOHA. Thus while a stronger gyre may result in decreased access to nutrients at the center of the gyre (Di Lorenzo et al., 2008), the positive NPGO may result in increased access to nutrients at the edge. Furthermore, changes of biological and physical properties at The expansion of the topography-deﬁned gyre positively correlated with the NPGO, and negatively correlated with MEI, PDO, and the gyre deﬁned by the 0.07 mg chl a m−3 threshold. Isolation of Station ALOHA (the distance to the NE boundary of the most oligotrophic seascape) also was related to the NPGO (was positively correlated but led geostrophic expansion by 7 months) and exhibited a weak trend toward increased isolation April 2018 \| Volume 5 \| Article 130 Frontiers in Marine Science \| www.frontiersin.org 10 Aloha From the Edge Kavanaugh et al. from 0.7 to 0.4% year−1) may contribute to a greater extent. Nevertheless, the gyre as deﬁned by dynamic topography also appears to be expanding, and relatedly, increasing the isolation of Station ALOHA. Physical gyre expansion was fastest prior to 1998, but appeared to increase throughout the altimetry record even with adjustments made for steric sea level rise (e.g., Gille, 2014). Note that surface topography in our analysis has not been corrected for interannual variability in atmospheric pressure; for this reason our reported trends based on SSH must be viewed as preliminary. Frontiers in Marine Science \| www.frontiersin.org CONCLUSION Figure S4 \| Cross correlation analysis of climate indices and in situ surface properties at station ALOHA. Table S1 \| Trends of subtropical expansion across geographic metrics, chl a algorithms, and time-series duration. Italicized values denote no signiﬁcant trend over the duration measured. The effect of climate oscillations has not been removed. Table S1 \| Trends of subtropical expansion across geographic metrics, chl a algorithms, and time-series duration. Italicized values denote no signiﬁcant trend over the duration measured. The effect of climate oscillations has not been removed. CONCLUSION The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmars. 2018.00130/full#supplementary-material Through the lens of pelagic seascape ecology, we have quantiﬁed diﬀerent geographic systems in the North Paciﬁc that expand and contract on seasonal and interannual scales and have embedded long-term Eulerian in situ observations from Station ALOHA in a dynamic mosaic of the NPSG. Over a three- decade time-series, we were able to quantify subtle secular trends and strong oscillations in gyre-scale geographic patterns and in situ conditions at Station ALOHA. Due to a longer data record and improved sensitivity of satellite algorithms to low-level chl a, we observe secular trends that are weaker than previously reported, but evident in both physical and biological deﬁnitions of the gyre, physical changes at Station ALOHA, and changes in phytoplankton standing stock and NPP, primarily in the deeper euphotic zone. Finally, we observe that conditions at Station ALOHA shift analogously to strong interannual oscillations in gyre-scale oligotrophy as deﬁned by multivariate seascapes, with shoaled mixed layer and declines in phytoplankton biomass and production occurring with an expanded subtropical seascape. Figure S1 \| Seascape determination of dynamic gyre geography. (A) Variance explained by satellite and model seascapes and the percent of pixels that have the same classiﬁcation in space and time between satellite and modeled seascapes. (B) Cross-correlation of model and satellite time series of subtropical seascape expansion. (C) Cross-correlation of model and satellite time series of isolation of Station ALOHA. Figure S2 \| Cross-correlation of MEI and PDO with NPGO. Figure S2 \| Cross-correlation of MEI and PDO with NPGO. Figure S3 \| Cross-correlation of geographic indicators. (A) Areal extent of low level chl a, extent deﬁned by geostrophy, and isolation distance of Station ALOHA in relation to variations in subtropical seascape extent. (B) Areal extent of low level chl a, extent deﬁned by geostrophy, and subtropical seascape extent in relation to variations in the isolation distance of Station ALOHA within the subtropical seascape. Figure S3 \| Cross-correlation of geographic indicators. (A) Areal extent of low level chl a, extent deﬁned by geostrophy, and isolation distance of Station ALOHA in relation to variations in subtropical seascape extent. (B) Areal extent of low level chl a, extent deﬁned by geostrophy, and subtropical seascape extent in relation to variations in the isolation distance of Station ALOHA within the subtropical seascape. ACKNOWLEDGMENTS We acknowledge support from the National Science Foundation through the Center for Microbial Oceanography: Research and Education (C-MORE), an NSF Science and Technology Center (EF-0424599), and the Hawaii Ocean Time-series (OCE- 1260164; MC and DK), and the National Aeronautics and Space Administration Ocean Biology and Biogeochemistry Program (NASA NNX14AM36G, SD and MK) and the Biodiversity and Ecological Forecasting Program (NNX14AP62A and 80NSSC18K0412, MK). Additional support was provided by the Gordon and Betty Moore Foundation (#3794; DK) and the Simons Foundation’s SCOPE project (#329104 ; DK). AUTHOR CONTRIBUTIONS methods (e.g., Quay et al., 2010; Juranek and Quay, 2013), optical assessments of size structure (White et al., 2015) and gross carbon accumulation (White et al., 2017) along with 1-D modeling (e.g., Luo et al., 2012) will be helpful to determine how long-term changes in physical and chemical parameters are aﬀecting the ecosystem structure and function at Station ALOHA. methods (e.g., Quay et al., 2010; Juranek and Quay, 2013), optical assessments of size structure (White et al., 2015) and gross carbon accumulation (White et al., 2017) along with 1-D modeling (e.g., Luo et al., 2012) will be helpful to determine how long-term changes in physical and chemical parameters are aﬀecting the ecosystem structure and function at Station ALOHA. MK: Devised the original question with input from CD and RL, created the study design, and analyzed the data with input from SD; MC and DK: Contributed data and historical context to the manuscript. All authors contributed to the writing and editing of the manuscript. Our results also suggest that long-term satellite and 3-D modeling studies, particularly those that parameterize multiple species and currencies of production (e.g., Luo et al., 2012; Nicholson et al., 2014) are necessary to understand the spatial and temporal scales of physical forcing and ecological responses in the NPSG. We note that, although the 30+ year record may be suﬃcient to examine many oscillations, a modeling study determined that ∼40 years was necessary to detect secular trends in the subtropics (Henson et al., 2010). However, the relationships between the geographic indicators and conditions at Station ALOHA revealed in this study may allow for future hypotheses to be tested, particularly those that relate to the role of mesoscale variability in modulating seasonal to interannual change and the role of Station ALOHA as an early indicator of gyre responses to climate oscillations. DISCUSSION Irwin and Oliver (2009) reported general increases through time in the most oligotrophic provinces in their classiﬁcation, with oscillatory behavior found across all oligotrophic provinces. Using a longer record and linear regression analyses, Signorini et al. (2015) found mostly secular declines in surface chl a over large, subjectively chosen regions, but also discussed the inﬂuence of mixed layer depth changes and subsequent photoacclimation on chl a in the subtropics. In our re-analysis of geographic trends in the North Paciﬁc using the latest processing of MODIS and SeaWiFS, we observe ENSO- and PDO-inﬂuenced oscillations of areal extent of regions with chl a <0.07 mg m−3 over a weak secular trend (Figure 3A, Table 1); however, we derive much slower rates of expansion than previously reported. Reprocessing and subsequent lowering of blue water chl a concentrations in the early SeaWiFS record (https://oceancolor. gsfc.nasa.gov/reprocessing/r2014/seawifs/) may be one reason for this discrepancy in expansion rate (2–1.1%). However, the chl a algorithm used (a decline of 1.1–0.7% year−1) and a data record length that could account for climate oscillations (a decline April 2018 \| Volume 5 \| Article 130 11 Aloha From the Edge Kavanaugh et al. Frontiers in Marine Science \| www.frontiersin.org REFERENCES S., et al., (2009). Nutrient and salinity decadal variations in the central and eastern North Paciﬁc. Geophys. Res. Lett. 36:L14601. doi: 10.1029/2009GL038261 Kavanaugh, M.T., Emerson, S. R., Lockwood, D. M., Quay, P.D., and Letelier, R. M. (2014a). Physicochemical and biological controls on primary and net community production across NE Paciﬁc seascapes. Limnol. Oceanogr. 59, 2013–2027. doi: 10.4319/lo.2014.59.6.2013 DiTullio, G. R., and Laws, E. A. (1991). 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Environ. 135, 77–91. doi: 10.1016/j.rse.2013.03.025 Copyright © 2018 Kavanaugh, Church, Davis, Karl, Letelier and Doney. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Copyright © 2018 Kavanaugh, Church, Davis, Karl, Letelier and Doney. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Signorini, S. R., Franz, B. A., and McClain, C. R. (2015). Chlorophyll variability in the oligotrophic gyres: mechanisms, seasonality and trends. Front. Mar. Sci. 2:1. Frontiers in Marine Science \| www.frontiersin.org REFERENCES doi: 10.3389/fmars.2015.00001 April 2018 \| Volume 5 \| Article 130 Frontiers in Marine Science \| www.frontiersin.org 14
https://openalex.org/W4311316799	https://link.springer.com/content/pdf/10.1007/s00723-022-01511-w.pdf	English	null	High-field/High-frequency EPR Spectroscopy in Protein Research: Principles and Examples	Applied magnetic resonance	2,022	cc-by	46,927	Applied Magnetic Resonance (2023) 54:207–287 https://doi.org/10.1007/s00723-022-01511-w Applied Magnetic Resonance (2023) 54:207–287 https://doi.org/10.1007/s00723-022-01511-w Applied Magnetic Resonance REVIEW REVIEW Abstract During the last decades, the combined efforts of biologists, chemists, and physi- cists in developing high-field/high-frequency EPR techniques and applying them to functional proteins have demonstrated that this type of magnetic resonance spectros- copy is particularly powerful for characterizing the structure and dynamics of stable and transient states of proteins in action on biologically relevant time scales rang- ing from nanoseconds to hours. The review article describes how high-field EPR methodology, in conjunction with site-specific isotope and spin-labeling strategies, is capable of providing new insights into fundamental biological processes. Specifi- cally, we discuss the theoretical and instrumental background of continuous-wave and pulse high-field EPR and the multiple-resonance extensions EDNMR, ENDOR, TRIPLE, ESEEM, PELDOR, and RIDME. Some emphasis is placed on a bal- anced description of both the historical spadework and the achieved performance of advanced EPR at 95 GHz and 360 GHz. This culminates in a coherent treatment of state-of-the-art research of high-field EPR in terms of both instrumentation develop- ment and application to representative protein complexes such as cofactor binding sites in photosynthesis. High‑field/High‑frequency EPR Spectroscopy in Protein Research: Principles and Examples Klaus Möbius1 · Anton Savitsky2 Received: 7 October 2022 / Revised: 10 October 2022 / Accepted: 9 November 2022 / Published online: 13 December 2022 © The Author(s) 2022 Received: 7 October 2022 / Revised: 10 October 2022 / Accepted: 9 November 2022 / Published online: 13 December 2022 © The Author(s) 2022 * Anton Savitsky anton.savitsky@tu-dortmund.de * Klaus Möbius moebius@physik.fu-berlin.de * Klaus Möbius moebius@physik.fu-berlin.de * Anton Savitsky anton.savitsky@tu-dortmund.de 1 Department of Physics, Free University Berlin, Arnimallee 14, 14195 Berlin, Germany 2 Faculty of Physics, Technical University Dortmund, Otto‑Hahn‑Str. 4a, 44227 Dortmund, Germany 1 Department of Physics, Free University Berlin, Arnimallee 14, 14195 Berlin, Germany 1 Introduction During the last 30 years or so, high-field/high-frequency EPR spectroscopy is rap- idly growing. Application of ever higher magnetic fields and microwave frequen- cies results in spectacular improvements of both spectral and time resolution. 456789) 3 208 K. Möbius, A. Savitsky Thereby, more complex spin systems can be studied by EPR, very much in anal- ogy to what has happened in modern NMR spectroscopy decades earlier. The rea- sons for this time delay between EPR and NMR developments are obvious: EPR has to cope with resonance frequencies three orders of magnitude larger than in NMR and correspondingly shorter electron spin relaxation times. The resulting require- ments in adequate microwave technology were challenging and needed their time to be solved. It was only after the lifting of the East/West “Iron Curtain” in the early 1990’s when low-noise mm microwave sources, fast switches and detectors, which had been developed in the military domain, became available also for unclassified research in the public domain, for instance at universities and basic-research insti- tutions. These devices deliver and process microwave power in the range of sev- eral 10 milliwatt at frequencies up to several 100 GHz necessary for fast pulsed high-field EPR. Nowadays, we witness multifrequency and multi-dimensional EPR benchmark experiments at the technical limits of quasioptical sub-mm microwave bridges and sweepable wide-bore cryomagnets with homogeneous Zeeman fields of up to 10 Tesla and beyond [1]. Thereby, more complex spin systems can be studied by EPR, very much in anal- ogy to what has happened in modern NMR spectroscopy decades earlier. The rea- sons for this time delay between EPR and NMR developments are obvious: EPR has to cope with resonance frequencies three orders of magnitude larger than in NMR and correspondingly shorter electron spin relaxation times. The resulting require- ments in adequate microwave technology were challenging and needed their time to be solved. It was only after the lifting of the East/West “Iron Curtain” in the early 1990’s when low-noise mm microwave sources, fast switches and detectors, which had been developed in the military domain, became available also for unclassified research in the public domain, for instance at universities and basic-research insti- tutions. These devices deliver and process microwave power in the range of sev- eral 10 milliwatt at frequencies up to several 100 GHz necessary for fast pulsed high-field EPR. 1 3 1 Introduction Nowadays, we witness multifrequency and multi-dimensional EPR benchmark experiments at the technical limits of quasioptical sub-mm microwave bridges and sweepable wide-bore cryomagnets with homogeneous Zeeman fields of up to 10 Tesla and beyond [1]. Our review contribution to this Special Issue of Applied Magnetic Resonance is intended as an introduction for students and experts alike. It is offering guiding prin- ciples of high-field/high-frequency EPR spectroscopy and the characteristics of its specific instrumentation. This goes along with illustrative examples, most of them from our own research work in biochemistry and molecular biology, supplemented by a few examples from dedicated laboratories around the world other laboratories as well. We hope that the mixture of introductory and specialized sections of the article will be interesting for both specialists and newcomers in this fascinating area of research and instrumentation development. Our research described was performed in collaboration projects with numerous internationally renowned spectroscopy groups, and this in teamwork with under- graduate and graduate students, postdocs and senior scientists from around the world. A stronghold of the projects was photochemistry and photobiology, with emphasis on the cofactors of photosynthesis in interaction with their immediate molecular environment of the “solvent matrix”. In more recent years, our cooperation network developed to a Platonic octahe- dron with six groups at the cornerstones (the vertices): Novosibirsk, Kazan, Mos- cow, Bologna, Berlin, and Mülheim (Ruhr). This network was 2018 extended by a seventh group in Dortmund (Anton Savitsky, previously in Berlin and Mülheim) resulting in a more complex polyhedron of collaborations that can be visualized as an augmented triangular prism with seven vertices (see Fig. 1). The collaborations focus on light-initiated electron-transfer processes in natural and artificial photo- synthesis. They require a variety of advanced multifrequency, multiresonance EPR techniques to be characterized in detail [2–18].i The unique potential of high-field/high-frequency EPR spectroscopy for study- ing complex spin systems was recognized by a few research groups already more than 45 years ago, and Yakov S. Lebedev and his group at the NN Semenov Institute of Chemical Physics in Moscow were the first to start a dedicated high-field/high- frequency research and development program. They had realized the distinct advan- tages of high-field/high-frequency EPR compared to conventional X-band EPR. In a 1 3 High‑field/High‑frequency EPR Spectroscopy in Protein… 209 Fig. 1 Introduction 1 Left: Platonic octahedron of EPR collaborations between six groups (the vertices, the names of the respective group leaders are given in parentheses) in Novosibirsk (Renad Sagdeev), Kazan (Kev Salikhov), Moscow (Alexey Semenov), Bologna (Giovanni Venturoli), Berlin (Klaus Möbius), and Mül- heim/Ruhr (Wolfgang Lubitz). Right: Since 2018, the octahedron was extended by Dortmund (Anton Savitsky) to a more complex polyhedron of collaborations visualized as an augmented triangular prism with seven vertices Fig. 1 Left: Platonic octahedron of EPR collaborations between six groups (the vertices, the names of the respective group leaders are given in parentheses) in Novosibirsk (Renad Sagdeev), Kazan (Kev Salikhov), Moscow (Alexey Semenov), Bologna (Giovanni Venturoli), Berlin (Klaus Möbius), and Mül- heim/Ruhr (Wolfgang Lubitz). Right: Since 2018, the octahedron was extended by Dortmund (Anton Savitsky) to a more complex polyhedron of collaborations visualized as an augmented triangular prism with seven vertices nutshell, the advantages are enhanced spectral resolution and orientational selectiv- ity even for disordered molecules with small g-tensor anisotropy, enhanced spectral separation of radicals with only slightly different g-values, enhanced time resolution for tracing transient radical intermediates, enhanced detection sensitivity for small samples with low spin concentration, increased sensitivity toward molecular motion conformational changes and associated relaxation effects by transforming motion- ally narrowed EPR spectra into the slow-motion regime. In 1976, they completed a versatile continuous-wave (cw) high-field EPR spectrometer employing a 150 GHz microwave source and a 5 T superconducting magnet. In the following, the advan- tages of the high-field EPR will be discussed in some detail. 1.1 Why EPR at High Magnetic Fields/Microwave Frequencies? In general, EPR spectroscopy has proven to be a very useful technique for studying both stable and transient radical-pair intermediates in liquid and solid phases. For large low-symmetry spin systems, however, standard X-band EPR (9.5 GHz) soon reaches its limits of useful information content, unless single-crystal samples are available. Unfortunately, large molecular complexes are often available only as dis- ordered samples. Their X-band EPR spectra are poorly resolved, and the information on magnetic parameters and molecular orientations is hidden under broad lines due to strong inhomogeneous line broadening. By turning to higher and higher magnetic fields and microwave frequencies, for example to EPR at W-band (95 GHz) or even at 360 GHz, at least five important features, (i), (ii)–(v), are emerging from the EPR spectra: (i) enhanced spectral resolution; (ii) enhanced orientational selectivity in disordered samples; (iii) enhanced low-temperature electron spin polarization; (iv) enhanced detection sensitivity for restricted-volume samples such as small protein single crystals, and (v) enhanced sensitivity for probing fast motional dynamics, i.e., 1 3 210 K. Möbius, A. Savitsky high-frequency EPR makes a faster ’’snapshot’’ for characterizing complex molecu- lar motions. high-frequency EPR makes a faster ’’snapshot’’ for characterizing complex molecu- lar motions. Ad (i): The strategy for spectral resolution enhancement is similar in EPR and NMR: With increasing external Zeeman field the field-dependent spin interactions in the spin Hamiltonian are separated from the field-independent ones (see Fig. 2). In high-field EPR, the g-factor resolution is increased in relation to the hyperfine couplings, in high-field NMR the chemical-shift resolution is increased in relation to the spin–spin couplings. Resolution problems due to inhomogeneous line broadening also arise when sev- eral radical species or different magnetic sites of rather similar g-values are present in the sample. Or when a small g-tensor anisotropy of the paramagnetic system does not allow canonical orientations of the powder EPR spectrum to be resolved.i For "real" high-field EPR experiments, properties of the spectrometer have to be related with properties of the sample: For all cases of delocalized spin sys- tems, in which unresolved hyperfine interactions dominate the inhomogeneous EPR linewidth, a real high-field experiment must fulfill the condition: (1) Δg giso ⋅B0 > ΔB, (1) Fig. 2 Enhanced spectral resolution by high-field EPR, taking the cofactor radical ions in bacterial pho- tosynthetic reaction centers as example, with P the primary donor, QA and QB the quinone acceptors. 1.1 Why EPR at High Magnetic Fields/Microwave Frequencies? The spin Hamiltonian in the inset describes two radicals in an external Zeeman field B0 and contains the Zee- man interactions of the two electron spins S1 , S2 and their hyperfine interactions with the nuclear spins Ii , Ij in the radicals. For details, see text Fig. 2 Enhanced spectral resolution by high-field EPR, taking the cofactor radical ions in bacterial pho- tosynthetic reaction centers as example, with P the primary donor, QA and QB the quinone acceptors. The spin Hamiltonian in the inset describes two radicals in an external Zeeman field B0 and contains the Zee- man interactions of the two electron spins S1 , S2 and their hyperfine interactions with the nuclear spins Ii , Ij in the radicals. For details, see text 1 High‑field/High‑frequency EPR Spectroscopy in Protein… 211 i.e., the anisotropic electron Zeeman interaction, described by the difference Δg of principal g-tensor components, must exceed the EPR inhomogeneous linewidth ΔB . On the other hand, when ∆B is reduced by isotope labeling, e.g., by perdeuteration of the nitroxide spin-label molecule and/or 15 N-substitution of 14 N, lower B0 fields may already be sufficient to meet the condition for true high-field EPR, Eq. (1). In certain systems, the reduction of ΔB by isotope labeling may be the only way to enhance the spectral resolution when field-dependent g-strain effects become the linewidth-determining parameter at higher Zeeman fields, see [1].i i.e., the anisotropic electron Zeeman interaction, described by the difference Δg of principal g-tensor components, must exceed the EPR inhomogeneous linewidth ΔB . On the other hand, when ∆B is reduced by isotope labeling, e.g., by perdeuteration of the nitroxide spin-label molecule and/or 15 N-substitution of 14 N, lower B0 fields may already be sufficient to meet the condition for true high-field EPR, Eq. (1). In certain systems, the reduction of ΔB by isotope labeling may be the only way to enhance the spectral resolution when field-dependent g-strain effects become the linewidth-determining parameter at higher Zeeman fields, see [1].i i Ad (ii): The important feature of enhanced orientation selectivity by high-field EPR on randomly oriented spin systems becomes essential for organic radicals with only small g-anisotropy (see Fig. 3). For example, below room temperature the over- all rotation of a protein complex often becomes so slow that powder-type EPR spec- tra are obtained. 1.1 Why EPR at High Magnetic Fields/Microwave Frequencies? However, if the anisotropy of the leading interaction tensor in the spin Hamiltonian is larger than the inhomogeneous linewidth, the canonical orienta- tions of the interaction tensor can be resolved. As a consequence, single-crystal-like information on the hyperfine interactions can be extracted by performing orienta- tion-selective ENDOR at the field values of resolved spectral features. In the case of transition-metal complexes, however, the hyperfine anisotropy of the metal ion may provide this orientation selectivity from the entire orientational distribution of the molecules. Their g-anisotropy is often large enough to allow for distinct orien- tational selectivity already in X-band EPR and single-crystal like ENDOR spectra [19–21]. For a detailed elucidation of the molecular structure and orientation of large biological complexes, such as membrane proteins, this is an important strategy because preparation of single crystals is often difficult or even impossible. Fig. 3 Enhanced orientation selectivity by high-field EPR, taking the anion radical of the ubiquinone acceptor cofactor in frozen-solution bacterial photosynthetic reaction centers as example Fig. 3 Enhanced orientation selectivity by high-field EPR, taking the anion radical of the ubiquinone acceptor cofactor in frozen-solution bacterial photosynthetic reaction centers as example 3 212 K. Möbius, A. Savitsky Ad (iii): Because of the enhanced low-temperature electron spin polarization at sufficiently large Zeeman fields, the absolute sign of the zero-field splitting param- eter, D , of a two-spin system such as biradicals or triplet states can be determined. At high fields, considerable thermal spin polarization can be achieved already well above helium temperature. The necessary condition is that the sample temperature becomes comparable with the Zeeman temperature, Tz = g ⋅휇B ⋅B0∕kB ( g : elec- tron g-factor, 휇B : Bohr magneton, B0 : Zeeman field; kB : Boltzmann constant). At T ≫TZ , the characteristic triplet powder EPR spectrum (see Fig. 4) is symmetric at its low- and high-field sides and, hence, contains no information of the sign of D. At T < TZ , the Boltzmann distribution leads to increased populations of the low- energy levels, resulting in asymmetric line shapes from which the absolute sign of D can be directly read off. 1.1 Why EPR at High Magnetic Fields/Microwave Frequencies? Thermal spin polarization as a means to determine the absolute sign of D in high-spin systems has been used at a variety of EPR frequen- cies, for example at 9.5 GHz ( TZ ≈0.4 K) [22], at 95 GHz ( TZ ≈4 K) [23], 140 GHz ( TZ ≈6.5 K) [24], 360 GHz ( TZ ≈15.5 K) [25]. Ad (iv): With respect to detection sensitivity and its enhancement with increas- ing microwave frequencies, one has to distinguish between the absolute and rela- tive (concentration) sensitivities. The absolute sensitivity is defined by the mini- mum detectable number of spins in the sample, Nmin ; the relative sensitivity is given by Nmin∕VS , i.e., is scaled by the sample volume, VS . This is limited by the amount of sample that can be placed into the cavity of high-field EPR spectrom- eters which, of course, is usually significantly smaller than standard X-band cavities. Consequently, if the amount of sample available is limited like in single crystals of proteins, the sensitivity of high-field EPR can be superior by orders of magnitude Fig. 4 Enhanced thermal spin polarization by high-field EPR, taking mechanically generated radical pairs in a donor–acceptor mixture as example. For details, see [23] Fig. 4 Enhanced thermal spin polarization by high-field EPR, taking mechanically generated radical pairs in a donor–acceptor mixture as example. For details, see [23] High‑field/High‑frequency EPR Spectroscopy in Protein… 213 because the absolute sensitivity grows with increasing frequencies much stronger than the relative sensitivity does. Under certain experimental conditions, for con- stant incident microwave power and unsaturated EPR lines, one obtains theoretical expressions for the absolute sensitivity, Nmin ∝휔−9∕2 0 , and for the relative sensitivity, Nmin∕VS ∝휔−3∕2 0 (see the detailed sensitivity discussions in refs. [26, 27]). because the absolute sensitivity grows with increasing frequencies much stronger than the relative sensitivity does. Under certain experimental conditions, for con- stant incident microwave power and unsaturated EPR lines, one obtains theoretical expressions for the absolute sensitivity, Nmin ∝휔−9∕2 0 , and for the relative sensitivity, Nmin∕VS ∝휔−3∕2 0 (see the detailed sensitivity discussions in refs. [26, 27]). 0 Ad (v): The faster ’’snapshot’’ capability for complex motional dynamics with increasing EPR frequency can be used in a multifrequency continuous-wave (cw) EPR approach at the same temperature to probe fast internal modes of motion and to discriminate them from the slow restricted motion of a macromolecule in solution. 1.1 Why EPR at High Magnetic Fields/Microwave Frequencies? In high-frequency cw EPR spectra, slow motions appear to be frozen out, whereas fast motions dominate the observed spectral lineshape [28–31]. 1.2 NMR Versus EPR For many decades after the invention of EPR and NMR in the 1940s, the popularity of EPR has been hopelessly behind its famous (though younger) sister NMR, and it is only during the last decades that the chemistry, biology and physics commu- nities appreciate the dramatic catching up of EPR in modern magnetic resonance spectroscopy. The reasons for EPR’s large-scale jump ahead are to be found in the remarkable technological breakthroughs in pulsed microwave technology, sweepa- ble cryomagnet fabrication and fast data acquisition and handling instrumentation. Modern advanced EPR is apparently booming, rather similar to what had happened with NMR 15–20 years earlier, both exhibiting unique and complementary capabili- ties in elucidating structure and dynamics of complex (bio)chemical systems in the fluid, glassy, or solid state. l The technical requirements for pulsed NMR and pulsed EPR are dramatically dif- ferent and related to the different time scales of the NMR and EPR phenomena. They are a consequence of the vastly different magnetic moments of nuclei and electrons (for example for 1H, the magnetic moment ratio is 1.5⋅10–3, for 14 N it is 1.1⋅10–4). Thus, the time scales are determined by the nuclear and electron resonance frequen- cies, in the radiofrequency (rf) and microwave (mw) domains, respectively. And the characteristic frequency separations in the respective resonance spectra (Hz versus MHz) and the relaxation times T1 , T2 (ms versus ns) are vastly different. Because of the long nuclear T1 and T2 times in diamagnetic molecules, NMR 90° and 180° pulses need not be shorter than 10 μs, which to generate and detect coherently does not pose technical problems. The electronic transverse relaxation times ( T2 ), how- ever, are typically in the 100 ns range and, consequently, in EPR, the mw 90° and 180°pulses have to be as short as a few ns. To generate and detect them coherently poses great technical problems even today. This holds, for example, for the mw sources with adequate output power, for fast mw switches and mixers as well as for fast electronic semiconductor components and computers for controlling the pulse trains, likewise for detecting and handling the transient signals in the ns time scale. Nowadays, pulse NMR has completely replaced cw NMR, culminating in multi- dimensional Fourier Transform (FT) spectroscopy. High-field steady-state cryomag- nets and nuclear resonance frequencies up to 1 GHz (for protons) have dramatically 1 3 214 K. Möbius, A. 1.2 NMR Versus EPR Savitsky improved the detection sensitivity and chemical-shift separations. And even for- merly exotic nuclei have become routinely observable by now. Despite the spectacu- lar breakthroughs in mm and sub-mm microwave technologies in the last decade, in EPR the cw versus pulse situation is still very different from that in NMR. In fact, the general prognosis is that coexistence between cw and pulse EPR will con- tinue to persist. Which option to choose will be determined entirely by the sample under study. The specific sample properties and relaxation times ultimately dictate the preference for either a cw or pulse experiment to be performed. Regarding spectral resolution and detection sensitivity, both modern EPR and NMR spectroscopies follow similar strategies: to apply higher and higher static Zee- man fields to separate field-dependent from field-independent spin interactions in the molecular system. By this strategy not only otherwise overlapping lines can be disentangled, but also the population difference and quantum energy of the driven transitions between electron and nuclear spin energy levels will be increased allow- ing for the detection of fewer and fewer spins. 1.3 Chronological Account transform NMR has an enormous potential for recording multi-dimensional spectra of complex biosystems in the liquid and solid state, and for applications in medical imaging such as MRI diagnostics. It took almost a decade before R. J. Blume at Columbia University [32] observed, for the first time, electron spin echoes analogous to Hahn’s nuclear spin echoes. And it took many more years before electron spin echo-detected EPR (ESE) methodolo- gies gained sufficient experimental and theoretical backing to revolutionize FT-EPR spectroscopy, see A. Schweiger and G. Jeschke [33]. Pulse EPR spectroscopy has many discoverers, both from the theoretical and the experimental side. In the early 1970s, K. M. Salikhov at Akademgorodok (Novosi- birsk) [34] laid the theoretical foundation for several advanced pulse EPR methods. For example, he developed the theory of electron spin phase relaxation by stochas- tic modulation of the dipole–dipole interaction between paramagnetic centers and its effect on the ESE decay [35]. He suggested the first pulse ELDOR (PELDOR) protocol to observe the modulation of the ESE signal due to the electron–electron dipolar interaction of weakly coupled biradical systems in disordered solids [36]. Some years later, K. M. Salikhov theoretically predicted new spin phenomena, such as quantum beats of the EPR line intensity [37] and out-of-phase spin echoes of correlated radical pairs [38]. These spin phenomena should be observable in time- resolved EPR spectra of transient spin-polarized radical-pair intermediates. And, indeed, such new spin phenomena were observed in several laboratories shortly after they had been predicted. A prominent example of application was the bacterial pho- tosynthetic reaction center with its donor photoexcited to the singlet state by short laser flashes leading to spin-correlated donor–acceptor radical pairs (for a review of these early experiments, see [39]. A rich variety of mw pulse sequences and sophis- ticated experiments is found in more recent reviews and text books, for example that of A. Schweiger and G. Jeschke [33]. Pioneering work was done, among others, by W. B. Mims at Bell Telephone Laboratories, Yu. D. Tsvetkov in Novosibirsk, J. H. Freed at Cornell University and A. Schweiger at ETH Zurich. It was a milestone in the history of magnetic resonance spectroscopy when, in 1956, G. Feher at Bell Labs [40] invented ENDOR (electron–nuclear double res- onance). 1.3 Chronological Account EPR and NMR phenomena were originally discovered in radiofrequency spec- troscopy experiments employing cw electromagnetic fields, EPR in 1944 by E. K. Zavoisky at Kazan University, NMR in 1946 by E. M. Purcell, H. G. Torrey and R. V. Pound at Harvard University and, independently, by F. Bloch, W. Hansen and M. E. Packard at Stanford University. These classic NMR experiments were honored as early as 1952 by the Nobel Prize in Physics to Bloch and Purcell. Zavoisky’s discovery of EPR, on the other hand, was only inadequately recognized on the West side of the Iron Curtain—in contrast to the East side: In 1957, E. K. Zavoisky was awarded the Lenin Prize, the highest sign of recognition in the former USSR for his discovery of ’’The Electron Paramagnetic Resonance Phenomenon’’. It was not until 1977 that E. K. Zavoisky was finally also honored internationally. Though not by a Nobel Prize, but at least by the prestigious ISMAR Award of the International Soci- ety of Magnetic Resonance. The Award was conferred in May 1977 at the ISMAR Conference in Banff, Canada—alas posthumously [18], because he had already died in Moscow in October 1976, just after having been informed about the decision of the international ISMAR Prize Committee. Up to the 1960’s, both NMR and EPR remained to be cw methods, i.e., the sam- ples in the static magnetic field (the Zeeman field B0 ) were irradiated with con- tinuous radiofrequency (rf) and microwave (mw) fields to drive NMR and EPR transitions, respectively. But it was as early as 1949, when E. L. Hahn at Urbana University applied rf pulses and invented the nuclear “spin-echo” detection. This, together with the introduction of powerful fast computers for “Fast Fourier transfor- mation” in the late 1970s, opened the arena for FT-NMR spectroscopy. R. R. Ernst was awarded the Nobel Prize in Chemistry in 1991 for his contributions toward the development of Fourier transform nuclear magnetic resonance spectroscopy while at Varian Associates and ETH Zurich (Richard R. Ernst, Nobel Lecture, Nuclear Magnetic Resonance Fourier Transform Spectroscopy, December 9, 1992). Fourier 1 3 High‑field/High‑frequency EPR Spectroscopy in Protein… 215 transform NMR has an enormous potential for recording multi-dimensional spectra of complex biosystems in the liquid and solid state, and for applications in medical imaging such as MRI diagnostics. 1.3 Chronological Account His ingenious concept was to apply simultaneously two electromagnetic fields, one in the mw, the other in the rf range, to drive EPR and NMR transitions having an energy level in common. Thereby, the advantages of EPR (high detec- tion sensitivity) are combined with those of NMR (high resolution capability). Feher’s first cw ENDOR experiment was technically feasible only because the sam- ple—phosphorus doped silicon—was kept at low temperature, where all the relaxa- tion times are sufficiently long to easily saturate both EPR and NMR transitions, which is a necessary condition for cw ENDOR. The cw ENDOR technique was later extended to ESE-detected pulse versions for solid-state samples by W. B. Mims (1965) at Bell Labs [41] and E. R. Davies (1974) at Clarendon Laboratory [42]. W.B. Mims is widely acknowledged to be the driving force in pulse EPR in general, and in pulse ENDOR in particular [33]. In contrast to solid-state ENDOR at low temperatures, for radicals in liquid solu- tion the electronic and nuclear relaxation times are much shorter—in the order of 10–5 to 10–7 s. Consequently, cw ENDOR-in-solution experiments are technically 1 3 216 K. Möbius, A. Savitsky much more demanding since much larger saturating mw and rf fields have to be applied. This is the reason why liquid-state ENDOR experiments took many more years than solid-state ENDOR experiments before they were successful. The pio- neering work was performed by A. L. Cederquist at Washington University [43] in 1963, who studied metal ammonia solutions. And by J. S. Hyde and A. H. Maki at Varian Associates and Harvard University, respectively, in 1964 [44], who studied a stable organic radical dissolved in n-heptane solution. The further development of cw ENDOR-in-solution spectroscopy was highly stimulated by J. H. Freed at Cor- nell University, whose general theory of saturation and double resonance proved to be adequate for describing amplitude, width, and shape of ENDOR lines in great detail (see, for example, [45]). There are apparent weaknesses of ENDOR in comparison to EPR concerning sensitivity (typically one order of magnitude lower) and relative line intensities (they do no longer reflect the number of contributing nuclei). These weaknesses can be overcome by extending ENDOR to electron–nuclear–nuclear triple resonance. For the special case of only one set of hyperfine-coupled nuclei, such a triple resonance extension was proposed early on by G. Feher [46] and J. H. Freed [47]. 1 3 1.3 Chronological Account He applied a mw spin-echo pulse train with varying inter-pulse separation and observed, on top of the exponential echo-decay trace, echo-amplitude modulations from hyperfine and quadrupole interactions. To obtain detectable echo modulations, efficient mixing of the nuclear and electron spin eigenfunctions by the dipolar hyperfine interaction is mandatory. Consequently, the strength of the external magnetic field has to be properly chosen to balance the Zeeman splitting of the nuclear sublevels and the respective hyperfine splitting (’’cancelation condition’’). driven either directly by rf fields as in ENDOR or, as a more indirect alternative, by ESEEM (electron spin echo modulation). This single-resonance technique was introduced by W. B. Mims [56] in 1972. He applied a mw spin-echo pulse train with varying inter-pulse separation and observed, on top of the exponential echo-decay trace, echo-amplitude modulations from hyperfine and quadrupole interactions. To obtain detectable echo modulations, efficient mixing of the nuclear and electron spin eigenfunctions by the dipolar hyperfine interaction is mandatory. Consequently, the strength of the external magnetic field has to be properly chosen to balance the Zeeman splitting of the nuclear sublevels and the respective hyperfine splitting (’’cancelation condition’’). Concerning the detection of short-lived transient states or reaction interme- diates, besides pulsed EPR also specific cw EPR strategies can be used to obtain time-resolved signals. Employing field modulation at a frequency as high as 1 MHz, the time resolution could be extended to the µs range. A decisive step forward to drastically higher time resolution was achieved for fast photoreactions by abandon- ing field modulation at all but generating the time-dependent EPR signal via wave- length-selective pulsed laser excitation. Subsequent direct, i.e., broadband detec- tion of the transient EPR signal at a fixed Zeeman field value is accomplished by employing sufficiently fast data acquisition systems. The Zeeman field is stepped through the spectrum establishing a time-resolved transient (TR) technique known as ’’TREPR’’. The pioneering experiment was done by S.I. Weissman and co-work- ers [57] at Washington University in 1979. In TREPR, the inherent loss of sensitivity for broadband detection of transient paramagnetic states can often be compensated by accumulation of the spectra after each light flash. Moreover, an orders-of-magni- tude signal enhancement via electron spin polarization effects can be utilized which occur in many photoreactions. 1.3 Chronological Account Its experi- mental realization, however, had to wait until 1974, when K. P. Dinse in the Möbius group at Free University (FU) Berlin accomplished ’’Special TRIPLE’’ on radicals in liquid solution [48]. In cw Special TRIPLE, the two frequency-swept rf fields are applied at frequencies always symmetrically placed around the Larmor frequency of the respective nucleus. This variant of triple resonance enhances the signal inten- sity and allows to relate relative line intensities to the number of responsible nuclei. Thereby, the assignment of ENDOR lines to molecular positions is made possible, which is a vital task, but notoriously difficult in ENDOR spectroscopy [49–51]. fi About a year later, it was demonstrated by R. Biehl in the same group at FU Berlin [52], that additional information about relative signs of hyperfine couplings of radicals in solution—and thereby about their assignment—can be obtained by generalizing the triple resonance experiment to include NMR transitions of differ- ent nuclei in the radical ("General TRIPLE"). In cw General TRIPLE, two rf fields with independently variable frequencies are applied, one pumping a selected NMR transition while the other is swept through the ENDOR spectrum. From the result- ing characteristic intensity changes in the ENDOR spectrum, the relative signs of the hyperfine couplings can be directly read off. The analog of this experiment for solid-state samples at low temperature (77 K) was performed earlier by R. J. Cook and D. H. Whiffen (1964) at Teddington National Physical Laboratory [53]. They called it ’’double ENDOR’’, and applied it to X-irradiated organic crystals to determine relative signs of hyperfine couplings. The advantages of TRIPLE over ENDOR—enhanced sensitivity and resolution, information about multiplicity and relative signs of hyperfine couplings from line intensity variations—justify the extra experimental efforts inherent in the triple resonance spectroscopy. It was shown to be extremely powerful in elucidating the electronic structures not only of organic radicals in solution [49] but also of transient cofactor radical–ion intermediates in primary photosynthesis [54, 55].i To measure the electron–nuclear hyperfine and nuclear quadrupole interac- tions by combinations of EPR and NMR techniques, the nuclear transitions can be 1 3 High‑field/High‑frequency EPR Spectroscopy in Protein… 217 driven either directly by rf fields as in ENDOR or, as a more indirect alternative, by ESEEM (electron spin echo modulation). This single-resonance technique was introduced by W. B. Mims [56] in 1972. 1.3 Chronological Account They appear in reactions with instantaneously gener- ated excited states and subsequent fast detection of the transient reaction intermedi- ates, e.g., triplets, radicals, radical pairs, before spin–lattice relaxation can thermal- ize them (for overviews of electron spin polarization effects, see for example [39, 58, 59]. By now, the time resolution of TREPR has been pushed to the 10 ns range, and TREPR has proven to perform extremely well over a wide range of mw frequencies, from S-band (4 GHz), X-band (9 GHz), K-band (24 GHz), Q-band (35 GHz) up to the high-field EPR frequencies 95 GHz, 120 GHz and 240 GHz [39, 60–62]. For many applications in photochemistry, a multifrequency approach of TREPR experi- ments with a wide range of Zeeman fields turned out to be essential for the detailed analysis of spin-polarized spectra in case of competing polarization mechanisms. Long-range distance measurements with the scale of a few nm in chemical and biological systems are an important application for pulse EPR spectroscopy [63]. They are based on the electron–electron dipolar coupling between two spin-car- rying domains, which is a function of their interspin distance and relative orienta- tion. The main advantage of pulse versus cw EPR techniques in this endeavor is the ability to separate the electron–electron coupling from other interactions, such as electron–nuclear hyperfine interactions, and to reduce inhomogeneous line broadening. Thereby, the distance range that can be probed is extended to about 8 nm. Yu. D. Tsvetkov and his co-workers at Novosibirsk [36, 64] established the 3-pulse electron–electron double resonance (PELDOR, also called DEER, double 1 3 3 218 K. Möbius, A. Savitsky electron–electron resonance) technique in 1981. Later it was extended to a 4-pulse sequence for dead-time free detection [33, 65]. Other powerful pulse sequences for measuring electron–electron frequencies and, thereby, distances have been invented, for example the single-frequency techniques DQC (double-quantum-coherence EPR) by J.H. Freed and co-workers at Cornell [66] and RIDME (relaxation-induced dipolar modulation enhancement) by Yu. D. Tsvetkov and co-workers in Novosi- birsk [67].i At this point, we would like to emphasize that the development of high-field/ high-frequency EPR marked another leap forward of the capabilities of EPR spec- troscopy on (bio)chemical complexes. As many major developments in science, also high-field EPR spectroscopy has several fathers who independently took the decisive actions in a similar period of time. 1.3 Chronological Account Thorough historical overviews on such developments are provided in the literature, see for instance, [13, 68, 69]. First publications on this subject appeared around 1970 [70–73]. But it was Ya. S. Lebedev in Moscow who was the first to start a long-term research and develop- ment program on high-field/high-frequency EPR in physical chemistry, starting in the 70’s [69]. In his group, Oleg Grinberg and Alexander Dubinskii were primar- ily involved in the instrument development [69]. They were inspired by the earlier preliminary experiments of D. J. E. Ingram at the University of Keele [70, 71]. The construction of their high-field/high-frequency EPR started in 1973 in close coop- eration with the Physical-Technical Institute in Donetsk (Ukraine). In the following years, several prototypes of the spectrometer were constructed. Different microwave sources including klystrons, BWOs, diffraction generators and solid-state oscillators were tested. Single-mode resonators, oversized cavities and Fabry–Perot resonators, as well as different detectors, were compared in their performance. The first working version of the 140 GHz cw EPR spectrometer, finished in 1979, reached a sensitivity of 4⋅108 spins/mT at 1 Hz bandwidth, which was about three orders of magnitude higher than for X-band EPR at that time. In parallel with the development of EPR instrumentation, the Lebedev group performed a series of EPR experiments approv- ing the advantages of high-field/high-frequency EPR for application to physical, chemical and biological systems. Yakov Lebedev’s contributions certainly set quality benchmarks and widened the horizons in EPR spectroscopy. His early death in 1996 at the age of only 61 was a tragic loss for the whole EPR community, his family and friends, see [6]. In the early 1980s, the Möbius group at Free University Berlin started with their 95 GHz EPR and ENDOR projects [74], and extended them in subsequent years to 360 GHz EPR and ENDOR. These projects focused on applications to protein complexes and model systems (for an overview, see [1]). Only a few laboratories have developed the instrumentation for millimeter and submillimeter high-field EPR spectrometers, thus ploughing the ground for a prom- ising new field of molecular spectroscopy of complex systems. 1.3 Chronological Account Until the second half of the 1990s, laboratory-built high-field EPR spectrometers have been described for continuous-wave (cw) microwave irradiation at 95 GHz ( λ ≈ 3 mm), for example by the Möbius group at FU Berlin: [74–77], around 150 GHz ( 휆≈ 2 mm) [78–80], at 250 GHz ( 휆≈ 1 mm) [81–83], and in the sub-mm region [84–86], even reaching 360 GHz/14 T EPR [87]. A few other EPR spectrometers operating at frequencies 1 3 High‑field/High‑frequency EPR Spectroscopy in Protein… 219 above 200 GHz have been described until the end of the 90s [88–91]. High-field EPR spectrometers with pulsed microwave irradiation have also been described dur- ing this time, operating at wavelengths of 3 mm [60, 92, 93] and 2 mm [80, 94, 95].i above 200 GHz have been described until the end of the 90s [88–91]. High-field EPR spectrometers with pulsed microwave irradiation have also been described dur- ing this time, operating at wavelengths of 3 mm [60, 92, 93] and 2 mm [80, 94, 95].i The extension to high-field ENDOR experiments has been realized at the 3 mm [60, 74, 96, 97] and 1 mm [98] microwave frequency bands. Whereas W-band high- field cw ENDOR was accomplished first in Berlin [96], high-field pulse ENDOR was accomplished first in Leiden [93, 99].i i After these early years of high-field EPR spectroscopy, several groups continued and other groups started to build their dedicated high-field/high-frequency instru- mentation. To give a few examples:i The research group of J. H. Freed at Cornell University was the first to push EPR instrumentation to far-infrared technology, and pioneered the use of quasioptical mw techniques in EPR. Details of their 250 GHz EPR spectrometer were published in 1988 [81]. The Cornell group also developed specially designed Fabry–Perot reso- nators for which high detection sensitivity was achieved even for aqueous biologi- cal samples [100]. The Frankfurt group of Thomas Prisner built a pulsed 180 GHz spectrometer with a quasioptical circulator and single-mode cavity; the instrument is designed for optional pulsed ENDOR and PELDOR experiments [101]. In 1989, the group of L.-C. Brunel at the Grenoble High-Magnetic-Field Labo- ratory realized an EPR system operating up to 525 GHz and magnetic fields up to 20 T [85]. A couple of years later, L.-C. Brunel moved to the National High Mag- netic Field Laboratory (NHMFL), Tallahassee, Florida, where he, together with H. 1.3 Chronological Account van Tol, developed a 25 Tesla cw EPR machine that can operate up to 700 GHz for a Lande factor g = 2 system [91, 102]. And around 2008, the research group of G. M. Smith and co-workers in St Andrews published their first spectacular results of their ambitious HIPER design concepts of a high-power, wideband 94 GHz spectrometer [103]. The HIPER instrumentation project, entitled “Bringing the NMR Paradigm to EPR”, was a top ranked research proposal in the UK and, later, turned into a UK high-field EPR facility [104]. This spectrometer has state-of-the-art sensitivity and is now a UK EPSRC facility and part of a European network on high-field EPR instrumentation and research. Commercially available EPR and ENDOR spectrom- eters are mostly operating at W-band frequencies. They were introduced at the end of the 1990s, the major manufacturer was, and still is, Bruker Biospin, Germany [105, 106]. Appropriate references to the laboratories which completed the con- struction of mm- and sub-mm high-field EPR spectrometers since the end of the 90s are included in recent overview articles, for instance [1, 29, 107–118]. Time-resolved EPR spectroscopy is an important issue in general, and under high- field conditions in particular. The arena of time-resolved high-field EPR was opened in 1989 by the first pulsed W-band EPR spectrometer built in the Schmidt group in Leiden [77, 99]. Soon after, T. F. Prisner and M. Rohrer in the Möbius group in Berlin completed a versatile pulsed EPR spectrometer at W-band, which served also for the first high-field echo-detected TREPR experiment on pulsed laser-generated transient radicals in photosynthetic reaction centers [60]. However, the introduction of time resolution capability of the sub-mm high-field EPR at 360 GHz with qua- sioptical microwave bridge had to wait until 2004. The design of this spectrometer in Berlin started with the cw mode of operation [87] using a solid-state 120 GHz 1 3 3 3 220 K. Möbius, A. Savitsky source with subsequent tripling. The 360 GHz output power at the Gaussian horn antenna is only 1 mW, thus limiting the B1 field at the sample in the Fabry–Perot resonator to values too small for fast pulsing. 1.3 Chronological Account As an unconventional novel approach to time-resolved sub-mm high-field EPR, a dedicated 360 GHz pulsed Orotron (’’Oro’’ refers to Russian abbreviations for ’’open resonator plus reflecting grid’’ in the high-voltage vacuum-tube generator) was constructed and integrated in the quasioptical microwave bridge of the heterodyne induction-mode EPR spectrometer in Berlin [119]. As mentioned above, under certain conditions, ESEEM can be a competitive alternative to ENDOR for measuring hyperfine and quadrupole interactions [56]. First successful W-band high-field ESEEM measurements of nitrogen hyperfine and quadrupole interactions in disordered powder samples were performed in Berlin in 1998 [120]. More recently, this work was largely extended by elaborate W-band ESEEM studies on nitroxide spin-label molecules to explore the sensitivity of the g-, hyperfine- and quadrupole-tensors for probing polarity and proticity effects of the solvent matrix [121].ii In 2006/2007 pulsed high-field electron dipolar spectroscopy, specifically PEL- DOR or DEER, was introduced for resolving the relative orientation of weakly coupled biradical partners in a frozen-solution sample, in addition to measur- ing their distance. This extension of electron dipolar spectroscopy, established at X-band frequencies for determining large inter-radical distances (see recent over- views [122–124], to high frequencies and fields was accomplished independently by the group of T. F. Prisner (Frankfurt) [116, 125, 126] at 180 GHz, the group of G. Jeschke (Konstanz) [127] at 95 GHz and the group of K. Möbius (Berlin) at 95 GHz [128]. A more detailed chronological account of the landmark developments in advanced EPR spectroscopy and its multifrequency extensions can be found in [1]. Figure 5 shows the microwave and radiofrequency irradiation schemes of a vari- ety of cw and pulse high-field EPR techniques that have been applied by various laboratories also at high Zeeman fields. Several of them are discussed in consider- able detail in the present article. Concluding this Introduction section, we point out that a major challenge of molecular biology, biochemistry, and biophysics is to understand function and reac- tion mechanism of highly specialized proteins on the level of their molecular and electronic structure. We take the view that the arsenal of modern EPR techniques in general, and of high-field EPR in particular, provides powerful and versatile tools highly needed for such an endeavor. The molecular information obtained is comple- mentary to what can be learned from other biophysical techniques established in the field. 2 Principles of High‑Field/High‑Frequency EPR In the following, we present a rather phenomenological description of the theoreti- cal principles of EPR techniques that stood the test for high-field applications in biophysics and biochemistry. For a more in-depth theoretical treatment of advanced EPR spectroscopy we refer to renowned textbooks, for example [1, 33, 58, 129, 1 3 High‑field/High‑frequency EPR Spectroscopy in Protein… 221 Fig. 5 Microwave (mw) and radiofrequency (rf) cw and pulse irradiation schemes of various time- resolved EPR techniques. The initial laser excitation pulse h휈 starts the photoreaction with paramagnetic intermediates. For stable paramagnetic systems, the laser pulse is omitted. cw continuous wave, TR tran- sient, ENDOR electron–nuclear double resonance, TRIPLE electron–nuclear–nuclear triple resonance, ESE electron spin echo, HYSCORE hyperfine sublevel correlation spectroscopy, DF dual frequency, pulsed ELDOR PELDOR: pulsed electron–electron double resonance, EDNMR ELDOR-detected NMR, HTA high turning angle. For details, see text and [1]. In this ref., additional pulse irradiation schemes of various time-resolved EPR techniques are discussed Fig. 5 Microwave (mw) and radiofrequency (rf) cw and pulse irradiation schemes of various time- resolved EPR techniques. The initial laser excitation pulse h휈 starts the photoreaction with paramagnetic intermediates. For stable paramagnetic systems, the laser pulse is omitted. cw continuous wave, TR tran- sient, ENDOR electron–nuclear double resonance, TRIPLE electron–nuclear–nuclear triple resonance, ESE electron spin echo, HYSCORE hyperfine sublevel correlation spectroscopy, DF dual frequency, pulsed ELDOR PELDOR: pulsed electron–electron double resonance, EDNMR ELDOR-detected NMR, HTA high turning angle. For details, see text and [1]. In this ref., additional pulse irradiation schemes of various time-resolved EPR techniques are discussed 130]. We also refer to rather recent overview articles on high-field/high-frequency EPR in the biosciences [107–110, 115, 116, 118, 131–136]. We will focus on high- field EPR studies of biochemical systems that were performed in our laboratory at FU Berlin in teamwork with undergraduate and graduate students, postdocs and sen- ior scientists from over the world. 2.1 Spin Hamiltonians for EPR at High Magnetic Fields We first describe the basic spin Hamiltonians with interaction terms that determine the energy levels and EPR transition frequencies and, thereby, the characteristic fea- tures of the EPR spectrum of an open-shell system. 2.1.1 Organic Radicals and Low‑Spin Transition‑Metal Ions ( S = 1∕2) In molecular radicals or transition-metal ion complexes with unpaired electron spins S = 1∕2 , the electron and nuclear spins will align with respect to the total magnetic field they experience. This is the vector sum of the external Zeeman field, ⃗B0 , the local field originating from the residual orbital angular momentum of the unpaired electron and spin–orbit coupling (leading to effective g-tensor 1 3 222 K. Möbius, A. Savitsky components shifted from the free-electron value), and the additional local hyper- fine and quadrupole fields from nearby magnetic nuclei. For example, 14N nuclei with I = 1 or protons, 1H, with nuclear spin I = 1∕2 . They are located in the radical molecule or in the solvent micro-environment of the radical (’’matrix’’ nuclei). ̂ For such S = 1∕2 systems, the static spin Hamiltonian, ̂H0 , that describes the time-independent spin-interaction energies, consists of the terms (2) H0 h = 𝜇B h ⋅⃗B0 ⋅g ⋅S − ∑ i gni ⋅𝜇N h ⋅⃗B0 ⋅Ii + ∑ i S ⋅Ai ⋅Ii + ∑ i Ii ⋅Pi ⋅Ii, (2) i.e., ̂H0 contains the field-dependent electron and nuclear Zeeman interactions as well as the field-independent electron–nuclear hyperfine and nuclear quadrupole interactions with magnetic nuclei (for the quadrupole interaction to exist, nuclei with I > 1∕2 in an asymmetric electronic environment are required). Here, ⃗B0 is the external magnetic field (a vector), ̃g , ̃A and ̃P are the corresponding interac- tion tensors (matrices) of the electron Zeeman, hyperfine and quadrupole interac- tions ( h : Planck constant; 휇B and 휇N : Bohr and nuclear magnetons; gn : nuclear g-factors; ̂S , ̂I : electron and nuclear spin vector operators; the summation is over all nuclei). The quadrupole interaction between a nuclear electric quadrupole moment, Q , and an electric field gradient at the position of the nucleus is described by the spin Hamiltonian ̂I ⋅̃P ⋅̂I . 2.1.1 Organic Radicals and Low‑Spin Transition‑Metal Ions ( S = 1∕2) e For nucleus i , the Hamiltonian of the anisotropic part of the electron–nuclear hyperfine interaction, the electron–nuclear dipolar (END) term, ̂HSI , can be written as [137] For nucleus i , the Hamiltonian of the anisotropic part of the electron–nuclear hyperfine interaction, the electron–nuclear dipolar (END) term, ̂HSI , can be written as [137] (6) ̂HSI h = 휇0 4휋⋅h ⋅ge ⋅휇B ⋅gn ⋅휇N ⋅(A + B + C + D + E + F). (6) The six terms A, B, C, D, E, F represent products of electron and nuclear spin operators and angular functions in spherical coordinates with the polar angles 휃 and 휑 describing the orientation between the dipolar axis and the external Zeeman field ⃗B0: (7) A = ̂Sz ⋅̂Iz ⋅(3cos2휃−1), B = 1 4 ⋅ ( ̂S+ ⋅̂I−+ ̂S−⋅̂I+ ) ⋅(3cos2휃−1), C = 2 3 ⋅(̂S+ ⋅̂Iz + ̂Sz ⋅̂I+) ⋅sin휃⋅cos휃⋅e−i휑, D = 2 3 ⋅ ( ̂S−⋅̂Iz + ̂Sz ⋅̂I− ) ⋅sin휃⋅cos휃⋅ei휑, E = 3 4 ⋅̂S+ ⋅̂I+ ⋅e−2i휑, F = 3 4 ⋅̂S−⋅̂I−⋅e2i휑. (7) Here, the x- and y-components of the spin operators are expressed in terms of the raising and lowering shift operators. Depending on the specific way these spin operators are acting on a state of the unperturbed Hamiltonian (the electron Zeeman interaction, being the leading term in X-band EPR and even more so in high-field EPR) the A, B, C, D, E, F terms are classified as secular ( A term), pseudo-secular ( B term) and non-secular (C, D, E, F terms). Only the secular and pseudo-secular parts in the END term contribute in first order to the dipolar splitting of the unper- turbed energy levels. In EPR (or NMR or ENDOR), the coherent microwave (or radiofrequency) fields are normally applied in a direction perpendicular to the static external Zeeman field so that the selection rules ΔmS = ±1 , ΔmI = ±1 hold. As a consequence, the non-secular terms are unimportant for determining the line posi- tions of the spectra to first-order perturbation theory. Higher-order satellites due to small state admixtures will have vanishingly small intensities as long the Zeeman terms are much larger than the C, D, E, F terms. This does not mean, however, that the non-secular END parts, when becoming time dependent, are unimportant for the electron and nuclear relaxation pathways. 2.1.1 Organic Radicals and Low‑Spin Transition‑Metal Ions ( S = 1∕2) In its principal axes system, the quadrupole tensor, ̃P , is trace- less, and the quadrupole Hamiltonian can be written as (3 ̂HQ h = Pxx ⋅̂I2 x + Pyy ⋅̂I2 y + Pzz ⋅̂I2 z = e2 ⋅q ⋅Q 4I ⋅(2I −1) ⋅h ⋅ [ 3 ⋅̂I2 z + 2I(I + 1) + 휂⋅ ( ̂I2 x −̂I2 y )] , (3) where e ⋅q is the electric field gradient of the electron plus nuclear charge distribu- tion along the z-direction, and 휂= (Pxx −Pyy)∕Pzz is the asymmetry parameter of the charge distribution with \|Pzz\| ≥\|Pyy\| ≥\|Pxx\| and 0 ≤ 휂 ≤ 1 [33, 130]. The largest principal value of the quadrupole tensor is given by Pzz = e2⋅q⋅Q 2I⋅(2I−1)⋅h. Commonly, the quadrupole-tensor components for any given value I ≥1 are characterized by the two quantities e2 ⋅q ⋅Q∕h (in linear frequency units) and 휂 ; for quantifying the magnitude of the interaction the factor 2I(2I −1) has to be included. The dipolar hyperfine interaction between an electron and a nuclear spin, ̂S ⋅̃A ⋅̂I in Eq. (2), can be written as the sum of the isotropic (or Fermi contact) interaction, ̂HFermi h = Aiso ̂S ⋅̂I ; with (in SI units) Commonly, the quadrupole-tensor components for any given value I ≥1 are characterized by the two quantities e2 ⋅q ⋅Q∕h (in linear frequency units) and 휂 ; for quantifying the magnitude of the interaction the factor 2I(2I −1) has to be included.i ̂ ̃ ̂ The dipolar hyperfine interaction between an electron and a nuclear spin, ̂S ⋅A ⋅̂I in Eq. (2), can be written as the sum of the isotropic (or Fermi contact) interaction, ̂HFermi h = Aiso ̂S ⋅̂I ; with (in SI units) (4) Aiso = 2휇0 3h ⋅ge ⋅휇B ⋅gn ⋅휇N ⋅\|Ψ(0)\|2, (4) and the anisotropic dipole–dipole interaction between the magnetic moments of the electron and nuclear spins (END interaction) 1 3 High‑field/High‑frequency EPR Spectroscopy in Protein… 223 (5) HSI h = 𝜇0 4𝜋⋅h ⋅ge ⋅𝜇B ⋅gn ⋅𝜇N ⋅ ⎡ ⎢ ⎢ ⎢⎣ 3 S ⋅⃗r ⃗r ⋅I r5 − S ⋅I r3 ⎤ ⎥ ⎥ ⎥⎦ , (5) where 휇0 is the vacuum permeability, \|Ψ(0)\|2 the electron density at the nucleus, r is the distance between the electron and nuclear spins, ge is the free-electron g-factor. 2.1.1 Organic Radicals and Low‑Spin Transition‑Metal Ions ( S = 1∕2) They are induced by the pertaining ran- domly fluctuating local fields, for instance owing to Brownian motion of the radicals in liquid-state samples. Then, the time-dependent non-secular interactions induce 1 3 224 K. Möbius, A. Savitsky relaxation transitions of the electron spins ( We ) and nuclear spins ( Wn ) as well as coupled ’’flop-flop’’ cross-relaxation transitions ( Wx2). ̃ ̃i relaxation transitions of the electron spins ( We ) and nuclear spins ( Wn ) as well as coupled ’’flop-flop’’ cross-relaxation transitions ( Wx2). ̃ ̃i ll The tensors ̃g , ̃A, and ̃P of the electron Zeeman interaction, the hyperfine inter- action, and the quadrupole interaction probe the electronic structure (i.e., the elec- tron wave function and energy) of the molecule either globally (g-tensor) or locally (hyperfine- and quadrupole-tensors). The g- and A-tensors contain isotropic and anisotropic contributions, whereas the P-tensor is traceless, i.e., contains only ani- sotropic contributions. In isotropic fluid solution at temperatures high enough for fast molecular tumbling, the anisotropic interaction components are averaged out so that only the isotropic values, 1∕3 ⋅Tr(̃g) and 1∕3 ⋅Tr(̃Ai) , contribute to the observed line positions. In frozen solutions, powders or single crystals, also anisotropic tensor contributions become observable, providing that the necessary spectral resolution conditions prevail, i.e., the separations of the lines are larger than their linewidths. For this situation, the information content of the EPR spectra is, of course, consider- ably enhanced by spatial information and, for example, molecular orientations with respect to ⃗B0 or electron–nuclear distances can be extracted.ii In the strong-field approximation, the energy eigenvalues of Eq. (2) are classified by the magnetic spin quantum numbers, mS and mI , and are given (without quadru- pole contribution), to first order (in frequency units) by (8) E(mS, mI ) h = g ⋅휇B h ⋅B0 ⋅mS − ∑ i gni ⋅휇N h ⋅B0 ⋅mIi + ∑ i Ai ⋅mS ⋅mIi, (8) where the scalar interaction parameters g , A are the square root values of the squared tensors and contain the desired information about magnitude and orientation of the interaction tensors [130]. To keep the energy expression simple, the contribu- tions due to the quadrupole interaction, P, are omitted for the moment. In thermal equilibrium between the spin system and the lattice, the energy levels are populated according to the Boltzmann distribution. 2.1.1 Organic Radicals and Low‑Spin Transition‑Metal Ions ( S = 1∕2) When irradiating the sample with micro- waves at a fixed frequency and sweeping the external field through the resonance region, EPR transitions occur according to the first-order selection rules ΔmS = ±1 , ΔmIi = 0 . This leads to an EPR spectrum of absorption lines that is characteristic for the electron spin interactions of the sample molecules. The intensity distribution of the hyperfine lines is determined by the number of symmetry-equivalent nuclei. Hence, the intensity ratio of the hyperfine lines is a valuable aid for assigning the lines to specific nuclear positions in the molecule. From Eq. (8) it follows that the purely nuclear spin interactions, i.e., the nuclear Zeeman and quadrupole interac- tions, do not show up in the EPR spectrum as long as the first-order approximation for the selection rules holds. Second-order contributions from the nuclear Zeeman and quadrupole energy terms can often be neglected (see Fig. 2). However, this is only justified as long as the nuclear spin terms are considerably smaller than the other two terms. This might be no longer the case at high Zeeman fields or for sub- stantial quadrupole couplings. where the scalar interaction parameters g , A are the square root values of the squared tensors and contain the desired information about magnitude and orientation of the interaction tensors [130]. To keep the energy expression simple, the contribu- tions due to the quadrupole interaction, P, are omitted for the moment. In thermal equilibrium between the spin system and the lattice, the energy levels are populated according to the Boltzmann distribution. When irradiating the sample with micro- waves at a fixed frequency and sweeping the external field through the resonance region, EPR transitions occur according to the first-order selection rules ΔmS = ±1 , ΔmIi = 0 . This leads to an EPR spectrum of absorption lines that is characteristic for the electron spin interactions of the sample molecules. The intensity distribution of the hyperfine lines is determined by the number of symmetry-equivalent nuclei. Hence, the intensity ratio of the hyperfine lines is a valuable aid for assigning the lines to specific nuclear positions in the molecule. From Eq. (8) it follows that the purely nuclear spin interactions, i.e., the nuclear Zeeman and quadrupole interac- tions, do not show up in the EPR spectrum as long as the first-order approximation for the selection rules holds. 2.1.1 Organic Radicals and Low‑Spin Transition‑Metal Ions ( S = 1∕2) Second-order contributions from the nuclear Zeeman and quadrupole energy terms can often be neglected (see Fig. 2). However, this is only justified as long as the nuclear spin terms are considerably smaller than the other two terms. This might be no longer the case at high Zeeman fields or for sub- stantial quadrupole couplings. As a simple example, a doublet radical ( S = 1∕2 ) is considered, containing four symmetry-equivalent protons (each with I = 1∕2 ) in a strong B0 field. Figure 6 shows the energy level scheme according to Eq. (8) for the case that the radical is 1 3 High‑field/High‑frequency EPR Spectroscopy in Protein… 225 dissolved in isotropic fluid solution. In this situation, only scalar interactions prevail provided that fast molecular tumbling occurs. As a consequence of the strong-field EPR selection rules, five EPR lines are observed with binominal intensity distribu- tion owing to the first-order transition-frequency degeneracies of equivalent nuclei. Their isotropic hyperfine coupling constant (hfc) Aiso = 1∕3 ⋅Tr(̃Ai) can be directly read off from the separation of the hyperfine lines in the EPR spectrum. F i l l l f h di l h l d h fi Fig. 6 High-field spin energy levels of a radical ( S = 1∕2 ) with a group of four equivalent protons ( I = 1∕2 ) dissolved in fluid solution. The allowed EPR and ENDOR transitions are marked by arrows. The energy splittings are drawn according to Eq. (8) with A > 0 . The total nuclear quantum number of the group is Mi = ∑ i mIi. Fig. 6 High-field spin energy levels of a radical ( S = 1∕2 ) with a group of four equivalent protons ( I = 1∕2 ) dissolved in fluid solution. The allowed EPR and ENDOR transitions are marked by arrows. The energy splittings are drawn according to Eq. (8) with A > 0 . The total nuclear quantum number of the group is Mi = ∑ i mIi. Fig. 6 High-field spin energy levels of a radical ( S = 1∕2 ) with a group of four equivalent protons ( I = 1∕2 ) dissolved in fluid solution. The allowed EPR and ENDOR transitions are marked by arrows. The energy splittings are drawn according to Eq. (8) with A > 0 . The total nuclear quantum number of the group is Mi = ∑ i mIi. 2.1.1 Organic Radicals and Low‑Spin Transition‑Metal Ions ( S = 1∕2) dissolved in isotropic fluid solution. In this situation, only scalar interactions prevail provided that fast molecular tumbling occurs. As a consequence of the strong-field EPR selection rules, five EPR lines are observed with binominal intensity distribu- tion owing to the first-order transition-frequency degeneracies of equivalent nuclei. Their isotropic hyperfine coupling constant (hfc) Aiso = 1∕3 ⋅Tr(̃Ai) can be directly read off from the separation of the hyperfine lines in the EPR spectrum.i dissolved in isotropic fluid solution. In this situation, only scalar interactions prevail provided that fast molecular tumbling occurs. As a consequence of the strong-field EPR selection rules, five EPR lines are observed with binominal intensity distribu- tion owing to the first-order transition-frequency degeneracies of equivalent nuclei. Their isotropic hyperfine coupling constant (hfc) Aiso = 1∕3 ⋅Tr(̃Ai) can be directly read off from the separation of the hyperfine lines in the EPR spectrum.i fi For single-crystal samples of the same radical, the complete g- and hyperfine- tensor information, i.e., both the isotropic and anisotropic contributions, can be extracted from the angular dependence of the EPR lines ("rotation patterns") when the crystal is rotated about three orthogonal axes. Note that there exists no quadru- pole interaction with protons or other nuclei with I = 1∕2 . If the tensors ̃g and ̃Ai are collinear, i.e., have the same principal axes system ( 훼, 훽, 훾 ), their rotation patterns will have the same angular dependence. When the crystal is mounted on a proper wedge in such a way that the crystal rotation axes are parallel to the molecular axes system ( x, y, z ) and remain under rotation perpendicular to the B0 field direction, the actual rotation occurs in a molecular plane defining an angle Θ between the rotation axis and the field direction. Iterated measurements of the angular dependence of the apparent g- and A-values at different orientations give the elements of the tensors ̃g2 and ̃A2 , which can be transformed to principal axes. One could begin by rotating the field in the xy plane, followed by rotations in the yz and zx planes. Then, the depend- ence of the apparent g-value on the rotation angle Θij in the ij plane takes the form [130, 138] (notice that g2 ij denotes the components of the squared g-tensor): (9) g ( Θxy ) = ( g2 xy ⋅sin2Θxy + g2 yy ⋅cos2Θxy + g2 xy ⋅sin2Θxy )1∕2 . Fig. 6 High-field spin energy levels of a radical ( S = 1∕2 ) with a group of four equivalent protons ( I = 1∕2 ) dissolved in fluid solution. The allowed EPR and ENDOR transitions are marked by arrows. The energy splittings are drawn according to Eq. (8) with A > 0 . The total nuclear quantum number of the group is Mi = ∑ i mIi. 2.1.1 Organic Radicals and Low‑Spin Transition‑Metal Ions ( S = 1∕2) (9) An analogous expression holds for the hyperfine coupling value Ai of nucleus i in the limit of small g-anisotropy, as is typical for bioorganic systems as long as they do not contain paramagnetic transition-metal ions. For the other two molecular planes, yz and zx , the corresponding tensor components are found by cyclic replace- ment y →z →x →y , i.e., 3 K. Möbius, A. Savitsky 226 (10) g ( Θyz ) = ( g2 yy ⋅sin2Θyz + g2 zz ⋅cos2Θyz + g2 yz ⋅sin2Θyz )1∕2 , (10) (11) g(Θzx ) = (g2 zz ⋅sin2Θzx + g2 xx ⋅cos2Θzx + g2 zx ⋅sin2Θzx )1∕2, (11) with the rotation angles Θij defined as the respective angles between the i- and j-axis and the field direction. For many bioorganic samples, in particular in frozen solution, conventional X-band EPR runs into problems with spectral resolution. This is because several radical species or different magnetic sites with rather similar g-values may be pre- sent or because a small g-tensor anisotropy does not allow canonical orientations of the powder spectrum to be resolved. From the spin Hamiltonian Eq. (2) one sees that some interactions are magnetic field-dependent (the Zeeman interactions), while others are not (the hyperfine and quadrupole interactions). Consequently, if paramagnetic species with different g-factors or with anisotropic g-tensor compo- nents are present, the difference in resonance field positions ΔB0 is proportional to the frequency of the electromagnetic irradiation: (12) ΔB0 = h ⋅휈 휇B ⋅ ( 1 g1 −1 g2 ) . (12) This shows, in analogy to modern NMR spectroscopy, that higher mw EPR fre- quencies, 휈 , and corresponding resonance fields, B0, should lead to enhanced spec- tral resolution, at least as long as the linewidths do not increase with field. Except for transition-metal complexes, for many bioorganic systems g ≅2 , and relative g-value variations Δg∕g rarely exceed 10–4 – 10− 3. At X-band frequencies, therefore, the line separation due to g-value differences is only ΔB0 = 0.03 – 0.3 mT, which can easily be masked in disordered solid-state samples with typical linewidths around 1 mT. An increase of the mw frequency by, for instance, a factor of 10 (W-band, 95 GHz) improves the spectral resolution accordingly. This is a consequence of the increasing importance of the g-tensor components in the electronic Zeeman inter- action as the magnetic field is increased. 2.1.1 Organic Radicals and Low‑Spin Transition‑Metal Ions ( S = 1∕2) Moreover, the spectral analysis is gener- ally simpler in high-field EPR because the first-order approximation for the energies often applies.i The question arises, how large the Zeeman field ought to be for maximum spec- tral information for a particular sample? The answer for all cases of delocalized spin systems, in which unresolved hyperfine interactions dominate the inhomogeneous EPR linewidth, is that a successful high-field experiment must fulfill the condition (13) Δg g ⋅B0 > ΔBhf 1∕2, (13) which relates the Zeeman field B0 of the spectrometer with properties of the sample, i.e., the anisotropic electron Zeeman interaction must exceed the inhomogeneous linewidth, ΔBhf 1∕2 . Apparently, there are two options to fulfill this condition, either to make the Zeeman interaction large enough or to reduce the linewidth sufficiently. 1 High‑field/High‑frequency EPR Spectroscopy in Protein… 227 Hence, instead of fixing a minimum field/frequency value to meet the ’’high-field EPR’’ benchmark, one should ensure that the chosen B0 value renders the Zeeman splitting to be larger than the inhomogeneous linewidth. Nevertheless, it has become common practice in the EPR community to use the term ’’high-field EPR’’ for microwave frequencies in the W-band or higher and Zeeman fields produced by superconducting magnets. For example, for deuterated samples, Q-band EPR might already fulfill the high-field condition Eq. (13) in the case of semiquinone radicals with a rather large g-anisotropy [139–141], whereas for protonated samples with inherently larger linewidths, it does not. On the other hand, in the case of chloro- phyll ion radicals, due to their very small g-anisotropy, even W-band EPR might not meet the high-field condition for protonated samples. Then deuteration of the sam- ple will be necessary or, as an alternative, a further increase of the mw frequency and B0 field is required, for instance by resorting to 360 GHz EPR. Fortunately, for many protein systems with no paramagnetic transition-metal ion sites or, at least, no substantial spin density at such a site, the increase of line separation ∆B0 with increasing Zeeman field directly translates into an increase of spectral resolution, because often no noticeable line broadening due to ’’g-strain’’ effects occurs with increasing B0. For the primary donor cation radical in reaction centers (RCs) from Rb. sphaeroides, for example, up to 24 T were applied [142], and g-strain broaden- ing was found to be negligible. 2.1.1 Organic Radicals and Low‑Spin Transition‑Metal Ions ( S = 1∕2) The term ’’g-strain’’ is used to describe a spread of principal g-factors caused by heterogeneities of the local environment of the spins and leading to additional line broadening that increases linearly, or approximately linearly with the Zee- man field. This phenomenon is well known in the case of paramagnetic transi- tion-metal complexes such as metallo-proteins [143, 144] where large spin–orbit coupling can produce dramatic variations in the g-values with varying crystal fields. Such effects are, however, expected to be small in the case of organic radi- cals in proteins for which small g-anisotropies are the rule. Apparently, the pri- mary donor cation radical in RCs from Rb. sphaeroides is not an exception of this rule.i In addition to the improved g-resolution mentioned above, high-field EPR can also improve the detection sensitivity. In this respect one has to distinguish between the absolute and relative (or concentration) sensitivities because the amount of sam- ple that can be introduced into the cavity of high-field EPR spectrometers is usu- ally significantly smaller than in standard X-band spectrometers. Consequently, the amount of sample available must be considered. When this is limited like in single crystals of proteins, the sensitivity of high-field EPR can be superior by orders of magnitude. For a quantitative discussion of the sensitivity problems, we refer to [1]. 2.1.2 Triplet States and High‑Spin Transition‑Metal Ions ( S > 1∕2) Also for high-spin systems ( S > 1∕2 ), such as enzymatic proteins with one or sev- eral transition-metal cofactors, EPR spectroscopy at high magnetic fields might be advantageous. For such systems "fine-structure" and exchange terms have to be added to the spin Hamiltonian, i.e., Eq. (2) has to be extended by 1 3 K. Möbius, A. Savitsky 228 (14) ̂HZFS h + ̂Hexchange h = ̂S ⋅̃D ⋅̂S −J ⋅ ( ̂S2 −1 ) , (14) with the total spin S = (S1 + S2 + … ) . Here, ̃D is the traceless zero-field splitting (ZFS) tensor, and J is the isotropic exchange interaction parameter (in standard con- vention, J < 0 corresponds to antiferromagnetic coupling, J > 0 to ferromagnetic coupling). In the principal axes system of ̃D , the anisotropic part in Eq. (14) is nor- mally rewritten in terms of the zero-field parameters D , E: (15) ̂HZFS h = ̂S ⋅̃D ⋅̂S = D ⋅ ( ̂S2 z −1 3 ⋅S ⋅(S + 1) ) + E ( ̂S2 x −̂S2 y ) , (15) with D = 3∕2 ⋅Dzz and E = (Dxx−Dyy )∕2 , where Dxx , Dyy and Dzz are the principal values of the ZFS tensor. For a triplet state, they are related to the zero-field energy eigenvalues (energy levels) of the triplet spin eigenfunctionsTx⟩,Ty⟩ , Tz⟩ as follows [138]: (16) Ex∕h = 1 3 ⋅D −E, Ey∕h = 1 3 ⋅D + E, Ez∕h = −2 3 ⋅D. (16) Obviously, it might happen that EPR transitions of high-spin systems with large zero-field splitting cannot be observed at all at standard X-band frequencies because the energy of the mw quantum is too small. For such cases, the higher quantum energy of high-frequency microwaves can drive the transitions [145, 146]. An exam- ple for such a biological high-spin system is metmyoglobin with S = 5∕2 ferric heme, for which EPR transitions at 130 GHz became observable that had been unde- tectable at X-band due to the large zero-field splitting [88].ii i Although in the spin Hamiltonian the fine-structure term is not field-dependent it leads, in combination with the electronic Zeeman term, to a field-dependent mix- ing of the electron spin eigenfunctions. At zero field, the triplet spin eigenfunctions, Tx⟩ , Ty⟩ , Tz⟩ , are quantized along the molecular axes system ( x, y, z ). 2.1.2 Triplet States and High‑Spin Transition‑Metal Ions ( S > 1∕2) At high field, the magnetic spin quantum number, mS = +1, 0, −1 , is a good quantum number, and the spin eigenfunctions become T+1⟩ , T0⟩ , T−1⟩ . If the external field values B0 are such that the electron Zeeman and the fine-structure splitting are comparable in magnitude, the spin functions become mixed functions of both bases, the degree of mixing depending on B0 and the relative orientation of the molecule with respect to the field. As a consequence, the triplet energy eigenvalues of the different elec- tron wave functions are not linearly related to the strength of B0 . This intermediate region requires more complicated calculations to analyze the EPR spectrum. Hence, for high-spin systems ( S > 1∕2 ), there is another reason for choosing EPR at high Zeeman field, which is to simplify the analysis of spectra.i i There is an additional benefit: The EPR lines of high-spin systems usually get narrower at higher magnetic fields than in X-band EPR spectra, again because of second-order effects. If we take Mn2+ centers ( S = 5∕2, I = 5∕2 ) in disordered pro- tein samples as an example, the EPR transitions are strongly broadened by contribu- tions from the zero-field tensor components. Their linewidth, ΔB1∕2 , is determined 1 3 1 3 High‑field/High‑frequency EPR Spectroscopy in Protein… 229 by second-order contributions from the zero-field coupling D , ΔB1∕2 ∝D2 B0 . No won- der, therefore, that "needle sharp" manganese hyperfine lines are normally observed in the 95 GHz high-field EPR spectra of Mn2+ containing protein complexes, such as photosystem II of oxygenic photosynthesis, even in disordered frozen-solution samples.i This is exploited by high-field EPR spectroscopists who use Mn2+ ions doped into MgO powder as reference sample for magnetic field calibration and precise g-factor measurements [74]. Up to second order, the EPR resonance fields of the six Mn2+ hyperfine components,mI = 5∕2, ... + 5∕2 , are given by (17) BmI = B0 −Aiso ⋅ [ mI −Aiso 2B0 ⋅(I ⋅(I −1) −m2 I )] , (17) where Aiso is the isotropic Mn2+ hyperfine coupling in field units. The elec- tronic g-factor is contained in the Zeeman field B0 = F ⋅휈∕g ( 휈 frequency, F = h∕휇B= 71.447751 mT⋅GHz−1). 2.1.2 Triplet States and High‑Spin Transition‑Metal Ions ( S > 1∕2) The high-precision reference data are: g (Mn2+) = 2.00101 ± 0.00005 and Aiso(Mn2+) = − (8.710 ± 0.003) mT [74].i To underline it once again: High-field EPR is particularly useful for half-integer high-spin systems ( S = (2n + 1)∕2, n = 1, 2, ... ), such as Mn(II) and Fe(III) with S = 5∕2 , and Gd(III) with S = 7∕2 . For such systems the inhomogeneous linewidth of orientationally disordered samples, ΔB1∕2 , of the central \| −1∕2 →\|1∕2 EPR transition is determined by second-order contributions from the zero-field coupling. Hence, when g ⋅𝜇B ⋅B0 h ≫D the broadening becomes negligible and narrow sig- nals are obtained also in orientationally disordered samples. This leads to increased sensitivity and resolution. In this case the first-order approximation is valid and the resonance fields are given by: B(mS →mS + 1) = B0 − h g ⋅휇B ⋅Aiso ⋅mI + h g ⋅휇B ⋅(2mS + 1) ⋅휈D (18 휈D = D 2 ⋅[(3cos2휃−1) + 휂⋅sin2휃⋅cos2휑], B(mS →mS + 1) = B0 − h g ⋅휇B ⋅Aiso ⋅mI + h g ⋅휇B ⋅(2mS + 1) ⋅휈D (18) 휈D = D 2 ⋅[(3cos2휃−1) + 휂⋅sin2휃⋅cos2휑], (18) where Aiso is the isotropic hyperfine coupling of 55Mn (in frequency units), mI is the corresponding nuclear spin projection and 휂= 3 ⋅E∕D . The angles 휃 and 휑 describe the orientation of the magnetic field relative to the principal axes system of the ZFS tensor.i Another important aspect of high-field EPR on high-spin systems, for exam- ple organic triplet states and radical pairs with S = 1 , is the possibility to deter- mine the absolute sign of the zero-field parameter D by sufficient thermal polari- zation of the triplet levels already at moderately low temperatures [22, 23]. If the temperature is low enough to fulfill the condition kBT < g ⋅𝜇B ⋅B0 , the lowest spin level, corresponding to mS = −1∕2 , is predominantly populated, and the EPR spectra of disordered samples become asymmetric. The ’’Zeeman temperature’’, TZ = g ⋅휇B ⋅B0∕kB , is defined accordingly as the temperature around which this asymmetry becomes pronounced: A higher line intensity is observed either on the 1 3 3 230 K. Möbius, A. Savitsky high-field side (D > 0) or on the low-field side (D < 0) of the spectrum. 2.1.2 Triplet States and High‑Spin Transition‑Metal Ions ( S > 1∕2) Approximate values of the Zeeman temperature at different EPR frequencies are: TZ = 0.4 K (at 9.5 GHz), 4 K (at 95 GHz), 6.5 K (at 140 GHz), and 15.5 K (at 360 GHz). The sign of D is indicative of the shape of the dipolar tensor. Organic triplet states generally have disk-shaped dipolar tensors ( D > 0), whereas weakly coupled radical pairs have cigar-shaped ones ( D < 0). 2.2 High‑field ENDOR, TRIPLE, ESEEM, HYSCORE, EDNMR, PELDOR, RIDME We now turn to several extensions of high-field/high-frequency EPR spectroscopy in some more detail to clarify what can be learned additionally about (bio)chemi- cal and biological systems when going beyond conventional X-band techniques. The topics selected are ENDOR, TRIPLE, ESEEM, HYSCORE, EDNMR, PELDOR, and RIDME. We will group these techniques under two headings: Electron–nuclear hyperfine spectroscopy; Electron–electron dipolar spectroscopy. 1 3 2.2.1 Electron–Nuclear Hyperfine Spectroscopy Thorough accounts of high-field/high-frequency ENDOR and TRIPLE spectroscopy have recently been published, and we suggest them for further reading [1, 118, 147, 148]. 2.2.1.1 cw ENDOR For large low-symmetry radicals with the unpaired electron delocalized over many spin-carrying nuclei, for example the cofactor ion radicals occurring in photosynthetic electron transfer, with each set of inequivalent nuclei the number of EPR lines increases in a multiplicative way, according to the EPR selec- tion rules ΔmS = ±1, ΔmIi = ±1 . This results in strong inhomogeneous broadening of the EPR spectra because individual hyperfine lines can no longer be resolved in the available spectral range. For g = 2 systems, this is restricted to ca. 3 mT due to the normalization condition for the unpaired electron spin density. y For such cases, by resorting to ENDOR techniques the spectral resolution can be greatly improved. This is because ENDOR is inherently a variant of NMR on paramagnetic systems, the unpaired electron serving as highly sensitive detector for the NMR transitions, mIi = ±1 . Each group of equivalent nuclei—no matter how many nuclei are involved and of what value their individual nuclear spin is—con- tributes only two ENDOR lines at 휈± because, within an mS manifold, the hyperfine levels are equidistant to first order (see Fig. 6). Hence, in ENDOR, with each set of inequivalent nuclei the number of resonance lines increases merely in an addi- tive way. Double resonance excitation, thus, offers the advantage of NMR in terms of high resolution via reduced number of redundant hyperfine lines in conjunction with the advantage of EPR in terms of detecting low-intensity rf transitions via high- intensity mw transitions, i.e., by means of quantum transformation via the gyromag- netic ratios. In cw ENDOR, the sample is irradiated simultaneously by two electromagnetic fields, a mw field (to drive EPR transitions ΔmS = ±1 ) and an rf field (to drive NMR 1 3 1 3 High‑field/High‑frequency EPR Spectroscopy in Protein… 231 transitions ΔmIi = ±1 ). Under appropriate experimental conditions, which are more stringent for cw than for pulse irradiation schemes [33, 129, 149], ENDOR sig- nals are observed by monitoring the changes of EPR line intensities when sweep- ing the rf field through the nuclear resonance frequencies. In cw Special TRIPLE resonance [48] the two rf fields are applied at frequencies symmetrically placed around the nuclear Larmor frequency to enhance the signal intensity. 2.2.1 Electron–Nuclear Hyperfine Spectroscopy In cw General TRIPLE resonance [52], two rf fields with independently variable frequencies are applied, one pumping a selected ENDOR transition while the other is swept through the ENDOR spectrum. From the resulting characteristic intensity changes in the ENDOR spectrum, the relative signs of the hyperfine couplings can directly be read off.i f In the pulsed version of triple resonance [150], the first rf pulse (pump pulse) with fixed frequency pumps a specific nuclear transition, while the frequency of the second rf pulse is swept to cover the resonance region. The time separation between the two rf pulses can be varied. Again, assignment of hyperfine couplings and deter- mination of their relative signs are the main goals. When the frequencies of the two rf pulses are varied independently, a two-dimensional triple resonance spectrum is obtained [151]. Apparently, the gain in resolution of ENDOR versus EPR, becomes very pro- nounced for low-symmetry molecules with increasing number of groups of symme- try-related nuclei. The resolution enhancement becomes particularly drastic when nuclei with different magnetic moments are involved. Their ENDOR lines appear in different frequency ranges and, providing that their Larmor frequencies are sepa- rated at the chosen Zeeman field value B0 , the different nuclei can be immediately identified. In the case of an accidental overlap of ENDOR lines from the different nuclei at X-band (9.5 GHz, 0.34 T) the lines can be separated when working at higher Zeeman fields and mw frequencies, for instance at 3.4 T, 95 GHz [96] or even at 12.9 T, 360 GHz [152]. This disentangling of ENDOR lines by different field/fre- quency settings for the EPR condition is depicted in Fig. 7. In biological molecules with several non-proton magnetic nuclei this separation of accidentally overlapping ENDOR lines is of great help for analyzing complex spin systems by means of their nuclear Zeeman and hyperfine interactions.i i In the pulsed version of triple resonance [150], the first rf pulse (pump pulse) with fixed frequency pumps a specific nuclear transition, while the frequency of the second rf pulse is swept to cover the resonance region. The time separation between the two rf pulses can be varied. Again, assignment of hyperfine couplings and deter- mination of their relative signs are the main goals. When the frequencies of the two rf pulses are varied independently, a two-dimensional triple resonance spectrum is obtained [151]. 2.2.1 Electron–Nuclear Hyperfine Spectroscopy Apparently, the gain in resolution of ENDOR versus EPR, becomes very pro- nounced for low-symmetry molecules with increasing number of groups of symme- try-related nuclei. The resolution enhancement becomes particularly drastic when nuclei with different magnetic moments are involved. Their ENDOR lines appear in different frequency ranges and, providing that their Larmor frequencies are sepa- rated at the chosen Zeeman field value B0 , the different nuclei can be immediately identified. In the case of an accidental overlap of ENDOR lines from the different 1 3 232 K. Möbius, A. Savitsky Fig. 7 Gain in ENDOR resolution for doublet-state systems ( S = 1∕2, g = 2 ) with increasing Zeeman field B0 and corresponding microwave resonance frequency 휈 . Spectral lines of typical nuclei in organic biomolecules, largely overlapping at traditional X-band ENDOR ( B0 = 0.34 T, 휈 = 9.5 GHz), become completely separated at 360 GHz/12.9 T ENDOR Fig. 7 Gain in ENDOR resolution for doublet-state systems ( S = 1∕2, g = 2 ) with increasing Zeeman field B0 and corresponding microwave resonance frequency 휈 . Spectral lines of typical nuclei in organic biomolecules, largely overlapping at traditional X-band ENDOR ( B0 = 0.34 T, 휈 = 9.5 GHz), become completely separated at 360 GHz/12.9 T ENDOR nuclei at X-band (9.5 GHz, 0.34 T) the lines can be separated when working at higher Zeeman fields and mw frequencies, for instance at 3.4 T, 95 GHz [96] or even at 12.9 T, 360 GHz [152]. This disentangling of ENDOR lines by different field/fre- quency settings for the EPR condition is depicted in Fig. 7. In biological molecules with several non-proton magnetic nuclei this separation of accidentally overlapping ENDOR lines is a great help for the analysis of complex spin systems by means of their nuclear Zeeman and hyperfine interactions.i i For a doublet radical with electron–nuclear hyperfine interaction, but without nuclear quadrupole interactions, according to its spin Hamiltonian only two ENDOR lines of a particular group of equivalent nuclei with I = 1∕2 , appear, to first order, at (19) 휈± ENDOR = \|휈n ± Ai∕2\|, (19) where the nuclear Larmor frequency is given by 휈n = gn ⋅휇N ⋅B0∕h , and the hyper- fine coupling parameter A contains isotropic and anisotropic contributions. Obvi- ously, the two ENDOR lines are symmetrically displayed about 휈n or A∕2 , which- ever is larger. 2.2.1 Electron–Nuclear Hyperfine Spectroscopy In isotropic solution, in steady-state ENDOR, the hyperfine couplings (hfcs) are given by Aiso = 1∕3 ⋅Tr(̃A). High‑field/High‑frequency EPR Spectroscopy in Protein… 233 At this point, it seems appropriate to give a brief phenomenological explana- tion of liquid-phase cw steady-state ENDOR signal intensities and their alteration by applying a second rf field in TRIPLE resonance experiments. In isotropic liq- uid solution, at sufficiently elevated temperatures, the anisotropic contributions of the g-tensor are effectively averaged out. Nevertheless, EPR and ENDOR studies at higher frequency/field settings than for standard X-band EPR can be of great advan- tage: If in the sample several radical species with only slightly different isotropic g-values are present, for example as intermediate or final reaction products with g1 and g2, they can be spectroscopically resolved and identified at sufficiently high Zee- man fields according to Eq. (12). i A detailed theoretical study of steady-state multiresonance experiments in the liquid phase has been carried out by J. H. Freed and co-workers in a series of papers [153–155] using the density matrix formalism and Redfield’s approximate treatment of relaxation. In one of these papers [155], subtle line shape effects— broadening and splitting—were described that are due to the coherent nature of the applied strong rf and mw fields. A specific coherence effect is particularly interest- ing because it can be exploited to assign ENDOR lines to molecular positions, i.e., when applying ENDOR as an analytical tool. It requires nuclear spins I > 1∕2 or a set of at least two equivalent nuclei of I = 1∕2 . The magnitude of the coherence splitting is dependent on the hyperfine transitions being mw saturated and on the rf field strength.f i This coherence effect was optimized by K. P. Dinse et al. [156, 157] to assign hyperfine splittings in ENDOR-in-solution spectra of various low-symmetry radicals by counting the number of protons contributing to a specific ENDOR line. A cylin- drical ENDOR cavity (TE011 mode) was constructed to achieve cw rf fields up to 3 mT (rotating frame). The internal NMR coil is part of the power stage of a 1 kW cw rf transmitter station. To secure thermal stability of the cavity frequency, effec- tive water cooling was employed both for the cavity body and the two-loop NMR coil [157].l On the basis of Freed’s relaxation theory for radicals in fluid solution, M. Plato, W. Lubitz, and K. 2.2.1 Electron–Nuclear Hyperfine Spectroscopy Möbius [158] carried out a systematic investigation of the cw ENDOR effect, i.e., the ENDOR sensitivity, of various hetero-nuclei (nuclei other than protons) in organic radicals. Optimum ENDOR conditions, such as temperature and viscosity of the solvent, mw and rf field strengths, were formulated as a function of a few nuclear and molecular properties. They include relaxation rates due to fluc- tuating spin-rotation interaction, electron–nuclear dipolar and nuclear quadrupolar couplings and Heisenberg spin exchange. The theoretical results were found to be in good agreement with experimental observations on 2H, 13C, 14/15 N, 19F, 31P and alkali nuclei in different molecular systems, thus allowing predictions to be made on the ENDOR detectability of other chemically interesting nuclei, such as 10/11B, 17O, 27Al, 29Si, 33S and 35/37Cl. In the meantime, most of these nuclei have indeed been detected by cw ENDOR in solution [49, 159, 160]. 2.2.1.2 cw TRIPLE Resonance as an Extension of Steady‑State ENDOR From many applications in chemistry, biology and physics it became clear that steady-state cw ENDOR in solution, though extremely powerful in resolving complex hyperfine 1 3 3 234 K. Möbius, A. Savitsky structures of low-symmetry radicals, is suffering from sensitivity problems: Only less than 10% of the EPR intensity is normally observed as steady-state ENDOR effect which has to be maximized by carefully controlling temperature and viscosity of the solvents, thereby optimizing a delicate interplay between electron and nuclear relaxa- tion rates, W훼훽 [158]. Additionally, cw ENDOR suffers from problems of assign- ing the measured hyperfine couplings to molecular positions. This is because the ENDOR line intensities are determined primarily by electron and nuclear relaxa- tion rates and not by the multiplicity of the NMR transitions. These drawbacks were the main motivation to extend liquid-solution ENDOR to electron–nuclear–nuclear TRIPLE resonance [48, 52] in which two high-power rf sources are connected to the NMR coil inside the EPR cavity. Figure 8 shows the energy level diagram and transition scheme of induced mw and rf as well as relaxation driven EPR and NMR transitions for the simplest case S = 1∕2 , I = 1∕2 . According to the different irradiation schemes involving only one nucleus or two inequivalent nuclei, we distinguish between Special TRIPLE and General TRIPLE resonance. 2.2.1 Electron–Nuclear Hyperfine Spectroscopy Special TRIPLE Resonance: There are mainly two drawbacks of the cw ENDOR method: (i) In the frequently occurring case of a Wn bottleneck, i.e., whenWn ≪We , the ENDOR effect becomes very weak if cross relaxation is absent. (ii) The intensity pattern of ENDOR lines generally does not reflect the number of nuclei involved in the various transitions. Both drawbacks can, at least in part, be overcome by apply- ing two NMR rf fields at a frequency separation 휈± of the hfc of a particular set of equivalent nuclei. This Special TRIPLE resonance experiment can be under- stood with the aid of the four-level scheme of Fig. 8. In addition to the first NMR rf field at, for instance, frequency 휈− (1 ↔ 3), a second NMR rf field at frequency 휈+ Fig. 8 Four-level diagram of a spin system with S = 1∕2 , I = 1∕2 (left, a positive sign of the isotropic hyperfine coupling constant is assumed) with indicated (right) induced EPR and NMR transitions together with spin relaxation rates W훼훽 . Only one EPR- and one NMR-induced transition is shown. The Greek letters 훼훼N , 훼훽N ,… denote the values of the electron and nuclear magnetic spin quantum numbers mS and mI , respectively. The wavy lines indicate the relaxation transitions with their rates for electron ( We ), nuclear ( Wn ), and electron–nuclear cross relaxation ( Wx1 , Wx2 ). For details, see [1] and references given in the text Fig. 8 Four-level diagram of a spin system with S = 1∕2 , I = 1∕2 (left, a positive sign of the isotropic hyperfine coupling constant is assumed) with indicated (right) induced EPR and NMR transitions together with spin relaxation rates W훼훽 . Only one EPR- and one NMR-induced transition is shown. The Greek letters 훼훼N , 훼훽N ,… denote the values of the electron and nuclear magnetic spin quantum numbers mS and mI , respectively. The wavy lines indicate the relaxation transitions with their rates for electron ( We ), nuclear ( Wn ), and electron–nuclear cross relaxation ( Wx1 , Wx2 ). For details, see [1] and references given in the text 1 3 High‑field/High‑frequency EPR Spectroscopy in Protein… 235 short-circuits the Wn bottleneck (2 ↔ 4). Thereby, the efficiency of the NMR-induced relaxation bypass is enhanced. Provided that both rf fields are applied at saturating levels, a considerable increase in the signal intensity can be achieved and, addition- ally, the line intensities become rather independent ofWn . For Wn ≪We , this results in an intensity pattern which, similar to NMR, is dominated by the number of nuclei involved in a particular transition. This facilitates the assignment of hfc’s to specific molecular positions. Furthermore, Special TRIPLE has the advantage of narrower lines, i.e., at a given power level the effective NMR saturation, which determines the observed linewidth, is smaller in TRIPLE than in ENDOR. Linewidth reductions of typically 30–50% are observed in agreement with model calculations [161]. General TRIPLE Resonance: We have shown that in the special triple resonance version, both rf fields are applied at a separation of the hfc, Aiso , (denoted as " a " in Fig. 9) of the same nucleus. Triple resonance can, however, be generalized to several inequivalent nuclei with the aim to obtain hfc’s together with their relative signs [52]. If we consider, for example, two inequivalent protons, the first-order energy levels in the basis memI1mI2⟩ can be arranged to form the eight corners of a cube, see Fig. 9. In such a three-dimensional representation, the various desatura- tion bypasses for a pumped electron transition, involving the NMR transitions of the two nuclei, can be visualized more clearly than in the conventional two-dimensional transition schemes. In Fig. 9, this energy level arrangement is depicted for the two different cases, a1 , a2 > 0 and a1 > 0, a2 < 0. Every two corners are connected by the various relaxation and induced transitions, the EPR transitions occurring vertically, Fig. 9 Topology of ENDOR and TRIPLE resonance experiments for the three-spin system S = 1∕2 , I1 = 1∕2 , I2 = 1∕2 . The NMR transitions with their frequencies are given for the two cases of equal and opposite signs of the hyperfine couplings. For simplification, the EPR transitions are not distinguished. The graph shows the TRIPLE amplification factor V as function of the ratio We∕Wn , obtained by analyz- ing the electric-circuit analog of the various relaxation networks. 1 3 The curves shown are valid for induced NMR rates 100 times larger than Wn ; cross relaxation was neglected. For details, see ref. [48] Fig. 9 Topology of ENDOR and TRIPLE resonance experiments for the three-spin system S = 1∕2 , I1 = 1∕2 , I2 = 1∕2 . The NMR transitions with their frequencies are given for the two cases of equal and opposite signs of the hyperfine couplings. For simplification, the EPR transitions are not distinguished. The graph shows the TRIPLE amplification factor V as function of the ratio We∕Wn , obtained by analyz- ing the electric-circuit analog of the various relaxation networks. The curves shown are valid for induced NMR rates 100 times larger than Wn ; cross relaxation was neglected. For details, see ref. [48] 1 1 3 236 K. Möbius, A. Savitsky the NMR transitions horizontally. All transitions are doubly degenerate to first order. If we now consider the level populations, the different multiple-resonance experi- ments can be represented by different geometrical figures (see left part of Fig. 9). These figures are derived from the cubes by contracting those corners that are con- nected by induced NMR transitions. They represent the limiting case of highly sat- urated transitions where the populations of the connected levels are equalized. In this representation, an ENDOR experiment forms a prism, and a Special TRIPLE experiment forms a square. If we consider the case that both rf fields drive the low- frequency transitions ( 휈− 1 and 휈− 2 ), in General TRIPLE two different cases have to be distinguished depending on the relative signs of the hfc’s. If they have the same sign, all the NMR transitions are saturated in the same plane resulting in a pyramid. If the hfcs have opposite signs, the NMR transitions for the two nuclei are satu- rated in the opposite planes and a tetrahedron is formed. The sign of the hyperfine coupling is an important parameter in molecular spectroscopy. For example, in the case of the isotropic hyperfine constant, Aiso , the sign provides additional insight into the electronic structure when finding an answer to the question: how is unpaired electron density produced at the nucleus? Either by a spin-polarization mechanism ( Aiso < 0, for example α-protons) or by conjugation or hyperconjugation mecha- nisms ( Aiso > 0, for example β-protons).i The significance of these topological games is visualized using Fig. 9. 1 3 It shows the result of a theoretical analysis of the relaxation networks of the various geo- metrical figures for a wide range of We∕Wn values [52]. This analysis was performed within the approximation of the electric-circuit analog of rate equations (mentioned above) applying Kirchhoff’s laws for branching networks. When a TRIPLE ampli- fication factor, V , is defined as the ratio of TRIPLE and ENDOR line amplitudes, always V > 1 for a "tetrahedron" experiment and V < 1 for a "pyramid" experi- ment. The difference between pyramid and tetrahedron becomes particularly pro- nounced in cases where Wn is much smaller than We . Such cases are typical for many ENDOR-in-solution experiments. In the extreme situation We∕Wn > > 1, Special TRIPLE can even reach 100% EPR sensitivity! Obviously, relative signs of hfc’s can easily be determined from intensity changes in General TRIPLE spectra. As a rep- resentative example, Fig. 10 shows for the fluorenone·−/Na+ ion pair in fluid solu- tion how the intensity patterns of proton and sodium lines change in a characteristic way when extending ENDOR to General TRIPLE. From such intensity patterns the signs of the various hfcs are revealed relative to the sign of the hfc belonging to the pumped transition [162]. Different relative signs of the hfcs of different types of protons and of the sodium counter ion as a function of temperature are reflected by inversion of the amplitude ratios and, thereby, inform about the planarity of the ion pair [162]. To conclude this section on cw mode ENDOR and TRIPLE resonance on sys- tems in fluid solution, we point out that pulsed ENDOR and TRIPLE on solid-state systems is a completely different story, which to tell would require a separate sec- tion. This would go beyond the scope of this article and, hence, we mention this issue only in passing and refer to excellent presentations in the literature instead, see for instance [1, 33, 163]. 3 High‑field/High‑frequency EPR Spectroscopy in Protein… 237 Fig. 10 ENDOR and General TRIPLE spectra of the fluorenone anion radical (solvent: tetrahydrofurane, counter ion: Na+, T = 226 K). For details, see [162] Fig. 10 ENDOR and General TRIPLE spectra of the fluorenone anion radical (solvent: tetrahydrofurane, counter ion: Na+, T = 226 K). 1 3 For details, see [162] The basis of the various techniques in pulsed EPR spectroscopy and its multitude of combinations of electron–nuclear and electron–electron resonances, is the detec- tion of the resulting electron spin echoes—in analogy to the spin-echo detection in NMR spectroscopy introduced by E.L. Hahn already in 1950 (“Hahn echoes”) (Fig. 11). Translated to pulsed EPR, in a Hahn electron spin echo (ESE) experiment, a microwave 90° (π/2) pulse at resonance frequency 휈 produces a signal that decays away (FID). If the EPR spectrum is inhomogeneously broadened, we can recover this disappeared signal with another microwave pulse at the same frequency 휈 , but of pulse length of 180° (π): after an inter-pulse time delay of 휏 a Hahn echo is pro- duced, see [33]. Echoes are important in EPR because FIDs of very broad spectra decay away very quickly and often escape detection. Fig. 11 Hahn Spin Echo in NMR (or EPR). The NMR signal observed following an initial mw excitation π/2 pulse decays in the xy plane in the rotating frame (FID, Free Induction Decay) with time due to both spin relaxation and any inhomogeneous effects which cause the spins in the sample to precess at different speeds. The first of these effects, relaxation, leads to an irreversible loss of magnetization. But the inho- mogeneous dephasing can be removed by applying a π inversion pulse that inverts the magnetization vec- tors. Examples of inhomogeneous effects include a magnetic field gradient and a distribution of chemical shifts. If the inversion pulse is applied after a period τ of dephasing, the inhomogeneous evolution will rephase to form an echo at time 2τ Fig. 11 Hahn Spin Echo in NMR (or EPR). The NMR signal observed following an initial mw excitation π/2 pulse decays in the xy plane in the rotating frame (FID, Free Induction Decay) with time due to both spin relaxation and any inhomogeneous effects which cause the spins in the sample to precess at different speeds. The first of these effects, relaxation, leads to an irreversible loss of magnetization. But the inho- mogeneous dephasing can be removed by applying a π inversion pulse that inverts the magnetization vec- tors. Examples of inhomogeneous effects include a magnetic field gradient and a distribution of chemical shifts. 1 3 If the inversion pulse is applied after a period τ of dephasing, the inhomogeneous evolution will rephase to form an echo at time 2τ 1 3 238 K. Möbius, A. Savitsky For technical reasons, presently signals cannot be detected during an approxi- mately80 ns period after the microwave pulse. This period of time is called the dead- time of the spectrometer. If the FID is very short, it will disappear before the dead- time ends. If we make τ long enough, we can ensure that the echo appears after the deadtime. There are different pulse schemes for different types of echoes, e.g., the “dead-time free echo detection” and the “stimulated-echo detection” (for references, see [33]). Both of them have become common practice in modern NMR/EPR spec- troscopy and magnetic resonance imaging (MRI). Whereas a Hahn spin echo arises from the action of two radiofrequency/microwave pulses, a stimulated echo occurs from the action of three or more radiofrequency/microwave pulses. How does the echo bring back our magnetic resonance signal? The decay of the FID is due to the different spin packet resonance frequencies in the EPR spectrum causing the magnetization to fan out in the x–y plane of the rotating frame. When we apply the π pulse, we flip the magnetization about the x axis. The magnetization still rotates in the same direction and speed. The higher frequency spin packets will have traveled further than the lower frequency spin packets after the first pulse. However, because the higher frequency spin packets are rotating more quickly, they will even- tually catch up with the lower frequency spin packets along the + y axis after the second pulse [33]). After the spin packets had bunched up, they will dephase again just like an FID. If we Fourier transform the FID, we obtain the EPR spectrum. The Fourier transform is a mathematical function that decomposes a waveform, which is a function of time, into the frequencies that make up the waveform. Hence, the Fourier transform is the primary tool for analyzing signals in the frequency domain, especially when signals are sampled. According to Fourier theory, “broad” in the time domain means “narrow” in the frequency domain. 1 3 2.2.1.3 Pulse ENDOR In addition to what has been said concerning relaxation effects on the cw ENDOR signal strength, it should be noted that also pulse ENDOR tech- niques are sensitive to nuclear spin relaxation to some degree, but as a general rule, cw techniques to a greater, pulse techniques to a lesser extent. The cw ENDOR enhancement effect requires a delicate balance of saturation and desaturation of elec- tron and nuclear sublevel transitions at a particular working temperature. For specific sample conditions this is sometimes difficult to do owing to unfavorable electron and nuclear relaxation times T1 and T2 . In such a situation, pulsed ENDOR techniques are often the solution of the problem. But also pulsed ENDOR might be impeded by too short T2 relaxation times, while the T1 times can generally be adjusted by lowering the temperature. Pulsed ENDOR techniques require the nuclear T1·T2 product to be sufficiently long to coherently rotate the nuclear magnetization by the rf pulses used for detection. Hence, the sample properties dictate whether cw or pulse ENDOR is the preferable method. To quote from the Schweiger-Jeschke “Scripture”: “In most practical situations, cw ENDOR is the method of choice for the measurement of small hyperfine couplings in liquid solution, whereas in solids pulse ENDOR is often superior” [33]. For solid-state samples, ESE-detected pulse ENDOR versions were introduced by W. B. Mims (1965) at Bell Labs [41] and E. R. Davies (1974) at Clarendon [42], see below. Pulsed ENDOR spectroscopy offers several distinct advantages over the 1 3 1 3 High‑field/High‑frequency EPR Spectroscopy in Protein… 239 conventional cw technique: The ENDOR effect can be as large as the electron spin echo intensity itself (cw ENDOR reaches only 1–10% of EPR); it requires no critical balance of rf-driven and relaxation transition rates (a condition which has to be met in cw ENDOR); it is less susceptible to artifacts as there is neither an rf nor a mw field applied during the detection period (high-power cw ENDOR often suffers from rf pick-up problems distorting or even ruining the detected signals); it gives imme- diate access to all relaxation times of a spin system (electron T1 and T2 , nuclear T1 and T2 , cross relaxation Tx ). Therefore, pulse ENDOR has been the commonly used technique in many laboratories in the last decades, applying pulse sequences pre- dominantly developed by Mims [41] and Davies [42]. 1 3 The two techniques are very similar to each other in their implementation; however, they are sort of complemen- tary in their results and usefulness, specifically concerning handling of experimental artifacts such as “blind spots” in the spectrum [164]. The Mims-type ENDOR technique is based on a stimulated electron spin echo (ESE) sequence, using two π/2 preparation mw pulses to invert the electron spin population, and a final π/2 mw pulse after the mixing period for stimulated echo signal detection. Between the preparation pulses and the final pulse, an rf π pulse is used to invert the nuclear spin population, resulting in polarization transfer between the nuclear and electronic transitions in the mixing period. The echo intensity is subsequently measured as a function of the rf frequency to give the characteristic ENDOR spectrum. The Mims ENDOR technique is most suited for weakly coupled nuclei, i.e., nuclei with small hyperfine coupling constants. i In Davies-type ENDOR, a preparation mw π pulse is used in order to invert the magnetization of the electron spins in the applied static B0 field. This essentially cre- ates a hole in the EPR spectrum, whose width and depth depend on the length of the pulse applied, with a long pulse producing narrow holes. During the mixing period, an rf π pulse is applied, and only if the rf frequency is resonant with an NMR transi- tion, magnetization will be transferred to the other mS electron spin manifold. Oth- erwise, no mixing will occur to fill in the hole that the inversion pulse creates. Dur- ing the detection period, the z-component of the magnetization is measured using a mw two-pulse echo sequence, π/2, π, and one detects an inverted echo, i.e., the EPR signal is restored during the mixing period. The Davies ENDOR technique is well suited to detect nuclei with large hyperfine couplings.f i All pulse ENDOR techniques suffer from detectability problems at certain fre- quencies (“blind spots”). The important difference between the Mims and Davies pulse sequences, however, is that in Davies ENDOR there are no blind spots. Blind spots are directly correlated to the size of the hyperfine interactions of the coupled nuclei. 1 3 Hence, when using pulsed ENDOR as a tool for structure determination, one has to make sure that no artifacts have occurred owing to the specific pulse strategy applied which might mask certain hyperfine couplings and lead to wrong interpreta- tion of the spectra [164]. Powerful pulse strategies have been developed in various EPR laboratories to either avoid or overcome blind-spot artifacts, for references, see [33, 165]. Here we only want to mention, as a promising pulse strategy for high-field pulse EPR spectroscopy, the FID detection of EPR and ENDOR spectra [166] owing to the short deadtime that can be achieved at high microwave frequencies. This FID detection is particularly attractive for EPR and ENDOR of paramagnetic species that 1 3 3 240 K. Möbius, A. Savitsky exhibit inhomogeneous EPR line broadening and short dephasing times [33], for example metallo-proteins [33, 167]. 2.2.1.4 HYSCORE A very powerful technique for measuring electron–nuclear hyper- fine couplings of complex molecular systems is HYSCORE (Hyperfine sublevel cor- relation spectroscopy), which is essentially a two-dimensional ESEEM experiment in which correlation is transferred from one electron spin manifold mS (e.g., mS = +1∕2 ) to nuclear frequencies in the other mS manifold (e.g., mS = −1∕2 ). It is based upon the COSY (Correlation Spectroscopy) NMR experiment and was introduced by P. Höfer, M. Mehring and co-workers [168] at the University of Stuttgart in 1986. This 2D experiment improves the spectral resolution by spreading overlapping peaks over two dimensions and provides information which is difficult to obtain in a 1D ESEEM experiment. Hence, HYSCORE has become the standard experiment for the measure- ment of complex hyperfine and quadrupole spectra [33] and produces complimentary and comparable information to the ENDOR experiment. In a HYSCORE experiment, the time between the second π/2 and π pulse is varied in one dimension and the time between the π and third π/2 pulse is varied in a second dimension. A two-dimensional Fourier transformation then gives the spectra, and examples from protein chemistry are given, for instance, in [13]. 2.2.1.5 ESEEM Hyperfine Spectroscopy A very important class of echo experiments is ESEEM (Electron Spin Echo Envelope Modulation, (ESEEM) [56]). The electron spins interact with the nuclei in their vicinity and this interaction causes a periodic oscillation in the echo height superimposed on the normal echo decay. The modula- tion or oscillation is caused by periodic dephasing by the nuclei. 1 3 If we subtract the decay of the spin echo and Fourier transform the oscillations, we obtain the splittings due to the nuclei. Armed with this information, we can identify nearby nuclei and their distances from the electron spin and shed light on the local environment of the radical or metal ion. Thus, when a single-frequency microwave spin-echo pulse train with varying pulse spacing is applied to a molecular system with hyperfine-coupled electron and nuclear spins, amplitude modulations of the exponentially decaying echo signal can be observed. This ESEEM phenomenon requires certain conditions to be fulfilled by the spin system, as will be shown below. The modulated echo-decay time trace is normally analyzed by means of fast Fourier transformation into the frequency domain to extract hyperfine and quadrupole couplings in a way that is complemen- tary to ENDOR.i In the following, we summarize a recent high-field EPR and ESEEM investiga- tion of the 14 N quadrupole interaction of nitroxide spin labels in disordered solids [121]. The aim was to explore the experimental and theoretical background for using the quadrupole-tensor components of nitroxide spin labels for probing polarity and proticity effects of their micro-environment. It is an attempt to obtain an additional handle for separating polarity from proticity effects, which is expected to comple- ment the matrix information obtained from using the g- and nitrogen hyperfine- tensor components of the nitroxide spin probe. We consider the high-field ESEEM 1 3 High‑field/High‑frequency EPR Spectroscopy in Protein… 241 experiments on nitroxide spin labels in organic frozen solutions as a step toward dif- ferentiation between polarity and proticity matrix effects on the biological function of proteins. experiments on nitroxide spin labels in organic frozen solutions as a step toward dif- ferentiation between polarity and proticity matrix effects on the biological function of proteins. For protein systems that have exclusively diamagnetic states of their reactants, e.g., the intermediate states of the light-controlled proton pump bacteriorhodopsin, EPR techniques can still serve for probing environmental effects on the process effi- ciency. This is possible by resorting to site-directed spin-labeling (SDSL) mutagen- esis techniques, using specific nitroxide spin-label side chains as reporter groups, for example the MTS [(1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) methanethiosul- fonate] spin label [169, 170]. SDSL has matured to an extremely important branch of bio-EPR spectroscopy, and tailor-made nitroxide side chains can be introduced at almost any desired site in a protein. 1 3 It has been shown (for reviews, see [171, 172] that the isotropic and anisotropic components of the g- and hyperfine-tensors of the nitroxide spin label can be used to reveal the polarity and proticity properties of the immediate environment of the reporter group. Since single crystals of membrane proteins are often difficult to prepare, if at all, frozen-solution protein preparations, lacking long-range order, are commonly used for EPR studies of matrix effects. f For nitroxide spin-labeled molecules with rather small anisotropies of their spin interactions, the application of high-field EPR techniques with correspondingly high microwave frequencies is preferable compared to the standard X-band EPR techniques to ensure high spectral and time resolution as well as high orientational selectivity [115, 173]. Such a magnetoselection can be further exploited using dou- ble resonance techniques at high fields, e.g., ENDOR or pulsed ELDOR (PELDOR) or ESEEM techniques. They are capable of providing single-crystal like information from orientationally selected fractions of molecules in the disordered samples. By combination of high-field EPR and SDSL techniques, subtle changes of the polarity and proticity profiles could be measured, for example along proton transfer pathways in proteins embedded in natural and artificial membranes [173]. This information was obtained by resolving the gxx and Azz components of the nitroxide interaction tensors of a series of molecules with the spin label attached to specific molecular sites. The linear correlation plots of gxx vs Azz are theoretically predicted [174–178] and experimentally established [179, 180]. Moreover, different slopes occasionally observed in the gxx vs Azz plots were assigned to either polarity or proticity effects on the magnetic parameters of the spin label from its local protein or membrane environment.i In contrast to the g- and nitrogen hyperfine-tensors, the 14 N ( I = 1 ) quadrupole interaction tensor of the nitroxide spin label has not been widely exploited in EPR for probing effects of the micro-environment of functional protein sites. Precise knowledge of the 14 N quadrupole coupling constant e2 ⋅q ⋅Q and the asymmetry parameter 휂 of the electric field gradient at the 14 N nucleus in the nitroxide would enlarge the arsenal of sensitive probes for environmental effects on specific sites of the molecule, both in terms of polarity [181] and hydrogen-bond effects [182]. f To measure directly the nuclear quadrupole interaction by advanced EPR tech- niques, the nuclear transitions are normally driven directly by rf fields as in ENDOR. 1 3 A more indirect though effective alternative is offered by ESEEM, i.e., by applying a mw single-frequency pulse train with varying pulse separation and 3 3 3 242 K. Möbius, A. Savitsky observing spin-echo modulations from hyperfine and quadrupole interactions. To obtain detectable modulations of the echo decays it is mandatory that ’’forbid- den’’ transitions, flipping the electron and nuclear spins simultaneously, become partially allowed [56]. This requires an efficient mixing of the nuclear and electron spin eigenfunctions by the dipolar hyperfine interaction. Consequently, the strength of the external magnetic field has to be properly chosen to approximately balance the Zeeman splitting of the nuclear sublevels and the respective hyperfine splitting ("cancelation condition") [183]. This means that an optimum Zeeman field value exists for each nucleus and hyperfine coupling, as has been demonstrated by mul- tifrequency ESEEM experiments on S = 1∕2 , I = 1∕2 as well as on S = 1∕2 , I = 1 systems [183–186]. Pulsed ENDOR and ESEEM techniques are complementary to each other [33, 187] concerning their ability to reveal large or small nitrogen dipolar hyperfine interactions ("geminate" or "distal" nitrogens), respectively. Only a few high-field nitrogen ESEEM experiments on single-crystalline [188] and disordered samples [186] have been reported in recent years. Below, we will review an example from our laboratory [121].i The aim of this work [121] was to investigate, by means of high-field EPR and ESEEM experiments in conjunction with DFT calculations, two important issues: (i) whether nitrogen quadrupole-tensor components can be determined with high accu- racy from frozen-solution samples and (ii) what kind of information on the polar- ity and proticity properties of the nitroxide spin-label environment can be extracted from the interaction between the electric field gradient (tensor with elements e⋅qij) at the site of the 14N nitrogen nucleus and its electric quadrupole moment ( e ⋅Q ). Specifically, the question was addressed whether the 14 N quadrupole information on matrix effects is similar or complementary to that obtained from the established spin-probe parameters, gxx and Azz. We focused on the spectroscopic and quantum-chemical aspects of measuring and calculating the quadrupole interaction parameters of the perdeuterated nitroxide radical R1 dissolved in frozen solutions of either non-polar, aprotic ortho-terphenyl or polar, protic glycerol, Fig. 12. Fig. 1 3 12 Molecular structure of the perdeuterated nitroxide radical (R1); the conventional principal axes of the g-tensor are indicated; ortho-terphenyl and glycerol hosts for the diluted nitroxide glassy solutions, see [121]. R1was studied both as R1-14N and R1-15N nitroxide radicals Fig. 12 Molecular structure of the perdeuterated nitroxide radical (R1); the conventional principal axes of the g-tensor are indicated; ortho-terphenyl and glycerol hosts for the diluted nitroxide glassy solutions, see [121]. R1was studied both as R1-14N and R1-15N nitroxide radicals Fig. 12 Molecular structure of the perdeuterated nitroxide radical (R1); the conventional principal axes of the g-tensor are indicated; ortho-terphenyl and glycerol hosts for the diluted nitroxide glassy solutions, see [121]. R1was studied both as R1-14N and R1-15N nitroxide radicals 1 3 High‑field/High‑frequency EPR Spectroscopy in Protein… 243 Fig. 13 a Experimental W-band cw EPR spectra of 1 mM R1-15N (upper spectrum) and R1-14N (lower spectrum) nitroxide radicals in frozen solution of ortho-terphenyl taken at 180 K. They are overlaid with the corresponding best-fit spectra (red lines) obtained without taking the quadrupole interaction into account. For the derived magnetic parameters, see text. b Expanded view of the gxx , gyy regions of the R1-14N spectrum. The best-fit spectra calculated without quadrupole contributions and with the quadru- pole couplings Pxx = 1.1 MHz; Pyy = 0.6 MHz are shown by red and dark-green lines, respectively. For details, see [121] Fig. 13 a Experimental W-band cw EPR spectra of 1 mM R1-15N (upper spectrum) and R1-14N (lower spectrum) nitroxide radicals in frozen solution of ortho-terphenyl taken at 180 K. They are overlaid with the corresponding best-fit spectra (red lines) obtained without taking the quadrupole interaction into account. For the derived magnetic parameters, see text. b Expanded view of the gxx , gyy regions of the R1-14N spectrum. The best-fit spectra calculated without quadrupole contributions and with the quadru- pole couplings Pxx = 1.1 MHz; Pyy = 0.6 MHz are shown by red and dark-green lines, respectively. For details, see [121] Figure 13 shows W-band cw EPR spectra of R1-14N and R1-15N nitroxide radi- cals in frozen ortho-terphenyl solution at 180 K. The spectra exhibit the typical powder-pattern lineshape expected for a dilute distribution of nitroxides. 1 3 The spectra are clearly resolved into three separate regions corresponding to the principal values of the g-tensor, gxx , gyy and gzz Moreover, due to the reduction of the inhomogene- ous linewidth by perdeuteration of the radicals, the nitrogen hyperfine splitting (dou- blets for R1-15N, I = 1∕2 , triplets for R1-14N, I = 1 ) is observed in all g-regions. The spectra were analyzed by numerical solution of the spin Hamiltonian given in Eq. (2). At first, the spectrum of R1-15N was considered. The best-fit spectrum is shown in Fig. 13a. Perfect agreement with the experimental spectrum is achieved by using the set of magnetic parameters, i.e., g-tensor, nitrogen hyperfine-tensor, homo- geneous EPR linewidth, orientation-dependent EPR inhomogeneous linewidths, as listened in [121]. In the next step, the spectrum of R1-14N was calculated using the parameters obtained from R1-15N, i.e., tentatively omitting quadrupole 1 3 244 K. Möbius, A. Savitsky contributions, but rescaling the 훾N and A values by the factor (1.4)−1 according to the ratio of the nitrogen nuclear g-values gn(15N)/gn(14N) = 1.4. Although there is good agreement of the positions of the EPR lines in all spectral regions of the g-tensor components (see Fig. 13a), the EPR signal intensities agree only in the gzz spectral region. This disagreement of intensities in the gxx and gyy regions cannot be improved by varying the corresponding linewidths. However, switching on the 14 N quadrupole interaction term in the spin Hamiltonian allows to reproduce the intensities of the experimental EPR spectrum, Fig. 13b. From the simulation, the values Pxx = + 1.1 MHz and Pyy = + 0.6 MHz were obtained. Thus, the quadrupole effects in the cw W-band EPR spectrum of perdeuterated 14N nitroxide radicals are directly observed. This is only possible because of the narrow EPR linewidth (below 10 MHz) of the perdeuterated spin label. 14 Although the 14N quadrupole interaction is observed in the cw W-band EPR spectra, the Pxx and Pyy values can be evaluated only with quite a large error. This is because they are obtained by a multiparameter fit of fine details of the experimental cw EPR spectra. Hence, we decided to resort to ESEEM experiments from which Fig. 14 W-band microwave pulse sequence for the stimulated high-field ESEEM experiment on the nitroxide radical R1. 1 3 Top: The echo-detected EPR spectrum of the R1-14N radical as well as the micro- wave excitation bandwidth for typical microwave pulse-length settings are shown ( τ = 40 ns, π/2-pulse length tp= 30 ns. The time T is stepped from T0 = 100 ns in 5 ns steps). Bottom left: Representative example of a nuclear modulation echo-decay trace at the indicated B0 position. Bottom right: Fourier- transformed (FT) spectrum of the ESEEM echo-decay trace. For details, see [121] Fig. 14 W-band microwave pulse sequence for the stimulated high-field ESEEM experiment on the nitroxide radical R1. Top: The echo-detected EPR spectrum of the R1-14N radical as well as the micro- wave excitation bandwidth for typical microwave pulse-length settings are shown ( τ = 40 ns, π/2-pulse length tp= 30 ns. The time T is stepped from T0 = 100 ns in 5 ns steps). Bottom left: Representative example of a nuclear modulation echo-decay trace at the indicated B0 position. Bottom right: Fourier- transformed (FT) spectrum of the ESEEM echo-decay trace. For details, see [121] 1 3 3 3 High‑field/High‑frequency EPR Spectroscopy in Protein… 245 Fig. 15 3D representation of the experimental ESEEM surface. In the frequency-B0 plane, the contour- plot representation of the ESEEM intensities is shown. This 3D representation is now commonly used in the literature. For details, see [121] Fig. 15 3D representation of the experimental ESEEM surface. In the frequency-B0 plane, the contour- plot representation of the ESEEM intensities is shown. This 3D representation is now commonly used in the literature. For details, see [121] the quadrupole interactions can be determined in a more straightforward way, see Figs. 14, 15). The stimulated ESE decays show distinct modulations for both the R1-15N and the R1-14N nitroxides. In the case of R1-15N, the echo modulation is dominated by a frequency of about 22.1 MHz in the whole spectral region of nitroxide EPR absorp- tion. This modulation is assigned to the nitroxide methyl deuterons, see Fig. 12. Additional ESEEM frequencies are detected in the gxx , gyy region. Their analysis and interpretation are thoroughly discussed in [121] and [1]. There, it is concluded that measurement of the quadrupole component Pyy can provide important contributions to the study of environmental effects. Additionally, it serves as a consistency check of polarity and/or proticity results obtained from the often employed gxx vs. 1 3 Azz cor- relation in spin-label EPR spectroscopy.i Polarity control in protein complexes from innermolecular electric fields have been widely investigated by observing shifts of Azz and/or gxx on NO spin labels in various environments. Whereas Azz reacts to polarity changes in non-bonding as well as H-bonding situations predominantly through changes in the spin density dis- tribution of the NO bond (as a consequence of charge displacements between N and O), gxx is also significantly affected by additional perturbations of the n–π energy gap of the O-atom in H-bonding situations. Thus, the observation of gxx shifts may be desirable for the detection of H-bond formation (proticity), but can also lead to ambiguous results in trying to quantitatively separate proticity from polarity effects. If measurement of Azz alone does not safely yield the desired information on polarity changes, measurement of Pyy is, therefore, the appropriate choice. Qualitatively, Pyy 1 3 246 K. Möbius, A. Savitsky has the same probing properties as Azz in detecting polarity and proticity effects, see [121]. 2.2.1.6 ELDOR‑Detected NMR (EDNMR) As we have seen, there are several pulsed EPR techniques that are capable of probing nuclear transition frequencies of para- magnetic compounds such as ESEEM-based techniques, as well as ENDOR tech- niques. There is another technique able to unravel congested nuclear spectra which has become very popular in recent years, and this is Pulsed ELDOR-detected NMR (EDNMR) [141], see Fig. 5. The acronym ELDOR stands for electron–electron dou- ble resonance, i.e., two microwave fields are involved. The EDNMR experiment was introduced by A. Schweiger and his co-workers at the ETH Zurich in 1994, it excels by its potential to determine small hyperfine interactions in disordered systems. In the original version of EDNMR, a strong and long selective preparation pulse (HTA) of microwave frequency 휈b excites allowed and forbidden transitions simultaneously, thereby burning spectral holes into the EPR line. The positions of the holes caused by the excitation of forbidden transitions correspond to the nuclear transition frequen- cies of the spin system. A selective detection pulse of frequency 휈a creates an FID with integrated intensity proportional to the magnetization at this frequency. The entire hole pattern is obtained by recording the integrated intensity of the FID while varying the frequency difference Δ휈= 휈b −휈a step by step. 1 3 More recent EDNMR experiments apply a Hahn-type primary echo pulse sequence for detection instead of using FID detection [288, 289].f g A few words concerning the fundamental difference between coherence-trans- fer techniques and polarization-transfer techniques seem to be appropriate at this point: ESEEM is a coherence-transfer technique in which the nuclear frequencies are obtained from the analysis of the time-dependent electron spin-echo modulation caused by the oscillation between allowed and forbidden electron coherences or by the evolution of nuclear coherences. ENDOR and EDNMR are polarization-transfer pulsed EPR experiments, that is, the nuclear frequencies are detected by manipu- lating the polarizations of electron and nuclear levels. The principal difference between ENDOR and EDNMR is the way in which the population of the nuclear levels is changed. In pulse ENDOR, the nuclear polarization is inverted by driving the allowed NMR transition ( ΔmS = 0 ; ΔmI = ±1 ) with an rf π-pulse. In contrast, in EDNMR, the nuclear transitions of the spin manifold are probed indirectly by using a second strong and long microwave pulse (also called high-turning-angle (HTA) pulse), which drives forbidden electron transitions, ( ΔmS = 0 ; ΔmI = ±1 ) that is, transitions where both the electron and nuclear spin change their projection direc- tion. Such “forbidden” transitions in the spin manifold are “only weakly allowed” in the presence of an anisotropic hyperfine interaction or a nuclear quadrupole interac- tion. The frequency 휈2 of the pumping HTA pulse is swept around the fixed reso- nance frequency, 휈1 of the detection Hahn echo pulse sequence. At mw frequencies, where the HTA pulse coincides with a forbidden transition the observed primary echo signal of an allowed EPR transition decreases due to population transfer via the forbidden transitions. These 휈mw dependent changes in population differences and concomitant signal changes are observed as spectral lines, which correspond to the nuclear transitions of the spin manifold. For the simple case of a 4-level scheme 1 3 High‑field/High‑frequency EPR Spectroscopy in Protein… 247 Fig. 16 Comparison of pulsed Davies-type ENDOR a with pulsed ELDOR-detected NMR, EDNMR b phenomenological description of spin populations after the respective pulses have flipped the spin pro- jections of the electron and nucleus for the case S = 1∕2 , I = 1∕2 . Polarization transfer in the Davies- type pulse ENDOR experiment involving allowed electron and nuclear spin transitions. 1 3 Polarization transfer in the EDNMR experiment involving allowed and forbidden transitions of the hyperfine-coupled electron and nuclear spins. For details, see ref. [13]. The labels πa, πRF and πHTA denote the π-pulses required for optimal polarization transfer. Note: In the figure, the radiofrequency pulses are denoted “RF”, while in the text they are denoted “rf” Fig. 16 Comparison of pulsed Davies-type ENDOR a with pulsed ELDOR-detected NMR, EDNMR b phenomenological description of spin populations after the respective pulses have flipped the spin pro- jections of the electron and nucleus for the case S = 1∕2 , I = 1∕2 . Polarization transfer in the Davies- type pulse ENDOR experiment involving allowed electron and nuclear spin transitions. Polarization transfer in the EDNMR experiment involving allowed and forbidden transitions of the hyperfine-coupled electron and nuclear spins. For details, see ref. [13]. The labels πa, πRF and πHTA denote the π-pulses required for optimal polarization transfer. Note: In the figure, the radiofrequency pulses are denoted “RF”, while in the text they are denoted “rf” ( S = 1∕2 , I = 1∕2 with hyperfine interaction), Fig. 16 depicts the different forms of pulse-driven polarization transfer in Davies ENDOR (Fig. 16a) and EDNMR (Fig. 16b). ( S = 1∕2 , I = 1∕2 with hyperfine interaction), Fig. 16 depicts the different forms of pulse-driven polarization transfer in Davies ENDOR (Fig. 16a) and EDNMR (Fig. 16b). g Pulsed ENDOR uses a selective mw pulse train at a frequency, which is in res- onance with an allowed electron spin transition, to flip the electron spins and to observe the change of electron polarization which had occurred as a result of the change in nuclear polarization by flipping the nuclear spins by the rf pulse with the frequency of an allowed nuclear spin transition. In pulsed ELDOR-detected NMR, following the HTA pulse at a chosen mw frequency 휈2 , a selective pulse train with fixed mw frequency 휈1 is applied probing an allowed electron spin transition. The frequency 휈b of the HTA pulse is varied, and once it matches one of the forbid- den “cross” transitions ( ΔmS = ±1 ; ΔmI = ±1 ), a population transfer occurs which is manifested in a change of the Hahn echo intensity (this population transfer can 1 3 248 K. Möbius, A. Savitsky alternatively be observed as a change of FID intensity [189]). 1 3 The EDNMR experi- ment can principally be performed at any microwave frequency bands where the “forbidden” cross transitions are observable by EPR. It turned out, however, that it works best at Zeeman fields high enough to separate the NMR lines around the nuclear Larmor frequency well from the hole burnt into the EPR spectrum of allowed transitions by the HTA pulse, but small enough to keep the ratio of hyper- fine and Zeeman interactions large enough to retain substantial second-order terms in the electron transition probabilities. Apparently, such conditions working in oppo- site direction can often be fulfilled by high-field EDNMR at W-band [190–194], and this method has become very attractive for measuring nuclear frequencies for low γ nuclei exhibiting broad ENDOR lines.i Compared to ENDOR, high-field EDNMR presents several advantages for the investigation of low-γ nuclei coupled to the electron spin of metallo-proteins. EDNMR is more robust against fast electron spin–lattice relaxation, T1 , and spec- tral diffusion than ENDOR. This robustness is because no preparation of the elec- tron spin system prior to the HTA pulse is required and short HTA pulses can often be realized with the available microwave power. This results in high sensitivity and allows one to rapidly record 2D-EDNMR spectra with a sufficient signal-to-noise ratio. Moreover, the recorded EDNMR spectrum is not distorted by blind spots around the nuclear Larmor frequencies. These advantages have been demonstrated using a simple model system, MnII(H2 17O)6, see [195]. The spin Hamiltonian param- eters extracted are: Aiso = −7.7 MHz; Adip = [−1.5; −1.5;3.0] MHz; e2qQ h = −8.5 MHz; 휂= 1 . For details, see [195] and [13]. 1 Baute and Goldfarb [196] had shown that the 17O signals arising from the hyper- fine splitting of the 17O nucleus within the mS = ±1∕2 and ±3∕2 sublevels of the MnII electron spin manifold can be readily detected at W-band using Davies ENDOR for the corresponding EDNMR of the MnII(H2 17O)6 complex. For details, see [13]. Recently, the Goldfarb group (Rehovot) has extended EDNMR to a correlation method, referring to it with the acronym THYCOS (triple resonance hyperfine sub- level correlation spectroscopy) [191, 194]. It combines ENDOR and EDNMR in a manner similar to the pulsed TRIPLE method [151, 197, 198]. 1 3 1 3 In the THYCOS pulse sequence, the HTA mw pulse with frequency ν1 transfers population across a forbidden EPR transition ( mS = ±1 ; ΔmI = ±1 ). This reduces the population dif- ference between the corresponding allowed transition which is detected via an FID (or a spin echo) produced by a selective pulse (or an echo pulse sequence) with fre- quency ν2. A change in the FID intensity occurs when a rf pulse transfers population from any level affected by either of the mw pulses. The FID (or spin-echo) intensity decreases if the rf pulse excites the same nucleus as the HTA pulse, and it recovers if another nucleus is involved. Therefore, the THYCOS experiment correlates lines in the EDNMR spectrum with lines in the ENDOR spectrum of nuclei belonging to different electron spin manifolds mS . This facilitates the assignment of nuclear reso- nance frequencies to their respective mS manifold, and, hence, gives the sign of the hyperfine coupling. The feasibility of this new technique has been demonstrated for Cu2+-histidine complexes both in single-crystal and frozen-solution samples [191].i The following is an interesting example of high-field EDNMR and ENDOR in photosynthesis research [16]: 1 3 High‑field/High‑frequency EPR Spectroscopy in Protein… 249 Example: Local water sensing: Water exchange in bacterial photosynthetic reac- tion centersi By means of pulsed W-band (94 GHz) high-field multiresonance EPR spectros- copies, such as ELDOR-detected NMR and ENDOR, in conjunction with using spe- cifically isotope-labeled water (D2O and H2 17O), the biologically important issue of detection and quantification of local water in membrane proteins is addressed. A specifically engeneering mutant of the bacterial reaction center (bRC) from Rhodo- bacter sphaeroides R26 embedded into a trehalose glass matrix is used as the model system. This bRC mutant hosts the two native radical cofactor ions P⋅+ 865 ) (primary electron donor) and Q⋅− A (primary electron acceptor) as well as a nitroxide spin label site-specifically attached to a cysteine amino-acid site on the surface of the H-protein domain of the bRC. The resulting three paramagnetic reporter groups have distinctly different local environments. They serve as local probes to detect water molecules via hyperfine interactions (electron–nuclear hyperfine and quadrupole) with either 2H or 17O nuclei. The bRCs in the EPR sample tube were equilibrated in an atmos- phere of different relative humidities allowing to control precisely different hydra- tion levels of the protein. 1 3 As the main result, we showed that using oxygen-17-la- beled water, quantitative conclusions about the local and bulk water environment of the protein can be made. This approach is superior to the conventional strategy of using D2O water for aqueous matrix studies, which suffers from proton–deuterium exchange processes in the protein. From the experiments we also concluded that dry trehalose operates as an anhydrobiotic protein stabilizer, a conclusion that is in line with the “anchorage hypothesis” of anhydrobiotic bio-protection. It predicts selec- tive changes in the first solvation shell of the protein upon trehalose-matrix dehydra- tion with subsequent changes in the hydrogen-bonding network. Changes in hydro- gen-bonding patterns usually have an impact on the overall function of a biological system. After this brief summary of our 2017 study, we will present some informative details that seem advisable for a better understanding of the work [16]. Water plays an important, if not essential part in the chemistry of Life on Earth. In particular, it governs the internal dynamics of biological macromolecules, such as proteins. Unrestricted dynamics at a specific time scale is a crucial requirement for the specific biological activity of proteins, including enzyme activity, macromo- lecular recognition, ligand binding and participation in electron and proton transfer processes. Under physiological conditions these macromolecules fluctuate between different conformational states [199]. Their dynamics span an enormous time range, i.e., from sub-picosecond to tens of microseconds, and include a multitude of stochastic local and collective motions, from bond vibrations to domain motions. Despite significant efforts over the past decades, our microscopic understanding of protein dynamics remains rather limited [200–202]. Liquid water has some unusual physical and chemical properties that are impor- tant for controlling its potency as a solvent, its ability to form hydrogen bonds and its amphoteric nature, i.e., the ability to act either as an acid or a base. Neverthe- less, until rather recently, molecular biologists have regarded water essentially as the backdrop on which the molecular components constituting “Life” are arrayed and functionalized [203]. It is clear by now that water is an active constituent of 1 3 3 3 250 K. Möbius, A. Savitsky cell biology which has to be included in a meaningful molecular picture of life pro- cesses, see for example [204]. 1 3 Water drives protein folding through hydrophobic interactions (the “hydropho- bic effect”) [205–208] but also contributes to the stabilization of the 3D protein structure and modulates the dynamics of a protein in a variety of ways [209]. In biological systems water is usually divided into three distinctly different classes: (i) internal, strongly bound water that cannot be removed even upon lyophilization; it contributes to the stabilization of the native protein structure. Internal water mol- ecules that are hydrogen-bonded to specific amino-acid residues in a water pocket or are mobile along inner protein channels are often of key importance for protein function [210–212]; (ii) surface water in the hydration shell of the protein at the solute–solvent interface, and (iii) bulk water randomly distributed in the protein matrix. Water molecules in the protein hydration layer have restricted dynamics with respect to water molecules in the bulk. The thickness of the hydration layer at the solute–solvent interface is still a matter of debate, and the different results reported can often be rationalized by considering that different experimental meth- ods probe different dynamical ranges of molecular motion. Such methods include nuclear magnetic resonance (NMR) [209, 213–215], Dielectric Spectroscopy in the microwave region [216], Terahertz Absorption Spectroscopy [204, 217], Infra- red Spectroscopy (IR) [218, 219], and Neutron Spectroscopy [199]. Parallel to the development of new experimental methods for unraveling solvation dynamics of large molecules, powerful extensions of molecular dynamics simulation techniques have been recently reported [204, 220–222]. Solvated proteins exhibit complex con- formational dynamics at physiologically relevant temperatures fluctuating between a multitude of conformational substates in a rugged energy landscape that is hier- archically organized in energy tiers [206, 223]. For most proteins cooled below the glass-transition temperature (typically around 200 K) their biological function is blocked due to restricted conformational motion. The “freezing out” of conforma- tional dynamics by lowering the temperature is, thus, a common strategy for study- ing function–dynamics relationships in proteins. However, freezing proteins in the presence of a cryoprotectant (used to mini- mize freezing damage by ice crystals) is problematic because it aggravates disen- tanglement of the influences of solvent and temperature on the protein dynamics. An elegant alternative approach is to embed the protein into amorphous matrices formed by disaccharides like trehalose (α-d-glucopyranosil α-d-glucopyranoside) [224, 225]. This allows preservation of the native protein fold during extensive pro- tein dehydration, even at temperatures well above room temperature. 1 3 In nature, the extraordinary bio-protective capabilities of disaccharide glasses are exploited by specific organisms, which are able to survive extreme conditions of temperature and dehydration by entering a state of reversibly arrested metabolic activity, called anhy- drobiosis or cryptobiosis [226]. Extensive spectroscopic work, exploiting neutron scattering [227, 228], Raman [229], optical laser-flash [230], FTIR [231], and EPR [8] on different proteins incorporated into trehalose glasses have revealed a tight protein–matrix dynamic coupling at low water content. This implies that the protein conformational dynamics is controlled by that of the water–trehalose matrix coating the protein surface [8, 227–232]. At room temperature, the stepwise dehydration of 1 3 High‑field/High‑frequency EPR Spectroscopy in Protein… 251 the trehalose matrix results in increasingly inhibited dynamics of the embedded pro- tein. This was observed in both small globular proteins like myoglobin [224, 227] and large membrane proteins like bacterial photosynthetic reaction centers [225, 233] and photosystem I [234] of oxygenic photosynthesis.fi the trehalose matrix results in increasingly inhibited dynamics of the embedded pro- tein. This was observed in both small globular proteins like myoglobin [224, 227] and large membrane proteins like bacterial photosynthetic reaction centers [225, 233] and photosystem I [234] of oxygenic photosynthesis.fi Trehalose is the most efficient sugar for bio-protection against extreme dehydra- tion and osmotic stress. The exact mechanistic details of the trehalose efficiency are not clear yet, but likely involve an interplay between several factors that include its extraordinarily high glass-transition temperature (385 K) [235], its polymorphism adopting several crystalline and amorphous states [236], its distinct propensity for hydrogen-bonding and the pronounced rigidity of its dehydrated glass matrix [237]. There exist several mechanistic hypotheses for explaining the exceptional behavior of trehalose (for a recent review, see ref. [238]). Generally speaking, these hypoth- eses, which are not mutually exclusive, vary in the fate of water molecules close to the protein surface and on how much the first solvation shell is depleted in favor of H-bond forming trehalose molecules. A dehydration of the protein certainly shifts the hydration equilibrium, but it is not known to what extent. In this work, we applied pulsed multiresonance EPR techniques at high micro- wave frequency (W-band, 94 GHz) and a correspondingly high magnetic Zeeman field (3.4 T) to detect and quantify the accessibility of local water in bacterial photo- synthetic reaction centers (bRC) from Rhodobacter (Rb.) sphaeroides R26 that were embedded in trehalose glasses at distinct hydration levels. 1 3 The bRC is an integral membrane protein that catalyzes the initial photochemical processes to convert light energy into chemical free energy [239–241]. In the R26 mutant, the paramagnetic Fe2+ cofactor is replaced by diamagnetic Zn2+ to avoid excessive line broadening due to the paramagnetic Fe2+ ion [242]. The three bRC protein subunits, L, M, and H, host several cofactors [243] sequen- tially involved in light-driven electron transfer. Two bacteriochlorophyll a (BChl a) molecules near the periplasmic side of the membrane form the “special pair” P865. After photo-excitation to its singlet state, the subsequent electron transfer proceeds predominantly via the protein branch A [239] to the QA acceptor. In the present EPR study, the secondary electron transfer Q⋅− A → QB was blocked by QB depriva- tion (here, using stigmatellin [244]. The anion radical Q⋅− A is of particular concern. The nitroxide spin-label MTSSL was attached to Cys156 in the H-protein subunit. By EPR spectroscopy one can measure the electron Zeeman and electron–nuclear hyperfine interactions and thereby probe the unpaired electron spin density distribu- tion in the transient ionic radical states, P⋅+ 865 and Q⋅− A , which are created during light- induced charge separation. These paramagnetic states provide unique information about the electronic structure of the electron-transfer system and, hence, have been thoroughly characterized previously by both cw and pulse EPR, for review see refs. [1, 140]. P865 and QA are located close to the periplasmic and cytoplasmic sides of the membrane, respectively, and are, thus, well suited to be used to study local water accessibility in the protein near its surface. In the bRC preparation of this work, with no cytochrome present and electron transfer from Q⋅− A to QB blocked, the charge- separated state P⋅+ 865Q⋅− A recombines to P865QA within 100 ms by direct electron tun- neling [239]. Hence, continuous illumination generates a steady-state EPR signal of the P⋅+ 865Q⋅− A state that rapidly decays once the light is switched off. The W-band EPR 1 3 3 252 K. Möbius, A. Savitsky Fig. 17 a Top: Simulated rigid limit W-band EPR absorption spectra of P⋅+ 865 (blue trace) and Q⋅− A (red trace), the radical-ion states of the primary donor and acceptor. For the simulation, previously reported magnetic parameter values were used [245, 246]. Bottom: Simulated rigid limit W-band EPR absorption spectrum of the nitroxide radical R1 (green trace). 1 3 1 3 1 3 High‑field/High‑frequency EPR Spectroscopy in Protein… 253 In bRCs, the coating with trehalose affects the electron-transfer step between the pri- mary and secondary quinone acceptors [233] which is a conformationally gated pro- cess [253]. The trehalose matrix also influences the lifetime of the charge-separated radical-pair state, P⋅+ 865Q⋅− A . Upon progressive dehydration of the trehalose matrix, the kinetics of P⋅+ 865Q⋅− A charge recombination becomes faster and exhibits widely dis- tributed rate constants [225, 230, 252, 254]. Thus, it mimics at room temperature the bRC recombination kinetics observed at cryogenic temperatures in the water–glyc- erol system when frozen in the dark [255, 256].f A reasonable explanation for this matrix effect is the stronger dynamical coupling between sugar and protein via hydrogen-bonding networks in a dehydrated treha- lose matrix. It blocks the fluctuations between conformational substates of the bRC [230], as put forward in the “anchorage hypothesis” [232]. The progressive dehydration of sugar glasses requires a change of the water equi- libria between the first solvation shell and bulk water (and possibly also between strongly bound internal water). Two relative hydration levels, r, were chosen in this work that result in a different extent of slaving the protein dynamics to the embed- ding sugar matrix: (i) r = 11% for which the protein dynamics is arrested on the time scale of seconds [252], and (ii) r = 74% for which the dynamics is only mildly retarded as compared to solution [14]. Using EDNMR, we aim to probe directly if the dehydration of the sugar matrix results in a changed hydration level of the inner protein core surrounding the native cofactors, P⋅+ 865 and Q⋅− A . To trace the exchange of water between the sugar matrix and the interior of the protein, the use of isotope- labeled water is essential, with regard to both deuterium and oxygen-17. By high- field EPR, we can test how the hydration of the sugar matrix translates into water exchange between the sugar matrix and either protein surface or protein interior. Fig. 18 a W-band EDNMR spectra for SL-bRC/trehalose glass samples equilibrated for 70 h at r = 74% (NaCl/H2 17O) black trace; r = 11% (LiCl/H2 17O) magenta trace; and r = 11% (LiCl/H2O) red trace. The spectra were recorded at the same nitroxide spectral position (gzz, MI = 0). 1 3 The magnetic parameter values used for the spectrum simulation were previously reported for a nitroxide dissolved in a water/glycerol mixture [247]. b Chemi- cal structures of the pyrroline type MTSSL nitroxide radical R1 and the disaccharide trehalose (color figure online) Fig. 17 a Top: Simulated rigid limit W-band EPR absorption spectra of P⋅+ 865 (blue trace) and Q⋅− A (red trace), the radical-ion states of the primary donor and acceptor. For the simulation, previously reported magnetic parameter values were used [245, 246]. Bottom: Simulated rigid limit W-band EPR absorption spectrum of the nitroxide radical R1 (green trace). The magnetic parameter values used for the spectrum simulation were previously reported for a nitroxide dissolved in a water/glycerol mixture [247]. b Chemi- cal structures of the pyrroline type MTSSL nitroxide radical R1 and the disaccharide trehalose (color figure online) spectra of both P⋅+ 865 and Q⋅− A reveal rhombic g-tensors [245, 246] resulting in charac- teristic line shapes, see Fig. 17a.i The native bRC contains five cysteine residues which are buried within the pro- tein domains except for cysteine 156 which is moderately solvent exposed in subunit H. This residue can be site-specifically spin labeled with an external paramagnetic probe molecule. We used MTSSL (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl- methanethiosulphonate) as spin label [248, 249]. The nitroxide-labeled bRC protein [250] (SL-bRC) provides a third paramagnetic probe within the bRC in addition to the “natural” radical ions P⋅+ 865 and Q⋅− A . According to the known protein 3D struc- ture, the “artificial” nitroxide spin label experiences a water accessibility very differ- ent from that of the native cofactors, P865 and QA. Even under high-field (W-band) conditions, the close similarity of the g-tensor components results in strong overlap of the EPR spectra of P⋅+ 865 , Q⋅− A and nitroxide spin label embedded in their protein matrix environment. A powerful pulse EPR technique that can be employed also at W-band frequen- cies is EDNMR. [189] Using a second mw source, it overcomes the instrumental limitations of the ESEEM single-resonance technique at W-band. Recently, W-band EDNMR has been demonstrated to provide superior sensitivity as compared to ENDOR [12]. Also the solvation properties of large protein metal complexes [195, 251] could be successfully studied by W-band EDNMR.f The effect of bRC coating in a trehalose glass matrix has been thoroughly studied previously using laser-flash optical and EPR spectroscopies [8, 225, 230, 233, 252]. 1 3 When decreasing the mw field amplitude, the overall signal intensity decreases, but the matrix 17O line is affected to a larger extent. To suppress the matrix line almost completely, a two times lower mw field amplitude and shorter HTA pulse length were used, see blue trace in Fig. 18c.i Subsequently, W-band Davies ENDOR measurements were performed to confirm the 17O hyperfine coupling constant as extracted from the EDNMR spectrum, see green trace in Fig. 18c. In Davies ENDOR exclusively coupled water is probed since the signal from distant 17O nuclei is suppressed because of the blind spot at 휈n(17O). [33] The presence of the 17O line in the EDNMR spectrum of the dehydrated pro- tein/trehalose glass (r = 11%) unambiguously shows that, despite extensive dehydra- tion and the high rigidity of the trehalose, the water retained in the sugar and the water on the protein surface can exchange. We conclude: hi k d h ibili h i i i hi h We conclude: In this work, we compared the water accessibility at three protein sites within the reaction center from the purple bacterium Rb. sphaeroides R26 that hosts the two native radical cofactor ions P⋅+ 865 and Q⋅− A and the artificial nitroxide spin label. The three paramagnetic reporter groups have distinctly different local environments in the protein and can probe different types of water molecules, internal water, bulk water and surface water. The native cofactors buried within the protein report on strongly bound internal water and on bulk water in pools and/or channels, the nitrox- ide radical is surface attached and reports on the first and second solvation shell of the protein. The bRCs were embedded into a trehalose glass and equilibrated in the atmos- phere of either low (r = 11%) or high (r = 74%) relative humidity. The resulting sugar matrix has very different properties: At low humidity and corresponding low hydra- tion level the protein-matrix system is rigid with only 0.5 water molecules per sugar molecule; at higher humidity and hydration level the sugar matrix is less rigid and contains more water molecules as potential partners for hydrogen bonding to the protein and to the sugar matrix. 1 3 b The 17O EDNMR spectra for r = 74% (NaCl/H2 17O) (black trace) and r = 11% (LiCl/H2 17O) (magenta trace) samples. The gray dashed lines show the best-fit simulation of experimental recordings to a Gaussian line. c Microwave field ampli- tude dependence of 17O EDNMR spectra of SL-bRC/trehalose glasses equilibrated for three days at r = 11% (LiCl/H2 17O). The black spectra were recorded using an HTA pulse length of tHTA= 20 μs. The blue trace shows the EDNMR spectrum acquired with tHTA= 7 μs. The green line at the bottom shows the Davies ENDOR spectrum recorded at the same spectral position (color figure online) Fig. 18 a W-band EDNMR spectra for SL-bRC/trehalose glass samples equilibrated for 70 h at r = 74% (NaCl/H2 17O) black trace; r = 11% (LiCl/H2 17O) magenta trace; and r = 11% (LiCl/H2O) red trace. The spectra were recorded at the same nitroxide spectral position (gzz, MI = 0). b The 17O EDNMR spectra for r = 74% (NaCl/H2 17O) (black trace) and r = 11% (LiCl/H2 17O) (magenta trace) samples. The gray dashed lines show the best-fit simulation of experimental recordings to a Gaussian line. c Microwave field ampli- tude dependence of 17O EDNMR spectra of SL-bRC/trehalose glasses equilibrated for three days at r = 11% (LiCl/H2 17O). The black spectra were recorded using an HTA pulse length of tHTA= 20 μs. The blue trace shows the EDNMR spectrum acquired with tHTA= 7 μs. The green line at the bottom shows the Davies ENDOR spectrum recorded at the same spectral position (color figure online) 1 3 254 K. Möbius, A. Savitsky For details of the sample preparation, the controlled dehydration protocols, and the EDNMR/ENDOR experiments, see [16]. Similar to ENDOR, in EDNMR spec- tra the hyperfine or/and quadrupole coupled proton or deuterium nuclei yield signals centered at the nuclear Zeeman frequency 휈n(H) or 휈n(D), split by the hyperfine or quadrupole coupling constant.l In the following, the main results of this work will be briefly summarized: l Figure 18c shows EDNMR spectra recorded for SL-RC/trehalose at r = 74% (H2 17O) with variable mw field amplitudes. At the highest mw field amplitude used, the intensity of the 17O EDNMR line is about 0.1, and both distant and coupled 17O nuclei are detected. 1 3 Different hydration levels, therefore, affect the pro- tein dynamics differently: At r = 11%, the dynamics is blocked [9, 14, 252], at least on the timescale of P⋅+ 865-Q⋅− A charge recombination, but is not affected at r = 74%. This effect of the dry trehalose matrix, to inhibit internal protein dynamics (leading to bio-protection of proteins against thermal denaturation), was ascribed earlier to a strong “slaving” of the protein dynamics to that of the embedding matrix [206, 223]. The detailed molecular mechanism of bio-protection is still unclear and leaves open questions, for instance: Why does the hydration of different embedding disaccharide sugars has a strongly different effect on the solvation shell of the protein and on the 1 3 High‑field/High‑frequency EPR Spectroscopy in Protein… 255 dynamics of the water molecules inside the protein [14]? For a detailed discussion of this issue, see [16].ii dynamics of the water molecules inside the protein [14]? For a detailed discussion of this issue, see [16].ii As to the scientific significance of such studies, we want to emphasize that hydra- tion water plays a crucial role in protein dynamics and structural relaxation on all time scales. In the literature [206, 223], it is suggested that changes in the amount of hydration water affect not only the protein’s energy landscape but also significantly affect structural fluctuations between the “conformational substates” and, thereby, control biological function. Hence, understanding the functional difference between surface-bound water and bulk water is a key issue for controlling the biological function of macromolecules, including proteins, DNA, RNA, and photosynthetic reaction centers, right up to enabling anhydrobiotic food preservation and storage in dehydrated trehalose matrices at room temperature. Water is just far more than merely a solvent. So, is dehydrated trehalose the elixir of Life? As far as countering extreme stress conditions by evolutionary adapted living organism are concerned, probably yes. Apparently, trehalose works so well because it possesses an optimum mix of physi- cal and chemical parameters that Nature can provide and implement into a single molecular complex. None of them will be able to accomplish the task of anhydrobi- otic stabilization on its own. What works is probably a cumulative effect of all the mechanisms put forward already in the various theories in the literature. 1 3 The relative contribution of each parameter is adjusted according to the specific stress factor that has to be encountered since it endangers cellular survival in evolutionary selection.i What, at first glance, appears to be of mere academic interest in the biophysi- cal and biological sciences reveals, at second glance, a promising future for treha- lose applications in food and medical industries: Owing to its unique properties as allowing for anhydrobiotic survival under harsh conditions of dryness and heat trehalose proved to be an active stabilizer of enzymes, proteins, vaccines, pharma- ceutical preparations, and even organs for transplantation. And in molecular engi- neering departments of biological research institutions, scientists are trying to copy the genetic code of “resurrection plants” in deserts to adapt “normal” plants to the looming global warming of the climate catastrophe. Among the large variety of photosynthetic species are those with the astounding capability to survive extreme conditions of heat (higher than 60 °C) and dryness (more than 99% of their water body removed) as are typical for hot desert climates. An ultimate strategy by Nature for the survival of drought, in which an organism loses virtually all its free intercellular water, is anhydrobiosis in which the organism ceases metabolism for several years but remains capable of revival after rehydration. Hence, plants qualified for anhydrobiosis have been romanticized as resurrection plants. The pressing question is how anhydrobiosis is accomplished by the organ- isms. A common theme among such organisms is that during photosynthesis activ- ity they accumulate large quantities of non-reducing disaccharides, the most com- mon of which is trehalose. Survival in the dry state is strongly correlated with a high concentration of this sugar, but also sucrose is found in high concentration in certain organisms in their anhydrobiotic state. Anyhow, trehalose appeared to be particu- larly effective in stabilizing this state of “Life without water”. 3 256 K. Möbius, A. Savitsky This concept of fluctuations between a large number of different conformations in a hierarchically structured energy landscape has been thoroughly studied over the past decades, both theoretically and experimentally, for well-characterized pro- tein complexes, such as myoglobin as a paradigm system for globular proteins. And the Swiss-American physicist Hans Frauenfelder from the University of Illinois at Urbana-Champaign, later at Los Alamos National Laboratory, was a key figure in this field. 1 3 He was born July 28, 1922, he died July 10, 2022 in Tesuque, New Mex- ico, shortly before celebrating his 100th anniversary. To quote a wise contemporary of his, Richard Feynman (1918–1988): “… everything that living things do can be understood in terms of the jigglings and wigglings of atoms.” “… everything that living things do can be understood in terms of the jigglings and wigglings of atoms.” “… everything that living things do can be understood in terms of the jigglings and wigglings of atoms.” The control exerted on the RC dynamics and ET kinetics by the fluctuations of the cofactor environment could be clarified within the framework of the “unified model of protein dynamics” developed by Hans Frauenfelder and his co-workers. In particular, the RC from the purple bacterium Rb. sphaeroides provides a privileged model system for exploring the relationships between electron-transfer processes and protein conformational dynamics. 2.2.2 Electron–Electron Dipolar Spectroscopy The determination of distance and orientation of protein domains and their changes in the course of biological action is of primary concern in proteomics to elucidate the relation between structure, dynamics and function. A variety of biophysical techniques have been developed and applied to measure distances (and orienta- tions) between spin centers in large biosystems. Often, these systems are available only as disordered samples as frozen solutions so that X-ray crystallography is not applicable. For paramagnetic disordered systems, however, EPR spectroscopy offers powerful tools to obtain structural information over wide distance ranges. They are based on dominating anisotropic spin interactions. By this approach, established techniques like FRET (fluorescence energy transfer) or solid-state NMR are com- plemented in terms of distance ranges. For anisotropic electron–nuclear hyperfine interactions, as is preferentially measured by ENDOR techniques, the accessible dis- tance range stays well below 1 nm (10 Å). For dominating dipolar electron–electron interaction, however, when measured by specialized microwave pulse sequences in pulsed electron–electron double resonance (PELDOR) techniques, the distance range can be dramatically extended, in ideal cases to about 8 nm (80 Å) [257], in proteins realistically to about 5 nm (50 Å). This means that even distances between cofactor radicals and radical pairs across photosynthetic membranes can be meas- ured! This is good news for in-depth electron-transfer studies of primary photosyn- thesis (see below). For large distances between well-localized electron spins A and B in a radical pair, for which the point-dipole approximation holds and the exchange coupling, J , can be neglected, the electron–electron dipolar Hamiltonian, ̂Hss , is commonly writ- ten as 1 3 1 3 High‑field/High‑frequency EPR Spectroscopy in Protein… 257 ( ̂Hss h = −휇0 4휋⋅h ⋅ gA (휃A, 휑A ) ⋅gB (휃B, 휑B ) ⋅휇2 B r3 AB ⋅(A + B + C + D + E + F), (20) where gA (휃A, 휑A ) , gB (휃B, 휑B ) are the orientation-dependent g-values of radicals A and B selected by the external magnetic field B0 , 휇B is the Bohr magneton, 휇0 the vacuum permeability, and h the Planck constant. In analogy of the Hamiltonian of the anisotropic part of the electron–nuclear hyperfine interaction (see Eq. 2.2.2 Electron–Electron Dipolar Spectroscopy 7), the six terms A, B, ..., F represent products of spin-component operators and angular expres- sions in a spherical coordinate system ( rAB, 휃, 휑 ), in which r describes the radial distance of spin B from spin A located at the origin (or vice versa). The zenith and azimuth polar angles 휃 and 휑 describe the orientation of the radical-pair axis with respect to the external Zeeman field B0 [258]: (21) A = ̂SA z ⋅̂SB z ⋅(3cos2휃−1), B = 1 4 ⋅ ( ̂SA + ⋅̂SB −+ ̂SA −⋅̂SB + ) ⋅(3cos2휃−1), C = 2 3 ⋅(̂SA + ⋅̂SB z + ̂SA z ⋅̂SB +) ⋅sin휃⋅cos휃⋅e−i휑, D = 2 3 ⋅(̂SA −⋅̂SB z + ̂SA z ⋅̂SB −) ⋅sin휃⋅cos휃⋅ei휑, E = 3 4 ⋅̂SA + ⋅̂SB + ⋅e−2i휑, F = 3 4 ⋅̂SA −⋅̂SB −⋅e2i휑. (21) The ̂SA(B) x and ̂SA(B) y spin operators are expressed in terms of the raising and lower- ing shift operators ̂SA(B) + and ̂SA(B) − . Note the formal analogy of the electron–electron dipolar interactions with the electron–nuclear dipolar (END) interaction discussed above. Following the same arguments as used for the END hyperfine interaction [1], in high magnetic fields, when the dipolar coupling of the two unlike electron spins is small compared to the difference of their Zeeman interactions, the dipolar splitting of the EPR transitions is predominantly determined by the ’’secular’’ ̂SA z ⋅̂SB z term A in Eq. (7), whereas the ’’pseudo-secular’’ ̂SA +(−) ⋅̂SB −(+) term B can be neglected to first order. The ’’non-secular’’ terms C, D, E, and F are not important at all for the dipolar energy splitting [137]. The pseudo-secular term becomes important in the case of like spins, i.e., when the dipolar coupling is large compared to the difference of their Zeeman interac- tions. This leads to a scaling of the dipolar coupling frequency by a factor of 3/2 as compared to the case of unlike spins [124]. The intermediate case with compara- ble magnitudes of the dipolar coupling and Zeeman splitting is more complex, and adequate data analysis requires simulation of the spectra on the basis of the full spin Hamiltonian of the molecular system, including both secular and pseudo-secular terms of the dipolar interaction. 2.2.2 Electron–Electron Dipolar Spectroscopy When working at a particular microwave frequency, the validity of the unlike-spin limit is guaranteed only for a large enough distance of the spins, e.g., rAB ≥ 2 nm (20 Å) at X-band [124]. In the case of a pair of two nitroxide radicals, which are frequently used for distance measurements on doubly site-specifically labeled proteins, the two nitroxide spins usually have their Zeeman (and hyperfine) frequencies substantially different and, hence, fulfill the condition 1 3 258 K. Möbius, A. Savitsky of the unlike-spin limit. The difference in resonance frequencies of the two radicals arises from their different orientations with respect to the Zeeman field ⃗B0 , so that the effective g- and hyperfine-values of their g- and hyperfine- tensors are different. of the unlike-spin limit. The difference in resonance frequencies of the two radicals arises from their different orientations with respect to the Zeeman field ⃗B0 , so that the effective g- and hyperfine-values of their g- and hyperfine- tensors are different. fiif Neglecting the exchange coupling, in the unlike-spin limit the dipolar coupling frequency, 휈AB , is given by (22) 휈AB(휃) = 휈d ⋅(1 −3cos2휽), (22) ith the dipole–dipole coupling parameter 휈d = 휇0 4휋⋅h ⋅ 휇2 B⋅gA⋅gB r3 AB . with the dipole–dipole coupling parameter 휈d = 휇0 4휋⋅h ⋅ 휇2 B⋅gA⋅gB r3 AB . AB Here, the gA and gB values of the radical partners A and B are the weighted prin- cipal components of their g-tensors, i.e., weighted according to their orientations in the pair, rAB is their distance and 휃 the angle between the Zeeman field B0 and the interspin distance vector rAB . For an isotropic distribution of angles 휃 , i.e., iso- tropic frozen sample without orientational selectivity of the experiment, a charac- teristic powder-type spectrum, the ’’Pake pattern’’, is obtained. From this, rAB can be deduced, provided gA and gB are known from independent EPR experiments. At sufficiently good signal-to-noise ratio, the dipole–dipole coupling parameter νd can be directly read off from the singularities of the Pake pattern. f The methodological challenge is to devise an EPR strategy that separates the electron–electron coupling from other interactions, such as electron–nuclear hyper- fine interactions and inhomogeneous line broadening. Common to most elec- tron–electron dipolar EPR methods for weakly coupled radical-pair systems, in which the dipolar coupling is smaller than the cw EPR linewidth, is the electron spin echo (ESE)-detected mode of operation. 1 3 2.2.2 Electron–Electron Dipolar Spectroscopy It is an inherent strength of the ESE method that it is ’’blind’’ toward static energy contributions, thus eliminating the masking effects of any source of inhomogeneous EPR line broadening [259]. Only when the interactions become time-dependent and, thus, contribute to T2 relaxation of the spin system, they determine the spin-echo formation. In cases of anisotropic spin interac- tions, such as electron–nuclear dipolar or quadrupolar interactions or electron–elec- tron dipole interaction, the echo amplitude may become modulated (ESEEM, spin echo envelope modulation) when the time between the pulses in the echo sequence is varied. In a biradical with interspin distances less than 80 Å the modulation pattern is often determined by the electron–electron dipole coupling frequency. The inhomo- geneous broadening of nitroxide lineshapes from unresolved intramolecular proton or deuteron hyperfine interactions must be considered when extracting relaxation data from cw spectra, whereas the ESE measurements are independent of any source of inhomogeneous broadening. The pulse electron–electron dipolar spectroscopy was originally introduced 1981–1984 in Novosibirsk as a 3-pulse method employing two microwave frequen- cies [64, 260–264] to measure weak electron–electron dipolar couplings from ESE decays. It soon received the acronym PELDOR (pulsed electron–electron dou- ble resonance). About a decade later, in 1993, the first publication on pulse elec- tron–electron dipolar spectroscopy appeared from outside Novosibirsk [265]; the authors discussed orientational selection and created the acronym DEER (dou- ble electron–electron resonance). In the 4-pulse DEER method [65], an additional 1 3 High‑field/High‑frequency EPR Spectroscopy in Protein… 259 refocusing mw pulse is introduced that provides dead-time free data collection. As the evolution of electron double quantum coherence (DQC) also depends on the electron dipole–dipole interaction, a powerful 6-pulse DQC method was introduced by the Freed group at Cornell [266, 267]. Commonly used pulse EPR schemes as well as dedicated cw EPR experiments for distance measurements are described in many review articles and books, for example in the anthology Distance measure- ments in biological systems by EPR, edited by L. J. Berliner, G. R. Eaton and S. S. Eaton [63]. In this book, experts in the field report in detail about the pros and cons of distance measurements by cw and pulse EPR methods when applied to a broad variety of biological two-spin systems. They range from organic radicals and transi- tion-metal ions to dipolarly coupled nitroxide spin labels, to donor–acceptor cofactor radical-ion pairs in photosynthetic reaction centers. 2.2.2 Electron–Electron Dipolar Spectroscopy An important book covering in depth the subject of distance measurements by electron dipolar spectroscopy is the monograph Principles of pulse electron paramagnetic resonance by A. Schweiger and G. Jeschke [33]. Several excellent reviews have appeared in the last few years covering both methodologies and applications of pulse dipolar EPR spectroscopy, primarily on structural biology of spin-labeled systems which have attracted grow- ing attention, for example [122–124, 268–273].i The high-field extension of 3-pulse PELDOR, which was developed with the aim to resolve the relative orientation of the radical-pair partners, is very powerful for large protein systems [128]. Also the conceptually related, but single-frequency dipolar spectroscopy method RIDME (relaxation-induced dipolar modulation enhancement) [67] has been extended to high magnetic fields and microwave fre- quencies. Interesting RIDME applications have been reported already, for example at 130 GHz on nitroxide biradicals [274] or at 95 GHz on donor–acceptor radical pairs in photosynthetic reaction centers [128].l Here, we will briefly describe a few aspects of PELDOR and RIDME at high Zeeman fields that are pertinent for measuring both distance and relative orienta- tion of radical-pair partners. Orientation resolving high-field PELDOR (or DEER) experiments have been pioneered independently by three research groups: the Fig. 19 The most important dual-frequency pulse schemes of pulse electron–electron double resonance. a 3-pulse constant-time PELDOR [64]; b 3-pulse variable-time PELDOR; c 4-pulse constant-time PEL- DOR (DEER) [65]; d 4-pulse variable-time PELDOR, all based on a stimulated-echo sequence [128, 276] Fig. 19 The most important dual-frequency pulse schemes of pulse electron–electron double resonance. a 3-pulse constant-time PELDOR [64]; b 3-pulse variable-time PELDOR; c 4-pulse constant-time PEL- DOR (DEER) [65]; d 4-pulse variable-time PELDOR, all based on a stimulated-echo sequence [128, 276] 1 3 3 260 K. Möbius, A. Savitsky Prisner group in Frankfurt [125, 126, 275], the Jeschke group, then in Konstanz [127], and the Möbius group in Berlin [128, 152]. Prisner group in Frankfurt [125, 126, 275], the Jeschke group, then in Konstanz [127], and the Möbius group in Berlin [128, 152]. 2.2.2.1 PELDOR Figure 19 shows the most prominent pulse schemes typically employed for dual-frequency PELDOR (or DEER) experiments. The 3-pulse version was first introduced by A. D. Milov, K. M. Salikhov, M. D. Shirov [261], see Fig. 19a. It is analogous to the SEDOR (spin-echo double resonance) sequence used in solid- state NMR to detect the coupling between two nuclear spins [277]. 2.2.2 Electron–Electron Dipolar Spectroscopy In this sequence, a 2-pulse Hahn echo sequence at 휈a with a fixed pulse separation time 휏1 is involved to selectively detect the echo intensity of the radical A of the A-B radical pair. An addi- tional microwave pulse at time 휏2 after the first pulse and at the microwave frequency νb flips the B spin by 180°. If the spins A and B are coupled by dipolar interaction, the detected echo intensity becomes modulated when varying 휏2 . The interaction fre- quency 휈AB is obtained by Fourier transformation of the echo time trace.i Our strategy for high-field PELDOR spectroscopy is as follows: We use the 3-pulse stimulated-echo (SSE) sequence, in which the mw pulses are frequency adjusted to the observer spins A, and apply within the mixing period between the SSE pulses an additional mw π-pulse in resonance with the partner spins B to flip them, see Fig. 19d. The PELDOR-time trace is obtained measuring the SSE echo intensity at different 휏 values and using the “reference-signal deconvolution” tech- nique. The reference time trace is obtained by simply skipping the π-pulse at 휈b . Dividing the trace with the additional π-pulse by the reference trace yields the pure dipolar evolution function. When performed at stepped resonance field positions of both excitation sites within the spectra of the coupled radicals A and B, this strat- egy allows to find the particular field positions in the EPR spectrum that are con- jugated by dipolar interaction between the A and B spins of the radical pair. For a detailed discussion of this strategy, see [1]. Orientational information on radicals in disordered solids is, of course, only available at a sufficient degree of orientation selectivity in the EPR spectrum. Therefore, PELDOR experiments in high-field EPR spectroscopy with adequate Zeeman magnetoselectivity appear to be a promising approach for structure determination of disordered protein systems [128]. An obvious problem is the excitation bandwidth which must exceed the dipolar coupling, since both EPR lines of the dipolar doublet of a given A or B spin have to be excited. This means that in dual-frequency (DF) PELDOR experiments, the pump pulse at the second resonance frequency 휈b must excite a significant fraction of B spins that are dipole-coupled to the observer spins A, whose resonance fre- quency is 휈a . 2.2.2 Electron–Electron Dipolar Spectroscopy In high-field EPR, the spectral width of the two radicals in the weakly coupled pair increases in proportion to their difference in g-values and applied Zee- man field. Fortunately, the bandwidth of the EPR resonator also increases with high mw frequency. Even for a single-mode cavity with high Q value, a bandwidth of 100 MHz was achieved at 95 GHz (W-band). This is sufficient for accommodating both 휈a and 휈b for donor–acceptor radical pairs in photosynthesis, but not for nitrox- ide spin-labeled radical pairs. W-band EPR spectra of nitroxide radicals are typically spread over a range of 400 MHz as compared to 200 MHz at X-band. We, thus, see that DF-PELDOR at W-band is applicable for donor–acceptor ion radical pairs in 1 3 High‑field/High‑frequency EPR Spectroscopy in Protein… 261 photosynthesis, and detailed information of interspin distance and orientation of the charge-separated cofactors in the photocycle could be obtained [128]. photosynthesis, and detailed information of interspin distance and orientation of the charge-separated cofactors in the photocycle could be obtained [128]. Here is an interesting example: Example: Conformational changes during light-induced electron transfer in photosynthesis The chosen example of applications of high-field EPR and PELDOR spectros- copy mirrors our fascination for one of the great intellectual challenges of biology, the separation of charges induced by light, which is essential for photosynthesis and, hence, Life on Earth. New research advances the understanding of microbial photo- synthesis provides new insight into the atomic structures and synthesizing mecha- nisms of key photosynthetic proteins. The capture of the sun’s energy through pho- tosynthesis in distinct photosynthetic organisms is the fundamentals of almost all life on Earth. Under the current circumstances of global climate change and food/ energy crisis, there has been a revival of considerable interest in studying how pho- tosynthetic bacteria and plants trap and convert solar energy in nature. The reaction center (RC) of the purple photosynthetic bacterium mutant Rhodo- bacter (Rb.) sphaeroides R26 is a pigment–protein complex that is capable of con- verting light energy to chemical energy with quantum yield approaching 1. Electron transfer (ET) in this RC has been extensively studied; the structure and spectro- scopic features of the complex are well known, the complex is very stable, and a large variety of mutants is available in addition to the wild type (WT). This RC also serves as a model system for understanding protein–cofactor interactions and the Fig. 2.2.2 Electron–Electron Dipolar Spectroscopy 20 a X-ray structural model of the RC from the photosynthetic purple bacterium Rb. sphaeroides R26 [279] composed of the protein subunits L, M, H and the cofactors P865, BChl, BPhe, Q and Fe (P865, primary donor “special pair” of bacteriochlorophyll a (BChl a); BPhe, bacteriopheophytin a; Q, ubiqui- none acceptors QA, QB; Fe, non-heme iron Fe2+). Light-induced electron transfer (ET) for charge separa- tion proceeds predominantly along the A branch of the protein-embedded cofactors ("unidirectionality" enigma) despite the approximate C2 symmetry of the cofactor arrangement. For the EPR experiments reported here, the non-heme high-spin iron Fe2+ in the RC was replaced by diamagnetic Zn2+ to avoid magnetic interaction with the iron. b The ET time constants range from 2 ps to 100 μs in the cascade of transmembrane charge-separation steps. For details and references, see overview [1] Fig. 20 a X-ray structural model of the RC from the photosynthetic purple bacterium Rb. sphaeroides R26 [279] composed of the protein subunits L, M, H and the cofactors P865, BChl, BPhe, Q and Fe (P865, primary donor “special pair” of bacteriochlorophyll a (BChl a); BPhe, bacteriopheophytin a; Q, ubiqui- none acceptors QA, QB; Fe, non-heme iron Fe2+). Light-induced electron transfer (ET) for charge separa- tion proceeds predominantly along the A branch of the protein-embedded cofactors ("unidirectionality" enigma) despite the approximate C2 symmetry of the cofactor arrangement. For the EPR experiments reported here, the non-heme high-spin iron Fe2+ in the RC was replaced by diamagnetic Zn2+ to avoid magnetic interaction with the iron. b The ET time constants range from 2 ps to 100 μs in the cascade of transmembrane charge-separation steps. For details and references, see overview [1] 1 3 262 K. Möbius, A. Savitsky role that protein plays in ET [278]. The RC from Rb. sphaeroides comprises 3 pro- tein subunits, H, M, and L. As shown in Fig. 20, the RC complex binds 9 cofac- tors that form 2 potential ET chains (referred to as A and B) in a C2 symmetric arrangement. The “special pair” (P865) is a dimer of bacteriochlorophyll (BChl) a molecules and is located on the periplasmic side of the cytoplasmic membrane. Two monomeric BChls (BChlA and BChlB, with the subscripts denoting which chain the cofactor belongs to) are present on either side of P865. These are followed by 2 bac- teriopheophytin (BPhe) molecules (BPheA and BPheB). 2.2.2 Electron–Electron Dipolar Spectroscopy A non-heme iron Fe and 2 quinones (QA and QB) are near the cytoplasmic side of the RC. When P is excited by actinic light, an electron is transferred through the A branch cofactors, and then to QB. In the WT RC, the times for ET from P to BPheA to QA to QB are 3 ps, 200 ps, and 200 μs, respectively. The transfer from P to BPheA is thought to be via BChlA, but this is still a controversially debated issue. Photosynthesis generally starts with photon absorption by light-harvesting antenna. The absorbed energy is then rapidly and efficiently transferred to a reaction center (RC) for charge separation. Purple bacteria are the oldest photosynthetic spe- cies and carry out anaerobic photosynthesis in diverse ecological niches. In purple bacteria, light-harvesting 1 (LH1) complexes often associate with the RC to form a RC-LH1 core supercomplex, containing a high content of non-covalently attached pigments such as bacterial chlorophylls and carotenoids, to increase the effective cross section for light absorption of each RC. This then supports photosynthesis to operate effectively over a wide range of environmental light intensities and wave- lengths. For example, purple bacteria are highly efficient in utilizing green and far- red light of wavelengths above 750 nm that plants and algae do not utilize. Due to the simplicity of the photosynthetic machinery and ease of growth, anaerobic purple photosynthetic bacteria have been used to study the fundamental mechanisms of photosynthesis. For decades, X-ray crystallography has been used by structural biologists to solve the structural composition of water-soluble “globu- lar” protein complexes. But not of water-insoluble membrane proteins, which were considered non-crystallizable. In 1982, Hartmut Michel (Munich) succeeded in crystallizing the photosynthetic reaction center of the purple bacterium Rhodop- seudomonas viridis, thus creating the basis for a molecular X-ray crystal structure analysis of a membrane protein complex. The Nobel Prize in Chemistry 1988 was awarded to H. Michel, J. Deisenhofer, and R. Huber for their contributions to crys- tallize the membrane protein and determine the high-resolution X-ray structure of the RC of a photosynthetic purple bacterium. It is noted that over the past few years, the cutting-edge cryo-electron microscopy (cryo-EM) technology has revolutionized the structural understanding of photosynthetic RC-LH1 complexes in a variety of purple bacteria.l The flexibility of the quinone binding site in the RC of Rb. 2.2.2 Electron–Electron Dipolar Spectroscopy sphaeroides R26 has initiated speculations about its functional role in the charge-separation/charge- recombination electron-transfer cycle. For example, such speculations related to potential structural changes associated with QA reduction were fostered by an early observation by D. Kleinfeld, M. Y. Okamura and G. Feher [255] who showed by optical spectroscopy that the rate of recombination from the transient radical-pair 1 3 1 3 High‑field/High‑frequency EPR Spectroscopy in Protein… 263 state P⋅+ 865Q⋅− A to the ground state P865QA differs in RCs cooled to cryogenic tempera- tures in the dark (dark-adapted RCs) compared to RCs cooled under continuous illu- mination (light-adapted RCs) [255]. The authors explained the slower recombination kinetics in the light-adapted sample by suggesting changes of the donor–acceptor average distance (by ≈1 Å) and of its distribution. However, by using FTIR spec- troscopy Breton and co-workers showed that near room temperature QA does not move significantly upon reduction with respect to its protein binding site [280]. To obtain structural information at lower temperatures transient and pulsed EPR stud- ies were performed [281, 282]. No significant changes in the donor–acceptor dis- tance and its distribution were observed by Zech et al. [281] using X- and Q-band transient direct-detection cw EPR and out-of-phase electron spin echo (ESE) experi- ments. On the other hand, Borovykh et al. [282] deduced by X-band electron spin echo envelope modulation (ESEEM) that the donor–acceptor distance in the sample frozen under illumination is slightly larger (≈ 0.4 Å) than that in the sample frozen in the dark. Potential light-induced structural changes associated with the charge- separated state, P⋅+ 865Q⋅− A , have also been investigated by X-ray diffraction on single- crystal RCs frozen either in the dark or under continuous illumination [283]. In con- trast to the secondary quinone QB (see ref. [284]) the X-ray crystallography results for QA showed no significant changes, within the error margin (≈ 0.2 Å), neither in the donor and primary acceptor positions nor in their orientations [283, 285]. A few years ago, Heinen et al. [286] proposed from quantum-beat oscillations of transient Q-band EPR signals ( T = 70 K) of radical pairs P⋅+ 865Q⋅− A in dark-adapted RCs that an unprecedented reorientation of Q⋅− A by as much as 60° upon light-induced charge separation occurs. 2.2.2 Electron–Electron Dipolar Spectroscopy The authors concluded that this large difference in orientations reflects a rotation of the quinone in its ring plane that is caused by structure accom- modation to the charged configuration of the acceptor binding site. At variance with this suggested model [286], Savitsky et al. [128] and Flores et al. [287] concluded from their orientation-resolving W-band PELDOR and Q-band ENDOR studies (at T = 150 K) of the transient P⋅+ 865Q⋅− A radical pair that no large rearrangement at the QA site occur under illumination, see below. We want to point out that we believe that the discrepancy between the studies of refs. [128, 286, 287] is rooted in the inherent sign ambiguity of the measured squares of any spin-interaction tensors and degeneracy of structure solutions, and that in ref. [286] the wrong sign was chosen. The important issue of finding a unique solution for the spin-interaction tensor ori- entation is thoroughly discussed in [1] to which we refer for further reading. The problem of finding the unique structure solution in the specific case of the state P⋅+ 865Q⋅− A to the ground state P865QA differs in RCs cooled to cryogenic tempera- tures in the dark (dark-adapted RCs) compared to RCs cooled under continuous illu- mination (light-adapted RCs) [255]. The authors explained the slower recombination kinetics in the light-adapted sample by suggesting changes of the donor–acceptor average distance (by ≈1 Å) and of its distribution. However, by using FTIR spec- troscopy Breton and co-workers showed that near room temperature QA does not move significantly upon reduction with respect to its protein binding site [280]. To obtain structural information at lower temperatures transient and pulsed EPR stud- ies were performed [281, 282]. No significant changes in the donor–acceptor dis- tance and its distribution were observed by Zech et al. [281] using X- and Q-band transient direct-detection cw EPR and out-of-phase electron spin echo (ESE) experi- ments. On the other hand, Borovykh et al. [282] deduced by X-band electron spin echo envelope modulation (ESEEM) that the donor–acceptor distance in the sample frozen under illumination is slightly larger (≈ 0.4 Å) than that in the sample frozen in the dark. Potential light-induced structural changes associated with the charge- separated state, P⋅+ 865Q⋅− A , have also been investigated by X-ray diffraction on single- crystal RCs frozen either in the dark or under continuous illumination [283]. 2.2.2 Electron–Electron Dipolar Spectroscopy In con- trast to the secondary quinone QB (see ref. [284]) the X-ray crystallography results for QA showed no significant changes, within the error margin (≈ 0.2 Å), neither in the donor and primary acceptor positions nor in their orientations [283, 285]. A few years ago, Heinen et al. [286] proposed from quantum-beat oscillations of transient Q-band EPR signals ( T = 70 K) of radical pairs P⋅+ 865Q⋅− A in dark-adapted RCs that an unprecedented reorientation of Q⋅− A by as much as 60° upon light-induced charge separation occurs. The authors concluded that this large difference in orientations reflects a rotation of the quinone in its ring plane that is caused by structure accom- modation to the charged configuration of the acceptor binding site. At variance with this suggested model [286], Savitsky et al. [128] and Flores et al. [287] concluded from their orientation-resolving W-band PELDOR and Q-band ENDOR studies (at T = 150 K) of the transient P⋅+ 865Q⋅− A radical pair that no large rearrangement at the QA site occur under illumination, see below. We want to point out that we believe that the discrepancy between the studies of refs. [128, 286, 287] is rooted in the inherent sign ambiguity of the measured squares of any spin-interaction tensors and degeneracy of structure solutions, and that in ref. [286] the wrong sign was chosen. The important issue of finding a unique solution for the spin-interaction tensor ori- entation is thoroughly discussed in [1] to which we refer for further reading.ii The problem of finding the unique structure solution in the specific case of the quinone acceptor orientation after light-driven charge separation was solved by com- paring the PELDOR and ENDOR spectra of RCs frozen under illumination with those of RCs frozen in the dark before illumination [128, 281, 282, 287], and doing so under otherwise identical conditions. While PELDOR gives information of the global structure of the dipolarly interacting radical pairs, ENDOR gives direct infor- mation on the local environment of the radicals via their electron–nuclear hyperfine interactions. As has been shown previously [140, 141, 288–290], ENDOR is exqui- sitely sensitive to probe protons H-bonded to quinones in bacterial RCs. 1 3 264 K. Möbius, A. Savitsky Fig. 21 W-band PELDOR spectra of the spin-correlated radical pair P⋅+ 865Q⋅− A in Zn-substituted RCs from Rb. 2.2.2 Electron–Electron Dipolar Spectroscopy sphaeroides at 90 K, a in the sample frozen in the dark, b frozen under continuous illumination. Only those PELDOR responses are shown that were used to probe the spectral position, BQ\|\| , within the Q⋅− A EPR spectrum corresponding to the parallel dipolar frequency, 휈\|\| . The observer mw frequency is fixed at the value corresponding to the field value B′ , while the pump mw is swept through the field region B′′ . Upper part: The individual EPR spectra of P⋅+ 865 and Q⋅− A are shown for referring to the spectral positions. Middle part: Contour plot of the positive Fourier amplitudes of the PELDOR echo decays. Lower part: The contour-plot amplitude (at the slice position) vs magnetic field is shown (dots). The inhomogeneously broadend (intrinsic) EPR linewidth (red line) is 0.29 mT (note that the RC is fully deu- terated allowing for such a narrow linewidth). The broadening of the PELDOR lines (widthΔB1∕2 ) is due to the orientational distribution of Q⋅− A . For further explanations, see refs. [128, 291] Fig. 21 W-band PELDOR spectra of the spin-correlated radical pair P⋅+ 865Q⋅− A in Zn-substituted RCs from Rb. sphaeroides at 90 K, a in the sample frozen in the dark, b frozen under continuous illumination. Only those PELDOR responses are shown that were used to probe the spectral position, BQ\|\| , within the Q⋅− A EPR spectrum corresponding to the parallel dipolar frequency, 휈\|\| . The observer mw frequency is fixed at the value corresponding to the field value B′ , while the pump mw is swept through the field region B′′ . Upper part: The individual EPR spectra of P⋅+ 865 and Q⋅− A are shown for referring to the spectral positions. Middle part: Contour plot of the positive Fourier amplitudes of the PELDOR echo decays. Lower part: The contour-plot amplitude (at the slice position) vs magnetic field is shown (dots). The inhomogeneously broadend (intrinsic) EPR linewidth (red line) is 0.29 mT (note that the RC is fully deu- terated allowing for such a narrow linewidth). The broadening of the PELDOR lines (widthΔB1∕2 ) is due to the orientational distribution of Q⋅− A . For further explanations, see refs. [128, 291] In the following, we briefly summarize the results of such PELDOR and ENDOR experiments on P⋅+ 865Q⋅− A and Q⋅− A in RCs from Rb. sphaeroides [291]. 2.2.2 Electron–Electron Dipolar Spectroscopy The experi- ments are part of a larger research project related to the important question of whether the high quantum yield of photosynthetic primary electron-transfer pro- cesses are due to light-induced structural changes of the cofactor binding sites. In Fig. 21, the PELDOR results for the dark-adapted and light-adapted RCs are summarized. The parameters 휈\|\| and BQ\|\| , which are highly specific for the radi- cal-pair structure ( 휈\|\| for the distance, BQ\|\| for the orientation, see ref. [128]), can be directly read off from the PELDOR spectra. The values 휈\|\| = 4.11 ± 0.02 MHz and BQ\|\| = 3383.08 ± 0.02 mT for the dark-adapted sample fully agree with 휈\|\| = 4.10 ± 0.02 MHz and BQ\|\| = 3383.09 ± 0.03 mT for the light-adapted sample. Also, the angular distribution width, ΔB1∕2 , is the same within experimental error. This implies that neither the interspin distance in the radical pair nor the relative orienta- tion of donor and acceptor ions is different for the different illumination-freezing 1 3 High‑field/High‑frequency EPR Spectroscopy in Protein… 265 protocols. In other words, from PELDOR we learn that there is no conformational redistribution of QA under light-driven reduction. 1 This conclusion is fully supported by the Davies-type pulsed Q-band 1H ENDOR experiment [291]: In the dark-adapted and light-adapted RCs, the ENDOR spec- tra of Q⋅− A are identical within experimental error. The ENDOR results irrefutably show that independent of the history of the freezing and illumination of the RCs, Q⋅− A remains linked to the protein by two asymmetrical H-bonds from His M219 and Ala M260 to the carbonyl oxygens. This conclusion is consistent with earlier ENDOR measurements [141] and DFT calculations [292] of the H-bonding network of the quinone acceptor. For further arguments in support of this conclusion, the reader is referred to ref. [291]. Clearly, further studies will be needed to fully understand the underlying strategies for fine-tuning the efficiency of primary electron transfer in photosynthesis.i p y As we have seen, high-field DF-PELDOR at W-band is well suited to elucidate the three-dimensional structure of the charge-separated radical pair P⋅+ 865Q⋅− A in pri- mary photosynthesis. The situation may be different for two-spin systems of nitrox- ide biradicals for which W-band EPR spectra are typically spread over a range of 400 MHz. For such systems, an alternative to high-field DF-PELDOR would be W-band field-jump (FJ)-PELDOR. 2.2.2 Electron–Electron Dipolar Spectroscopy The fraction of B spins flipping an odd number of times is given by the inversion factor [67] (23) qinv = 1 2 ⋅ ( 1 −e −T T1 ) , (23) where T1 is the spin–lattice relaxation time. Under the conditions T1 >> 𝜏 and ( T1 ⋅휈AB)2 > > 1, the stimulated-echo amplitude as a function of inter-pulse time 휏 is given [67] by (24) s−y(휏) = A1(휏) ⋅A2(T) ⋅[1 −qinv + qinv ⋅cos(2휋⋅휈AB ⋅휏)], (24) where the relaxation factors A1(휏) and A2(T) describe the echo decay because of transversal and longitudinal relaxation of the observer spins. Thus, measuring the echo amplitude will exhibit a periodic oscillation with dipolar frequency 휈AB . Unfor- tunately, hyperfine modulations can obscure the RIDME effect. Therefore, RIDME experiments are best performed at high magnetic fields and microwave frequencies, where hyperfine modulations are strongly reduced [128, 274].fi i If the RIDME experiment is performed successively at different field positions within the partially Zeeman-resolved EPR spectrum of the two radicals of a coupled pair, it can reveal the specific field positions at which the dipolar modulation occurs at the principal values, 휈\|\| = 휈(휃= 0) and 휈⟂= 휈(휃= 휋∕2) , of the dipolar frequency. Magnetoselection at these field positions establishes correlations between the polar angles (휂A(B), 휙A(B)) that define possible orientations of the dipolar axis in the molec- ular frame of radicals in the pair. The main advantage of the RIDME method is its simplicity as compared to PEL- DOR because it is based just on a single-frequency stimulated-echo pulse sequence. It can be performed with any pulsed EPR spectrometer without needing dual-fre- quency or field-jump extensions. Moreover, a large modulation depth, even at high- field EPR, can be reached because the inversion factor qinv depends only on the time T in the stimulated-echo sequence. The main disadvantage of the RIDME method is the partial loss of orientational selectivity. It can be a difficult task to assign the observed modulations to a specific radical in the radical pair, especially if the radicals are identical or their EPR spectra strongly overlap. Additionally, the success of a RIDME experiment depends on the proper choice of experimental conditions, in particular of the sample temperature. The dipolar modulations are enhanced in the case of a pure T1 relaxation process, see Eq. 2.2.2 Electron–Electron Dipolar Spectroscopy Logically, in our laboratory at FU Berlin, we have built a powerful field-jump unit and attached it to our W-band spectrometer [1, 276]. The pulsed magnetic field of 16 mT at the sample inside the TE011 EPR cav- ity, made from Ti–6Al–4V alloy to reduce unavoidable eddy currents, is generated by a pulsed electric current (70 A) flowing through a pair of Helmholtz coils that are fixed outside the cavity and replace the field modulation coils. The screening field from eddy currents in the cavity was found to decay with a time constant of 110 ± 10 ns in the Ti–6Al–4V cavity, i.e., an order of magnitude faster than in the brass cavity of the same geometry. Thus, to satisfy the requirements for FJ-PEL- DOR experiments (to achieve a stable peak-field value and to avoid residual fields after switching off) with the Ti–6Al–4V cavity, a duration of the field jump of about 700 ns and an after-pulse delay of 500 ns are estimated which determine a minimum stimulated-spin-echo (SSE) mixing time interval T of about 1 μs. This pulse pattern already allows FJ-PELDOR experiments in high magnetic fields to be made for the determination of nanometer distance and relative orientation of nitroxide spin labels in disordered samples [276]. 2.2.2.2 RIDME In the 3-pulse stimulated-echo sequence in resonance with the observer spins A, the partner spins B can flip also spontaneously by their longitudinal relaxation that occurs during the mixing period (interval T between the 2nd and 3rd microwave pulses). This relaxation mechanism is exploited in RIDME spectroscopy which can, thus, be considered as a single-frequency variant of dipolar spectroscopy in which T1 relaxation substitutes the coherent mw irradiation in PELDOR to flip the B spins by 180° (π). Similar to PELDOR, the RIDME experiment yields an echo- decay time trace in which the dipolar coupling between the A and B spins shows up as a modulation with the period of the inverse dipolar frequency. 1 3 266 K. Möbius, A. Savitsky Certain conditions have to be met for the RIDME experiment to operate as a function of the preparation time 휏 : The fixed mixing time T should be long enough to allow the longitudinal spin relaxation to flip the partner spins in the pair, but short enough to avoid a considerable reduction of the echo signal caused by the longitu- dinal spin relaxation of the observer spins. 3 Instrumentation EPR spectroscopy, like any other spectroscopic method, must meet three main requirements to be useful as an analytical tool: (i) detection, (ii) identification, and (iii) characterization of the specimen. That means, first of all, the method should be sensitive enough to detect fingerprint signals of the system under investigation. The second step is the assignment of the observed signals to one or more species in the sample or, vice versa, identification of the species from the observed signals. The third step is the characterization of the species in their molecular environment using specific spin-interaction parameters that are extracted from the observed EPR signals.i g Historically, EPR spectroscopy had to go a long way to fulfill these requirements. Sensitive detection methods were being developed but needed decades to reach the present standard. The systematic investigations of a large number of the para- magnetic species provided the knowledge about their spectroscopic properties and allowed to understand the molecular interactions suitable for characterizing the spin system. Finally, sophisticated data analysis techniques were developed and approved which allow EPR spectroscopists to extract the interaction of interest from the com- plex spectra. In the meantime, EPR spectroscopy is approaching the level of techni- cal development necessary to become a powerful analytical tool, ready to be applied to characterize the investigated systems to hitherto unprecedented details.i While we were writing on this high-field EPR “Instrumentation” section, we real- ized that a full description of this success story would go far beyond the scope of the present review article. Instead, we decided to refer here to our 2009 book on High- field EPR spectroscopy on proteins and their model systems [1], which documents in detail the instrumentation of modern high-field EPR and its chronological develop- ment. 2.2.2 Electron–Electron Dipolar Spectroscopy (23), but the decay of stimulated-echo intensity can be governed by addi- tional mechanisms, for example spectral diffusion [293]. For details concerning our 95 GHz high-field PELDOR and RIDME experiments on transient radical pairs in bacterial photosynthetic reaction centers, see [128] and [1]. 1 3 High‑field/High‑frequency EPR Spectroscopy in Protein… 267 3 Instrumentation From this monograph, we will quote only a few distinctive examples.ii There are five basic requirements for a high-field/high-frequency EPR spectrom- eter: (i) A strong homogeneous and stable magnetic field; (ii) Suitable low-noise microwave sources; (iii) The microwave power must be transferred with minimal losses to a resonator with the sample and from the resonator to the detector by means of a suitable mw transmission line; (iv) The resonator must concentrate the incident microwave radiation onto the sample and allow to detect the small amount of energy absorbed when the EPR resonance condition is met; (v) The detector must be able to measure, with high signal-to-noise ratio, the variation of the mw power level in case of EPR absorption. All considerations concerning the detection schemes at X- or Q-band generally hold also for high-frequency EPR. However, the degradations in the performance of specific spectrometer parts at high mw frequencies put limitations on the spectrom- eter design and require a careful choice of the components. In [1] individual sections are dedicated to the microwave sources, resonators, transmission lines and magnet systems which have been successfully tested and employed in high-field spectrom- eter designs of various laboratories. For additional information we refer to review articles dealing in detail with aspects of instrumentation development of high-fre- quency EPR [108, 110, 114, 131, 294–298]. 1 3 268 K. Möbius, A. Savitsky 3.1 The 95 GHz Spectrometer Built at FU Berlin The W-band high-field EPR spectrometer at FU Berlin was designed as a mul- tipurpose instrument for cw, pulsed and time-resolved EPR, as well as for double resonance experiments (ENDOR, ELDOR). The instrumental development started around 1983. The significant steps forward in the performance and capabilities of the spectrometer up to its present version have been taken predominantly by diploma and PhD students as well as postdoctoral co-workers of the Möbius group. In par- ticular we mention (in chronological order) E. Haindl [75], O. Burghaus [74, 96, 299–301], T. Götzinger [302], M. Rohrer [74, 303–305], R. Klette [245, 306], T. F. Prisner [60, 298, 307], A. Schnegg [308], M. Fuhs [309–311], A. Savitsky [61, 128, 312], and Yu. Grishin [276]. The EPR spectrometer consists of the microwave bridge, the magnet system, the probehead, the cryostat, the control electronics. The mw bridge combines the transmitter, which produces the mw power for continuous or pulsed excitation of the sample, with the receiver for detecting the EPR signal. The external magnetic field is provided by a superconducting magnet which is sweepable to match the EPR resonance condition. The EPR probehead includes the mw cavity, sample holder, mw tuning mechanics, field modulation coils for cw EPR, rf coils for ENDOR. Each component is described in detail in [1]. Particular attention has been paid to the development of probeheads for 95 GHz EPR spectroscopy and its double resonance extensions ENDOR and PELDOR. When striving for high spectrometer sensitivity at mm wavelengths, incorpora- tion of a microwave resonance structure in the probehead is indispensable. There is no a priory preference for a single-mode cavity, which is commonly used in X- and Q-bands, or a multi-mode Fabry–Perot (FP) resonator, which is commonly used in the submillimeter and optical regions. The choice between these two reso- nance structures depends strongly on physical and technical considerations related to the type of EPR experiment to be performed. Consequently, both FP resona- tors and cylindrical single-mode cavities have been designed for our 95 GHz spectrometer. 3.1.1 Fabry–Perot probeheads Fabry–Perot (FP) resonators have been constructed and tested in several high-field EPR laboratories. Most experimental set-ups use cryomagnets and FPs with verti- cal B0 and resonator axes. This configuration, however, is inconvenient for sam- ple access, light irradiation and extension to ENDOR. Hence, we have placed the axis of the FP resonator perpendicular to the B0 axis [74]. Such a configuration allows to use vertically or horizontally mounted sample capillaries. The simulta- neously used Mn2+ standard sample for precision magnet field measurements can be adjusted in its position within the FP (see Fig. 22). It consists of two concave spherical mirrors (diameter 13 mm and 19 mm with corresponding curvature radii of 15 mm and 20 mm) in an approximately confocal arrangement (radius of cur- vature equal to mirror distance). The mirror distance can be tuned by a fine thread to adjust the resonator frequency to the mw source frequency (10 MHz frequency 1 3 High‑field/High‑frequency EPR Spectroscopy in Protein… 269 Fig. 22 W-band Fabry–Perot resonator with goniometer with mounted single crystal (shown orientation B0 \|\| crystal c axis. a mirrors (diameter 19 mm, distance ca. 10 mm); b mirror supports (synchronously movable for frequency-tuning); c WR10 waveguide; d microwave coupling unit with e drive shaft; f goniometer arm with gear for rotation of the arm about the resonator axis; g arm support (movable for adjusting the sample in the center of the resonator); h rotatable sample holder; i thread passing through arm f to sample holder h for sample-axis rotation; k quartz capillary with single crystal; l independently adjustable quartz capillary with Mn2+ standard sample; m modulation coils; n holder; o guide rod with right/left thread for symmetrical mirror adjustment (frequency-tuning). Rotation axes: axis 1, gear-driven rotation about the resonator axis; axis 2, thread-driven rotation about the sample capillary axis. For details, see [245, 306] Fig. 22 W-band Fabry–Perot resonator with goniometer with mounted single crystal (shown orientation B0 \|\| crystal c axis. a mirrors (diameter 19 mm, distance ca. 3.1.1 Fabry–Perot probeheads 10 mm); b mirror supports (synchronously movable for frequency-tuning); c WR10 waveguide; d microwave coupling unit with e drive shaft; f goniometer arm with gear for rotation of the arm about the resonator axis; g arm support (movable for adjusting the sample in the center of the resonator); h rotatable sample holder; i thread passing through arm f to sample holder h for sample-axis rotation; k quartz capillary with single crystal; l independently adjustable quartz capillary with Mn2+ standard sample; m modulation coils; n holder; o guide rod with right/left thread for symmetrical mirror adjustment (frequency-tuning). Rotation axes: axis 1, gear-driven rotation about the resonator axis; axis 2, thread-driven rotation about the sample capillary axis. For details, see [245, 306] variation for 1 μm distance variation). In most EPR and ENDOR experiments, the fundamental TEM00q with q set from 6 to 9 is preferred. For optimizing the detection sensitivity, the microwave coupling between resonator and waveguide is important. Sufficient coupling dynamics of the FP is achieved by moving a thin dielectric plate (MACOR) partially over the iris in one of the mirrors (for details of construction, see [74]). The basic realization of the FP probehead allows only rotation of the sample around the sample axis with an accuracy of ± 1°. In a next step, a FP probehead for single-crystal investigations with rotation around three axes by means of a goniometer was developed, Fig. 22. It allows to rotate the sample by 240° around the resonator axis and by 360° around the other two axes with an angular accu- racy of better than 0.5° [245]. The FP probehead is equipped with two orthogonal quartz fiber bundles for uniform light excitation while rotating the single-crystal sample. 3.1.2 TE011 Cavity Probeheads The disadvantages of FP resonators in terms of low filling and conversion factors become critical for pulsed high-field EPR experiments with only rather moder- ate mw power available at high frequencies. This was our motivation behind the 3 3 K. Möbius, A. Savitsky 270 Fig. 23 a Exploded view of the 95 GHz EPR TE011 optical transmission cavity with microwave-coupling mechanism and light-access chimneys (with minimum mw radiation losses) to insert the quartz-fiber light pipe. b Exploded view of TE011 (ENDOR variant) probehead with frequency-tuning mechanism and field modulation coils. For details, see [74, 302] Fig. 23 a Exploded view of the 95 GHz EPR TE011 optical transmission cavity with microwave-coupling mechanism and light-access chimneys (with minimum mw radiation losses) to insert the quartz-fiber light pipe. b Exploded view of TE011 (ENDOR variant) probehead with frequency-tuning mechanism and field modulation coils. For details, see [74, 302] construction of W-band EPR and ENDOR probeheads with cylindrical single-mode cavities. The TE011 mode has high filling and conversion factors, and its resonance frequency can easily be tuned by changing the cavity length. The development of cylindrical single-mode cavities for W-band started in our group in 1988 [302]. The first-generation construction design proved to be very reliable in terms of frequency and coupling stability. This design concept is still applied for fabricating new gen- erations of W-band cylindrical cavities without major modifications. y ji Figure 23a shows exploded views of the W-band EPR probehead. The main ele- ments are the cylindrical TE011 cavity optimized for 95 GHz, the frequency-tuning mechanism, the mw coupling mechanism and the field modulation coils. They are mechanically isolated from the cavity in order to avoid microphonics. At 10 kHz they supply up to 2 mT of field modulation amplitude at the sample. The cavity diameter is 4.16 mm. The cavity length can be adjusted to 4.16 ± 1.5 mm (corre- sponding to TE011 mode resonance frequencies from 92 to 104 GHz for an empty cavity). This is done by changing the separation of the two sliding pistons in the cav- ity by means of a gear drive of high mechanical precision, Fig. 23b. The tuning drive consists of two cam followers 180° out of phase, thereby guaranteeing the maximum of the mw field to stay in the center of the cavity. This is an important requirement for experiments with small single-crystal samples and light excitation. 3.1.2 TE011 Cavity Probeheads For the dif- ferent capillary diameters (ID 0.1–1.0 mm), different pairs of pistons are used. On the top of the coupling needle (DELRIN) a small silver ball of 0.7 mm diameter is painted, using a suspension of fine silver flakes (about 8 μm). The coupling needle 1 3 High‑field/High‑frequency EPR Spectroscopy in Protein… 271 can be moved in the upper part of the W-band (WR10) waveguide by means of a vertically sliding mechanism with a high-precision thread drive, Fig. 23a. The iris coupling hole in the middle of the cavity wall has a diameter of 0.8 mm, the wall thickness around the iris is less than 0.1 mm. For different EPR experiments it was necessary to construct different types of cavities. They are commented on in the following:i For pulsed W-band EPR experiments, in which a high B1 field at the sample position is required, a special TE011 cavity with high Q is used. The cavity body was machined from gold (99.9%). Additionally, the brass pistons for the frequency adjustment of the cavity were gold plated (100 µm) at the front side. A room-tem- perature unloaded QU = 7400 was measured without sample. This is close to the cal- culated QU value of 8600 when accounting for the ohmic losses in the cavity walls only. At 90 K, QU = 12,000 was determined which is consistent with the room- temperature value taking the known temperature dependence of gold resistivity into account.i For specific EPR experiments in which light irradiation of the sample is required, the standard cw cavity is replaced by an optical transmission cavity, Fig. 23a. It has two symmetrically placed 0.6 mm holes in the cavity wall for the light access fur- nished with mw chimneys and holding a 0.8 mm thick quartz fiber [304].i i For ENDOR experiments, the cw variant of the probehead had to be modified [303]. The standard cw cavity is replaced by a gold-plated bronze TE011 cavity, the body of which is slotted with regular slots of 0.3 mm width and 0.6 mm separa- tion to reduce rf-induced eddy currents. The slots slightly reduce QU to a typical value of 4000. We use two variants of the ENDOR cavity, one with and the other without light-access holes. The bronze pistons are replaced by pistons machined from MACOR ceramics with gold-plated end faces. 3.1.2 TE011 Cavity Probeheads The rf ENDOR-coil holder is mounted on the top of the upper field modulation coil. The rf saddle coils around the cavity center produce a rf magnetic field perpendicular to both the external and mw magnetic fields [74].ii i In field-jump PELDOR experiments [276], the field modulation coils are replaced by field-jump coils of the same geometry. The cavity with dimensions that are adapted from the ENDOR variant is machined from a titanium/aluminum/vana- dium alloy and then gold plated [276]. The ENDOR pistons are used for frequency- tuning. The unloaded quality factor of such a cavity configuration is about 3000. 3.2.1 Quasioptical Microwave Propagation The microwave propagation losses in normal metallic waveguides rise rapidly at high frequencies, making standard waveguides unusable above 150 GHz except for very short distances of a few cm. Dielectric fibers and similar "light-pipe" structures can work very well for frequencies in the visible and near-visible spec- tral region. Unfortunately, they have significant dielectric losses at millimeter waves leading to high damping of the propagated wave. Free space is a low-loss alternative. The damping of millimeter waves in air due to molecular absorption is quantified in dB/km in contrast to dB/m or even dB/cm in classical waveguide systems. The techniques for the propagation of electromagnetic waves over free space are well developed for radiation with wavelengths of less than a micron. Established techniques exist for optical systems for which the characterictic dimensions are many thousand times larger than the wavelength. These systems can be designed and analyzed using the traditional methods of geometrical optics with only rare resort to wave optics. In order to treat millimeter wave systems in the geometrical optics way, one would have to handle optical elements with at least one meter in diameter. Gaussian quasioptics offers a solution to this prob- lem. Quasioptics can be considered as a specific branch of microwave science and engineering. This term is used to characterize methods and tools devised for handling electromagnetic waves propagating in the form of narrow directed beams, whose width w is greater than the wavelength 휆 , but smaller than the cross-section size, D , of the limiting apertures and guiding structures:𝜆< w < D . Normally, D < 100 ⋅𝜆 , but also devices as small as D = 3 ⋅휆 can be analyzed using quasioptical principles. In contrast to geometrical optics, which requires D > 1000 ⋅𝜆 , quasioptics consider both ray-like and diffraction-causing optical phenomena when tracing the beams as they interact with the surfaces of optical elements and the matter in the pathway. The simplest form of an electromagnetic wave propagating through free space is the Gaussian beam. The fundamental-mode beam has a Gaussian distribution of the electric field amplitude perpendicular to the z-axis of propagation [315, 316] (25) \|E(z, r)\| = \|E(0, r)\| ⋅e−r2∕w2, (25) where r is the distance from the propagation axis and w is the beam radius. 3.2 The 360 GHz Spectrometer Built at FU Berlin The instrumental development of the 360 GHz EPR spectrometer at FU Berlin has been started in 1995. Several PhD students and postdoctoral co-workers of the Möbius group were involved in this development, in particular M. Fuchs [25, 87, 313], A. Schnegg [152, 314], Yu. Grishin [119, 152] and T. F. Prisner [87]. Dur- ing the last decade several design improvements of the spectrometer were realized including the pulse EPR and cw ENDOR extensions. The most notable difference of the 360 GHz (λ ≈ 0.8 mm) spectrometer in relation to our W-band spectrometer 1 3 272 K. Möbius, A. Savitsky is the use of quasioptical microwave components and a corrugated waveguide in the transmission line to the probehead. is the use of quasioptical microwave components and a corrugated waveguide in the transmission line to the probehead. 3.2.1 Quasioptical Microwave Propagation The beam radius w will have a minimum value w0 at a specific place along the beam axis, which defines the beam waist. For a beam of wavelength 휆 at a distance z along the beam, as measured from the beam waist, the variation of the beam radius is given by (26) w(z) = w0 ⋅(1 + (z∕z0)2)1∕2, (26) and the radius of curvature R of the wavefront comprising the beam is and the radius of curvature R of the wavefront comprising the beam is 1 3 High‑field/High‑frequency EPR Spectroscopy in Protein… 273 R(z) = z ⋅[1 + (z0∕z)2], (27) R(z) = z ⋅[1 + (z0∕z)2], (27) where z0 = 휋⋅w2 0∕휆 is the confocal distance (also called Rayleigh length). Quasiop- tical elements for propagation of Gaussian beams are waveguides, lenses or mirrors to refocus the Gaussian beam, and antennas or feed-horn systems to transform the waveguide modes to Gaussian modes of propagation. The actual diameters of quasi- optical elements are determined by the boundary values of still tolerable diffraction and beam truncation effects. An element with a single aperture D = 2 ⋅w has a cou- pling efficiency of 98.8%, i.e., a beam-power loss of -0.05 dB when transmitting a fundamental-mode beam. The propagation of radiation in a Gaussian beam in free space is independent of its polarization. Thus, generation of beams with different polarizations and manipu- lation of them with polarization-dependent elements is possible. For example, wire grids with very thin conducting filaments, that are separated by distances less than a wavelength, are very effective polarizers. For radiation with the electric field in the directions of the wires currents are induced, and the grid acts as a reflector. A perpendicularly oriented electrical field does not induce currents, and the radia- tion passes the grid without attenuation. Proper manipulation of the Gaussian beam Fig. 24 The 360 GHz EPR spectrometer setup at FU Berlin [313]. The cw Gunn source (green) can be exchanged for the pulsed Orotron source (green) by replacing the first off-axis elliptical mirror for a Teflon lens. In the beam path of the quasioptical bridge (orange), beam propagation directions are indicated by dashed arrows (red) and polarization directions by solid arrows (green). Inside the magnet (gray), the corrugated waveguide, the modulation coil and the semiconfocal Fabry– Perot resonator with mesh coupling are indicated. 3.2.2 Microwave‑Bridge Design The general design of the 360 GHz microwave bridge is depicted in Fig. 24. There are three mw sources for sample excitation available. The Orotron source is described in detail in [1]. For pulsed 360 GHz EPR operation, the cw Gunn diode source in the spectrometer can be exchanged for a novel pulsed 360 GHz vacuum- tube source named Orotron (Gycom). The Orotron was tailor-made for our 360 GHz EPR studies in a joint German-Russian pilot project supported by the DFG [119]. The acronym Orotron was originally introduced as an abbreviation of the Russian words describing a device with an open resonator, generally a Fabry–Perot resona- tor, and a reflecting diffraction grating. Conceptually, the Orotron is a non-relativis- tic free-electron laser using the stimulated Smith–Purcell radiation of a flat electron beam interacting with a periodic grating structure. This acts as one of the mirrors in an over-sized high-quality Fabry–Perot resonator structure to achieve feedback for high output power and frequency stability. The electromagnetic field present in the open FP resonator bunches the electron beam which leads to coherent oscilla- tion [319]. Such open resonator generators were independently developed by F. S. Rusin and G. D. Bogomolov in Moscow, USSR [320] and K. Mizuno and S. Ono in Sendai, Japan [321]. The present tailor-made design extends the traditional cw Oro- tron by a gate electrode and a high-voltage pulsing unit to control the electron beam current. The generated pulses at 360 GHz have pulse lengths from 100 ns to 10 µs and a pulse power of up to 30 mW. Within a 10 ms time slot, incoherent pulse trains of arbitrary duration can be generated. The pulsed Orotron has been incorporated in the quasioptical microwave bridge of our heterodyne induction-mode 360 GHz EPR spectrometer. First free-induction decay (FID) measurements at 360 GHz and a magnetic field of 12.9 T on a polycrystalline perylene-ion sample were very prom- ising for future applications of the Orotron in EPR spectroscopy under very high- frequency/high-field conditions [119].i i In the cw configuration [95, 313], the microwave at 360.03 GHz is gener- ated either by a phase-locked tripled 120 GHz Gunn source (Farran Technology) or by a quadrupled 90 GHz Gunn oscillator (Radiometer Physics). The second cw source can be phase-locked to a 10 MHz oscillator or swept between 358.8 GHz and 361.2 GHz, which allows the precise frequency-tuning of the resonator contain- ing the sample. 3.2.1 Quasioptical Microwave Propagation The EPR-induced microwave signal reflected from the polarizer grid is focused onto the horn of the subharmonic mixer detector (blue). Light excitation is intro- duced into the probehead via a light guide (red) (color figure online) 1 3 Fig. 24 The 360 GHz EPR spectrometer setup at FU Berlin [313]. The cw Gunn source (green) can be exchanged for the pulsed Orotron source (green) by replacing the first off-axis elliptical mirror for a Teflon lens. In the beam path of the quasioptical bridge (orange), beam propagation directions are indicated by dashed arrows (red) and polarization directions by solid arrows (green). Inside the magnet (gray), the corrugated waveguide, the modulation coil and the semiconfocal Fabry– Perot resonator with mesh coupling are indicated. The EPR-induced microwave signal reflected from the polarizer grid is focused onto the horn of the subharmonic mixer detector (blue). Light excitation is intro- duced into the probehead via a light guide (red) (color figure online) Fig. 24 The 360 GHz EPR spectrometer setup at FU Berlin [313] Th G Fig. 24 The 360 GHz EPR spectrometer setup at FU Berlin [313]. The cw Gunn source (green) can be exchanged for the pulsed Orotron source (green) by replacing the first off-axis elliptical mirror for a Teflon lens. In the beam path of the quasioptical bridge (orange), beam propagation directions are indicated by dashed arrows (red) and polarization directions by solid arrows (green). Inside the magnet (gray), the corrugated waveguide, the modulation coil and the semiconfocal Fabry– Perot resonator with mesh coupling are indicated. The EPR-induced microwave signal reflected from the polarizer grid is focused onto the horn of the subharmonic mixer detector (blue). Light excitation is intro- duced into the probehead via a light guide (red) (color figure online) 3 274 K. Möbius, A. Savitsky polarization allows to construct the quasioptical analogs of corresponding wave- guide-based microwave elements such as circulators, directional couplers etc., which become either extremely lossy or even impossible to realize for frequencies above 150 GHz. Detailed descriptions of such quasioptical elements can be found in the monographs by Lesurf [316] and Goldsmith [315]. The engineering and design con- cepts of quasioptical EPR systems are considered in detail by Gulla and Budil [317], and a transfer matrix method for characterizing and optimizing the performance of quasioptical EPR sample resonators is described by Earle, Zeng and Budil [318]. 1 3 3.2.2 Microwave‑Bridge Design The output power at 360 GHz is about 1 mW for both cw sources. For detection a heterodyne mixer scheme is employed. The central component of the receiver (Farran Technology) is a subharmonic mixer detector with a detection bandwidth of 100 MHz. In contrast to a fundamental mixer, in a subharmonic mixer 1 3 High‑field/High‑frequency EPR Spectroscopy in Protein… 275 the incoming mw signal is mixed not with the fundamental frequency of the local oscillator (LO) input but with a higher harmonic. The local oscillator can, there- fore, operate at a lower frequency with all the advantages of lower frequency com- ponents. The disadvantage, of course, is the less efficient mixing process. The LO is provided by a phase-locked, doubled 90.3 GHz Gunn oscillator. The necessary reference signal is supplied directly from the 10 MHz source of the transmitter con- trol module. With the subharmonic mixer running at the 2nd LO harmonic, the EPR signal at 360 GHz is down-converted with a LO frequency of 361.2 GHz, so the resulting IF frequency is 1.2 GHz. This is then passed through a low-noise amplifier with 35 dB gain and a video filter. The noise figure of the complete receiver module is 15 dB. This performance is comparable to that of the most sensitive InSb hot- electron bolometer detectors nowadays available. The signal at 1.2 GHz is down- converted by a quadrature IF mixer (Anaren Microwave). The LO for this mixer is provided by a 1.21 GHz dielectric resonator oscillator (DRO) which again is phase- locked to the 10 MHz master oscillator. Since in a quadrature mixer both in-phase and out-of-phase signal components are generated simultaneously, both the absorp- tive and dispersive signal components can be detected in a single experimental run. In the 360 GHz spectrometer [87, 313], we employ a quasioptical transmission line setup in which a Gaussian beam is launched into free space via a corrugated horn antenna. If free-space propagation is not feasible, cylindrical corrugated over- sized waveguides are used. Conventional smooth-walled waveguides are inferior to corrugated waveguides due to excitation of higher-order modes, generation of stand- ing waves at transitions from and to fundamental-mode waveguide sections, and dis- tortion of the propagated beam polarization [322]. In analogy to geometrical optics, to refocus a diverging beam one can employ either curved mirrors or lenses. 3.2.2 Microwave‑Bridge Design The rotation stage is slightly tilted with respect to the optical axis to avoid the build-up of standing waves in the system. In the cylindrically symmetric Fabry–Perot resonator, the excitation mw repre- sents a composition of two circularly polarized modes with opposite polarization. Of those two modes only one interacts with the spin system and, therefore, becomes partially attenuated when magnetic resonance absorption occurs. Recombined with the unattenuated component, this yields elliptically polarized mw radiation that is reflected back into the corrugated waveguide. When hitting the wire-grid polarizer, only the component orthogonal to the excitation mw is reflected onto the receiver antenna, while the excitation power itself is passed to the transmitter tripler and attenuated there. Essential in this context is the use of the cylindrically symmetric Fabry–Perot resonator as a bimodal cavity. Since all directions of polarization in the resonator are degenerate, both the excitation microwave and the EPR induction component, polar- ized orthogonally to the excitation component, have to be supported by the resonator structure. This also applies to the oversized waveguide transmission line. The use of a single-mode cavity, despite many advantages, is not possible within an induction- mode detection scheme. The maximum obtainable isolation of the excitation from the detection arm is ultimately limited by the cross-polarized microwave component that is induced by the off-axis mirrors. This effect is partly corrected by the polarizer grid that is passed after the first mirror. Overall, this induction-mode setup provides an attenua- tion of the excitation power with respect to the EPR signal power of 20–30 dB. The superconducting magnet is a Teslatron H system (Oxford Instruments). It can sustain a magnetic field of up to B0 = 14 T. The experiments are typically run with a central magnetic field B0 = 12.846 T, the resonance field for the free-electron g-value and a microwave frequency of 360 GHz. The lowest accessible g-value with a 14 T central field is g = 1.84. i Integrated into the main coil assembly is a superconducting sweep coil with a sweep range of ± 100 mT. This way the magnet can be swept with a sweep rate of up to 70 mT/min, while the main coil remains in persistent mode. The homogeneity of the magnet is specified to 3 ppm in a 10 mm sphere. 3.2.2 Microwave‑Bridge Design Lenses can be the origin of standing waves in the system and cause dielectric losses. There- fore, all focusing elements in our quasioptical transmission line are metallic off-axis elliptical mirrors. To couple the microwave to the Fabry–Perot sample resonator, the output beam needs to be focused to a beam waist of 1.0 mm. This is achieved by a corrugated tapered waveguide section, made of gold-plated copper, at the end of the cylindri- cal waveguide. The corrugated waveguide itself consists of 100 mm long electro- formed sections soldered to an overall length of 1080 mm. The diameter is 23 mm, being reduced by the taper over a length of 20 mm to 3 mm. The corrugation depth is 0.6 mm and the slot/metal pitch 0.25 mm. The material of the main waveguide section is German silver, which is preferred to copper since it has a lower coefficient for heat conduction. The linear polarization of the Gaussian beam is utilized in an induction-mode detection scheme that was first described by Teaney et al. [323]. Later examples of this detection scheme at high magnetic fields were introduced for transmission mode by Prisner et al. [95] and for reflection mode by Smith et al. [90]. The Gauss- ian mw beam launched by the transmitter horn antenna has an initial beam waist w0 of 1.48 mm and is linearly polarized. As is indicated in Fig. 24, the first off- axis mirror focuses the widened beam onto the upper end of the corrugated wave- guide to a beam waist of 7.24 mm. On its way, the beam passes through a wire- grid polarizer that is oriented with an angle of 45° with respect to the optical axis. 1 3 276 K. Möbius, A. Savitsky The free-standing wires (12 wires/mm, 25 µm diameter) allow mw radiation that is polarized perpendicular to the direction of the wires to pass because in this direction no electric currents are induced, while radiation polarized along the wire direction is effectively reflected. Behind the polarizer, the residual radiation with polarization along the wires is attenuated by about 20 dB compared to the unattenuated compo- nent. By mounting a second grid on a rotation stage in front of the first, one obtains a polarizer/analyzer setup that acts as a variable attenuator with a dynamic range of 20 dB. 1 3 3.2.2 Microwave‑Bridge Design This was verified with an NMR Gaussmeter with a deuterium probe that was moved along the symmetry axis of the magnet. The linearity of the field sweep has also been tested with an NMR gaussme- ter for a full sweep. The sweep is not linear and after a half cycle, a remanence field of 4 mT can be observed. After a full cycle the field offset was 0.05 mT. This neces- sitates the use of a standard sample for magnetic field calibration. We normally use Mn2+/MgO dissolved in a polystyrene film as the standard sample whose magnetic interaction parameters are knows with high precision [74]. 1 3 High‑field/High‑frequency EPR Spectroscopy in Protein… 277 Fig. 25 Tunable rf circuit (left) together with a cross-section and an exploded view of the 360 GHz/550 MHz EPR/ENDOR probehead (right). The resonant rf circuit consists of a single-loop coil and two trimmer capacitors, which provide frequency-tuning and -matching. The rf is generated by a sweepable generator, a power amplifier and a single-turn coil, which generates a B2 field perpendicular to the microwave field B1 . The sample is placed on the gold-coated spherical quartz mirror. Note that for the EPR/ENDOR experiment the mw coupling mesh as the flat mirror of a Fabry–Perot resonator was removed. For details, see [152, 314] Fig. 25 Tunable rf circuit (left) together with a cross-section and an exploded view of the 360 GHz/550 MHz EPR/ENDOR probehead (right). The resonant rf circuit consists of a single-loop coil and two trimmer capacitors, which provide frequency-tuning and -matching. The rf is generated by a sweepable generator, a power amplifier and a single-turn coil, which generates a B2 field perpendicular to the microwave field B1 . The sample is placed on the gold-coated spherical quartz mirror. Note that for the EPR/ENDOR experiment the mw coupling mesh as the flat mirror of a Fabry–Perot resonator was removed. For details, see [152, 314] The warm bore of the cryomagnet has a diameter of 88 mm. This allows to employ a cryostat with the rather large diameter of 62 mm to accommodate the probehead. The cryostat is a helium-cooled static-flow cryostat (Oxford Instruments, CF1200) which covers a temperature range of 3.8–300 K. In a static-flow design, the probehead is not cooled directly by the He vapor. 3.2.2 Microwave‑Bridge Design Instead, the probehead space is closed off and filled with a buffer gas (e.g., argon) which in turn is cooled by a cop- per heat exchanger shield that is in direct contact with the helium. This setup avoids stability problems which often arise in direct-flow cryostat designs. 3.2.3 Probeheads Several probeheads have been developed for EPR and ENDOR measurements at 360 GHz [152, 313]. The ENDOR version is shown in Fig. 25. Both EPR and ENDOR probeheads are based on the plane-concave Fabry–Perot resonator oper- ated in the TEM006 mode. With a resonant mirror distance d = 2.58 mm and cur- vature radius R = 8.06 mm, one obtains a confocal distance of 3.76 mm. A typical value for the finesse of the resonator with sample is F = 160, giving for the loaded quality factor QL= 800. Coupling to the tapered end of the corrugated waveguide is achieved through the flat mirror, which is a highly reflective metallic mesh (typically 30 wires/mm). The mesh consists of electro-formed copper and is stretched to an exchangeable mesh holder, where it is fixed onto a circular frame. Since the mesh 1 3 278 K. Möbius, A. Savitsky holders are exchangeable, the mesh can be replaced by another mesh with differ- ent reflectivity, thus allowing for variable coupling for different samples. Tuning is achieved by translating the spherical mirror along three guide rods via a micrometer screw on top of the corrugated waveguide outside the magnet. The field modula- tion coils frame the spherical mirror. The modulation amplitudes can be raised up to 3 mT without generating excessive microphonics. A quartz fiber integrated into the probehead allows for photo-generation of radicals or triplet states inside the resona- tor by laser excitation. The overall detection sensitivity of the spectrometer equipped with the FP resonator has been measured to be 1.5⋅1010 spins/mT at 1 Hz detection bandwidth by evaluating the signal-to-noise ratio of a known number of spins in a Mn2+ sample. Figure 25 shows a cross section and an exploded view of the 360 GHz EPR/ ENDOR probehead together with the resonant rf circuit for ENDOR experiments. The sample is placed on the gold-coated quartz mirror and irradiated by a mw field B1 and a perpendicular rf field B2 that is produced in a single-loop ENDOR coil. The ENDOR coil is part of the resonant circuit (center frequency 547 MHz; bandwidth 60 MHz) which can be frequency-tuned and impedance-matched by two trimmer capacitors mounted on the rf circuit plate close to the ENDOR coil. The rf power is provided by a digital frequency generator (Hewlett Packard, 8648B) and ampli- fied by a 500 W power amplifier (Amplifier Research, 500HB). Declarations Conflict of Interest The authors declare no competing financial interests. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Com- mons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licen ses/by/4.0/. Author contributions All authors equally contributed to the writing of the manuscript. Funding Open Access funding enabled and organized by Projekt DEAL. Over the years, our high-field EPR activities have received enduring support from the Deutsche Forschungsgemeinschaft (DFG) in the frame of DFG priority programs (SFB 337, SFB 498, III P 5-MO 132/14-1, SPP 1051, MO 132/19-2, Cluster of Excellence RESOLV EXC 1069); by Volkswagenstiftung priority programs (I/70 382, I/73 145, I/73 146); and by the European Union network programs HCM (CHRX-CT9-30 328), TMR (FMRX- CT98-0214), INTAS (01-483) and COST (P15), which is gratefully acknowledged. K. M. is particularly thankful for the long-standing support of his scientific work by the Freie Universität Berlin (Department of Physics) and the Max Planck Gesellschaft (Max Planck Institute for Chemical Energy Conversion). 3.2.3 Probeheads To reduce stand- ing waves in the rf transmission line, a high-power rf circulator is used. The maxi- mum B2 generated by the ENDOR coil is 0.8 mT at 20 W incident rf power, for which unwanted heating effects are avoided. ENDOR spectra are typically recorded at a fixed external field B0 and mw field B1, while sweeping the frequency of the rf field B2 . The B2 field is frequency modulated, and the ENDOR signals are recorded using a lock-in amplifier. For more details of the 360 GHz ENDOR experiments, see [152].i To conclude this section: Many options for exploiting the strength of high-field EPR spectroscopy in terms of spectral and temporal resolution critically rely on the availability of powerful cw and pulsed mm and sub-mm microwave sources. Natu- rally, the technical difficulties and limitations rise with rising microwave frequen- cies. Nevertheless, the 360 GHz spectrometer could already be used to perform successful studies on the structure of protein systems such as the cofactors of photo- synthesis and DNA photolyase in their binding pockets. Examples of pertinent refer- ences include: [324–327]. Acknowledgements Over the years, many co-workers in the Möbius group—diploma and PhD students, postdocs and senior scientists—have contributed to the high-field EPR work reviewed in this article. In the course of these activities, it was their enthusiasm and tenacity which allowed to solve the numerous technical problems in developing high-field EPR, ENDOR, ESEEM, EDNMR and PELDOR instrumen- tation operating at 95 GHz or 360 GHz, and to apply these techniques to novel molecular systems from biology and biochemistry. In this context, we specifically acknowledge the former students (in chrono- logical order): Olaf Burghaus, Anna Toth-Kischkat, Thomas Götzinger, Robert Klette, Martin Rohrer, Moritz Knüpling, Jens Törring, Gordon Elger, Michael Fuhs, Ingo Köhne, Andreas Bloeß, Martin Fuchs, Marcus Gallander, Alexander Schnegg; and the former postdocs (in alphabetical order): Edmund Haindl, Martina Huber, Chris Kay, Wolfgang Lubitz, Martin Plato, Thomas Prisner, Anton Savitsky and Stefan Weber. K.M. wants to thank them all for their eminent contributions to exciting experiments of high-field EPR spectroscopy. 1 3 High‑field/High‑frequency EPR Spectroscopy in Protein… 279 Author contributions All authors equally contributed to the writing of the manuscript. Author contributions All authors equally contributed to the writing of the manuscript. References 1. K. Möbius, A. 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https://openalex.org/W1990077566	https://europepmc.org/articles/pmc2877419?pdf=render	English	null	Emergence of Pediatric Melioidosis in Siem Reap, Cambodia	The American journal of tropical medicine and hygiene	2,010	cc-by	6,292	MATERIALS AND METHODS Setting and patients. The study was conducted at the Angkor Hospital for Children (AHC), an non-governmental organiza- tion (NGO)-funded teaching hospital in Siem Reap, situated in the province of Siem Reap, northwest Cambodia. The AHC provides free outpatient, inpatient, emergency, surgical, medical, ophthalmological, and dental care, and it maintains 50 inpatient beds spread across high-, medium-, and low-intensity care areas. The outpatient department sees an average of approximately 400 children each day from an unrestricted catchment area. The study protocol was reviewed and approved by the Ethical Review Board of the Angkor Hospital for Children. A laboratory-based study was conducted between October 2005 and December 2008 to identify children presenting to inpatient or outpatient departments with one or more micro- biological samples positive for B. pseudomallei . A retrospec- tive review of case records was performed at the end of this period to collect information on age, gender, area of residence, sample(s) positive for B. pseudomallei , known sites of organ involvement, results of laboratory and radiological investiga- tions, antimicrobial treatment, procedures, duration of hospi- tal stay, outcome at hospital discharge, and details of follow-up. Sites of infection were established based on a record of history and examination findings in the medical notes together with investigation reports and procedure notes. Melioidosis is a leading cause of bacterial sepsis in northeast Thailand. 5– 8 Childhood infection accounts for around 10% of cases overall in this setting, and acute suppurative parotitis accounts for one-third of pediatric cases. 9 Adjacent to north- east Thailand is Laos to the east and southeast and Cambodia to the south. Melioidosis was first reported from Laos in 2001 after the development of a diagnostic microbiology laboratory at Mahosot Hospital, Vientiane, 10 and B. pseudomallei was subsequently isolated from the surrounding environment. 11 B. pseudomallei has also been isolated from rice paddies in Siem Reap province in northwest Cambodia, 12 and a recent report of two cases of melioidosis in adults from southern Cambodia has confirmed the presence of melioidosis in the indigenous population. 13 A seroprevalence study of children presenting to a hospital in Siem Reap detected antibodies to B. pseudomallei in 16% of cases, 12 but melioidosis has not been detected previously in this pediatric population. Here, we report the identification of 39 cases of melioidosis at the Angkor Hospital for Children in Siem Reap, the first reported Melioidosis was classified as localized or disseminated. * Address correspondence to Sharon J. Peacock, Department of Medi- cine, University of Cambridge, Addenbrooke’s Hospital, Cambridge CB2 0QQ, United Kingdom. E-mail: sp10@sanger.ac.uk Emergence of Pediatric Melioidosis in Siem Reap, Cambodia Yos Pagnarith , Varun Kumar , Janjira Thaipadungpanit , Vanaporn Wuthiekanun , Premjit Amornchai , Lina Sin , Nicholas P. Day , and Sharon J. Peacock * Lina Sin , Nicholas P. Day , and Sharon J. Peacock * Angkor Hospital for Children, Siem Reap, Cambodia; Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; Center for Clinical Vaccinology and Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, Churchill Hospital, Oxford, United Kingdom; Department of Medicine, University of Cambridge, Addenbrooke’s Hospital, Cambridge, United Kingdom Abstract. We describe the first cases of pediatric melioidosis in Cambodia. Thirty-nine cases were diagnosed at the Angkor Hospital for Children, Siem Reap, between October 2005 and December 2008 after the introduction of microbiol- ogy capabilities. Median age was 7.8 years (range = 1.6–16.2 years), 15 cases were male (38%), and 4 cases had pre-existing conditions that may have pre-disposed the patient to melioidosis. Infection was localized in 27 cases (69%) and dissemi- nated in 12 cases (31%). Eleven cases (28%) were treated as outpatients, and 28 (72%) cases were admitted. Eight chil- dren (21%) died a median of 2 days after admission; seven deaths were attributable to melioidosis, all of which occurred in children receiving suboptimal antimicrobial therapy and before bacteriological culture results were available. Our find- ings indicate the need for heightened awareness of melioidosis in Cambodia, and they have led us to review microbiology procedures and antimicrobial prescribing of suspected and confirmed cases. INTRODUCTION cases in Cambodian children. Their identification followed the introduction of diagnostic microbiology capabilities, highlight- ing one of the many benefits of laboratory strengthening in this region. Melioidosis is a serious infectious disease caused by the Gram-negative bacterium Burkholderia pseudomallei . 1, 2 This organism is present in the environment in a defined geographic distribution including much of south and east Asia, northern Australia, and areas of South America, where infection is thought to be acquired after bacterial inoculation, ingestion, or inhalation. 1– 3 The clinical presentation is highly variable and ranges from a mild localized infection to acute fulminant sepsis with widespread bacterial dissemination. 1, 2 The clinical diagnosis of melioidosis is notoriously inaccurate, and diag- nostic confirmation relies on culture of B. pseudomallei . 4 Lack of microbiological services in many of the countries predicted to be affected by melioidosis is likely to result in gross under- reporting of cases and an underestimate of the global burden of this infection. MATERIALS AND METHODS Localized infection was defined as a single, discrete, culture- positive focus of infection in the absence of a positive blood culture or clinical and/or microbiological evidence of dissemi- nation to a second site. Disseminated infection was defined as the presence of infection in two or more discrete body sites and/or the presence of B. pseudomallei in blood. Microbiological methods. Specimens from patients with suspected melioidosis were processed using standard laboratory procedures with the addition of Ashdown selective agar for 1106 1107 MELIOIDOSIS IN CAMBODIA culture of samples from colonized sites. 14 Identification of B. pseudomallei was made on the basis of Gram stain, resistance to gentamicin and colistin, a positive latex agglutination test, 15, 16 and the API 20NE profile. 16 Susceptibility testing was performed by disk diffusion assay for ceftazidime, meropenem, amoxicillin- clavulanate, and doxycycline and by E-test for trimethoprim- sulfamethoxazole (TMP-SMX). Isolates were stored in Tryptone Soya broth (TSB) with 20% glycerol at −80°C. spleen on ultrasound ( N = 1), or pneumonia associated with respiratory failure and septic shock ( N = 1). This last case with respiratory secretions positive for B. pseudomallei had neither positive blood cultures nor a second site of infection, but was classified as having disseminated infection based on the sys- temic response and severity of illness. Eight of thirty-nine children (21%) died a median of 2 days after admission (range, day of admission to day 23), all of whom were in the disseminated infection group (8/12; 67%). Seven deaths were attributable to melioidosis, whereas the eighth case had both tuberculosis meningitis and B. pseudo- mallei pneumonia and had responded to 14 days of ceftazi- dime by the time of death on day 23 because of intracranial hypertension secondary to tuberculosis meningitis. A posi- tive blood culture was associated with a very poor prognosis, and death occurred in 7 of 9 cases (78%). Both children with a total white blood cell (WBC) count of less than 5.0 × 10 9 /L (1.6 × 10 9 /L and 2.6 × 10 9 /L, respectively) died. Molecular characterization of B. pseudomallei . Genomic DNA was extracted from a 1-mL overnight culture of B. pseudomallei with an optical density of 1.0 at 600 nm using the High Pure PCR Template Preparation Kit (Roche Applied Science, Mannheim, Germany). Multilocus sequence typing (MLST) was performed as described pre viously. MATERIALS AND METHODS 17 Sequence-type (ST) assignment was based on the sequence of the alleles at each locus of seven housekeeping genes using the MLST database ( www.mlst.net ). Sequences that were not in the database were checked by resequencing, and then assigned as new alleles and deposited in the MLST allele database. All 39 B. pseudomallei isolates were susceptible to ceftazi- dime, meropenem, amoxicillin-clavulanate, TMP-SMX, and doxycycline, the most common drugs used for the treatment of adult melioidosis. MLST was performed on 39 B. pseudomallei isolates cul- tured from children with melioidosis described in this study and 14 B. pseudomallei isolates cultured previously from soil in Cambodia, of which 12 were isolated in Siem Reap province during 2006 12 and 2 were isolated in Phnom Penh in south- ern Cambodia in 1996. MLST data on B. pseudomallei iso- lates from Thailand that had originated in our laboratory and/ or had been reported previously 17– 19 were downloaded from www.mlst.net ( N = 462). Neighbor-joining trees were recon- structed using the two-parameter method of distance estima- tion as implemented in MEGA 4.0. Clinical management. Eleven children (28%) were treated in the outpatient department alone for localized B. pseudo- mallei infection (parotid abscess, 4; superficial soft-tissue abscess, 7). Incision and drainage of pus was performed in 10 of these cases, and all 11 cases were prescribed one or more antimicrobial drugs for a median of 4 weeks (range, 5 days to 17 weeks; IQR = 1–6 weeks). The antimicrobial drugs prescribed were amoxicillin-clavulanate alone ( N = 2), amoxicillin-clavulanate in combination with TMP-SMX ( N = 2), amoxicillin-clavulanate in combination with cipro- floxacin ( N = 2), or cloxacillin alone ( N = 5). Six of eleven children did not attend for follow-up, and their outcome was unknown. Five children followed-up for a period of 4–17 weeks were documented as having resolution of clinical features of infection. RESULTS Overview of cases. Thirty-nine cases of culture-proven melioidosis were identified in children presenting to the AHC between October 2005 and December 2008. No case clustering was observed, and cases were distributed throughout the study period as follows: 2005 (3 months), N = 2; 2006, N = 9; 2007, N = 13; 2008, N = 14. The geographic location of the village or town in which the cases lived is shown in Figure 1 . These observations suggest that infections were sporadic and not related to one or more outbreaks. Twenty-eight children (72%) were admitted to the AHC; 16 cases had localized infection, and 12 cases had disseminated infection. Duration of admission for the 20 in-patient survivors ranged from 1 to 31 days (median = 15.5 days; IQR = 12.0– 17.5 days). Management is presented separately for localized and disseminated infection. Admitted with localized infection ( N = 16). All cases had an incision and drainage procedure. Fifteen cases were started on empiric parenteral antimicrobial drugs (ceftriazone or cloxacillin) for suspected localized bacterial infection at the time of admission. The remaining case was a child with a pharyngeal abscess who was treated with incision and drainage under general anesthetic and discharged the following day with a 7-day course of oral cloxacillin for suspected S. aureus infection before culture results were available. This child was lost to follow-up. The remaining 15 cases were still in the hospital when the B. pseudomallei culture result became available, and therapy was changed to ceftazidime ( N = 14) or parenteral amoxicillin-clavulanate ( N = 1) for a median of 11 days (range = 1–15 days; IQR = 9–14 days). A switch to oral antibiotics was made prior to ( N = 5) or on the day of discharge ( N = 10). All 15 children were discharged on oral amoxicillin-clavulanate, which was prescribed for a median of 7 weeks (range = 2–23 weeks; IQR = 4–13 weeks). One or more follow-up appointments were attended by 13 of 15 cases Summary data for the 39 cases are shown in Table 1 . Median age was 7.8 years (range = 1.6–16.2 years), and the male/female ratio was 1:1.6. Four children (10%) had pre-existing condi- tions that may have pre-disposed them to infection: juvenile rheumatoid arthritis (1 case), thalassemia and renal disease (1 case), Pott’s disease (tuberculosis of the spine; 1 case), and chronic otitis media with B. RESULTS pseudomallei meningitis (1 case). Infection was defined as localized in 27 children (69%) and disseminated in 12 children (31%); 21 of 27 localized infec- tions (78%) were in the region of the head and neck, includ- ing 15 cases of acute suppurative parotitis. The disseminated infection group included nine children with blood cultures positive for B. pseudomallei in association with clinical and radiographic evidence of pneumonia ( N = 2), clinical meningi- tis and a history of chronic suppurative otitis media for whom the cerebrospinal fluid (CSF) culture was negative ( N = 1), or no other identified focus of infection ( N = 6) and three chil- dren who did not have a positive blood culture with multiple skin pustules ( N = 1), mastoiditis and abscesses in the liver and 1108 PAGNARITH AND OTHERS hole country map ( A ) and zoomed view showing the place of residence (triangles) of 39 children with melioid l for Children ( B ). H denotes this hospital. ole country map ( A ) and zoomed view showing the place of residence (triangles) of 39 children with melioido for Children ( B ). H denotes this hospital. Figure 1. Whole country map ( A ) and zoomed view showing the place of residence (triangles) of 39 children with melioidosis presenting to the Angkor Hospital for Children ( B ). H denotes this hospital. 1109 MELIOIDOSIS IN CAMBODIA a novel allele at gmhD . The remaining 19 STs have been described previously in relation to isolates obtained from the environment and/or human disease in at least one Asian country. Table 1 Table 1 Summary data for 39 children with melioidosis Variable Number Male gender 15 38% Age (years), median (range, IQR * ) 7.8 1.6–16.2; 4.1–12.4 Underlying disease present 4 10% Source of B. pseudomallei isolate Blood † 9 23% Pus 29 74% Respiratory secretions 1 3% Severity of infection ‡ Localized 27 69% Disseminated 12 31% Type/site of infection Acute suppurative parotitis 15 38% Superficial soft-tissue abscess 7 18% Blood culture positive and no focus identified 6 15% Lymph-node abscess 4 10% Pneumonia 3 8% Meningitis 1 3% Other § 3 8% Admission WBC (×10 9 cells); median (range, IQR) 16.7 1.6–33.3; 9.6–20.7 Died during admission 8 21% Death attributable to melioidosis 7 18% Time to death (days); median (range) 2 day of admission to day 5 * IQR, interquartile range. DISCUSSION during antimicrobial therapy, and clinical features of infection were documented to have resolved in all of these cases. This is the first description of pediatric melioidosis in Cambodia. It was predicted that melioidosis would occur in our patient population, because the incidence of melioidosis in neighboring northeast Thailand is high. 5– 8 B. pseudomallei has been isolated from soil in the province of Siem Reap, 12 Admitted with disseminated infection ( N = 12). The seven attributable deaths in this group occurred before the culture results were available, and these patients were receiving ceftriaxone at the time of death. All 7 cases were admitted to the intensive care unit where management included ventilation for respiratory failure ( N = 1), inotropes for circulatory failure ( N = 2), or both treatment modalities ( N = 4). Of note, two of four survivors in the disseminated group also had ventilatory failure requiring ventilation (1 case) or continous positive airway pressure (CPAP) therapy (1 case), and one of these cases also required inotropes. Empiric antimicrobial therapy (ceftiaxone) was also given to the four survivors before culture results, and after culture results were known, this therapy was followed by parenteral ceftazidime (1 case), amoxicillin- clavulanate plus ceftriaxone (2 cases), or ciprofloxacin (1 case). The duration of parenteral therapy in this group ranged from 10 to 31 days. Oral amoxicillin-clavulanate was given to all four cases after discharge and continued for between 5 and 23 weeks. One or more follow-up appointments were attended by the four cases during this treatment period, and clinical features of infection were documented to have resolved in all of these cases. Figure 2. Neighbor-joining tree using concatenated sequences of all seven loci for Cambodian B. pseudomallei isolates from pediatric cases or soil ( N = 53) together with concatenated sequences down- loaded from the MLST website ( www.mlst.net ) for B. pseudomal- lei isolates from Thailand that had originated in our laboratory and/ or had been reported previously 18– 20 ( N = 462). Red circles denote Cambodian isolates, and blue circles denote Thai isolates. Bacterial genotyping. The 39 B. pseudomallei isolates from the study patients were resolved by MLST into 33 different STs (0.85 ST per isolate). Of these, 28 STs occurred one time, 4 STs occurred two times, and one ST (ST 70) occurred three times. RESULTS † One child also had B. pseudomallei isolated from urine ‡ Localized infection was defined as a single, discrete culture-positive focus of infection in the absence a positive blood culture or clinical and/or microbiological evidence of dissemina- tion to a second site. Disseminated infection was defined as the presence of infection in two or more discrete body sites and/or the presence of B. pseudomallei in blood. § Psoas muscle abscess (1 case), mastoiditis (1 case), and pharyngeal abscess (1 case). Summary data for 39 children with melioidosis Fourteen B. pseudomallei isolates cultured previously from soil in Cambodia were also examined using MLST, of which 12 were isolated in Siem Reap province during 2006 12 and 2 were isolated in Phnom Penh in southern Cambodia in 1996. These were resolved into 14 different STs, including an additional 6 unique STs that have not been described previously and were not represented in the invasive-strain collection described above. Of the remainder, four STs were also represented in the collection of 39 pediatric isolates, and four STs were not found in the pediatric collection but had been isolated previ- ously in Asia. The large number of STs found in this evaluation (including a total of 20 novel STs) is consistent with studies elsewhere in Asia and Australia, 17– 20 and is based on a high rate of recom- bination of a pool of largely known alleles. A neighbor-joining tree was constructed using concatenated sequences of all seven loci for Cambodian invasive and soil isolates ( N = 53) together with concatenated sequences downloaded from the MLST website ( www.mlst.net ) for B. pseudomallei isolates from Thailand that had originated in our laboratory and/ or had been reported previously 17– 19 ( N = 462). This showed that isolates from Cambodia and Thailand were highly related ( Figure 2 ). * IQR, interquartile range. † One child also had B. pseudomallei isolated from urine ‡ Localized infection was defined as a single, discrete culture-positive focus of infection in the absence a positive blood culture or clinical and/or microbiological evidence of dissemina- tion to a second site. Disseminated infection was defined as the presence of infection in two or more discrete body sites and/or the presence of B. pseudomallei in blood. § Psoas muscle abscess (1 case), mastoiditis (1 case), and pharyngeal abscess (1 case). * IQR, interquartile range. † One child also had B. pseudomallei isolated from urine ‡ Localized infection was defined as a single, discrete culture-positive focus of infection in the absence a positive blood culture or clinical and/or microbiological evidence of dissemina- tion to a second site. Disseminated infection was defined as the presence of infection in two or more discrete body sites and/or the presence of B. pseudomallei in blood. § Psoas muscle abscess (1 case), mastoiditis (1 case), and pharyngeal abscess (1 case). DISCUSSION The clinical features of the cases described here are very sim- ilar to those described elsewhere in Asia. Childhood melioido- sis is predominantly a disease of apparently immunocompetent persons, which contrasts with disease in adulthood when at least 80% of those affected have at least one risk factor that affects host immunity. Our finding that two-thirds of disease was local- ized is consistent with the published literature from Asia, 30– 34 and the proportion of localized infections in the region of the head and neck is also consistent. 31, 33 A previous report describ- ing acute suppurative parotitis in children caused by B. pseudo- mallei in northeast Thailand suggested that this syndrome may prove to be a sensitive indicator of the presence of melioi- dosis within a given geographic area. 9 This has proven to be the case for childhood infection in Asia, although this form of the disease is rare in Thai adults (occurring in 1% of adults with melioidosis in northeast Thailand) and is not observed in Australia. 35 We speculate that parotitis and infection of the regional lymph nodes of the neck may relate to the presence of contaminated water in the mouth related to bathing, swim- ming, and drinking of untreated water, whereas localized infec- tion of the lower limbs may relate to inoculation events. Overall death rates in previous pediatric case series have varied from 10–38%, but common to all studies were the findings that no deaths occurred in children with localized disease and that bac- teremia was associated with a high mortality rate. 30– 35 Ceftriaxone and other cephalosporins, including cefotaxime, are active against B. pseudomallei in vitro . In a study of 100 B. pseudomallei isolates from northern Australia, the min- imum inhibitory concentration (MIC) 50 and MIC 90 were 4 μg/mL and 8 μg/mL, respectively, with a MIC range of 2–8 μg/mL 25 (MIC for susceptible Gram-negative bacte- ria is ≤ 8 μg/mL). The comparative MIC values for ceftazi- dime in the same study were 2 μg/mL and 4 μg/mL for the MIC 50 and MIC 90 , respectively, with a range of 1–8 μg/mL 25 (MIC for susceptible B. pseudomallei is ≤ 8 μg/mL). 26 This suggests that ceftazidime may have an advantage over cef- triaxone in vivo . The dose of ceftriaxone used at the AHC is 50–75 mg/kg given one time daily or in two divided doses. DISCUSSION ST 70 is the most frequent clone isolated in Thailand and has also been identified previously in Laos and Hong Kong. Fourteen STs (42%) had not been identified previously and were unique to this study, one of which also included Figure 2. Neighbor-joining tree using concatenated sequences of all seven loci for Cambodian B. pseudomallei isolates from pediatric cases or soil ( N = 53) together with concatenated sequences down- loaded from the MLST website ( www.mlst.net ) for B. pseudomal- lei isolates from Thailand that had originated in our laboratory and/ or had been reported previously 18– 20 ( N = 462). Red circles denote Cambodian isolates, and blue circles denote Thai isolates. 1110 PAGNARITH AND OTHERS and seropositivity in the pediatric population has been already documented. 12 However, we are able to draw several impor- tant lessons from our study. tality rate in 528 patients given empiric ceftazidime therapy on admission was 41.7% compared with a mortality rate of 71% in 167 patients who received cefotaxime or ceftriaxone. 28 This study had several possible confounders; for example, the cefotaxime/ceftriaxone group had a higher rate of positive blood cultures than patients in the ceftazidime group, and this would be predicted to be associated with a higher death rate. However, this finding supports the use of ceftazidime in pref- erence to other third-generation cephalosporins in patients with suspected or confirmed melioidosis. y This report highlights the value of diagnostic microbiol- ogy in defining the epidemiology of infectious diseases in developing world settings. Patterns of infectious diseases and resistance rates of prevalent pathogens may vary both within and between countries, and microbiology provides relevant, region-specific information on which to base effective empiric antimicrobial prescribing policies. Information generated by diagnostic microbiology also provides a feedback loop to phy- sicians on the accuracy of presumptive admission diagnoses. Melioidosis emerges when diagnostic microbiology facilities become available in areas where B. pseudomallei is present in the environment, but this is just one of many examples of the benefits associated with culture capabilities at the AHC. For example, we have recently identified children infected with multi-resistant community-associated methicillin resis- tant Staphylococcus aureus (CA-MRSA) 21 and are currently defining rates of CA-MRSA carriage in the community. Our laboratory is also providing information for the region on the burden of childhood disease from vaccine-preventable infec- tions such as those caused by Streptococcus pneumoniae and Haemophilus influenzae . DISCUSSION p Ceftazidime has been made available at the AHC and is now the parenteral treatment of choice for children with con- firmed melioidosis, followed by a course of oral amoxicillin- clavulanate. Deciding on best practice for empiric treatment is less clear cut, because melioidosis is rare (it accounted for 0.3% of all admissions during the study period), and B. pseudomallei ranks low on the list of bacterial pathogens isolated in our laboratory. Ceftriaxone would be the pre- ferred choice over ceftazidime for several of the more com- mon pathogens. Our current policy is to use ceftriaxone as the first-line empiric drug until culture results become available but to use ceftazidime or amoxicillin-clavulanate as the first- line choice in children admitted with acute suppurative paro- titis, in cases with multiple abscesses in the liver and/or spleen, and in cases suspected melioidosis based on other grounds. All deaths attributable to melioidosis occurred before cul- ture results became available, and more rapid diagnostic tests are required to improve time to effective therapy. We are reviewing the turnaround time of culture results and the pos- sibility of introducing a direct immunofluorescence test that can be used on pus and other material from suspected cases. This test can provide a presumptive same-day diagnosis and has a reported sensitivity and specificity of 66% and 99.5%, respectively. 29 The empiric antimicrobial drug of choice for children admit- ted to the AHC with suspected bacterial infection is ceftriax- one. This has broad-spectrum activity against a wide range of Gram-positive and Gram-negative bacteria, but is not recom- mended for melioidosis. Current guidelines for the antimicro- bial therapy of melioidosis are based on trials conducted in adult patients. 22– 24 They consist of an intensive phase of intra- venous antimicrobial agents (ceftazidime or a carbapenem drug) for a minimum of 10–14 days followed by an eradica- tion phase of oral antimicrobial agents (TMP-SMX with or without doxycycline or amoxicillin-clavulanate for pregnant women, children, or adults who cannot tolerate TMP-SMX) to complete a course of treatment lasting 12–20 weeks. A system- atic evaluation of the antimicrobial therapy of children has not been published. This is an important knowledge gap, because localized infection may be cured with shorter courses of anti- biotics, particularly when infection is mild and localized and a procedure is performed to drain pus. DISCUSSION A possible disadvantage of one-time daily dosing is that the plasma concentration may fall below the MIC of the organ- ism; average reported plasma concentrations in healthy adults 24 hours after a single intravenous infusion of 0.5, 1, or 2 gm were reported to be 5, 9, and 15 μg/mL, respectively. 27 A ret- rospective review of empiric treatment in 1,353 adult patients with melioidosis in northeast Thailand reported that the mor- Melioidosis is not considered in the differential diagnosis in the majority of health-care settings in Cambodia, and the level of local knowledge about this condition is low. Commonly 1111 MELIOIDOSIS IN CAMBODIA sis in Cambodia . Trans R Soc Trop Med Hyg 102 (Suppl 1) : S21 – S25 . used empiric antimicrobial therapies will rarely include first- line therapy for melioidosis. The identification of melioidosis at the AHC has led to an increased awareness and understand- ing of this infection in our practice, and this will be followed by improvements in patient care. 14. Peacock SJ , Chieng G , Cheng AC , Dance DA , Amornchai P , Wongsuvan G , Teerawattanasook N , Chierakul W , Day NP , Wuthiekanun V , 2005 . Comparison of Ashdown’s medium, Burkholderia cepacia medium, and Burkholderia pseudomallei selective agar for clinical isolation of Burkholderia pseudomal- lei . J Clin Microbiol 43: 5359 – 5361 . Received January 15, 2010. Accepted for publication March 2, 2010. Received January 15, 2010. Accepted for publication March 2, 2010. 15. Wuthiekanun V , Anuntagool N , White NJ , Sirisinha S , 2002 . Short report: a rapid method for the differentiation of Burkholderia pseudomallei and Burkholderia thailandensis . Am J Trop Med Hyg 66: 759 – 761 . Acknowledgments: We are grateful for the assistance given by the director and staff of the Angkor Hospital for Children and the Mahidol Oxford Tropical Medicine Research Unit. The authors thank Uri Gilad, who drew the maps shown. Acknowledgments: We are grateful for the assistance given by the director and staff of the Angkor Hospital for Children and the Mahidol Oxford Tropical Medicine Research Unit. The authors thank Uri Gilad, who drew the maps shown. 16. Amornchai P , Chierakul W , Wuthiekanun V , Mahakhunkijcharoen Y , Phetsouvanh R , Currie BJ , Newton PN , van Vinh Chau N , Wongratanacheewin S , Day NP , Peacock SJ , 2007 . REFERENCES The epidemiology of melioidosis in Ubon Ratchatani, northeast Thailand . Int J Epidemiol 23: 1082 – 1090 . 23. Chierakul W , Anunnatsiri S , Short JM , Maharjan B , Mootsikapun P , Simpson AJ , Limmathurotsakul D , Cheng AC , Stepniewska K , Newton PN , Chaowagul W , White NJ , Peacock SJ , Day NP , Chetchotisakd P , 2005 . Two randomized controlled trials of ceftazidime alone versus ceftazidime in combination with trimethoprim-sulfamethoxazole for the treatment of severe melioidosis . Clin Infect Dis 41: 1105 – 1113 . p 7. Leelarasamee A , Trakulsomboon S , Kusum M , Dejsirilert S , 1997 . Isolation rates of Burkholderia pseudomallei among the four regions in Thailand . Southeast Asian J Trop Med Public Health 28: 107 – 113 . 8. Chierakul W , Rajanuwong A , Wuthiekanun V , Teerawattanasook N , Gasiprong M , Simpson A , Chaowagul W , White NJ , 2004 . The changing pattern of bloodstream infections associated with the rise in HIV prevalence in northeastern Thailand . Trans R Soc Trop Med Hyg 98: 678 – 686 . f 24. Chaowagul W , Chierakul W , Simpson AJ , Short JM , Stepniewska K , Maharjan B , Rajchanuvong A , Busarawong D , Limmathurotsakul D , Cheng AC , Wuthiekanun V , Newton PN , White NJ , Day NP , Peacock SJ , 2005 . Open-label randomized trial of oral trimethoprim-sulfamethoxazole, doxycycline, and chloram- phenicol compared with trimethoprim-sulfamethoxazole and doxycycline for maintenance therapy of melioidosis . Antimicrob Agents Chemother 49: 4020 – 4025 . p yg 9. Dance DA , Davis TM , Wattanagoon Y , Chaowagul W , Saiphan P , Looareesuwan S , Wuthiekanun V , White NJ , 1989 . Acute suppu- rative parotitis caused by Pseudomomas pseudomallei in chil- dren . J Infect Dis 159: 654 – 660 . 10. Phetsouvanh R , Phongmany S , Newton P , Mayxay M , Ramsay A , Wuthiekanun V , White NJ , 2001 . Melioidosis and Pandora’s box in the Lao People’s Democratic Republic . Clin Infect Dis 32: 653 – 654 . g 25. Ashdown LR , 1988 . In vitro activities of the newer beta-lactam and quinolone antimicrobial agents against Pseudomonas pseudomallei . Antimicrob Agents Chemother 32: 1435 – 1436 . 11. REFERENCES 19. McCombie RL , Finkelstein RA , Woods DE , 2006 . Multilocus sequence typing of historical Burkholderia pseudomallei iso- lates collected in southeast Asia from 1964 to 1967 provides insight into the epidemiology of melioidosis . J Clin Microbiol 44: 2951 – 2962 . 1. Wiersinga WJ , van der Poll T , White NJ , Day NP , Peacock SJ , 2006 . Melioidosis: insights into the pathogenicity of Burkholderia pseudomallei . Nat Rev Microbiol 4: 272 – 282 . p 2. Cheng AC , Currie BJ , 2005 . Melioidosis: epidemiology, pathophys- iology, and management . Clin Microbiol Rev 18: 383 – 416 . 20. Cheng AC , Godoy D , Mayo M , Gal D , Spratt BG , Currie BJ , 2004 . Isolates of Burkholderia pseudomallei from northern Australia are distinct by multilocus sequence typing, but strain types do not correlate with clinical presentation . J Clin Microbiol 42: 5477 – 5483 . 3. Currie BJ , Dance DA , Cheng AC , 2008 . The global distribution of Burkholderia pseudomallei and melioidosis: an update . Trans R Soc Trop Med Hyg 102 (Suppl 1) : S1 – S4 . 21. Chheng K , Tarquinio S , Wuthiekanun V , Sin L , Thaipadungpanit J , Amornchai P , Chanpheaktra N , Tumapa S , Putchhat H , Day NP , Peacock SJ , 2009 . Emergence of community-associated methicillin- resistant Staphylococcus aureus associated with pediatric infection in Cambodia . PLoS One 4: e6630 . 4. Wuthiekanun V , Peacock SJ , 2006 . Management of melioidosis . Expert Rev Anti Infect Ther 4: 445 – 455 . 5. Chaowagul W , White NJ , Dance DA , Wattanagoon Y , Naigowit P , Davis TM , Looareesuwan S , Pitakwatchara N , 1989 . Melioidosis: a major cause of community-acquired septicemia in northeast- ern Thailand . J Infect Dis 159: 890 – 899 . 22. Simpson AJ , Suputtamongkol Y , Smith MD , Angus BJ , Rajanuwong A , Wuthiekanun V , Howe PA , Walsh AL , Chaowagul W , White NJ , 1999 . Comparison of imipenem and ceftazidime as therapy for severe melioidosis . Clin Infect Dis 29: 381 – 387 . 6. Suputtamongkol Y , Hall AJ , Dance DA , Chaowagul W , Rajchanuvong A , Smith MD , White NJ , 1994 . DISCUSSION Accuracy of Burkholderia pseudomallei identification using the API 20NE system and a latex agglutination test . J Clin Microbiol 45: 3774 – 3776 . Financial support: This study was funded by the Wellcome Trust and the Li Kha Shing Foundation. Financial support: This study was funded by the Wellcome Trust and the Li Kha Shing Foundation. Authors’ addresses: Yos Pagnarith, Varun Kumar, and Lina Sin, Angkor Hospital for Children, Siem Reap, Cambodia, E-mails: yospagnarith@yahoo.com , brownvk1@yahoo.com , and linasin2005@ yahoo.com . Janjira Thaipadungpanit, Vanaporn Wuthiekanun, Premjit Amornchai, and Nicholas P. Day, Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand, E-mails: janjira@tropmedres.ac , lek@tropmedres .ac , kung@tropmedres.ac , and nickd@tropmedres.ac . Sharon J. Peacock, Department of Medicine, University of Cambridge, Addenbrooke’s Hospital, Cambridge, United Kingdom, E-mail: sp10@sanger.ac.uk . 17. Vesaratchavest M , Tumapa S , Day NP , Wuthiekanun V , Chierakul W , Holden MT , White NJ , Currie BJ , Spratt BG , Feil EJ , Peacock SJ , 2006 . Nonrandom distribution of Burk- holderia pseudomallei clones in relation to geographical loca- tion and virulence . J Clin Microbiol 44: 2553 – 2557 . 18. Godoy D , Randle G , Simpson AJ , Aanensen DM , Pitt TL , Kinoshita R , Spratt BG , 2003 . Multilocus sequence typing and evolutionary relationships among the causative agents of melioidosis and glanders, Burkholderia pseudomallei and Burk- holderia mallei . J Clin Microbiol 41: 2068 – 2079 . REFERENCES Wuthiekanun V , Mayxay M , Chierakul W , Phetsouvanh R , Cheng AC , White NJ , Day NP , Peacock SJ , 2005 . Detection of Burkholderia pseudomallei in soil within the Lao People’s Democratic Republic . J Clin Microbiol 43: 923 – 924 . p g 26. National Committee for Clinical Laboratory Standards , 2000 . Per- formance Standards for Antimicrobial Disk Susceptibility Testing— 14th Informational Supplement M100-S14 . Wayne, PA : NCCLS. f pp y 27. Genentech Production Information, 2010. Rocephin® (ceftriaxone) full prescribing information. Available at: http://www.rocheusa .com/products/rocephin/pi.pdf . Accessed April 2, 2010. 12. Wuthiekanun V , Pheaktra N , Putchhat H , Sin L , Sen B , Kumar V , Langla S , Peacock SJ , Day NP , 2008 . Burkholderia pseudo- mallei antibodies in children, Cambodia . Emerg Infect Dis 14: 301 – 303 . p p p p p 28. Chaowagul W , Simpson AJ , Suputtamongkol Y , White NJ , 1999 . Empirical cephalosporin treatment of melioidosis . Clin Infect Dis 28: 1328 . 13. Overtoom R , Khieu V , Hem S , Cavailler P , Te V , Chan S , Lau P , Guillard B , Vong S , 2008 . A first report of pulmonary melioido- 1112 PAGNARITH AND OTHERS 29. Wuthiekanun V , Desakorn V , Wongsuvan G , Amornchai P , Cheng AC , Maharjan B , Limmathurotsakul D , Chierakul W , White NJ , Day NP , Peacock SJ , 2005 . Rapid immunofluores- cence microscopy for diagnosis of melioidosis . Clin Diagn Lab Immunol 12: 555 – 556 . 32. Lumbiganon P , Chotechuangnirun N , Kosalaraksa P , 2004 . Clinical experience with treatment of melioidosis in children . Pediatr Infect Dis J 23: 1165 – 1166 . f 33. How SH , Ng KH , Jamalludin AR , Shah A , Rathor Y , 2005 . Pediatric melioidosis in Pahang, Malaysia . Med J Malaysia 60: 606 – 613 . 30. Pongrithsukda V , Simakachorn N , Pimda J , 1988 . Childhood melioi- dosis in northeastern Thailand . Southeast Asian J Trop Med Public Health 19: 309 – 316 . 34. Sam IC , Puthucheary SD , 2006 . Melioidosis in children from Kuala Lumpur, Malaysia . Ann Trop Paediatr 26: 219 – 224 . 31. Lumbiganon P , Viengnondha S , 1995 . REFERENCES Clinical manifestations of melioidosis in children . Pediatr Infect Dis J 14: 136 – 140 . p y 35. Kandasamy Y , Norton R , 2008 . Paediatric melioidosis in north Queensland, Australia . J Paediatr Child Health 44: 706 – 708 .
https://openalex.org/W4237005655	https://www.researchsquare.com/article/rs-6298/latest.pdf	English	null	Effects of iron on the growth, biofilm formation and virulence of Klebsiella pneumoniae causing liver abscess	Research Square (Research Square)	2,020	cc-by	5,166	Research article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at BMC Microbiology on February 18th, 2020. See the published version at https://doi.org/10.1186/s12866-020-01727-5. Page 1/15 Abstract mellonella larvae indicated the virulence of liver abscess-causing K. pneumoniae (2/3) cultured in LB broth with additional iron was significantly higher than those under iron- restricted conditions (P<0.05). The relative expression levels of the four siderophore genes (iucB, B, irp1, entB) in K. pneumoniae strains isolated from liver abscesses cultured with additional iron were er than those under iron-restricted conditions (P<0.05). Conclusions: It is suggested by our research t iron in the environment can promote growth, biofilm formation and enhance virulence of K. umoniae causing liver abscesses. A lower expression of siderophore genes correlates with increased lence of liver abscess-causing K. pneumoniae. Further deeper evaluation of these phenomena is rranted. Abstract Background: Klebsiella pneumoniae is considered the most clinically relevant species of Background: Klebsiella pneumoniae is considered the most clinically relevant species of Enterobacteriaceae, known to cause severe infections including liver abscesses. To the best of our knowledge, a large proportion of iron in the human body is accumulated and stored in the liver. We hypothesize that increased iron availability is an important factor driving liver abscess formation and we therefore aim to understand the effects of iron on K. pneumoniae causing liver abscesses. Results: All tested K. pneumoniae clinical isolates, including those isolated from liver abscesses and other abdominal invasive infection sites, grew optimally when cultured in LB broth supplemented with 50 μM iron and exhibited the strongest biofilm formation ability under those conditions. Decreased growth and biofilm formation ability were observed in all tested strains when cultured with an iron chelator (P<0.05). The infection model of G. mellonella larvae indicated the virulence of liver abscess-causing K. pneumoniae (2/3) cultured in LB broth with additional iron was significantly higher than those under iron- restricted conditions (P<0.05). The relative expression levels of the four siderophore genes (iucB, iroB, irp1, entB) in K. pneumoniae strains isolated from liver abscesses cultured with additional iron were lower than those under iron-restricted conditions (P<0.05). Conclusions: It is suggested by our research that iron in the environment can promote growth, biofilm formation and enhance virulence of K. pneumoniae causing liver abscesses. A lower expression of siderophore genes correlates with increased virulence of liver abscess-causing K. pneumoniae. Further deeper evaluation of these phenomena is warranted. Background: Klebsiella pneumoniae is considered the most clinically relevant species of Enterobacteriaceae, known to cause severe infections including liver abscesses. To the best of our knowledge, a large proportion of iron in the human body is accumulated and stored in the liver. We hypothesize that increased iron availability is an important factor driving liver abscess formation and we therefore aim to understand the effects of iron on K. pneumoniae causing liver abscesses. Results: All tested K. pneumoniae clinical isolates, including those isolated from liver abscesses and other abdominal invasive infection sites, grew optimally when cultured in LB broth supplemented with 50 μM iron and exhibited the strongest biofilm formation ability under those conditions. Decreased growth and biofilm formation ability were observed in all tested strains when cultured with an iron chelator (P<0.05). The infection model of G. Background Klebsiella pneumoniae is considered the most clinically relevant species of Enterobacteriaceae, known to cause both community-acquired and nosocomial infections, including liver abscesses, pneumonia, urinary tract infections and bacteremia worldwide [1]. In the past two decades, a distinct hypervirulent variant of K. pneumoniae, characterized by its hypermucoviscous phenotype, was firstly isolated from liver abscesses in Asia and has emerged as a clinically significant pathogen responsible for highly invasive infections [2]. Unlike classical K. pneumoniae, hypervirulent K. pneumoniae (hvKp) can spread from the original site of infection to other organs. Once invasive dissemination occurs, patients often suffer severe and irreversible refractory sequelae, such as blindness and central nervous system damage [2,3]. The conditions of patients infected with hypervirulent K. pneumoniae causing liver abscess are serious, posing a great threat to public health and has attracted the attention of clinicians. The pathogenicity of K. pneumoniae mainly arise from various virulence factors which allow it to overcome innate host immunity and to maintain infection in a mammalian host. The main virulence factors that play an important role in pathogenicity are capsular polysaccharide, lipopolysaccharide, pili and siderophores [4]. Therein, K1 and K2 capsular types are considered to be highly pathogenic to human [5]. rmpA is an important activator of capsular production, resulting in the formation of hypermucoviscous phenotype and enhancement of virulence, while aerobactin (iucB), yersiniabactin (irp1), salmochelin (iroB) and enterobacterin (entB) are the four siderophores of K. pneumoniae [6,7]. Page 2/15 Page 2/15 Page 2/15 Therefore, understanding the virulence characteristics of K. pneumoniae and taking appropriate measures are critical for clinical assessment, controlling the prognosis and reducing mortality risk of patients with K. pneumoniae infection. It is recognized that most organisms require iron for a variety of metabolic and informational cellular pathways [8]. However, ferric ion (Fe3+) and its derivatives are poorly soluble, and cannot be utilized directly by most organisms. It is essential to synthesize and secrete siderophores to meet the demand for iron required for bacterial growth and metabolism. To repress microbial growth, iron availability within the human body is strictly limited [9]. One notable exception is the liver, in which iron concentration is used for assessing body iron stores [10]. These increased iron levels probably promote growth of K. Antimicrobial Susceptibility testing Five K. pneumoniae isolates (FK3065, FK3170, FK3226, FK3992, FK4003) were sensitive to conventional antimicrobials (levofloxacin, ciprofloxacin, cefepime, ceftazidime, imipenem, gentamicin, amikacin, tobramycin, ampicillin/sulbactam, piperacillin/tazobactam, trimethoprim/sulfamethoxazole, aztreonam, cefotetan, ertapenem, ceftriaxone). However, FK3087 was resistant to ampicillin/sulbactam, levofloxacin, ciprofloxacin, gentamicin and trimethoprim/sulfamethoxazole. Effect of iron on growth and biofilm formation of the K. pneumoniae isolates All K. pneumoniae isolates grew optimally in LB broth containing 50 μM iron, superior to the growth in LB broth containing 30 μM, 10 μM, and 0 μM iron, separately (P < 0.05). The growth of all tested strains in the iron-restricted environment caused by the iron chelating agent was worse (P < 0.05) (Figure1). In addition, we further found that iron promotes biofilm formation of K. pneumoniae in a concentration- dependent manner. All strains exhibited the strongest biofilm formation ability in LB broth supplemented with 50 μM iron, which was superior to the biofilm formation ability of the respective strains in LB broth containing 30 μM, 10 μM and 0 μM iron (P < 0.05). The strains showed a weaker biofilm formation ability in an iron-restricted environment (P < 0.05) (Figure 2). Background i i th li d id d t f li b f ti Th f t d It is recognized that most organisms require iron for a variety of metabolic and informational cellular pathways [8]. However, ferric ion (Fe3+) and its derivatives are poorly soluble, and cannot be utilized directly by most organisms. It is essential to synthesize and secrete siderophores to meet the demand for iron required for bacterial growth and metabolism. To repress microbial growth, iron availability within the human body is strictly limited [9]. One notable exception is the liver, in which iron concentration is used for assessing body iron stores [10]. These increased iron levels probably promote growth of K. pneumoniae in the liver and provide an advantage for liver abscess formation. Therefore, our study was designed to evaluate the effects of iron on the growth, biofilm formation and virulence of liver abscess- causing K. pneumoniae. umoniae in the liver and provide an advantage for liver abscess formation. Therefore, our study was gned to evaluate the effects of iron on the growth, biofilm formation and virulence of liver abscess- sing K. pneumoniae. The virulence factors of K. pneumoniae isolates The three liver abscess-causing K. pneumoniae isolates (FK3226, FK3992 and FK4003) carried at least nine virulence genes, including four kinds of siderophore genes (entB, iucB, iroB, irp1). The serotypes of these three isolates were K1, K2 and K1, respectively. The three non-liver abscess-causing K. pneumoniae isolates (FK3065, FK3087 and FK3170) carried four virulence genes, including the siderophore gene entB, Page 3/15 Page 3/15 and their serotypes did not belong to one of the common capsule types tested. The virulence genes of each strain were detailed in Table 1. Infection model of G. mellonella larvae The mortality rates of G. mellonella after infection with the liver abscess-causing K. pneumoniae isolates (FK3226, FK3992) and K. pneumoniae ATCC700603 cultured with additional iron were significantly higher than those cultured with iron chelator (P < 0.05). While the mortality rates of G. mellonella after infection with the non-liver abscess-causing K. pneumoniae isolates (FK3065, FK3087 and FK3170) and liver abscess-causing K. pneumoniae (FK4003) were not significantly different under these two conditions (Figure 3). Quantification of siderophore gene expression with qRT-PCR The relative expression levels of the four siderophore genes (iucB, iroB, irp1, entB) in the liver abscess- causing K. pneumoniae strains cultured with additional iron were lower than those cultured under iron- restricted conditions (P < 0.05). On the contrary, the relative expression levels of the siderophore gene entB in the non-liver abscess-causing K. pneumoniae isolates cultured with extra iron were higher than those cultured under iron-restricted conditions (P < 0.05) (Figure 4). Discussion The Gram-negative bacillus K. pneumoniae is a leading cause of healthcare-associated infections, including urinary tract infections, surgical sites infections, soft tissues infections, bacteremia and pneumonia [11]. In the 1980s, case reports from Taiwan described community acquired liver abscesses caused by hvKp in patients with serious concomitant endorgan manifestations, such as meningitis and endophthalmitis [12]. Since the first report in Taiwan, hvKPs have been observed with increasing frequency in many countries in Asia, Europe and America [12]. It is well known that hvKp can invade many organs in patient, including liver. Considering the different iron content between the liver and other tissues, we speculated that this higher iron level may be an important factor to affect the virulence of K. pneumoniae in the liver and provide an advantage for liver abscess formation. In this study, three liver abscess-causing K. pneumoniae isolates and three non-liver abscess-causing K. pneumoniae isolates were selected to perform growth, biofilm and virulence investigations under different iron conditions. In this research, we demonstrated that moderate amounts of iron promoted the growth of K. pneumoniae. A slower growth of K. pneumoniae isolates was discovered in iron-restricted environments. Furthermore, it was illustrated that iron promoted biofilm formation of K. pneumoniae, and the ability of bacterial biofilm formation was significantly attenuated by addition of an iron chelator. Previously, it has been demonstrated that the biofilm formation ability of Pseudomonas aeruginosa was significantly reduced in an iron-restricted environment, and biofilm formation increased significantly after iron complementation, indicating that iron plays an important role in the formation of P. aeruginosa biofilms [13]. The presence of an appropriate amount of iron is also conducive to the biofilm formation of Escherichia coli and Page 4/15 Staphylococcus aureus [14,15]. These findings, together with our investigation, indicate that iron plays a crucial role in the process of bacterial biofilm formation and growth. In this study, the virulence genes and capsular serotypes of seven experimental strains were detected by PCR. These three liver abscess-causing K. pneumoniae isolates carried more virulence genes, including all four siderophore genes iucB, iroB, irp1, entB, while the strains isolated from the other sites only carried enterobactin (entB) among their four virulence factors. Bacteria secrete siderophores to bind and import iron, which is required for growth. In this investigation, the effect of iron concentration on the relative expression of siderophore genes was detected by qRT-PCR. As expected, the tested K. Conclusions In summary, iron can promote the growth, biofilm formation and enhance virulence of K. pneumoniae causing liver abscess. Moreover, the diverse expression of siderophores genes may be one of the factors that regulate the variation in virulence. To further understand these phenomena, a deeper evaluation of the phenomena is warranted. Discussion pneumoniae strains causing liver abscesses showed lower relative expression levels of the four siderophore genes (iucB, iroB, irp1, entB) in media with high iron levels, compared to iron-restricted media. In the presence of the iron chelator, high-affinity iron binding siderophores are needed to obtain sufficient iron for growth, while at high iron concentrations less metabolically costly uptake mechanisms can be employed. In contrast, the single siderophore gene entB of the non-liver abscess-causing K. pneumoniae isolates was expressed relatively higher after addition of iron than during iron deficiency caused by iron chelating agent. This is a surprising finding, which might indicate that these strains rely on other iron uptake mechanisms apart from siderophores, which warrants further study. Previous studies have shown that different concentrations of iron could affect the surface characteristics of the pili and porins and further influence the virulence of Acinetobacter baumannii [16]. Therefore, We attempted to demonstrate that this higher iron level may be an important factor to affect the virulence of K. pneumoniae in the liver. To investigate the effect of iron on the virulence of K. pneumoniae, we determined the virulence of K. pneumoniae through the wax larvae infection model. Wax larvae are an invertebrate host model frequently used to detect the virulence of K. pneumoniae [17]. According to the results of the G. mellonella infection model, three strains caused higher mortality rate with extra iron than iron-restricted growth environment, indicating the potential virulence in vivo. The results of qRT-PCR indicate that a lower expression of siderophore genes correlates with increased virulence. Due to the various needs of different strains for iron, the expression of siderophore genes was not exactly the same. Other mechanisms by which iron affects the virulence of K. pneumoniae need to be further explored. Growth curves and biofilm formation The effect of iron on the growth of K. pneumoniae was measured following previous methods with some modifications [18,19]. In brief, overnight cultures of all K. pneumoniae clinical isolates (FK3065, FK3087, FK3170, FK3226, FK3992, FK4003) and K. pneumoniae ATCC 700603 were diluted 1:100 in Luria-Bertani (LB) broth supplemented with different iron concentrations (50 μM, 30 μM, 10 μM, 0 μM) and with 200 μM iron chelating agent (2,2'-Dipyridyl) +(-) 50 μM iron, respectively. Wherein, strains under LB + 200 μM iron chelating agent condition were used as control and LB + 200 μM iron chelating agent + 50 μM iron was set to produce an iron-restricted condition. The cultures were incubated at 37 °C with constant shaking at 180 rpm. Samples were collected at 0 h, 2 h, 4 h, 6 h, 8 h, 16 h, 24 h and the absorbance at 600nm was determined. Each sample was measured in triplicates and averages of absorbance values were used for analysis. The growth of K. pneumoniae was evaluated by plotting the values of OD600 against time. The biofilm assay was performed as published with some modifications [20]. Briefly, six K. pneumoniae clinical isolates and K. pneumoniae ATCC 700603 were grown overnight in LB broth. The overnight cultures were then diluted 1:100 in fresh LB broth supplemented with different iron concentrations (50 μM, 30 μM, 10 μM, 0 μM) and with 200 μM iron chelating agent +(-) 50 μM iron. A total of 100μL of each dilution were added to a 96-well polystyrene microtiter plate and incubated at 37 ℃ for 24 h. Wells containing media alone were used as blank. Planktonic cells were removed and the wells were washed twice with sterile water, then the wells were stained with 150 μL 0.1% crystal violet for 10 min and rinsed twice with sterile water. Stained biofilms were solubilized with 95% ethanol and quantified by measuring the OD600 using a microplate reader. Each sample was measured in triplicates and averages of absorbance values were used for analysis. Methods Bacterial strains and antimicrobial susceptibility profiling Page 5/15 Six K. pneumoniae clinical isolates (FK3065, FK3087, FK3170, FK3226, FK3992, FK4003) were collected from patients in the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China during 2016 and 2017. Herein, FK3226, FK3992, FK4003 were isolated from patients with liver abscess, and FK3065, FK3087, FK3170 were isolated from other abdominal invasive infection sites of non-liver abscess patients (ascites, biliary drainage fluid). Identification was conducted on all isolates using VITEK MS system (bioMérieux, Marcy L’Etoile, France). Antimicrobial susceptibility testing was performed by VITEK2 system (bioMérieux, Marcy L’Etoile, France) with AST-GN13 card. K. pneumoniae ATCC 700603 served as the control strain. Detection of virulence genes of isolates Virulence genes (magA, iucB, iroB, entB, irp1, iroN, kfuBC, rmpA, wcaG, alls, ybtA, ureA, uge, wabG, fimH and mrkD) and capsular serotypes (K1, K2, K5, K20, K54 and K57) of the six K. pneumoniae clinical isolates and K. pneumoniae ATCC 700603 were amplified by Polymerase Chain Reaction (PCR). Primers for the aforementioned genes are listed in Supplementary Table S1 [21,22]. Positive PCR products were Page 6/15 sequenced by Beijing Genomics Institute Technology Co. Ltd (Shanghai, China). Nucleotide sequences were compared using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Infection model of Galleria mellonella larvae G. mellonella killing assays were carried out on the six clinic as described previously, with minor modifications [17]. Eigh were randomly selected for each strain. A 10 μL of bacteria buffered saline (PBS) was injected into the last left proleg u bacterial suspension was prepared by culturing the strains i iron with 200 μM iron chelating agent for 24 h. Larvae inject insects were incubated at 37℃ in the dark and observed af considered dead when they repeatedly failed to respond to p insect model was rapidity and extent of mortality of G. mell and log-rank test. G. mellonella killing assays were carried out on the six clinical isolates and K. pneumoniae ATCC 700603 as described previously, with minor modifications [17]. Eight larvae weighing between 200mg-250mg were randomly selected for each strain. A 10 μL of bacterial suspension (108 CFU/mL) in phosphate- buffered saline (PBS) was injected into the last left proleg using a 25 μL Hamilton precision syringe. The bacterial suspension was prepared by culturing the strains in LB broth containing 50 μM iron, and 50 μM iron with 200 μM iron chelating agent for 24 h. Larvae injected with 10 μl PBS were used as control. The insects were incubated at 37℃ in the dark and observed after 24 h, 48 h and 72 h. Larvae were considered dead when they repeatedly failed to respond to physical stimuli. The primary outcome for the insect model was rapidity and extent of mortality of G. mellonella assessed with Kaplan-Meier analysis and log-rank test. Quantitative reverse transcription PCR (qRT-PCR) The effects of iron on the expression levels of K. pneumoniae siderophore genes (iucB, iroB, entB and irp1) were evaluated using quantitative reverse transcription PCR (qRT-PCR). For RNA extraction, K. pneumoniae isolates were grown in fresh LB medium with 50 μM iron, and 50 μM iron with 200 μM iron chelating agent at 37 ℃ for 24h. K. pneumoniae isolates grown in fresh LB medium were used as control. Total RNA was extracted using a RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. The extracted RNA samples were stored at −80 ℃. Purified RNA was reverse transcribed into cDNA for qRT-PCR analysis using a cDNA synthesis kit (TaKaRa, Tokyo, Japan) according to the manufacturer’s instructions. Gene expression levels were measured with qRT-PCR using a 7500 RT-PGE system (TOYOBO, Osaka, Japan) and SYBR Green qRT-PCR Kit (TOYOBO) with the specific primers listed in Supplementary Table S2 [23]. The rpoB gene was used as an internal control to normalize the data. Each sample was measured in triplicates and averages of Ct values were used for analysis. Gene expression levels were calculated using 2−△△Ct method. Statistical Analysis All experiments were conducted independently with at least two replicates on different days, and results were expressed as mean ± standard deviation or average. The total area under the curve was calculated for analysis on growth. Unpaired or two-tailed paired t-tests were used to evaluate the significance of differences between two groups. One-way analysis of variance (ANOVA) was performed to analyze the significance among more groups. Statistical significance was determined at P < 0.05. Statistical analyses were performed using SPSS version 17.0 statistical software. List Of Abbreviations Availability of data and materials The datasets used and analysed during the current study available from the corresponding author on reasonable request. Consent for publication Not applicable. Authors' contributions TC and GFD contributed equally to this study. TC and GFD wrote the manuscript under supervision of JMC, TLZ and QW. TC, GFD, SQZ, XCZ and YJZ performed the research. TC and GFD performed the data analysis. The research plan for this project was conceived based on several rounds of discussions among all co-authors. All authors read and approved the final version of the manuscript. Funding This work was supported by research grants from the National Natural Science Foundation of China (No. 81971986)and the Health Department of Zhejiang Province of the People's Republic of China (No.2019KY098).These two funding bodies provided funds for the purchase of consumption materials for the study but had no role in the design of the study and collection, analysis, and interpretation of data and writing of the manuscript. Competing interests The authors declare that they have no competing interests. Ethics approval and consent to participate Ethics approval and consent is deemed unnecessary in this research according to the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University. List Of Abbreviations Page 7/15 PCR: Polymerase Chain Reaction; qRT-PCR: Quantitative Reverse Transcription Polymerase Chain Reaction. PCR: Polymerase Chain Reaction; qRT-PCR: Quantitative Reverse Transcription Polymerase Chain Reaction. Acknowledgments The authors acknowledge the financial support of the National Natural Science Foundation of China (No.81741059) and the Health Department of Zhejiang Province of the People's Republic of China (No.2019KY098). The authors also want to give thanks to Dr. Andrea Rocker from Monash University for her help in paper writing. Page 8/15 Page 8/15 References 1. Paczosa MK, Mecsas J. Klebsiella pneumoniae: going on the offense with a strong defense. Microbiol Mol Biol Rev. 2016; 80(3): 629–661. 2. Prokesch BC, TeKippe M, Kim J, Raj P, TeKippe EM, Greenberg DE. Primary osteomyelitis caused by hypervirulent Klebsiella pneumoniae. Lancet Infect Dis. 2016; 16(9): e190–e195. 3. Shon AS, Bajwa RP, and Russo TA. Hypervirulent (hypermucoviscous) Klebsiella pneumoniae: a new and dangerous breed. Virulence. 2013; 4(2): 107–118. 4. Podschun R, Ullmann U. Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin Microbiol Rev. 1998; 11(4): 589–603. 5. Catalán-Nájera JC, Garza-Ramos U, Barrios-Camacho H. Hypervirulence and hypermucoviscosity: Two different but complementary Klebsiella spp. phenotypes? Virulence. 2017; 8(7): 1111-1123. 6. Fang CT, Chuang YP, Shun C, Chang SC, Wang JT. A novel virulence gene in Klebsiella pneumoniae strains causing primary liver abscess and septic metastatic complications. J Exp Med. 2004; 199(5): 697–705. 7. Hsieh PF, Lin TL, Lee CZ, Tsai SF, Wang JT. Serum-induced ironacquisition systems and TonB contribute to virulence in Klebsiella pneumoniae causing primary pyogenic liver abscess. J Infect Dis. 2008; 197(12): 1717-1727. 8. Miethke M, Marahiel MA. Siderophore-based iron acquisition and pathogen control. Microbiol Mol Biol Rev. 2007; 71(3): 413-451. 9. Cassat JE, Skaar EP. Iron in infection and immunity. Cell Host Microbe. 2013; 13(5): 509-519. 10. Kami M, Hamaki T, Kishi Y. Hepatic iron concentration and total body iron stores in thalassemia major. N Engl J Med. 2000; 343(22): 1657. 11. Corbella M, Caltagirone M, Gaiarsa S, et al. Characterization of an Outbreak of Extended-Spectrum beta-Lactamase-Producing Klebsiella pneumoniae in a Neonatal Intensive Care Unit in Italy. Microb Drug Resist. 2018; 24(8): 1128-1136. 12. Sanchez-Lopez J, Garcia-Caballero A, Navarro-San Francisco C, et al. Hypermucoviscous Klebsiella pneumoniae: A challenge in community acquired infection. IDCases. 2019; 17: 13. Wiens JR, Vasil AI, Schurr MJ, Vasil ML. Iron-regulated expression of alginate production, mucoid phenotype, and biofilm formation by Pseudomonas aeruginosa. MBio. 2014; 5(1): e01010-13. 13. Wiens JR, Vasil AI, Schurr MJ, Vasil ML. Iron-regulated expression of alginate production, mucoid phenotype, and biofilm formation by Pseudomonas aeruginosa. MBio. 2014; 5(1): e01010-13. 14. Alves JR, Pereira AC, Souza MC, et al. Iron-limited condition modulates biofilm formation and interaction with human epithelial cells of enteroaggregative Escherichia coli (EAEC). J Appl Microbiol. 2010; 108(1):246-255. 15. Lin MH, Shu JC, Huang HY, Cheng YC. Involvement of iron in biofilm formation by Staphylococcus aureus. PLoS One. 2012; 7(3):e34388. 16. References Eijkelkamp BA, Hassan KA, Paulsen IT, Brown MH. Investigation of the human pathogen Acinetobacter baumannii under iron limiting Conditions. BMC Genomics. 2011; 12(1): 126. Page 9/15 Page 9/15 17. Insua JL, Llobet E, Moranta D, et al. Modeling Klebsiella pneumoniae pathogenesis by infection of the wax moth Galleria mellonella. Infect Immun. 2013; 81(10): 3552-65. 18. Palacios M, Broberg CA, Walker KA, Miller VL. A Serendipitous Mutation Reveals the Severe Virulence Defect of a Klebsiella pneumoniae fepB Mutant. MSphere. 2017; 2(4). pii: e00341-17. 19. Pasqua M, Visaggio D, Lo Sciuto A, et al. Ferric Uptake Regulator Fur Is Conditionally Essential in Pseudomonas aeruginosa. J Bacteriol. 2017; 199(22). pii: e00472-17. 20. Wilksch JJ, Yang J, Clements A, et al. MrkH, a novel c-di-GMP-dependent transcriptional activator, controls Klebsiella pneumoniae biofilm formation by regulating type 3 fimbriae expression. PLoS Pathog. 2011; 7(8): e1002204. 21. Candan ED, Aksoz N. Klebsiella pneumoniae: characteristics of carbapenem resistance and virulence factors. Acta Biochim Pol. 2015; 62(4): 867-874. 21. Candan ED, Aksoz N. Klebsiella pneumoniae: characteristics of carbapenem resistance and virulence factors. Acta Biochim Pol. 2015; 62(4): 867-874. 22. Wasfi R, Elkhatib WF, Ashour HM. Molecular typing and virulence analysis of multidrug resistant Klebsiella pneumoniae clinical isolates recovered from Egyptian hospitals. Sci Rep. 2016; 6: 38929. 22. Wasfi R, Elkhatib WF, Ashour HM. Molecular typing and virulence analysis of multidrug resistant Klebsiella pneumoniae clinical isolates recovered from Egyptian hospitals. Sci Rep. 2016; 6: 38929. 23. Nadasy KA, Domiati-Saad R., Tribble MA. Invasive Klebsiella pneumoniae syndrome in North America. Clin Infect Dis. 2007; 45(3): e25–e28. 23. Nadasy KA, Domiati-Saad R., Tribble MA. Invasive Klebsiella pneumoniae syndrome in North America. Clin Infect Dis. 2007; 45(3): e25–e28. Tables Table 1. The virulence genes of K. pneumoniae Table 1. The virulence genes of K. pneumoniae Isolates Virulence genes FK3065 entB，uge，mrkD，fimH FK3087 entB，uge，mrkD，fimH FK3170 entB，uge，mrkD，fimH FK3226 iucB，iroB，entB，irp1，rmpA，wcaG，ybtA，magA ureA，uge，wabG，mrkD FK3992 iucB，iroB，entB，irp1，rmpA，ybtA，ureA，uge，wabG，mrkD FK4003 iucB，iroB，entB，irp1，rmpA，ureA，uge，wabG，mrkD ATCC700603 iucB，iroB，entB，irp1，rmpA，ybtA，ureA，wabG，mrkD e 1. The virulence genes of K. pneumoniae Isolates Virulence genes FK3065 entB，uge，mrkD，fimH FK3087 entB，uge，mrkD，fimH FK3170 entB，uge，mrkD，fimH FK3226 iucB，iroB，entB，irp1，rmpA，wcaG，ybtA，magA ureA，uge，wabG，mrkD FK3992 iucB，iroB，entB，irp1，rmpA，ybtA，ureA，uge，wabG，mrkD FK4003 iucB，iroB，entB，irp1，rmpA，ureA，uge，wabG，mrkD ATCC700603 iucB，iroB，entB，irp1，rmpA，ybtA，ureA，wabG，mrkD Virulence genes Page 10/15 Gene Sequence Reference iucB F: 5’-ATGTCTAAGGCAAACATCGT -3’ R: 5’- TTACAGACCGACCTCCGTGA -3’ This study iroB F: 5’- ACGACGGCGAACCCATTATT -3’ This study R: 5’- GACTTCACTGGCGGAATCCA -3’ entB F: 5’-ATATCCCGGCGAACAAGGTC-3’ This study R: 5’-CGGCGATATTAGCCACCACT -3’ irp1 F: 5’- CTCAGTGGCAACAACAGTGC -3’ This study R: 5’- GATGGCGCGGTGAATGTTAC -3’ rpoB F: 5’- AAGGCGAATCCAGCTTGTTCAGC -3’ [15] R: 5’-TGACGTTGCATGTTCGCACCCATCA -3’ Table 3. The virulence genes of K. pneumoniae Isolates Virulence genes FK3065 entB，uge，mrkD，fimH FK3087 entB，uge，mrkD，fimH FK3170 entB，uge，mrkD，fimH FK3226 iucB，iroB，entB，irp1，rmpA，wcaG，ybtA，magA ureA，uge，wabG，mrkD FK3992 iucB，iroB，entB，irp1，rmpA，ybtA，ureA，uge，wabG，mrkD FK4003 iucB，iroB，entB，irp1，rmpA，ureA，uge，wabG，mrkD ATCC700603 iucB，iroB，entB，irp1，rmpA，ybtA，ureA，wabG，mrkD Gene Sequence Reference iucB F: 5’-ATGTCTAAGGCAAACATCGT -3’ R: 5’- TTACAGACCGACCTCCGTGA -3’ This study iroB F: 5’- ACGACGGCGAACCCATTATT -3’ This study R: 5’- GACTTCACTGGCGGAATCCA -3’ entB F: 5’-ATATCCCGGCGAACAAGGTC-3’ This study R: 5’-CGGCGATATTAGCCACCACT -3’ irp1 F: 5’- CTCAGTGGCAACAACAGTGC -3’ This study R: 5’- GATGGCGCGGTGAATGTTAC -3’ rpoB F: 5’- AAGGCGAATCCAGCTTGTTCAGC -3’ [15] R: 5’-TGACGTTGCATGTTCGCACCCATCA -3’ Gene Sequence Reference iucB F: 5’-ATGTCTAAGGCAAACATCGT -3’ R: 5’- TTACAGACCGACCTCCGTGA -3’ This study iroB F: 5’- ACGACGGCGAACCCATTATT -3’ This study R: 5’- GACTTCACTGGCGGAATCCA -3’ entB F: 5’-ATATCCCGGCGAACAAGGTC-3’ This study R: 5’-CGGCGATATTAGCCACCACT -3’ irp1 F: 5’- CTCAGTGGCAACAACAGTGC -3’ This study R: 5’- GATGGCGCGGTGAATGTTAC -3’ rpoB F: 5’- AAGGCGAATCCAGCTTGTTCAGC -3’ [15] R: 5’-TGACGTTGCATGTTCGCACCCATCA -3’ Page 11/15 Table 3. The virulence genes of K. pneumoniae Isolates Virulence genes FK3065 entB，uge，mrkD，fimH FK3087 entB，uge，mrkD，fimH FK3170 entB，uge，mrkD，fimH FK3226 iucB，iroB，entB，irp1，rmpA，wcaG，ybtA，magA ureA，uge，wabG，mrkD FK3992 iucB，iroB，entB，irp1，rmpA，ybtA，ureA，uge，wabG，mrkD FK4003 iucB，iroB，entB，irp1，rmpA，ureA，uge，wabG，mrkD ATCC700603 iucB，iroB，entB，irp1，rmpA，ybtA，ureA，wabG，mrkD Figures Table 3. The virulence genes of K. pneumoniae Isolates Virulence genes FK3065 entB，uge，mrkD，fimH FK3087 entB，uge，mrkD，fimH FK3170 entB，uge，mrkD，fimH FK3226 iucB，iroB，entB，irp1，rmpA，wcaG，ybtA，magA ureA，uge，wabG，mrkD FK3992 iucB，iroB，entB，irp1，rmpA，ybtA，ureA，uge，wabG，mrkD FK4003 iucB，iroB，entB，irp1，rmpA，ureA，uge，wabG，mrkD ATCC700603 iucB，iroB，entB，irp1，rmpA，ybtA，ureA，wabG，mrkD Table 3. The virulence genes of K. pneumoniae le 3. The virulence genes of K. pneumoniae Isolates Virulence genes FK3065 entB，uge，mrkD，fimH FK3087 entB，uge，mrkD，fimH FK3170 entB，uge，mrkD，fimH FK3226 iucB，iroB，entB，irp1，rmpA，wcaG，ybtA，magA ureA，uge，wabG，mrkD FK3992 iucB，iroB，entB，irp1，rmpA，ybtA，ureA，uge，wabG，mrkD FK4003 iucB，iroB，entB，irp1，rmpA，ureA，uge，wabG，mrkD ATCC700603 iucB，iroB，entB，irp1，rmpA，ybtA，ureA，wabG，mrkD Figures Figure 1 The growth curves of K. pneumoniae. All K. pneumoniae isolates grew optimally in LB broth c 50 μM iron, superior to the growth in LB broth containing 30 μM, 10 μM, and 0 μM iron or 200 chelator +(-) 50 μM iron(P < 0.05). DP: 2,2'-Dipyridyl, added as an iron chelator. LB + 200 μM i chelating agent group was used as control Figure 1 The growth curves of K. pneumoniae. All K. pneumoniae isolates grew optimally in LB broth containing 50 μM iron, superior to the growth in LB broth containing 30 μM, 10 μM, and 0 μM iron or 200 μM iron chelator +(-) 50 μM iron(P < 0.05). DP: 2,2'-Dipyridyl, added as an iron chelator. LB + 200 μM iron chelating agent group was used as control Page 12/15 Page 12/15 Figure 2 Models of Galleria mellonella infected with K. pneumoniae. The virulence of FK3226, FK3992 and ATCC 700603 with additional iron was significantly higher than that of the strains with iron chelator (P<0.05). DP: 2,2'-Dipyridyl, added as an iron chelator. The PBS was control group. Figure 2 Figure 2 Models of Galleria mellonella infected with K. pneumoniae. The virulence of FK3226, FK3992 and ATCC 700603 with additional iron was significantly higher than that of the strains with iron chelator (P<0.05). DP: 2,2'-Dipyridyl, added as an iron chelator. The PBS was control group. Models of Galleria mellonella infected with K. pneumoniae. The virulence of FK3226, FK3992 and ATCC 700603 with additional iron was significantly higher than that of the strains with iron chelator (P<0.05). DP: 2,2'-Dipyridyl, added as an iron chelator. The PBS was control group. Page 13/15 Page 13/15 Page 13/15 Page 13/15 Page 14/15 Figure 3 Models of Galleria mellonella infected with K. pneumoniae. The virulence of FK3226, FK3992 700603 with additional iron was significantly higher than that of the strains with iron chelato DP: 2,2'-Dipyridyl, added as an iron chelator. The PBS was control group. Figure 3 Figure 4 Expression ratio of K. pneumoniae virulence genes. A. iucB gene. B. iroB gene. C. irp1 gene. D. entB gene. DP: 2,2'-Dipyridyl, added as an iron chelator. LB control was used to calculate relative expression. Expression ratio of K. pneumoniae virulence genes. A. iucB gene. B. iroB gene. C. irp1 gene. D. entB gene. DP: 2,2'-Dipyridyl, added as an iron chelator. LB control was used to calculate relative expression. Figure 3 Models of Galleria mellonella infected with K. pneumoniae. The virulence of FK3226, FK3992 and ATCC 700603 with additional iron was significantly higher than that of the strains with iron chelator (P<0.05). DP: 2,2'-Dipyridyl, added as an iron chelator. The PBS was control group. Page 14/15 Page 14/15 Page 14/15 Figure 4 Expression ratio of K. pneumoniae virulence genes. A. iucB gene. B. iroB gene. C. irp1 gene. D. entB gene. DP: 2,2'-Dipyridyl, added as an iron chelator. LB control was used to calculate relative expression. Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. SupplementaryTableS1.docx SupplementaryTableS2.docx Page 15/15 Page 15/15
https://openalex.org/W4319592734	https://www.degruyter.com/document/doi/10.1515/sai-2022-frontmatter2/pdf	Spanish; Castilian	null	Frontmatter	Sino-Ibero American interaction	2,022	cc-by	857	2022 · Volume 2 · Issue EDITOR-IN-CHIEF Chang Fuliang Beijing Foreign Studies University Interacción Sino- Iberoamericana Sino-Iberoamerican Interaction 2 2022 · Volume 2 · Issue 2 Interacción Sino- Iberoamericana Sino-Iberoamerican Interaction EDITOR-IN-CHIEF Chang Fuliang Beijing Foreign Studies University Sino-Iberoamerican Interaction provides a first-class academic platform for research on interactive relations between China and Spanish-Portuguese-speaking countries. From a bilateral or multilateral point of view, the journal primarily focuses on economic and trade relations, as well as political studies, cultural exchange and international relations. The extent of research goes from historical studies and studies on current situation to future vision, aiming to encourage exchange and cooperation between China and Spanish-Portuguese-speaking countries in an effective way, and to contribute to the idea of “a community with shared future for mankind”. The journal Sino-Iberoamerican Interaction is by far the only academic journal which is published in Spanish and Portuguese and is dedicated to research on interactive relations between China, Iberia and Latin America. La revista Interacción Sino-Iberoamericana proporciona una plataforma académica para estudiar las interacciones entre China y los países de habla hispana y portuguesa. Partiendo del punto de vista bilateral o multilateral, la revista se centra principalmente en las relaciones económicas y comerciales, así como en las interacciones políticas, los intercambios culturales y las relaciones internacionales. Las investigaciones incluidas cubren la historia, la situación actual y la visión futura de estas relaciones, con el objetivo de orientar eficazmente la práctica de intercambios amistosos y relaciones de cooper- ación, y construir una “comunidad de futuro compartido para la humanidad”. La revista Interacción Sino-Iberoamericana es, hasta ahora, la única revista académica, publicada en español y portugués, que se dedica al estudio de las interacciones entre China, Iberia y América Latina. e-ISSN 2747-7479 e-ISSN 2747-7479 All information regarding notes for contributors, subscriptions, back volumes and orders is available online at https://www.degruyter.com/SAI RESPONSIBLE EDITOR Shi Yan, Beijing Foreign Studies University, No. 2 North Xisanhuan Road, Beijing, 100089, China. E-mail: yan.shi@bfsu.edu.cn RESPONSIBLE EDITOR Shi Yan, Beijing Foreign Studies University, No. 2 North Xisanhuan Road, Beijing, 100089, China. E-mail: yan.shi@bfsu.edu.cn PUBLISHER Walter de Gruyter GmbH, Berlin/Boston, Genthiner Straße 13, 10785 Berlin, Germany PUBLISHER Walter de Gruyter GmbH, Berlin/Boston, Genthiner Straße 13, 10785 Berlin, Germany JOURNAL Coordinator Alexander Görlt, De Gruyter, Genthiner Straße 13, 10785 Berlin, Germany. Tel.: +49 (0)30 260 05–234, E-mail: alexander.goerlt@degruyter.com RESPONSIBLE FOR ADVERTISEMENTS Markus Kügel, De Gruyter, Rosenheimer Str. 143, 81671 München, Germany. Tel.: +49 89 76 902–424, E-mail: anzeigen@degruyter.com Published by Walter de Gruyter GmbH and Foreign Language Teaching and Research Publishing Co., Ltd. on behalf of © 2022 Beijing Foreign Studies University, China Typesetting TNQ Technologies, Chennai, India Contents Columna 1 Editorial Yan Shi Foro Sobre Relaciones Entre China y América Latina en el Contexto de la Comunidad de Futuro Compartido para la Humanidad 173 Columna 2 Relaciones políticas y diplomáticas Gonzalo Ghiggino China y América Latina en tiempos de pandemia: Bases para la construcción de una nueva gobernanza desde el Sur global 181 Eduardo Tzili-Apango La provisión de China del bien público global de la salud en México en la coyuntura de la Covid-19 199 Columna 3 Relaciones económicas y comerciales Huang Leping Cooperación Económica: China-América Latina y el Caribe ante “la Franja y la Ruta” 217 Columna 4 Sinología y estudios chinos Longxing Wang, Shuwei Xu y Juan Diego Zamudio Padilla La cobertura mediática sobre China en la prensa argentina durante el primer brote de Covid-19 en 2020. Un análisis de contenido de Clarín e Infobae 235 Esteban Poole Fuller La controversia jurídica en torno al incidente del barco María Luz (1872) y el establecimiento de relaciones diplomáticas de Perú con China y Japón en el contexto del cuestionamiento del régimen de extraterritorialidad 258 Sino-Iberoamer. Interac. 2022 \| Volume 2 \| Issue 2 Sino-Iberoamer. Interac. 2022 \| Volume 2 \| Issue 2 Sino-Iberoamer. Interac. Contents Columna 1 Editorial Columna 2 Relaciones políticas y diplomáticas Columna 2 Relaciones políticas y diplomáticas Gonzalo Ghiggino China y América Latina en tiempos de pandemia: Bases para la construcción de una nueva gobernanza desde el Sur global 181 Eduardo Tzili-Apango La provisión de China del bien público global de la salud en México en la coyuntura de la Covid-19 199 Columna 1 Editorial Yan Shi Foro Sobre Relaciones Entre China y América Latina en el Contexto de la Comunidad de Futuro Compartido para la Humanidad 173 Yan Shi Foro Sobre Relaciones Entre China y América Latina en el Contexto de la Comunidad de Futuro Compartido para la Humanidad 173 Foro Sobre Relaciones Entre China y América Latina en el Contexto de la Comunidad de Futuro Compartido para la Humanidad 173 Columna 5 Intercambio cultural Leticia-Tian Zhang y Qinwen Yu Promoción turística digital de España en China: estudio de caso de la cuenta de Turespaña en WeChat 278 Leticia-Tian Zhang y Qinwen Yu Promoción turística digital de España en China: estudio de caso de la cuenta de Turespaña en WeChat 278 Columna 4 Sinología y estudios chinos Sino-Iberoamer. Interac. 2022 \| Volume 2 \| Issue 2 Sino-Iberoamer. Interac. Columna 6 Reseñas de libros Radina Plamenova Dimitrova y Karla Sánchez Domínguez Historia del arte chino: la creación de capital cultural sobre China en América Latina 297
https://openalex.org/W3094920869	https://www.econstor.eu/bitstream/10419/258066/1/risks-08-00113.pdf	English	null	Exploiting Distributional Temporal Difference Learning to Deal with Tail Risk	Risks	2,020	cc-by	14,558	Provided in Cooperation with: MDPI – Multidisciplinary Digital Publishing Institute, Basel Suggested Citation: Bossaerts, Peter L.; Huang, Shijie; Yadav, Nitin (2020) : Exploiting distributional temporal difference learning to deal with tail risk, Risks, ISSN 2227-9091, MDPI, Basel, Vol. 8, Iss. 4, pp. 1-20, https://doi.org/10.3390/risks8040113 Terms of use: Documents in EconStor may be saved and copied for your personal and scholarly purposes. Die Dokumente auf EconStor dürfen zu eigenen wissenschaftlichen Zwecken und zum Privatgebrauch gespeichert und kopiert werden. Sie dürfen die Dokumente nicht für öffentliche oder kommerzielle Zwecke vervielfältigen, öffentlich ausstellen, öffentlich zugänglich machen, vertreiben oder anderweitig nutzen. You are not to copy documents for public or commercial purposes, to exhibit the documents publicly, to make them publicly available on the internet, or to distribute or otherwise use the documents in public. If the documents have been made available under an Open Content Licence (especially Creative Commons Licences), you may exercise further usage rights as specified in the indicated licence. Sofern die Verfasser die Dokumente unter Open-Content-Lizenzen (insbesondere CC-Lizenzen) zur Verfügung gestellt haben sollten, gelten abweichend von diesen Nutzungsbedingungen die in der dort genannten Lizenz gewährten Nutzungsrechte. https://creativecommons.org/licenses/by/4.0/ https://creativecommon Bossaerts, Peter L.; Huang, Shijie; Yadav, Nitin 1 The term “return” refers to the percentage investment gain/loss obtained over a certain time frame (e.g., daily). It should not be confused with the term “return” referred to in the RL literature. However, the two terms are related as they both refer to some feedback from the environment/market to agents/investors. Likewise, we should be aware of the context (ﬁnance/RL) for proper interpretation of the terms “payoffs” or “rewards.” Received: 23 September 2020; Accepted: 21 October 2020; Published: 26 October 2020 Received: 23 September 2020; Accepted: 21 October 2020; Published: 26 October 2020 Received: 23 September 2020; Accepted: 21 October 2020; Published: 26 October 2020 Abstract: In traditional Reinforcement Learning (RL), agents learn to optimize actions in a dynamic context based on recursive estimation of expected values. We show that this form of machine learning fails when rewards (returns) are affected by tail risk, i.e., leptokurtosis. Here, we adapt a recent extension of RL, called distributional RL (disRL), and introduce estimation efﬁciency, while properly adjusting for differential impact of outliers on the two terms of the RL prediction error in the updating equations. We show that the resulting “efﬁcient distributional RL” (e-disRL) learns much faster, and is robust once it settles on a policy. Our paper also provides a brief, nontechnical overview of machine learning, focusing on RL. Keywords: distributional reinforcement learning; markov decision process; leptokurtic distribution; tail risk; efﬁcient estimator risks Article Exploiting Distributional Temporal Difference Learning to Deal with Tail Risk Peter Bossaerts * , Shijie Huang and Nitin Yadav Brain Mind and Markets Lab, The University of Melbourne, 198 Berkeley Street, Parkville, VIC 3010, Australia harvey.huang@unimelb.edu.au (S.H.); nitin.yadav@unimelb.edu.au (N.Y.) * Correspondence: peter.bossaerts@unimelb.edu.au; Tel.: +61-3-3095-3257 Received: 23 September 2020; Accepted: 21 October 2020; Published: 26 October 2020 Abstract: In traditional Reinforcement Learning (RL), agents learn to optimize actions in a dynami context based on recursive estimation of expected values. We show that this form of machine learning fails when rewards (returns) are affected by tail risk, i.e., leptokurtosis. Here, we adapt a recen extension of RL, called distributional RL (disRL), and introduce estimation efﬁciency, while properly adjusting for differential impact of outliers on the two terms of the RL prediction error in the updating equations. We show that the resulting “efﬁcient distributional RL” (e-disRL) learns much faster and is robust once it settles on a policy. Our paper also provides a brief, nontechnical overview o risks Exploiting Distributional Temporal Difference Learning to Deal with Tail Risk Peter Bossaerts * , Shijie Huang and Nitin Yadav Peter Bossaerts * , Shijie Huang and Nitin Yadav Brain Mind and Markets Lab, The University of Melbourne, 198 Berkeley Street, Parkville, VIC 3010, Australia; harvey.huang@unimelb.edu.au (S.H.); nitin.yadav@unimelb.edu.au (N.Y.) * Correspondence: peter.bossaerts@unimelb.edu.au; Tel.: +61-3-3095-3257 Peter Bossaerts * , Shijie Huang and Nitin Yadav Brain Mind and Markets Lab, The University of Melbourne, 198 Berkeley Street, Parkville, VIC 3010, Australia; harvey.huang@unimelb.edu.au (S.H.); nitin.yadav@unimelb.edu.au (N.Y.) * Correspondence: peter.bossaerts@unimelb.edu.au; Tel.: +61-3-3095-3257 Peter Bossaerts , Shijie Huang and Nitin Yadav Brain Mind and Markets Lab, The University of Melbourne, 198 Berkeley Street, Parkville, VIC 3010, Australia; harvey.huang@unimelb.edu.au (S.H.); nitin.yadav@unimelb.edu.au (N.Y.) * Correspondence: peter.bossaerts@unimelb.edu.au; Tel.: +61-3-3095-3257 1. Introduction The weight on the prediction error is referred to as the “learning rate.” In this paper, we focus on one version of TD Learning, called SARSA,2 whereby the agent takes the action in the subsequent trial to be the one deemed optimal given the new state, i.e., the action that provides the maximum estimated Q value given the state. p g A more recent version of RL learns Q values, not through adaptive expectations, but by remembering the entire distribution of the rewards in a trial and estimated Q values in the subsequent trial.3 New estimates of the Q values of action-state pairs are then obtained by simply taking the expectation over this empirical distribution. This technique, referred to as Distributional RL (disRL), has been more successful than the traditional, recursive TD Learning, in contexts such as games where the state space is large and the relation states-action values is complex. See, e.g., Bellemare et al. (2017); Dabney et al. (2018); Lyle et al. (2019); Rowland et al. (2018). In a leptokurtic environment, TD Learning and certain versions of disRL are not robust. We show here that Q value estimates are very sensitive to outliers, and lead to frequent changes in estimated optimal policies, even after substantial learning. Consequently, if the learning rate decreases too fast, the agent’s policy is unlikely to be optimal. If the learning rate is allowed to decrease only if the optimal policy remains unaltered, then the learning rate may never decrease since outliers continue to affect the estimated Q values, and hence policy. p y We propose, and test, a solution. Exploiting the fact that disRL keeps track of the empirical distribution of estimated Q values for a given state-action pair,4 we propose not to estimate the true Q value by simply averaging over the distribution. Instead, we propose to use an efﬁcient estimator of the mean. Efﬁcient estimators are those that minimize the standard error. When rewards are gaussian, the sample mean is the most efﬁcient estimator of the true mean. If one posits that rewards are generated by a t distribution with low degrees of freedom, one of the canonical leptokurtic distributions, a much better estimator exists. This estimator weighs observations depending on how much they are in the tails of the empirical distribution. The weighting is chosen to maximize statistical efﬁciency. 1. Introduction Reinforcement Learning (RL) has been successfully applied in diverse domains. However, the domain of ﬁnance remains a challenge. A statistical feature central to ﬁnance is tail risk, or in technical jargon, leptokurtosis. For example, daily returns1 on the S&P500 index, despite its broad diversiﬁcation, follow a distribution with a kurtosis of 10 or higher, compared to the Gaussian kurtosis of 3. Kurtosis of individual stocks can be as high as 15 (Corhay and Rad 1994). In a leptokurtic environment, outliers, deﬁned as observations in the tails of the distribution, are frequent and salient. This contrasts with infrequent, non-salient outliers, known as “black swans” (Taleb 2007), or infrequent, salient outliers, known as “freak events” (Toenger et al. 2015). Statistically, the excessive mass in the tails of a leptokurtic distribution is compensated for by reducing mass about one standard deviation away from the mean (provided the standard deviation exists). As a result, besides outliers, small changes are also relatively more frequent than under the Gaussian distribution. Outliers are all the more salient since “typical” outcomes tend to be small. RL is a key technique in machine learning. The goal is for an artiﬁcial agent to learn the right actions in a dynamic context. The agent is to search for the optimal actions as a function of the state of the environment. Effectively, the agent performs stochastic dynamic programming. In the most popular version of RL, Temporal Difference (TD) Learning, the agent starts with estimates of the values of all possible actions in all possible states, referred to as “Q values.” The Q value of a state-action pair Risks 2020, 8, 113; doi:10.3390/risks8040113 www.mdpi.com/journal/risks Risks 2020, 8, 113 2 of 20 is the expected sum of future rewards conditional on taking suitable actions from the subsequent trial (trial 2) onwards. In each trial, the agent tries an action, observes an immediate reward, as well as the state of the subsequent trial. Using adaptive expectations, the agent updates the estimate of the (Q) value of the action it just took in the state it was in. The new estimate is a weighted average of the old estimate and the “prediction error.” The prediction error equals the difference between the old estimate, on the one hand, and the sum of the reward just obtained and the estimated value of the new state for a suitably chosen action, on the other hand. 3 The distribution can be remembered parsimoniously in various ways, e.g., as histograms, in terms of a speciﬁc set of quantiles or truncated expectations (“expectiles”). Compare, e.g., Bellemare et al. (2017); Dabney et al. (2018); Rowland et al. (2019). 4 Technically, this is not exactly true for many versions of disRL in the literature. Only parametrically ﬁtted histograms, quantiles or expectiles are remembered, reducing memory load. 2 SARSA is short for State-Action-Reward-State-Action. Risks 2020, 8, 113 Risks 2020, 8, 113 3 of 20 Efﬁcient estimation is not the only way disRL needs to be adjusted. Equally important is the following. The effect of leptokurtosis on experienced Q values decreases over time, as the agent re-visits trials in the same state and with the same action. At the same time, leptokurtosis continues to impact the distribution of immediate rewards. This calls for decoupling of the two terms in the prediction error used in updating. We propose a simple way to implement the de-coupling. We show that it works effectively. y We refer to our enhanced disRL as “efﬁcient” disRL, and use the abbreviation e-disRL. Using a simulation experiment, we prove the superiority of e-disRL over TD Learning and disRL. We also show superiority when rewards are drawn, not from a t distribution, but from the empirical distribution of daily returns on the S&P 500 index. We keep the environment in our experiment as simple as possible, in order to enhance transparency. We use a minimally complex environment, with two states and two possible actions. Optimal actions change with states. We envisage a situation where the artiﬁcial agent is asked to switch between two investments, while the optimal investments change with the state of the economy. Technically, our setting is a contextual two-arm bandit. The framework may appear simple, but it is generic. The contextual two-arm bandit can readily be extended to handle more involved, and hence, more realistic situations, by augmenting the state vector or the number of states, and/or increasing the number of control options beyond two (arms). The bandit does not have to be stationary; it can change randomly over time, to form a so-called restless bandit. Continuous states and large state spaces can be accommodated through deep learning (Mnih et al. 2013; Moravˇcík et al. 2017; Silver et al. 2016). We chose a simple, canonical setting, in order to illustrate how easy it is for traditional RL to fail under leptokurtosis, and how powerful our version of distributional RL is to address the failure. One could argue that there are other solutions to the problems leptokurtosis causes. This could be GARCH or stochastic volatility modelling (Simonato 2012), Monte Carlo approaches (Glasserman 2013), moment methods (Jurczenko and Maillet 2012), or parametric return process approximation and modelling (Nowak and Romaniuk 2013; Scherer et al. 2012). These procedures would effectively ﬁlter the data before application of RL. 1. Introduction The weighing does not simply truncate observations, but maps outliers back to the middle of the distribution. When the true mean can be estimated using maximum likelihood estimation (mle) and mle provides consistent estimates, the mle is the most efﬁcient possible. Technically, it reaches the “Cramér-Rao lower bound.” This is the case for the t distribution, so in our implementation, we use the mle estimator. In general, the mle estimator may not exist, and alternative estimators have to be found. We provide an example in the Conclusion. The importance of using efﬁcient estimators in ﬁnance, especially of mean returns, has been pointed out before. Madan (2017), for instance, shows how a kernel-based estimator generates lower standard errors than the usual sample average. Here, we propose to go for the best possible estimator, i.e., the one that maximizes efﬁciency (minimizes standard error). Risks 2020, 8, 113 But it is known that mere ﬁltering, while alleviating the impact of leptokurtosis, does not eliminate tail risk. Indeed, the ﬁltered risk appears to be best modeled with a t distribution (which we use here), or the stable Paretian distribution (for which variance does not even exist). These distributions still entail tail risk. See, e.g., Curto et al. (2009); Simonato (2012). More importantly, none of the aforementioned procedures deals with control, which is what RL is made for. The procedures aim only at forecasting. As such, they do not provided a good comparison to RL. RL is engaged in forecasting as well, but prediction subserves the goal of ﬁnding the best actions. The problem we address here is whether leptokurtosis affects discovery and maintenance of the optimal policy, not merely that of ﬁnding the best prediction of the future reward. Tail risk is a problem outside ﬁnance as well. In one very important context for RL, tail risk emerges when rewards occur only after potentially long chains of events (called “eligibility traces”; Sutton and Barto (2018)). The long chains cause the reward distribution to be leptokurtic. Singh and Dayan (1998) demonstrated that traditional TD Learning performs poorly when credit for rewards may have to be assigned to events that are at times too far in the past. Tail risk does not only plague learning by artiﬁcial agents. Evidence exists that humans, even professional traders, and despite continued vigilance, over-react to the frequent outliers that leptokurtosis entails (d’Acremont and Bossaerts 2016). For readers who may not be familiar with machine learning, we ﬁrst provide a nontechnical description of the various machine learning techniques, honing in on reinforcement learning (RL), which is what this paper is about. We then explain intuitively how our enhancement of disRL generates robustness when tail risk affects the rewards. In Section 3, we introduce RL in a technical way. Section 4 then discusses the implications of leptokurtosis for TD Learning and disRL. Section 5 explains our solution. Section 6 presents the results from our simulation experiments. Section 7 concludes. Risks 2020, 8, 113 4 of 20 The reader can replicate our results and study the code in the following github repo: https: //github.com/bmmlab/Distributional-RL-Tail-Risk. 5 It is also referred as semi-supervised learning as it sits in-between the above two types. 2.1. Machine Learning Broadly speaking, there exist three types of machine learning. All three were inspired, and have inspired, computational neuroscience, since the three are important to understand neural processes associated with learning in animals, including humans (Cichy and Kaiser 2019; Ludvig et al. 2011; Poggio and Serre 2013). The ﬁrst type is supervised learning, where the agent is given a dataset with cases (petal shape, color, etc.), described in terms of various features (e.g., ﬂower features). Each case is labelled (e.g., “tropical ﬂower,” “temperate-climate ﬂower”), and the goal is to learn the mapping from features to labels. The agent is given a limited set of cases with the correct labels (the “training set”), whence the term supervised learning. The mapping from features into labels can be highly nonlinear. A neural network with multiple (“deep”) layers allows the agent not only to ﬂexibly capture nonlinearity in the relationship between features and labels; it also provides a framework within which numerical optimization can be executed efﬁciently despite the nonlinearity and despite the high number of parameters. The second type is unsupervised learning, where the agent is given a dataset containing features but no labels. The goal is to ﬁnd structure, or patterns in the data. Techniques include factor analysis and cluster analysis. The retained factors or clusters help to identify hidden properties of the data, in the form of commonalities of features across data subsets. In other words, the agent is asked to come up with its own labelling system. It may end categorizing ﬂowers into “tropical” and “temperate-climate” unless there are more relevant ways to cluster them. 2.2. Reinforcement Learning We will be concerned here with a third type of machine learning, namely, reinforcement learning.5 There, an artiﬁcial agent is effectively asked to do stochastic dynamic programming, i.e., to ﬁnd, given the state of the environment, the actions which maximize total rewards (or minimize total losses) for the foreseeable future. An example is when the agent is asked, every month, to decide between a stock index or Treasury bonds, as a function of the state of the economy, with the goal of maximizing lifetime gains. In the sequel, we will only look at maximizing rewards, since minimization of losses is isomorphic. In stochastic dynamic programming, the agent recognizes that there are: states, maybe hidden; actions, which entail rewards and sometimes state transitions as well; and observables, including the rewards received. In its simplest form, the agent observes the states (observation may only reveal some properties of a state) and is asked to maximize the expected sum of (discounted) rewards in the future. Risk aversion can be built in by transforming rewards with a strictly concave function, as is standard in expected utility theory (Savage 1972). Under risk aversion, the agent maximizes the expected sum of nonlinearly transformed rewards. Here, we will assume risk neutrality, without loss of generality since we are focusing on learning. We will also assume that the states are fully observable. In a ﬁnance context, one could think of states as the general condition of the economy, measured through, e.g., change in industrial production. Stochastic dynamic programming is immensely difﬁcult even in the simplest cases. In theory, the agent has to ﬁnd a value function (referred to as the Bellman function) that maps the state into the expected sum of rewards for optimal choices of actions. A key result in stochastic dynamic 5 of 20 Risks 2020, 8, 113 programming is that, under certain condition, the optimal policy is to pick the action that maximizes the rewards over the immediate trial plus the (discounted) expectation of the value function for the subsequent trial. (We will ignore discounting since it is not relevant to our argument.) Given the state a given trial is in, the value function can then be calculated by adding, to the immediate reward, the expectation of the value function at the next trial, provided the agent picks the action that maximizes both terms. 2.2. Reinforcement Learning This is referred to as the “Bellman equation.” Across trials, states inevitably change, and hence, the mapping from states to values for a particular state can be traced by recording the realized optimal value in a trial when the environment is in that state. In general, agent actions may affect states. In our ﬁnance example, this would mean that the investment decisions of our agent would affect the state of the economy, which makes little sense. Therefore, we will assume throughout that actions do not affect states. A more elaborate discussion of the relation between actions and states in the context of ﬁnance can be found in Chapter 1 of Bossaerts (2005). Inspired by animal learning, and later conﬁrmed in neural processes induced by animal learning (Schultz et al. 1997), machine learning has developed a remarkably simple, yet surprisingly robust algorithm for the agent to learn to optimize in dynamic settings. The idea is actually straightforward. Remember that the value of a state, say s, can be obtained by considering a trial when the environment is that state s. Therefore, one could take the action deemed optimal for the trial, record the immediate reward, observe the resulting state in the subsequent trial, and add the previously recorded value for that state. But our agent does not know (yet) what the true optimal action is, nor does the agent know what the true value is in the state in the subsequent trial; however, it has observed immediate rewards from taking an action in previous trials where the state was the same, and it may have an estimate of what the value is of the state in the subsequent trial for some cleverly chosen action (we will discuss which action to choose later on). The sum of the two constitutes a “cue” for the agent of the value of taking the proposed action. We refer to this as the “Q” value of the proposed action given the state of the present trial.” Across trials where the same state is visited and the same action is taken, the estimated of the Q value can be updated by simple adaptive expectations: the agent updates the estimate using the prediction error. 2.2. Reinforcement Learning The prediction error equals the difference between, on the one hand, the sum of the newly recorded reward and Q value given the new state (in the subsequent trial), and, on the other hand, the old estimate of the Q value. The agent can do this across multiple trials. Trials are often arranged in episodes. This arrangement allows one to investigate what the agent has learned at pre-set points (episode ends). Because the Q values are updated by means of adaptive expectations, the learning technique is referred to as Temporal Difference Learning or “TD Learning.” The optimal value given a state is then obtained by choosing the action that maximizes the Q value for that state. For the technique to converge, i.e., for the Q values of the best action to converge, across states, to the true value function, two conditions are needed. First, all states have to be visited sufﬁciently often. Indeed, even if one knew what the optimal action was in a given state, the value of that state is the expected sum of all future rewards. Expectations can be learned through adaptive expectations, but one needs to experience enough observations for it to converge; a law of large numbers has to apply. The second, related, condition is that all actions are taken sufﬁciently often. If an action is rarely taken because it is deemed (estimated) to be sub-optimal, one may never learn that it is indeed sub-optimal; it may in fact be optimal! Because of the second condition, exploration is necessary. The agent must not decide prematurely that certain actions are inferior; the agent has to explore all actions, no matter how inferior they may seem. Several exploration strategies have been proposed. Here, we will use the simplest one, namely, the greedy strategy. In the greedy strategy, the agent picks what it considers to be optimal (based on current estimates of the Q values) with probability 1 −ϵ, while randomly picking any other action with probability ϵ. Here, ϵ may initially be a large number (less than 1), but it can be decreased over time, Risks 2020, 8, 113 6 of 20 as the agent learns, to ultimately converge to zero, at which point the agent stops learning. Choice of the exploration strategy will not resolve the issues with tail risk that we study here, which is why we stick to the simplest strategy. 2.2. Reinforcement Learning One more detail about TD Learning needs to be clariﬁed. It was mentioned before that the Q value of an action-state pair equals the sum of the immediate reward from the action plus the Q value of a suitably chosen action in the subsequent trial. In our application of TD Learning, we will choose the optimal action in the subsequent trial. Optimality is determined by the Q values across actions given the state in that trial. This approach is referred to as “SARSA,” which is short for State-Action-Reward-State-Action, indicating that only actions (deemed) optimal are taken. An alternative would be to use the Q value in the subsequent trial associated with an action which is optimal only with probability 1 −ϵ, i.e., an action that follows the greedy exploration policy. This choice was made in the original version of “Q Learning.” In TD learning, the Q value is learned through adaptive expectations. A recently suggested alternative would be to summarize all past observed Q values in the form of an empirical distribution (or a histogram),6 update this distribution in every trial where the same state occurs and the same action is taken, and use the mean of this empirical distribution as new estimate of the Q value. This approach is known as Distributional Reinforcement Learning, abbreviated disRL. See Bellemare et al. (2017). It has been shown to be far more effective than the recursive TD learning procedure, especially in strategic interaction (games). DisRL provides another advantage: it allows one to introduce neural network techniques in order to determine which elements of a set of states are relevant for optimal decision-making, and how Q values relate to these elements. Indeed, in a ﬁnance context, it could be that the Treasury bill rate and the dividend yield are potential candidates for optimally switching into and out of stocks, in addition to a change in industrial production, but only one is actually relevant. Therefore, we need a technique that determines which of the three is/are relevant. The technique that combines disRL with neural networks is referred to as deep RL. It is meant to simultaneously solve for the optimal action in a dynamic environment and solve the “credit assignment problem,” i.e., the question as to which aspects of the environment are relevant to determine optimal actions. We will not be concerned with deep RL here. 6 We focus here on techniques that use the empirical distribution, since it is a consistent estimate of the true distribution, while the histogram is not consistent as an estimate of the true density. 2.3. Our Contribution When tail risk affects the environment, i.e., when rewards are subject to frequent and large outliers, neither the recursive traditional TD learning nor the more recent Distributional RL are robust. This is because the estimate of the Q value for a state-action pair, whether obtained through adaptive expectations (i.e., recursively), or through the mean of the empirical distribution of past reward outcomes and Q estimates, is sensitive to these outliers. At its core, the method we propose is a simple, but profound improvement of distributional RL. The idea is not to estimate a Q value using the mean of the estimated empirical distribution, but to use the most efﬁcient estimator of the ﬁrst (signed) moment. Under leptokurtosis, the mean of the empirical distribution generally exhibits low efﬁciency, that is, its standard error is not the lowest. If a maximum likelihood estimator of the mean exists and is asymptotically efﬁcient, it will provide the most efﬁcient estimator, however (technically: it will reach the Cramér-Rao lower bound). We propose to use this estimator. The contribution is simpler to explain than to put into practice. This is because tail risk affects the two components of the TD prediction error differentially. Tail risk may always affect immediate rewards. However, eventually, after a suitably long learning period, it should no longer affect the Risks 2020, 8, 113 7 of 20 observed Q value in the subsequent trial since Q values are the expected sum of future rewards. Therefore, we account differentially for the two components: we apply efﬁcient disRL only to the reward term, implementing traditional recursive TD learning for the Q value term. How this is done technically is explained next. Appeal to maximum likelihood estimation requires the researcher to commit to a family of distributions from which the rewards (conditional on the state-action pair) are thought to have been generated. In the case of tail risk, i.e., under leptokurtosis, the t distribution provides a good working hypothesis. This is the family we use here. Even if the true distribution is not t, the approach gives good results; one can think of the use of the t distribution as “quasi maximum likelihood:” for the purpose of TD learning, it provides desired asymptotic properties. In the context of tail risk, the improvements can be dramatic, as we demonstrate with an experiment. 7 To simplify things, we suppress the stochastic index “ω” which is used in probability theory to capture randomness. ω only affects the rewards, and not the value function Q. This distinction will be important when we discuss de-coupling of the terms of the RL updating equations. 2.3. Our Contribution It deserves emphasis that these improvements emerge even if our approach does not increase the speed of convergence as a function of sample size (number of occurrences of the state-action pair); convergence will remain inversely proportional to the square root of the sample size. But the constant of proportionality will be decreased markedly, sufﬁciently so that our approach becomes robust to tail risk, while traditional TD Learning and distributional RL lack robustness. There are environments where our efﬁcient version of disRL affects also the speed of learning as a function of sample size. In the Conclusion section, we provide an example where the rewards are generated by a shifted-exponential distribution. There, the estimation error of the Q values, can be reduced, not in inverse proportion to the square-root of the sample size, but in inverse proportion of the sample size. 3.1. TD Learning We model the interaction of an agent with an environment in the traditional way, as a Markov Decision Process (MDP). An MDP is deﬁned by (S, A, R, P) where S denotes the set of states, and A denotes the set of the available actions. R is a random reward function that maps each state-action pair to a random reward that lives in an outcome space F, that is, R : S × A →F.7 P(s ′\|s, a), s, s ′ ∈S and a ∈A, denotes the state transition distribution from one trial to another. Primes ( ′) are used to denote “subsequent trials.” Let π(a\|s) be the policy, i.e., the probability of action a in state s. q ( \| ) p y p y We denote Qπ as the action-value function for a particular policy π when the initial (t = 0) state is s and the initial action is a. It is calculated as a discounted sum of expected future rewards if the agent follows the policy π from t = 1 onward: Qπ(s, a) := Eπ,P[ ∞ ∑ t=0 γtR(st, at)\|s0 = s, a0 = a] where st ∈S, at ∈A and γ ∈[0, 1) is the discount factor. At the same time, Qπ is a ﬁxed point of the Bellman Operator Tπ (Bellman 1957), where st ∈S, at ∈A and γ ∈[0, 1) is the discount factor. At the same time, Qπ is a ﬁxed point of the Bellman Operator Tπ (Bellman 1957), TπQ(s, a) := E[R(s, a)] + γ ∑ s′,a′ P(s ′\|s, a)π(a ′\|s ′)Q(s ′, a ′) 7 To simplify things, we suppress the stochastic index “ω” which is used in probability theory to capture randomness. ω only affects the rewards, and not the value function Q. This distinction will be important when we discuss de-coupling of the terms of the RL updating equations. Risks 2020, 8, 113 8 of 20 The Bellman operator Tπ is used in policy evaluation (Sutton and Barto 2018). We intend to ﬁnd an optimal policy π∗, such that Qπ∗(s, a) ≥Qπ(s, a) for all (s, a, π). where R(s, a) + γ max a′ Q(s ′, a ′) −Q(s, a) (1) (1) is referred as the prediction error, or “TD-error,” and α is the learning rate. The updating rule uses the estimate of the Q value in the subsequent trial for the action that is optimal given the state. TD Learning using this particular updating rule is called SARSA (State-Action-Reward-State-Action). Watkins and Dayan (1992) have shown that actions and action-state values converge to the true (optimal) quantities, provided the agent explores, and visits all states, sufﬁciently. To accomplish this, the agent has to ensure the learning rate α does not decrease too fast, and to use an exploration policy (choices of actions a) that tries out all possibilities sufﬁciently often. Here, we will use the greedy policy, whereby the agent chooses the optimal action in a state arg maxa Q(s, a) with chance 1 −ϵ and a randomly chosen sub-optimal action with chance ϵ, for some ϵ > 0 which is reduced over time. 3.1. TD Learning The optimal state-action value function Qπ∗is the unique ﬁxed point of the Bellman optimality operator T∗, T∗Q(s, a) := E[R(s, a)] + γ∑ s′ P(s ′\|s, a) max a′ Q(s ′, a ′) In general, we do not know the full model of the MDP, let alone the optimal value functions or optimal policy. Therefore, we introduce TD learning, which exploits the recursive nature of the Bellman operator (Sutton and Barto 2018). Different versions of TD Learning exist. Watkins and Dayan (1992) proposed the following updating Q(s, a) ←Q(s, a) + α(R(s, a) + γ max a′ Q(s ′, a ′) −Q(s, a)), 4. Leptokurtosis At the same time, rewards R(s, a) continue to be drawn from a leptokurtic distribution. As a result, the prediction error is a sum of a leptokurtic term and an asymptotically non-leptokurtic term. The resulting heterogeneity needs to be addressed. Measures to deal with leptokurtosis may inadversely affect the second term.8 The two terms have to be decoupled during updating. This is done neither in traditional TD Learning nor in disRL. 2. Heterogeneity of the prediction error. The prediction error (TD error) is the sum of two components, the period reward R(s, a) and the (discounted) increment in values γQ(s ′, a ′) − Q(s, a). As the agent has learned the optimal policy and the state transition probabilities, Q values converge to expectations of sums of rewards. These expectations should eventually depend only on states and actions. Since the distribution of state transitions is generally assumed to be non-leptokurtic (e.g., Poisson), the increment in Q values γQ(s ′, a ′) −Q(s, a) will no longer exhibit leptokurtosis. At the same time, rewards R(s, a) continue to be drawn from a leptokurtic distribution. As a result, the prediction error is a sum of a leptokurtic term and an asymptotically non-leptokurtic term. The resulting heterogeneity needs to be addressed. Measures to deal with leptokurtosis may inadversely affect the second term.8 The two terms have to be decoupled during updating. This is done neither in traditional TD Learning nor in disRL. 2. 3. 3. Non-stationarity of the distribution of Q values. As the agent learns, the empirical distribution of Q values shifts. These shifts can be dramatic, especially in a leptokurtic environment. This is problematic for implementations disRL that proceed as if it the distribution of Q values is stationary. Categorical disRL, for instance, represents the distribution by means of histogram deﬁned over a pre-set range. Outlier rewards may challenge the set range (i.e., outliers easily push the estimated Q-values beyond the set range). One could use a generous range, but this reduces the precision with which the middle of the distribution is estimated. We will illustrate this with an example when presenting results from our simulation experiments. Recursive procedures, like those used in the Kalman ﬁlter or in conventional TD learning, are preferred when a distribution is expected to change over time. Versions of disRL that ﬁx probability levels (e.g., by ﬁxing probability levels, as in quantile disRL; Dabney et al. 8 Maximum likelihood estimation of the mean of a leptokurtic distribution, the t distribution for instance, eliminates the inﬂuence of outliers by setting them (close to) zero, as we shall document later (see Figure 2). The resulting estimator is less efﬁcient than the simple sample average when the distribution is not leptokurtic, since observations are effectively discarded. 3.2. Distributional Reinforcement Learning (disRL) The mean Q value (given the state s and action a) is then computed by integrating over the auxiliary distribution: Q(s, a) = K ∑ i=1 pi(s, a\|θ)zi, Q(s, a) = K ∑ i=1 pi(s, a\|θ)zi, Q(s, a) = K ∑ i=1 pi(s, a\|θ)zi, where the zis are (evenly spaced) midpoints for the K bins of the histogram. 3.2. Distributional Reinforcement Learning (disRL) Similar to the Bellman operator in TD Learning, the distributional Bellman operator, Tπ D is formally deﬁned as: Similar to the Bellman operator in TD Learning, the distributional Bellman operator, Tπ D is formally deﬁned as: D ′ ′ deﬁned as: Tπ DZ(s, a) D= R(s, a) + γZ(s ′, a ′), (2) (2) where D= means equality in distribution (Bellemare et al. 2017; Rowland et al. 2019). States s ′ are drawn according to P(s ′\|s, a), actions a ′ are from the policy π, and rewards are drawn from the reward distribution corresponding to the state s and action a. One of the proposed algorithms, the categorical approach (Bellemare et al. 2017), represents distributions in terms of histograms. It assumes a categorical (binned) form for the state-action value distribution. As such, the categorical approach approximates the value distribution using a histogram with equal-size bins. The histogram is updated in two steps: (i) shifting the entire estimated probability distribution of estimated Q values using the newly observed reward, (ii) mapping the shifted histogram back to the original range. Notice that the range is ﬁxed beforehand. As we shall see, this is problematic especially in the context of tail risk. In disRL, the estimated distribution (in the form of, e.g., the histogram, as in categorical disRL) is mostly not used directly to obtain an estimate of the mean Q value. Instead, the distance of the estimated distribution from an auxiliary, parametric family of distributions is minimized. This allows for ﬂexible relationships between the Q value distributions and the states and actions. Often neural networks are ﬁtted, whence the term “deep RL.” Means of the ﬁtted distributions are then computed by simple integration. For instance, in categorical disRL, auxiliary probabilities pi(s, a\|θ) for each bin i (given the state s and action a) are obtained by minimizing the distance between them and the histogram. The relation between the state-action pairs (s, a) and the probability of the ith bin is ﬁt 9 of 20 Risks 2020, 8, 113 with a neural network with parameter vector θ using (nonlinear) Least Squares. 4. Leptokurtosis In principle (and as we shall see later through simulations), TD Learning and disRL are not well equipped to handle a leptokurtic reward environment. There are at least three reasons for this. 1. Leptokurtosis of the reward distribution. disRL simply integrates over the distribution in order to estimate the true Q value. Traditional TD Learning uses a recursive estimate. Both are inefﬁcient under leptokurtosis. Indeed, in general there exist much better estimators than the sample average, whether calculated using the entire sample, or calculated recursively. The most efﬁcient estimator is the one that reaches the Cramér-Rao lower bound, or if this bound is invalid, the Chapman-Robbins lower bound (Casella and Berger 2002; Schervish 2012). Under conditions where maximum likelihood estimation (MLE) is consistent, MLE will provide the asymptotically most efﬁcient estimator with the lowest variance: it reaches the Cramér-Rao lower bound (Casella and Berger 2002). Often, the MLE estimator of the mean is very different from the sample average. This is the case, among others, under leptokurtosis. 1. Leptokurtosis of the reward distribution. disRL simply integrates over the distribution in order to estimate the true Q value. Traditional TD Learning uses a recursive estimate. Both are inefﬁcient under leptokurtosis. Indeed, in general there exist much better estimators than the sample average, whether calculated using the entire sample, or calculated recursively. The most efﬁcient estimator is the one that reaches the Cramér-Rao lower bound, or if this bound is invalid, the Chapman-Robbins lower bound (Casella and Berger 2002; Schervish 2012). Under conditions where maximum likelihood estimation (MLE) is consistent, MLE will provide the asymptotically most efﬁcient estimator with the lowest variance: it reaches the Cramér-Rao lower bound (Casella and Berger 2002). Often, the MLE estimator of the mean is very different from the sample average. This is the case, among others, under leptokurtosis. 2. Heterogeneity of the prediction error. The prediction error (TD error) is the sum of two components, the period reward R(s, a) and the (discounted) increment in values γQ(s ′, a ′) − Q(s, a). As the agent has learned the optimal policy and the state transition probabilities, Q values converge to expectations of sums of rewards. These expectations should eventually depend only on states and actions. Since the distribution of state transitions is generally assumed to be non-leptokurtic (e.g., Poisson), the increment in Q values γQ(s ′, a ′) −Q(s, a) will no longer exhibit leptokurtosis. 5.1. Environment here are two states We create a canonical experimental environment that mimics typical decision-making in ﬁnancial markets. There are two states S : {s0, s1}, and two available actions A : {a0, a1}. So, agents’ actions have no effect on the states: P(s ′\|s, a) = P(s ′\|s). (We continue to use primes [ ′] to denote outcomes in the subsequent trial.) The state transition probability P(s ′\|s) is such that the probability of staying in the same state is higher than the probability of switching to another state. One can view our environment as a two-arm contextual bandit problem, with two discrete states and uncertain rewards. A graphical depiction in terms of a ﬁnite automaton is provided in Figure 1. { 0, 1}, { 0, 1} , g e states: P(s ′\|s, a) = P(s ′\|s). (We continue to use primes [ ′] to denote outcomes in the subsequent trial.) T ate transition probability P(s ′\|s) is such that the probability of staying in the same state is higher than t obability of switching to another state. One can view our environment as a two-arm contextual bandit proble ith two discrete states and uncertain rewards. A graphical depiction in terms of a ﬁnite automaton is provided gure 1. s0 s1 a0, a1\|0.3 a0, a1\|0.7 a0, a1\|0.7 a0, a1\|0.3 Figure 1. Environment, depicted in terms of a ﬁnite automaton. Figure 1. Environment, depicted in terms of a ﬁnite automaton. s0 s1 a0, a1\|0.3 a0, a1\|0.7 a0, a1\|0.7 a0, a1\|0.3 a0, a1\|0.3 a0, a1\|0.3 Figure 1. Environment, depicted in terms of a ﬁnite automaton. Figure 1. Environment, depicted in terms of a ﬁnite automaton. We consider three different reward distributions. One is the standard gaussian distribution; the second o a t distribution with low degrees of freedom, and hence, high leptokurtosis. The third one is the empiric stribution of daily returns on the S&P 500 index. We comment later how the second and third distributions a lated. Details are provided in Table 1. In all cases, the expected reward is the highest for action a0 in state We consider three different reward distributions. One is the standard gaussian distribution; the second one is a t distribution with low degrees of freedom, and hence, high leptokurtosis. The third one is the empirical distribution of daily returns on the S&P 500 index. We comment later how the second and third distributions are related. 4. Leptokurtosis (2018)) allow the range to ﬂexibly adjust, and therefore can accommodate nonstationarity. These would provide viable alternatives as well. Risks 2020, 8, 113 In this sectio 10 of 20 introduc 10 of 20 introduc 5. Proposed Solution ptokurtosis. In this section, we ﬁrst present a simple environment where leptokurtosis can easily be introduced. We subsequently propose our enhancement of disRL, aimed at imputing robustness into RL in the face of leptokurtosis. 1. Environment W i l i l i h i i i l d i i ki i ﬁ i l k 5.1. Environment here are two states Details are provided in Table 1. In all cases, the expected reward is the highest for action a0 in state s0 and action a1 in state s1. Consequently, the optimal policy is pure-strategy with optimal state-action pairs equal to (s0, a0) and (s1, a1). ction a1 in state s1. Consequently, the optimal policy is pure-strategy with optimal state-action pair a0) and (s1, a1). The traditional Gaussian reward structure provides the benchmark for the leptokurtic environments. F Table 1. Reward Distributions: Three Cases. The optimal policy (highlighted in bold) is π(si) = ai for i ∈{0, 1}. Nl,1 is the normal distribution with mean l and scale (standard deviation) 1, tl,1,1.1 is the location-scale student t-distribution with location l, scale 1, and degrees of freedom equal to 1.1, and µd is the empirical distribution of daily returns of the S&P 500 index. ake a t distribution with 1.1 degrees of freedom. This distrib tosis (????). The third case uses the empirical distribution o ocument later, when approximated using a t distribution, th Reward Distribution Rt(s, a) Gaussian Leptokurtic Empirical S&P 500 s0 s1 s0 s1 s0 s1 a0 N2.0,1 N1.5,1 t2.0,1,1.1 t1.5,1,1.1 µd + 2.0 µd + 1.5 a1 N1.5,1 N2.0,1 t1.5,1,1.1 t2.0,1,1.1 µd + 1.5 µd + 2.0 The traditional Gaussian reward structure provides the benchmark for the leptokurtic environments. For the first leptokurtic case, we take a t distribution with 1.1 degrees of freedom. This distribution has been popular in finance to model leptokurtosis (Alberg et al. 2008); (Franses et al. 2007); (Mittnik et al. 1998); (Bollerslev 1987). The third case uses the empirical distribution of daily returns on the S&P 500 index. As we shall document later, when approximated using a t distribution, the degrees of freedom are estimated to be about 3. Interestingly, the fourth moment (which tracks leptokurtosis) does not exist when the number of degrees of freedom equals 4 or less. In other words, leptokurtosis is extreme, even for an index as diversified as the S&P 500. We take the difference in mean rewards between optimal and sub-optimal actions to be equal to 0.5. In terms of returns, this implies that the difference in mean returns is a fraction of the reward 11 of 20 Risks 2020, 8, 113 standard deviation in the Gaussian case (or a fraction of the scale in the case of the t distribution). 5.2. Efﬁcient disRL (e-disRL) The key distinguishing features in our approach are that we (i) decouple the two terms of the prediction error in TD Learning, and (ii) use efficient estimation of the mean of the first term (R(s, a) in Equation (1)), exploiting, as in disRL, the availability of the entire empirical distribution, while (iii) applying standard recursive estimation on the second term of the prediction error (γQ(s ′, a ′) −Q(s, a) in Equation (1)). To disentangle the effect of separating the two terms of the prediction error and the use of efﬁcient estimation of the mean, we proceed in stages, and report results, ﬁrst, for an estimator that only implements the separation but continues to use the sample average as the estimator of the expected rewards, and second, for an estimator that both separates the components of the TD error and applies efﬁcient estimation when calculating the mean of the empirical distribution of rewards. We refer to the former as “e-disRL-,” and the latter as “e-disRL.” Summarizing: 1. e-disRL-: Rewards and discounted action-value updates are separated; standard recursive TD learning is applied to the latter, and standard disRL to the former (i.e., the mean is estimated by simple integration over the empirical reward distribution). 1. e-disRL-: Rewards and discounted action-value updates are separated; standard recursive TD learning is applied to the latter, and standard disRL to the former (i.e., the mean is estimated by simple integration over the empirical reward distribution). 2. e-disRL: Same as e-disRL-, but we use an efficient estimator for the mean of the reward distribution. 2. e-disRL: Same as e-disRL-, but we use an efficient estimator for the mean of the reward distribution. Algorithm 1 speciﬁes the updating in terms of pseudo code. The algorithms for e-disRL- and e-disRL only differ in how ˆE(Rt(s, a)) is computed. In both cases, per state-action pair, we record rewards received in a history buffer M(s, a) (see line 5 in Algorithm 1). In e-disRL-, ˆE(Rt(s, a)) = 1 nt nt ∑ i=0 Ri(s, a) ˆE(Rt(s, a)) = 1 nt nt ∑ i=0 Ri(s, a) where nt is the number of rewards recorded for state-action pair (s, a), or the dynamic length of the history buffer M(s, a), and Ri(s, a) is individual reward in the buffer. 5.1. Environment here are two states This is to best emulate experience in ﬁeld ﬁnancial markets: historically, the Sharpe ratio (expected reward to standard deviation ratio) of returns on ﬁnancial assets tends to be below 1.0, i.e., the expected return is a fraction of the return standard deviation. See, e.g., Bogle (2016). 5.2. Efﬁcient disRL (e-disRL) In case of e-disRL, we employ the Maximum Likelihood Estimation principle and obtain an estimate of the mean by applying the mle estimator to the history buffer M(s, a). Algorithm 1: Pseudo-Code for e-disRL- and e-disRL. Algorithm 1: Pseudo-Code for e-disRL- and e-disRL. 1 for episode in n episodes do 2 for step t in m steps do 3 a = ε-greedy(Q(s, a)); 4 Rt(s, a) = environment(a); 5 update M(s, a) by appending Rt(s, a); 6 Q(s, a) ←Q(s, a) + α( ˆE(Rt(s, a)) + γQ(s ′, a ′) −Q(s, a)) 7 end 8 end The mle estimator of the mean of a gaussian distribution equals the sample average, hence in the gaussian environment there is no difference between e-disRL- and e-disRL. However, when rewards are generated by the t distribution, the mle estimator differs. Here, we deploy the mle estimator of the mean when the scale parameter is unknown, but the number of degrees of freedom ﬁxed. See Liu and Rubin (1995). An analytical expression does not exist, but a common approach is to use 12 of 20 Risks 2020, 8, 113 an iterative procedure, the Expectation-Maximization (EM) algorithm. The algorithm ﬁrst performs an Expectation step (E) on the history buffer: ˆwi = [(v + 1)s2]/[vs2 t + (Ri(s, a) −¯x)2], ˆwi = [(v + 1)s2]/[vs2 t + (Ri(s, a) −¯x)2], ˆwi = [(v + 1)s2]/[vs2 t + (Ri(s, a) −¯x)2], where, initially, where, initially, , y, ¯x = 1 nt nt ∑ i=0 Ri(s, a) ¯x = 1 nt nt ∑ i=0 Ri(s, a) and and and s2 = 1 nt nt ∑ i=0 (Ri(s, a) −¯x)2. s2 = 1 nt nt ∑ i=0 (Ri(s, a) −¯x)2. This is followed by a Maximization step (M): This is followed by a Maximization step (M): ˆE(Rt(s, a)) = nt ∑ i=0 ˆwiRi(s, a)/ nt ∑ i=0 ˆwi, where v is the degrees of freedom, and nt is the length of the history buffer M(s, a) in trial t. The two steps can then be repeated until convergence, each time substituting the new estimate ˆE(Rt(s, a)) from step 2 for the old estimate of the mean in step 1 ( ¯x) and recomputing the sample variance using the same weights as for the mean: where v is the degrees of freedom, and nt is the length of the history buffer M(s, a) in trial t. 6.1. Methods To evaluate our approach, we ran TD Learning (SARSA version), categorical disRL, e-disRL- and e-disRL on the “Gaussian,” “Leptokurtic,” and the “Empirical S&P 500” reward settings in our canonical two-state environment. To implement disRL, we replicated the original categorical approach proposed in Bellemare et al. (2017), with slight modiﬁcations to make it suitable for our task. The number of bins (K) in the histogram was set to 100, and the midpoints were equispaced in a range from −30 to +30. We refrained from parametrically ﬁtting a family of distributions to the histograms because this step is used in disRL to learn an unknown relation between state-action values and states when the state space is complex. In our environment, there are only 2 discrete states, and hence, there is no need to learn the relationship Q values-states. Elimination of the parametric ﬁtting step allows us to focus on the action-value learning. We compute the mean of the histogram by integration over the histogram. October 22, 2020 ows us to focus on the action-value learning. We compute the mean of the histogram by integratio togram. n experiment consists of 100 game plays. Each game play contains 200 episodes, comprised each trials/periods). The episodes artiﬁcially divides game play into epochs of equal length, to evalua g An experiment consists of 100 game plays. Each game play contains 200 episodes, comprised each of 100 steps (trials/periods). The episodes artiﬁcially divides game play into epochs of equal length, to evaluate how much the agent has learned, and to control exploration and learning rates. Episodes 1–10 (1000 trials in total) are exploratory, whereby the agents uses the greedy algorithm with an exploration rate ε that decays exponentially from 0.9. After 10 episodes, exploration stops, and the agent implements the policy deemed optimal at the beginning of a new trial. The discount factor, γ = 0.9, and the learning rate, α = 0.1, are both ﬁxed throughout. We dynamically adjust the length of the buffer with historical rewards, which implies that the buffer grows across trials and episodes. Figure 3 displays a graphical depiction of the setup. he agent has learned, and to control exploration and learning rates. Episodes 1-10 (1000 trials in tot atory, whereby the agents uses the greedy algorithm with an exploration rate ε that decays expone 0.9. isRL /e disRL increase this percentage to 100% (in the gaussian case, e disRL and e DisRL are ide he Gaussian Environment 9 Traditional disRL approximates the empirical distribution of Q values with a parametric family of distribution, in order to capture complex relationships between a large state space and state-action values. This approximation complicates convergence proofs; see, e.g., Bellemare et al. (2017); Dabney et al. (2018); Rowland et al. (2018). 5.2. Efﬁcient disRL (e-disRL) The two steps can then be repeated until convergence, each time substituting the new estimate ˆE(Rt(s, a)) from step 2 for the old estimate of the mean in step 1 ( ¯x) and recomputing the sample variance using the same weights as for the mean: s2 = nt ∑ i=0 ˆwi(Ri(s, a) −¯x)2/ nt ∑ i=0 ˆwi. Importantly, the mle estimator does not simply truncate samples, as in many versions of robust estimation (e.g., Huber’s approach; see Sun et al. (2020)). The mle estimator down-weighs outliers in surprising ways; see Figure 2. The estimator compresses the outcomes into a ﬁnite interval, and even pushes outliers closer to the mean than non-outlier outcomes. The mle estimator equals the sample average of non-monotonically transformed outcomes. Figure 2. Transformation of outcomes in mle estimation of the mean of a t distribution with location 2.0, scale 1 and 1.1 degrees of freedom. Based on a sample of 500 observations. Outcomes in the tails of the distribution are not truncated; instead, they are mapped close to zero, eliminating their impact. Outcomes close to the (estimated) mean have maximum inﬂuence; they are mapped into the highest and lowest values. The transformation is non-monotonic. Figure 2. Transformation of outcomes in mle estimation of the mean of a t distribution with location 2.0, scale 1 and 1.1 degrees of freedom. Based on a sample of 500 observations. Outcomes in the tails of the distribution are not truncated; instead, they are mapped close to zero, eliminating their impact. Outcomes close to the (estimated) mean have maximum inﬂuence; they are mapped into the highest and lowest values. The transformation is non-monotonic. 13 of 20 Risks 2020, 8, 113 13 of 20 5.3. Convergence Convergence of estimated Q values to true ones is not an issue for e-disRL as it builds on a mix of disRL (for estimation of the expected immediate reward component of the prediction error) and TD learning (for the expected Q values in the state of the subsequent trial, conditional on optimal action). The efﬁcient estimators of the mean reward merely change speed of convergence, not convergence per se.9 10 The quantiles or expectiles versions of disRL are not subject this inﬂuence. Like the empirical distribution approach we use in e-disRL, unbounded ranges are possible. 6.1. Methods After 10 episodes, exploration stops, and the agent implements the policy deemed optimal ing of a new trial. The discount factor, γ = 0.9, and the learning rate, α = 0.1, are both ﬁxed throu namically adjust the length of the buffer with historical rewards, which implies that the buffer grows nd episodes. Figure 3 displays a graphical depiction of the setup. Episode 1 ϵ−greedy 10 200 j Step 1 100 Constant α Figure 3. Experiment Timeline. Figure 3. Experiment Timeline. Episode 1 ϵ−greedy 10 200 j Step 1 100 Constant α Figure 3. Experiment Timeline. Figure 3. Experiment Timeline. Episode 1 ϵ−greedy 10 200 j Step 1 100 Constant α Figure 3. Experiment Timeline. Figure 3. Experiment Timeline. Figure 3. Experiment Timeline Figure 3. Experiment Timeline. ll the results and analyses below are conducted solely on post-exploration data. That is, they reﬂect All the results and analyses below are conducted solely on post-exploration data. That is, they reﬂect actions and Q values after the 10 exploratory episodes (or 10 × 100 = 1000 trials). values after the 10 exploratory episodes (or 10 × 100 = 1000 trials). Other parameter conﬁgurations could be envisaged. We show below, however, that the parameter well in the baseline, gaussian environment. There, we ﬁnd that all learning approaches learn effe arning and (categorical) disRL report the correct policy at the end of ≈50% of episodes beyond e Other parameter conﬁgurations could be envisaged. We show below, however, that the parameter choice works well in the baseline, gaussian environment. There, we ﬁnd that all learning approaches learn effectively. TD Learning and (categorical) disRL report the correct policy at the end of ≈50% of episodes beyond episode 10. e-disRL-/e-disRL increase this percentage to 100% (in the gaussian case, e-disRL- and e-DisRL are identical). Risks 2020, 8, 113 14 of 20 14 of 20 6.2. The Gaussian Environment Under gaussian rewards, traditional TD Learning generates excellent performance, but e-disRL- (and equivalently, e-disRL) is more robust, generating 100% accuracy; see Table 2. disRL is very disappointing, but we hasten to add that this is because we implemented the categorical version, which estimates Q values distributions using histograms. As we shall discuss at the end of this section, this requires one to commit to a ﬁnite range of possible estimated Q values. But the range of immediate rewards is unbounded, and immediate rewards have a large inﬂuence on estimated Q values in early stages of learning. As a result, bounds on the range of estimated Q values generate biases.10 Table 2. Percentage of game plays where the artiﬁcial agent attained the optimal policy at the end of episodes 11-200. Convergence Robustness is the percentage of 100 game plays where the agent’s chosen policy is the optimal one at the end of episodes 11-200. Each episode consists of 100 trials. First 10 episodes are excluded because exploration takes place. Learning is allowed throughout all episodes. Learned Q values carry over across episodes. Percentages are averaged over the two states. The Convergence Robustness of e-disRL- and e-disRL in the Gaussian case is the same, because the maximum likelihood estimator of the mean (used in e-disRL) equals the sample average (used in e-disRL-). Table 2. Percentage of game plays where the artiﬁcial agent attained the optimal policy at the end of episodes 11-200. Convergence Robustness is the percentage of 100 game plays where the agent’s chosen policy is the optimal one at the end of episodes 11-200. Each episode consists of 100 trials. First 10 episodes are excluded because exploration takes place. Learning is allowed throughout all episodes. Learned Q values carry over across episodes. Percentages are averaged over the two states. The Convergence Robustness of e-disRL- and e-disRL in the Gaussian case is the same, because the maximum likelihood estimator of the mean (used in e-disRL) equals the sample average (used in e-disRL-). Convergence Robustness Gaussian Leptokurtic Empirical S&P 500 TD 67(%) 1(%) 42(%) disRL 5 1 1 e-disRL- e-disRL 100 16 80 100 100 6.3. The Leptokurtic Enviroment I: t-Distribution Convergence Robustness Gaussian Leptokurtic Empirical S&P 500 6.3. The Leptokurtic Enviroment I: t-Distribution 6.3. The Leptokurtic Enviroment I: t-Distribution With leptokurtic rewards, our results conﬁrm the importance of separately accounting for the two terms of the prediction error and using efﬁcient estimation on the term most affected by leptokurtosis. Table 2 shows that both TD Learning and disRL report the optimal policy at the end of all episodes 11-100 only in 1% of game plays. e-disRL-, which simply accommodates heterogeneity of the terms of the prediction error, increases this to 16%. With efﬁcient estimation of the mean of the reward distribution, e-disRL increases this further to an impressive 80%. Table 2 may display an overly tough criterion. Table 3 looks at average performance across episodes. Standard errors show that average performance is estimated with high precision. Only e-disRL generates high levels of performance: it attains the optimal policy on average in 95%/98% of episodes, though there are game plays where it reports optimal policy in none of the episodes. The latter may be attributed to the short duration of exploration (10 episodes). The other three learning protocols report optimal policies only in slightly more than half of the episodes. Performance improves when moving from TD Learning and disRL to e-disRL-, demonstrating that separately accounting for immediate rewards and subsequent Q values during updating is beneﬁcial. 15 of 20 Risks 2020, 8, 113 Table 3. Performance in the leptokurtic environment: Details. Robust Convergence: Percentage of 100 game plays the agent reports the optimal policy at the end of all of episodes 11-200; averages across states are reported in Table 2. Average Convergence: Percentage of episodes in a game play where optimal policy is reported at episode end. Episodes 1-10 are excluded. Mean, St Error and Min, Max are calculated over 100 game plays. Learning Robust Average Convergence Procedure State Convergence Mean St Error (Min, Max) TD Learning s0 1(%) 54(%) 3(%) (0(%), 100(%)) s1 1 56 3 (0, 100) disRL s0 1 55 4 (0, 100) s1 1 45 4 (0, 100) e-disRL- s0 20 59 4 (0, 100) s1 12 63 4 (0, 100) e-disRL s0 77 95 2 (0, 100) s1 83 98 1 (5, 100) Figure 4 displays histograms of the prediction errors in TD Learning and e-disRL for episodes 11 through 200 during a single random game play. The distributions for TD Learning are highly leptokurtic, as they inherit the leptokurtosis of the underlying reward distribution. 6.3. The Leptokurtic Enviroment I: t-Distribution In contrast, the distributions of prediction errors are concentrated around zero when using e-disRL. Notice that, for e-disRL, there are hardly any prediction errors for sub-optimal actions (a1 in state s0 and a0 in state 1). This is because (i) e-disRL learned the correct policy after 10 episodes, (ii) e-disRL rarely un-learned the optimal policy afterwards, (iii) the agent no longer explored (i.e., the agent always chooses the optimal action). State s0 State s1 Action a0 60 40 20 0 20 40 60 TD Errors 0 5 10 15 20 25 Frequency TD 0.05 0.00 0.05 0.10 TD Errors e-disRL 75 50 25 0 25 50 TD Errors 0 10 20 30 Frequency TD 0.0 0.2 0.4 0.6 0.8 1.0 TD Errors e-disRL Action a1 15 10 5 0 TD Errors 0.0 0.2 0.4 0.6 0.8 1.0 Frequency TD 0.0 0.2 0.4 0.6 0.8 1.0 TD Errors e-disRL 15 10 5 0 5 TD Errors 0 2 4 6 8 Frequency TD 0.06 0.04 0.02 0.00 0.02 0.04 TD Errors e-disRL Figure 4. Prediction error histograms for all trials during episodes 11-200 of a single game play, TD Learning and e-disRL. There are no observations, and hence, no histograms, for e-disRL in state-action pair (s0, a1), because the artiﬁcial agents reached the optimal policy at the end of the 10th episode, never switched policy afterwards, and stopped exploring. In one single trial, the e-disRL agent implemented the sub-optimal policy in state-action pair (s1, a0). State s0 Action a0 60 40 20 0 20 40 60 TD Errors 0 5 10 15 20 25 Frequency TD 0.05 0.00 0.05 0.10 TD Errors e-disRL Action a1 15 10 5 0 TD Errors 0.0 0.2 0.4 0.6 0.8 1.0 Frequency TD 0.0 0.2 0.4 0.6 0.8 1.0 TD Errors e-disRL State s1 75 50 25 0 25 50 TD Errors 0 10 20 30 Frequency TD 0.0 0.2 0.4 0.6 0.8 1.0 TD Errors e-disRL 15 10 5 0 5 TD Errors 0 2 4 6 8 Frequency TD 0.06 0.04 0.02 0.00 0.02 0.04 TD Errors e-disRL Figure 4. Prediction error histograms for all trials during episodes 11-200 of a single game play, TD Learning and e-disRL. Risks 2020, 8, 113 Risks 2020, 8, 113 16 of 20 16 of 20 Because it uses an efﬁcient estimator, e-disRL produces symmetric, concentrated distributions around the true values for the optimal state-action pairs (20).11 In the case of sub-optimal state-action pairs, the averages are below the true values (19.5).12 This can be attributed to the fact that the e-disRL agent rarely chooses sub-optimal actions after the exploration epoch (episodes 1-10), as discussed before. As a result, the estimated Q values are rarely updated beyond episode 10. Technically, they are based on erroneously chosen actions in the calculation of the estimated Q values in the subsequent trial. The actions are chosen erroneously because the agent has not learned yet to identify the optimal policy. The true Q values (19.5) are instead based on the true expected reward in the immediate reward and the expected (discounted) Q value across possible states in the subsequent trial, evaluated at the truly optimal actions.13 Incomplete learning also explains the higher variance of the estimated Q values in the case of suboptimal state-action pairs. The distributions of Q values are highly leptokurtic for TD Learning and e-disRL-, and in the case of TD Learning, signiﬁcantly left-skewed as well. State s0 Action a0 2000 0 Q-values 0 10 20 30 40 Frequency TD 100 0 100 Q-values e-disRL- 16 18 20 Q-values e-disRL Action a1 2000 1000 0 Q-values 0 10 20 30 Frequency TD 0 20 40 60 Q-values e-disRL- 14 16 18 20 Q-values e-disRL State s0 State s1 Action a0 2000 0 Q-values 0 10 20 30 40 Frequency TD 100 0 100 Q-values e-disRL- 16 18 20 Q-values e-disRL 2000 0 Q-values 0 10 20 30 Frequency TD 0 50 100 Q-values e-disRL- 16 18 20 Q-values e-disRL Action a1 2000 1000 0 Q-values 0 10 20 30 Frequency TD 0 20 40 60 Q-values e-disRL- 14 16 18 20 Q-values e-disRL 3000 2000 1000 0 Q-values 0 20 40 60 Frequency TD 20 0 20 40 60 Q-values e-disRL- 14 16 18 20 Q-values e-disRL Figure 5. Histograms of estimated Q values at the end of episode 200 in 100 game plays. TD Learning, e-disRL- and e-disRL. The effect of tail risk is marked under TD Learning, and is still quite noticeable when decoupling the immediate reward in the prediction error while estimating its mean using the sample average of past rewards (e-disRL-). Risks 2020, 8, 113 Only when an efﬁcient estimator of the mean is used (e-disRL) does the effect of tail risk disappear. 6.4. The Leptokurtic Environment II: Drawing Rewards from the Empirical Distribution of S&P 500 Daily Returns State s1 2000 0 Q-values 0 10 20 30 Frequency TD 0 50 100 Q-values e-disRL- 16 18 20 Q-values e-disRL 3000 2000 1000 0 Q-values 0 20 40 60 Frequency TD 20 0 20 40 60 Q-values e-disRL- 14 16 18 20 Q-values e-disRL Figure 5. Histograms of estimated Q values at the end of episode 200 in 100 game plays. TD Learning, e-disRL- and e-disRL. The effect of tail risk is marked under TD Learning, and is still quite noticeable when decoupling the immediate reward in the prediction error while estimating its mean using the sample average of past rewards (e-disRL-). Only when an efﬁcient estimator of the mean is used (e-disRL) does the effect of tail risk disappear. Figure 5. Histograms of estimated Q values at the end of episode 200 in 100 game plays. TD Learning, e-disRL- and e-disRL. The effect of tail risk is marked under TD Learning, and is still quite noticeable when decoupling the immediate reward in the prediction error while estimating its mean using the sample average of past rewards (e-disRL-). Only when an efﬁcient estimator of the mean is used (e-disRL) does the effect of tail risk disappear. 6.4. The Leptokurtic Environment II: Drawing Rewards from the Empirical Distribution of S&P 500 Daily Returns We now use the empirical distribution of daily open-to-close returns of the S&P500 index over the period of 1970–2019 as our reward distribution. We ﬁt a t distribution with mle to recover the degrees of freedom v and set the scaling factor equal to 1. We then use the ﬁtted v to implement efﬁcient estimation of the mean reward in e-disRL. The estimate of the degrees of freedom equalled 3.29. This is higher than the degrees of freedom we used in the second experiment, but still low enough for fourth moments not to exist, and hence, leptokurtosis (tail risk) to be extreme. When compared TD Learning and disRL, we again record a substantial improvement when compared to e-disRL. See Table 4. Evidently, most of the improvement appears to emerge because of decoupling of the immediate reward and the Q value in the subsequent trial: e-disRL- reaches the same convergence statistics as e-disRL. 12 For suboptimal state-action pairs, the Q values equal the immediate expected reward from a sub-optimal action, namely, 1.5, plus the expected inﬁnite sum of discounted rewards when switching to the optimal policy in the subsequent trial and beyond. That is, the Q value equals 1.5 + (0.9)(20) = 19.5. 11 The Q values of the optimal state-action pairs can readily be computed by taking the inﬁnite sum of maximal expected rewards (2.0) discounted with a discount factor equal to 0.9. That is, the Q values equal 2.0/(1 −0.9) = 20. y Q q 13 See previous footnote for calculations. 6.3. The Leptokurtic Enviroment I: t-Distribution There are no observations, and hence, no histograms, for e-disRL in state-action pair (s0, a1), because the artiﬁcial agents reached the optimal policy at the end of the 10th episode, never switched policy afterwards, and stopped exploring. In one single trial, the e-disRL agent implemented the sub-optimal policy in state-action pair (s1, a0). Figure 5 demonstrates that the leptokurtosis of the prediction errors for the TD Learning agent adversely impacts the estimates of the Q values. Shown are distributions of estimated Q values at the end of 200 episodes across 100 game plays. The impact is still noticeable when we merely split the prediction error and use the traditional sample average to estimate the expected reward in the immediate trial, as is the case for e-disRL-. y Q q ( )( ) 3 See previous footnote for calculations. Risks 2020, 8, 113 That is, in the case of a broadly diversiﬁed index such as S&P 17 of 20 Risks 2020, 8, 113 500, most of the issues with tail risk disappear by merely properly accounting for heterogeneity in the terms of the prediction error. Table 4. Performance against the S&P 500 daily return distribution: Details. Robust Convergence: Percentage of 100 game plays the agent reports the optimal policy at the end of all of episodes 11-200; averages across states are reported in Table 2. Average Convergence: Percentage of episodes in a game play where optimal policy is reported at episode end. Episodes 1-10 are excluded. Mean, St Error and Min, Max are calculated over 100 game plays. Learning Robust Average Convergence Procedure State Convergence Mean St Error (Min, Max) TD Learning s0 37(%) 99(%) <0.5(%) (95(%), 100(%)) s1 47 99 <0.5 (97, 100) disRL s0 1 69 3 (3, 100) s1 1 62 3 (1, 100) e-disRL- s0 100 100 0 (100, 100) s1 100 100 0 (100, 100) e-disRL s0 100 100 0 (100, 100) s1 100 100 0 (100, 100) 6.5. Impact of Outlier Risk on Categorical Distributional RL 6.5. Impact of Outlier Risk on Categorical Distributional RL 6.5. Impact of Outlier Risk on Categorical Distributional RL The categorical version of disRL does not perform well, even in the baseline, gaussian case. We attribute this to the agent’s setting of the range of potential Q values to a predetermined, ﬁxed (and ﬁnite) range. We had set the range equal to [−30, +30], based on knowledge of the optimal Q values (20 and 19.5 for optimal and sub-optimal state-action pairs, respectively) and of the reward distributions (mean 2 or 1.5). (In real-world implementation of disRL, this information may not be available!) However, in all treatments, the range of estimated Q values is unbounded since the immediate reward distribution is unbounded, so estimates of Q are unbounded too. Figure 6 illustrates how range constraints adversely affects inference in the leptokurtic treatment (t distribution). The ﬁgure shows that, at the end of episode 16 of the game play at hand, 100% of the distribution is assigned to the lowest bin in state-action pairs (s0, a1) and (s1, a0). The issue is resolved in the susbequent episode (17), i.e., after an additional 100 trials, in the case of (s1, a0). Risks 2020, 8, 113 However, it is not resolved for (s0, a1) and emerges anew in state-action pair (s1, a1). Intuitively, the results are to be expected because there is a high chance of an exceptionally large reward under a leptokurtic distribution such as that of daily returns on the S&P 500. State s0 State s1 Action a0 10 11 12 13 14 15 16 17 Q-value histogram 0.0 0.5 1.0 Probability Episode 16 3 2 1 0 1 2 3 Q-value histogram Episode 17 30 29 28 27 26 25 Q-value histogram 0.0 0.5 1.0 Probability Episode 16 3 2 1 0 1 2 3 Q-value histogram Episode 17 Action a1 30 29 28 27 26 25 Q-value histogram 0.0 0.5 1.0 Probability Episode 16 30 29 28 27 26 25 Q-value histogram Episode 17 10 11 12 13 14 15 16 17 Q-value histogram 0.0 0.5 1.0 Probability Episode 16 30 29 28 27 26 25 Q-value histogram Episode 17 Figure 6. Q value histograms for (Categorical) disRL, leptokurtic case. Shown are distributions at the end of Episodes 16 and 17 in one particular game play. Game play is chosen to highlight the impact of tail risk on categorical disRL. The distributions for Episode 17 are obtained from those in Episode 16 plus 100 trials of disRL learning (without exploration). Note that the scale of the horizontal axes is not uniform across state-action pairs and episodes. State s0 Action a0 10 11 12 13 14 15 16 17 Q-value histogram 0.0 0.5 1.0 Probability Episode 16 3 2 1 0 1 2 3 Q-value histogram Episode 17 Action a1 30 29 28 27 26 25 Q-value histogram 0.0 0.5 1.0 Probability Episode 16 30 29 28 27 26 25 Q-value histogram Episode 17 State s1 30 29 28 27 26 25 Q-value histogram 0.0 0.5 1.0 Probability Episode 16 3 2 1 0 1 2 3 Q-value histogram Episode 17 10 11 12 13 14 15 16 17 Q-value histogram 0.0 0.5 1.0 Probability Episode 16 30 29 28 27 26 25 Q-value histogram Episode 17 Figure 6. Q value histograms for (Categorical) disRL, leptokurtic case. Shown are distributions at the end of Episodes 16 and 17 in one particular game play. Game play is chosen to highlight the impact of tail risk on categorical disRL. 7. Conclusions Distributional RL improves on traditional RL by considering the entire distribution of action-values instead of just the mean. In a leptokurtic environment, availability of the distribution can be exploited to estimate the mean in a more efﬁcient way, ensuring that outliers do not cause policy non-convergence or policy instability. In addition, when tail risk affects the reward distribution and not the state transitions, it is beneﬁcial to decouple the two terms in the prediction error of RL, and apply efﬁcient estimation of the mean only to the immediate reward component. These two considerations are the essence of our proposal, e-disRL. In a simple, canonical investment problem, a contextual two-arm bandit problem, we demonstrated how e-disRL improves machine learning dramatically. We illustrated the importance of both efﬁcient estimation and decoupling of the components of the RL updating equations. From a broader perspective, our results underscore the importance of bringing prior domain-speciﬁc knowledge to machine learning algorithms using the tools of mathematical statistics. Leptokurtosis is a speciﬁc property of ﬁnancial data. Mathematical statistics has developed many useful principles and tools to tackle other problems as well. Distributional RL provides the appropriate setting to introduce those principles and tools. Performance is thereby improved substantially. An example beyond ﬁnance is an environment where rewards are generated by an exponential distribution with unknown lower bound (the so-called shifted-exponential distribution; see Rowland et al. (2019)). There, the sample average converges to the true mean at a speed equal to the square-root of the sample size. There exists an alternative estimator of the mean, computed from the minimum observed in the sample. This estimator converges much faster, at a rate equal to the sample size. The estimator is the most efﬁcient one; it reaches the Chapman-Robbins lower bound on the variance of any estimator.15 Mathematical statistics can be brought to bear on other widely encountered problems in reward distributions, such as skewness (another feature of ﬁnancial data), or small distributional shifts that become signiﬁcant only after substantial accumulation (“black swans”). In each domain, distributional reinforcement learning can be exploited to obtain the most efﬁcient way to estimate mean action-values across states, and hence, enhance control. In each case, however, it will be important to determine whether disRL should be applied to the entire prediction error, or whether one should de-couple immediate rewards from estimates of subsequent Q values. Author Contributions: Conceptualization, methodology and validation, P.B., S.H. Risks 2020, 8, 113 Risks 2020, 8, 113 18 of 20 As mentioned before, versions of disRL that do not ﬁx the range of possible edstimated Q values are immune to the negative inﬂuence of tail risk displayed in Figure 6. These include disRL techniques based on quantiles or expectiles. This does not mean that the alternatives fully accommodate tail risk. Rowland et al. (2019) shows how even quantile-based disRL cannot deal with tail risk in the shifted-exponential distribution. A disRL approach based on expectiles works better.14 ( y ) 15 The Cramèr-Rao lower bound does not exist for the shifted-exponential distribution, which is why we refer here to the Chapman-Robbins bound. 14 Expectiles are related to expected shortfall/Conditional Value at Risk (CVaR) in ﬁnance. CVaR is the expected loss in the tails of a distribution (Rockafellar and Uryasev 2000). 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https://openalex.org/W2524290243	https://projecteuclid.org/journals/electronic-journal-of-statistics/volume-10/issue-2/REML-estimation-with-intrinsic-Mat%c3%a9rn-dependence-in-the-spatial-linear/10.1214/16-EJS1125.pdf	English	null	REML estimation with intrinsic Matérn dependence in the spatial linear mixed model	Electronic journal of statistics	2,016	cc-by	18,824	REML estimation with intrinsic Mat´ern dependence in the spatial linear mixed model Somak Dutta Iowa State University, Ames, USA e-mail: somakd@iastate.edu and Debashis Mondal Oregon State University, Corvallis, USA e-mail: debashis@stat.oregonstate.edu Electronic Journal of Statistics Vol. 10 (2016) 2856–2893 ISSN: 1935-7524 DOI: 10.1214/16-EJS1125 Electronic Journal of Statistics Vol. 10 (2016) 2856–2893 ISSN: 1935-7524 DOI: 10.1214/16-EJS1125 Electronic Journal of Statistics Vol. 10 (2016) 2856–2893 ISSN: 1935-7524 DOI: 10.1214/16-EJS1125 Electronic Journal of Statistics Vol. 10 (2016) 2856–2893 ISSN: 1935-7524 DOI: 10.1214/16-EJS1125 1. Introduction In recent years, interest in connecting lattice-based Gaussian random ﬁelds with geostatistical models has increased signiﬁcantly, as researchers begin to explore the extent to which lattice-based models oﬀer an adequate ﬁt or explanation to continuum spatial variations we see in real data. At the forefront of these discussions are the works of McCullagh (2002), Besag (2002), Besag and Mon- dal (2005), McCullagh and Cliﬀord (2006), Lindgren et al. (2011), Dutta and Mondal (2015a), Mondal (2013), and many others. In particular, the work of McCullagh (2002) lays a theoretical foundation for the use of the de Wijs pro- cess or the logarithmic covariance model for spatial analysis. Besag and Mondal (2005) derive the connection between ﬁrst-order intrinsic autoregression and the de Wijs process in which the latter arises as the scaling limit of the for- mer. Using stochastic partial diﬀerential equation representations, Lindgren et al. (2011) (see also Gay and Heyde, 1990; Kelbert et al., 2005) provide useful Gaussian Markov random ﬁeld approximations to a subclass of Mat´ern covari- ance models, and present Galerkin methods for statistical computations. Dutta and Mondal (2015a), on the other hand, derive fast matrix-free computations for spatial mixed models based on Gaussian Markov random ﬁelds with nugget eﬀects, enhancing the computations developed in Lindgren et al. (2011). En- couraged by these developments, we undertake here the novel possibility of using the ﬁrst-order Gaussian intrinsic autoregression on the regular lattice as a building block for constructing fractionally diﬀerenced random ﬁelds. The latter, as we shall see, is unique in approximating continuum intrinsic Mat´ern random ﬁelds that have an important place in the geostatistical literature. It is these lattice-based fractional random ﬁelds that give a fresh perspective to the conceptual framework of spatial Mat´ern dependence analysis, whose full poten- tial has not been realized. We also develop novel, scalable, easy-to-implement and yet sophisticated matrix-free computations that allow us to argue for their wider relevance, and help us understand many of the strengths and weaknesses of intrinsic Mat´ern dependence models. Thus, our purpose in studying Mat´ern models is distinct from classical works of Mardia and Marshall (1984), Handcock and Stein (1993), Stein (1999), Dig- gle et al. (2003), Guttorp and Gneiting (2006), Minasny and McBratney (2007), Pardo-Ig´uzquiza et al. (2009), Anitescu et al. (2012) and many others on con- ventional ﬁtting of stationary Mat´ern covariances to spatial data. and Received April 2015. Received April 2015. and Abstract: We present a new matrix-free residual maximum likelihood (REML) analysis for irregularly spaced spatial data, where observations usually represent average values over very small regions that are inter- preted as points. The REML analysis is obtained after embedding the sam- pling locations in a ﬁne scale rectangular lattice, treating unobserved sites as missing data. The spatial random ﬁelds considered here are based on fractional Laplacian diﬀerencing on the lattice and they are unique in ap- proximating continuum intrinsic Mat´ern dependence. Here, using the h- likelihood method, we derive REML estimating equations that allow for singular precision matrices, estimation of covariate eﬀects, prediction of unobserved spatial eﬀects and REML estimation of precision parameters as a solution to an explicit gamma non-linear model. Furthermore, we de- vise a sophisticated computational algorithm that enables us to achieve scalable matrix-free statistical computations. In particular, these matrix- free computations include the use of (1) the two-dimensional discrete co- sine transformation that arises in the spectral decomposition of the preci- sion matrix of our spatial random ﬁelds and that allows fast matrix-free matrix-vector multiplication, (2) a matrix-free pre-conditioned Lanczos al- gorithm that solves non-sparse matrix equations with linear constraints, (3) a matrix-free Hutchinson’s trace estimator that stochastically approxi- mates the trace of a matrix, (4) a robust trust region method that always ﬁnds a local maximum of the non-concave residual log-likelihood function and (5) some preliminary computations of the log REML likelihood func- tion based on Taylor series approximation. Using computer experiments, we provide further understanding on not just the number and values but also the basins of attraction of the local and global maxima of the REML function. This understanding signiﬁcantly simpliﬁes the problem of ﬁnding global maxima. We further demonstrate through computer experiments that our matrix-free REML estimators attain both eﬃciency and geosta- tistical inference, and surpass the widely used INLA methods in computa- tional times. We provide an extensive application on mapping ground water arsenic concentration in Bangladesh, indicating numeric consistency of re- sults and robustness of inference to changes of lattice spacing. The paper closes with some discussions that include computations in the stationary case, conditional simulations and matrix-free MCMC computations. 2856 2857 REML estimation with intrinsic Mat´ern dependence Keywords and phrases: Arsenic contamination, discrete cosine trans- form, fractional diﬀerencing, Hutchinson’s trace approximations, incom- plete Cholesky, Lanczos algorithm, long range dependence, power vari- ogram, matrix-free computations, trust region method. 1. Introduction Furthermore, the purpose here is also distinct from studying Whittle’s spectral approxima- tions methods (see e.g., Guyon (1982), Dahlhaus and K¨unsch (1987), Kent and Mardia (1996), Fuentes (2007) and many others) for estimating parameters of 2858 S. Dutta and D. Mondal stationary covariances. Necessarily, we avoid direct modeling via covariances or spectral densities, and instead focus on the structure of the precision matrix (i.e., the canonical parameter) that arises through full conditional speciﬁcations and scaling limit connections. Furthermore, as geostatstics is largely about dif- ferencing and intrinsic random functions (Matheron, 1971, 1973; see also the review of Beran (1992) on long range dependence and the discussion of Dig- gle et al., 2010), we restrict our attention mostly to fractional diﬀerencing and approximations of intrinsic Mat´ern models. Corresponding methods for the sta- tionary case, if needed, can be obtained easily and will be discussed later in the paper. In geostatistical applications, observations usually represent average values over very small regions that are interpreted as points. Thus, for brevity of dis- cussion, we assume that we can embed the locations in a ﬁne scale rectangular lattice, treating unobserved sites as missing data. As we shall see in Sections 5 and 6, there is little loss in discretizing the space in the above way and in em- bedding irregularly spaced locations to a ﬁne regular grid. We can then focus on estimation of parameters in an overall mixed eﬀect formulation, which might in- clude covariate information as well as a white noise component. In other words, we consider a mixed linear model of the form y = Tτ + Fψ + ϵ. (1) (1) In the above y is the vector of observations around n sampling locations, T is an n × m matrix of some covariate values with τ as the coeﬃcients, ψ is the vector of latent spatial process coming from a ﬁne r × c regular array on which the sampling locations are embedded, F is a known incidence matrix indicating whether an observation belongs to a particular array cell so that Fψ gives back the vector of latent spatial variable values for the observed y and ϵ indicates the vector of residual ﬂuctuations that are left unexplained by the regularly varying spatial process or the covariate values. 1. Introduction We assume that the covariate values are so adjusted that T1 = F1 = 1 or F1 = 1 where 1 is the vector of all ones of appropriate dimension and 1 belongs to the column space of T. We interpret yi, the ith entry of the y vector, as the average value on the array cell on which the corresponding sampling location falls. Similarly, we interpret ψu,v, the (u, v)th entry of ψ, as the average value of the latent spatial random ﬁeld on the (u, v)th array cell. Furthermore, we embrace the residual ﬂuctuations as independent and identically distributed Gaussian random variables with an unknown precision parameter λy, and the random spatial eﬀect ψ as an intrinsic Mat´ern Gaussian random ﬁeld on an r × c array with singular precision matrix R. In other words, ϵ ∼(λy/(2π)) 1 2 n exp −1 2λyϵ Tϵ , ψ ∼\|R/(2π)\| 1 2 + exp −1 2ψ TRψ . where \|R\|+ denotes the product of nonzero eigenvalues of R. where \|R\|+ denotes the product of nonzero eigenvalues of R. Thus, what assumes importance is how we construct R and how we pursue subsequent statistical computations in order to draw meaningful statistical in- ference. In the case of ﬁrst-order Gaussian intrinsic autoregression on the two REML estimation with intrinsic Mat´ern dependence 2859 dimensional integer lattice, which is central to the Markov random ﬁeld ap- proach of spatial statistics, ψ is deﬁned via the following conditional mean and variance formula E (ψi,j \| ...) = 1 4(ψi,j−1 + ψi,j+1 + ψi−1,j + ψi+1,j), var (ψi,j \| ...) = 1, where the conditioning variables (namely ...) are all ψu,v such that (u, v) ̸= (i, j). When restricted on a ﬁnite r × c array, this intrinsic autoregression actually follows ψ ∼\|W/(2π)\| 1 2 + exp −1 2ψ TWψ , where the singular precision matrix W of ψ is the discrete (graph) Laplacian on the grid with rank(W) = q −1 and can be written in terms of a quadratic form as ψ TWψ = 1 4 (ψi,j −ψi−1,j)2 + 1 4 (ψi,j −ψi,j−1)2. In what follows, we shall draw upon the above construction of W to derive an explicit representation to R. 1. Introduction We oﬀer here new and interesting solutions to these challenges without resorting to dimension reduction, tapering, or procedures such as block averaging. We also demonstrate that there is little loss of statistical eﬃciency (in terms of achieving Cramer-Rao lower bound) due to our matrix-free scalable computations and we can obtain answers that are as good as exact answers. p g The rest of the paper is laid out as follows. In Section 2, we provide in- terpretation of (2) based on the fractional Laplacian diﬀerence equations and derive some justiﬁcations for its validity using both the frequency domain and the spatial domain results. Speciﬁcally, we show that 1) as the lattice spacing diminishes the eigenvalues of the singular precision matrix from (2) trace out the eigenvalues of an intrinsic Mat´ern process, and 2) the diﬀerences between the variograms of the fractionally Laplacian diﬀerenced process at a moderately ﬁne lattice and the continuum Mat´ern process are just a small constant. Thus, at a moderately ﬁne lattice scale, our approximations work remarkably well by the routine use of a random error term along with the spatial component. In Sections 3 and 4, we develop REML estimation procedure of the mixed model in (1). Speciﬁcally, we derive of matrix-free statistical computations of best lin- ear unbiased estimators (BLUEs) of the contrasts of ﬁxed eﬀect τ, best linear unbiased predictors (BLUPs) of the contrasts of random eﬀect ψ, and REML estimation of θ = (λy, λψ, ν)T. We also provide explicit characterization of the non-convex nature of REML estimation. Our computational steps consist of (1) matrix vector multiplications using the discrete cosine transformations (2) linear equations solving with a non-sparse Lanczos algorithm, (3) derivation of eﬀective matrix-free preconditioner using an incomplete Cholesky factor decom- position, (4) calculating traces of matrices using Hutchinson’s trace estimators and (5) optimization using a robust matrix-free trust region method. We also include some preliminary computations of the log REML likelihood function based on Taylor series approximation. In Section 5, we present through simula- tions the performance of the REML estimators. These simulation runs suggest that despite non-convex nature of REML optimization, the trust region method can pick the global estimates, at least when n = O(rc) and n is large. We also run simulations to demonstrate that there is little loss in embedding irregu- larly spaced locations to a ﬁne regular grid. 1. Introduction Speciﬁcally, we shall use R = λψWν, λψ > 0, ν ≥0, (2) (2) with a precision parameter λψ and a (long range) dependence parameter ν. The above may look rather simple, but as we shall see, it allows us to describe fractionally diﬀerenced random ﬁelds on regular arrays and, as lattice spacing diminishes, it allows us to approximate intrinsic Mat´ern models. Furthermore, it is the use of (2) that allows us to develop fast matrix-free REML compu- tations for the mixed model in (1) and, in the process, allow us to gain new understanding on the statistical estimation and inference of intrinsic Mat´ern models. It is worthwhile to point out that there is little literature on ﬁtting exact intrinsic Mat´ern mixed linear model besides McCullagh and Cliﬀord (2006). These exact REML estimation of McCullagh and Cliﬀord (2006) require O(n3) computations and O(n2) storage when data are observed at n spatial locations. It is also worthwhile to point out that in Lindgren et al. (2011) the focus was on the values ν = 1, 2, . . ., which correspond to a subclass of conditional autoregres- sions with sparse precision matrices and which allow them to use certain sparse matrix computations that are of O(n3/2). However, the sparse approximations and the fast computations of Lindgren et al. (2011) are not extensible for any arbitrary positive values of ν (e.g., for ν = 5/3 that arises in the study of turbu- lence). In comparison, the eﬀective order of matrix-free computations developed by Dutta and Mondal (2015a) for spatial mixed linear models based on a sparse precision matrix (i.e., ν = 1, 2, . . .) and nugget eﬀects is just O(n log n) with O(n) storage. In this paper, we not only extend the estimation to arbitrary ν > 0, but also the computations that we develop here for any ν > 0 eﬀec- tively require only O(n(log n)2) computations and O(n) storage. Other than non-sparsity, the statistical problem considered here has many challenges. As, 2860 S. Dutta and D. Mondal for example, the intrinsic Mat´ern dependence leads to not just a non-sparse singular precision matrix but also an ill-conditioned one and ﬁnding an useful preconditioning method is a challenge. Similarly, non-integer values of ν give rise to non-convexity in the log REML function which poses an important optimiza- tion challenge. 2. Fractionally diﬀerenced random ﬁelds and intrinsic Mat´ern processes The objective of this section is to provide some details of the fractional Lapla- cian diﬀerenced random ﬁelds on ﬁner and ﬁner regular arrays as approxima- tions to continuum intrinsic Mat´ern random ﬁelds. Here, we shall consider some frequency domain results, exact variogram calculations and precise algebraic eigenvalue expressions that lead to (2). We also discuss certain meaning and signiﬁcance to its use. First, as discussed by Mondal (2011) with regard to the paper of Lindgren et al. (2011), we consider a sequence of Gaussian random ﬁelds {Z(m)(u, v)} on regular two dimensional sub-lattices Z2 m with spacing 1/m, m = 1, 2, . . ., which have individual spectral densities of the form fm(ω, η) = σ2 m m2 sin2( 1 2mω) + sin2( 1 2mη) ν , (3) (3) with ω, η ∈(−πm, πm], σm > 0 and ν > 0. In the above, ν = 1 corresponds to the ﬁrst-order intrinsic autoregression, ν = 2 suggests a Whittle’s simultaneous intrinsic autoregression and so on. The integer values of ν, namely, ν = 1, 2, 3, . . . were the focus of Lindgren et al. (2011) and they correspond to various lattice- based intrinsic autoregressions with sparse precision matrices. In contrast, the non-integer values of ν, which is the primary focus of this paper, lead to frac- tionally diﬀerenced random ﬁelds. Typically, they correspond to random ﬁelds with non-sparse precision matrices and oﬀer greater ﬂexibility in modeling long range spatial dependencies. Speciﬁcally, following Duﬃn (1953), let Dm be the Laplace diﬀerence operator on the sub-lattice Z2 m, i.e., Dmf(u, v) = f(u, v) −1 4{f(u + 1 m, v) +f(u −1 m, v) +f(u, v + 1 m) +f(u, v −1 m)}, where f is any real valued function deﬁned at the lattice points of Z2 m. Then, by extending the results of Hosking (1981), we can represent {Z(m)(u, v)} as Dν/2 m Z(m) u,v = ξu,v, ν > 0, where ξu,v is a Gaussian white noise random ﬁeld on the sub-lattice Z2 m with var ξu,v = σ2 m/m2. Thus, {Z(m)(u, v)} can be interpreted as a fractional Lapla- cian diﬀerenced random ﬁeld on the sub-lattice Z2 m and they enjoy properties similar to those of fractionally diﬀerenced time series. 1. Introduction In particular, we show numeric consistency of results and highlight robustness of inference to changes of lattice spacing. We further demonstrate that our matrix-free REML estimators attain eﬃciency properties that are very close to those of the exact REML estimators. In terms of practical gain, we also report actual computational times that are better than those from Lindgren et al. (2011). In Section 6, we provide an exten- sive application to mapping ground water arsenic contamination in Bangladesh and again highlight robustness of statistical inference to the changes of scales. Finally, in Section 7, we close the paper with some discussions on computations of the stationary case, conditional simulations, and on potential challenges ahead REML estimation with intrinsic Mat´ern dependence 2861 in accommodating spatial anisotropy and heterogeneity within the framework developed here. 2. Fractionally diﬀerenced random ﬁelds and intrinsic Mat´ern processes Now, just like the fractionally diﬀerenced time series, the fractionally diﬀer- enced random ﬁelds {Z(m)(u, v)} can be thought of as discrete space analogue of certain continuum fractional random ﬁelds. In fact, these continuum random ﬁelds, say, {Z(u, v)} can be obtained by taking scaling limits of {Z(m)(u, v)}. 2862 S. Dutta and D. Mondal To elaborate on this, we assume that, as m →∞, mν−1σm →σ/2ν. Then, it is not diﬃcult to see that fm(ω, η) converges to f(ω, η) = σ2 (ω2 + η2)ν , σ > 0, (4) (4) The above gives the spectral density formula of a continuum Gaussian intrinsic Mat´ern random ﬁeld and thus, from the above convergence result, it follows that continuum Gaussian intrinsic Mat´ern random ﬁelds are scaling limits of fractionally diﬀerenced random ﬁelds. Furthermore, the above convergence re- sult explicitly describes the rescaling of parameters needed, particularly when we want to choose a suitable sub-lattice (e.g., to embed irregular sampling loca- tions into a grid or to approximate irregular regions by unions of grid cells when observations themselves are aggregates over such regions). As we shall see, this will become important to show numeric consistency and robustness of inference to changes of lattice spacing. The key question is, as approximations to the continuum intrinsic Mat´ern process, how good are the above lattice-based model {Z(m)(u, v)} for small values of m. For brevity of discussion, we focus on the case 1 < ν < 2. The intrinsic case with ν = 1 is discussed in details in Mondal (2005), Besag and Mondal (2005) and Dutta and Mondal (2015a). The intrinsic case with ν ≥2 requires use of higher-order contrasts and some calculations are outlined in Appendix and in the supplement to the paper. The intrinsic case with 0 < ν < 1 is similar to the case ν = 1 in the sense it also requires calculations using (contrasts of) averages over nun-null regions, and some calculations are given in the supplement to the paper. If 1 < ν < 2, following Matheron (1973), it is well known that the continuum intrinsic Mat´ern random ﬁeld {Z(u, v)} has the following exact power variogram formula γ(s, t) = σ2 R2 1 −cos(sω) cos(tη) 4π2(ω2 + η2)ν dωdη = − σ2π3/2Γ(ν −1 2) 4π2Γ(ν)Γ(2ν −1) sin(νπ) h2ν−2, where h = √ s2 + t2. 2. Fractionally diﬀerenced random ﬁelds and intrinsic Mat´ern processes On the other hand, the variogram of the fractional Lapla- cian diﬀerenced random ﬁeld on Z2 m at lag (s, t) is given by the double integral formula γm(s, t) = σ2 m mπ −mπ mπ −mπ 1 −cos(sω) cos(tη) 4π2m2 sin2 ω 2m + sin2 η 2m ν dωdη, which is computed numerically with great accuracy by a two-dimensional quad- rature method discussed in Dutta and Mondal (2015b). For large m, γm(s, t) will be very close to γ(s, t). However, we want to know whether one can work with {Z(m)(u, v)} for moderately small values of m instead of working with the continuum process {Z(u, v)} and essentially get the same result. To address this question, Figure 1 plots the diﬀerence {γ(s, t) −γm(s, t)} against the lag distance √ s2 + t2 for ν = 1.25 and ν = 1.5 and σ2 = 1. When ν = 1.25, we see that the diﬀerence decreases as m increases and this diﬀerence becomes almost REML estimation with intrinsic Mat´ern dependence 2863 Fig 1. Plots of the diﬀerences in semivariograms of the intrinsic Mat´ern process and the fractionally diﬀerenced process for ν = 1.25 (left) and 1.5 (right). We took σ2 = 1 and σ2 m = 4−νm2−2ν. Each diﬀerence is plotted against the lag distance h = √ s2 + t2 for lag (s, t). The values of γ(1, 0) for ν = 1.25 and ν = 1.5 are 0.304 and 0.159 respectively. Fig 1. Plots of the diﬀerences in semivariograms of the intrinsic Mat´ern process and the fractionally diﬀerenced process for ν = 1.25 (left) and 1.5 (right). We took σ2 = 1 and σ2 m = 4−νm2−2ν. Each diﬀerence is plotted against the lag distance h = √ s2 + t2 for lag (s, t). The values of γ(1, 0) for ν = 1.25 and ν = 1.5 are 0.304 and 0.159 respectively. a positive constant at m = 8. Thus, in this case it is to our advantage to add a small nugget eﬀect to the process {Z(m)(u, v)} so that statistical analyses based on the fractional Laplacian diﬀerenced random ﬁeld on sub-lattices Z2 m are essentially equivalent to the same based on continuum power variogram model on integer lattice Z2. 2. Fractionally diﬀerenced random ﬁelds and intrinsic Mat´ern processes ψ Furthermore, suppose for k = r or k = c that Mk is a k × k matrix corre- sponding to the discrete cosine transformation with entries m1,j = k−1 2 , mi,j = (2/k)−1 2 cos{π(i−1)(j−1 2)/k}, i = 2, . . . , k, j = 1, . . . , k. m1,j = k−1 2 , mi,j = (2/k)−1 2 cos{π(i−1)(j−1 2)/k}, i = 2, . . . , k, j = 1, . . . , k. Suppose also that Dk is a diagonal matrix with ith diagonal entry equal to dk,i = 2[1 −cos{π(i −1)/k}]. It then follows that Mk is orthogonal and M = Mc ⊗Mr diagonalizes W. Speciﬁcally, MWM T = 1 4Ic ⊗Dr + 1 4Dc ⊗Ir = D01 + D10, MWM T = 1 4Ic ⊗Dr + 1 4Dc ⊗Ir = D01 + D10, where D01 = 1 4Ic ⊗Dr and D10 = 1 4Dc ⊗Ir. Thus Wν has the spectral decom- position MWM T = 1 4Ic ⊗Dr + 1 4Dc ⊗Ir = D01 + D10, where D01 = 1 4Ic ⊗Dr and D10 = 1 4Dc ⊗Ir. Thus Wν has the spectral decom- position Wν = M T(D01 + D10)νM. It is now obvious that, for any m = 1, 2, . . ., the eigenvalues of R−, the Moore– Penrose inverse of R, exactly trace out the spectral density (3) at the discrete cosine frequencies, further justifying the validity of (2). It is also obvious that with the increase of m, i.e., as the lattice spacing diminishes, the eigenvalues of R−trace out the limiting continuum spectral density (4) better. The spatial formulation in (2) as representation of the inﬁnite lattice frac- tional random ﬁelds in (3) is enhanced further if we allow a few extra layers of conceptual ‘border’ grid cells when we embed sampling locations. The idea was put forward by Besag and Higdon (1999), purely as a computational ploy, to alleviate edge eﬀects, and, in practice, its implementation requires just a straightforward adjustment to F. 2. Fractionally diﬀerenced random ﬁelds and intrinsic Mat´ern processes On the other hand, when ν = 1.5, the diﬀerence of the variograms is negative, increases with respect to m and essentially becomes a small negative constant when m = 8. In fact, notice that this diﬀerence is only 1.5% of γ(0, 1) when m = 8. Thus, the lattice-based approximation would be good for moderate values of m even if a little nugget eﬀect is present in the data. In general, we have observed similar phenomena for other values of ν. In par- ticular, for other reasonable values of 1 < ν < 2, we found that the diﬀerence γ(s, t) −γm(s, t) is essentially a small constant (positive or negative) for mod- erately small values of m. Thus, it not unreasonable to anticipate that lattice based approximations are enhanced by routine use of a random error term along with the spatial component. In fact, these results are typical to the ones found in Mondal (2005), Besag and Mondal (2005) and Dutta and Mondal (2015a) and they can be further justiﬁed by writing down the asymptotic expansions of γm(s, t) using results from Duﬃn and Shaﬀer (1960), as done in Mondal (2005), Besag and Mondal (2005) and Dutta and Mondal (2015a) for the case of ν = 1. Overall, a fractional Laplacian diﬀerenced random ﬁeld plus a white noise at a reasonable sub-lattice is a very good approximation of the intrinsic Mat´ern plus white noise model on the actual lattice. Next we explore how the ﬁnite grid representation in (2) is connected with the inﬁnite lattice representation in (3). To this end, suppose that observations are made on a ﬁnite r × c regular array with r = p1 × m, c = p2 × m and lattice spacing 1/m. We can then approximate the Laplace diﬀerenced operator Dm by the discrete (graph) Laplacian matrix of this grid. However, note that the matrix −W in (2) is the Laplacian matrix on this ﬁnite grid. This leads to saying that the spatial lattice process Z(m) u,v on a ﬁnite integer lattice are approximately zero 864 S Dutta and D Mondal 2864 S. Dutta and D. Mondal mean Gaussian random variables with precision matrix σ−2 m Wν. This gives rise to (2) with λψ = σ−2 m . 3. Estimation for the mixed model The singularity of R presents some diﬃculty in interpreting the mixed model in (1). Put another way, (1) is interpreted in terms of contrasts of y, i.e., C0y = C0Tτ + C0Fψ + C0ϵ, where rows of C0 are vector of contrasts and rank(C0) is equal to rank(FR−FT). Thus, contrasts of τ are estimable and con- trasts of ψ are predictable. In such settings, typical REML estimation goes as follows. We estimate τ from marginal distribution of C0y. We estimate BLUPs of the contrasts of ψ using conditional mean formula given data diﬀerences C0y. Separate from the estimation of τ, we obtain estimates of λy, λψ and ν from maximizing the log REML function. However, unless carefully done, such estimation procedures based on data diﬀerencing do not lead to any simpliﬁca- tion nor do they provide any insight into the REML estimation problem. Thus, in what follows, we draw upon the work of Henderson (1950, 1975), Lee and Nelder (1996, 2001), and Dutta and Mondal (2015a) and derive exact REML estimation using h-likelihood formulation. REML estimation with intrinsic Mat´ern dependence 2865 3.1. Estimation of ﬁxed eﬀects and prediction of spatial eﬀects S. Dutta and D. Mondal 2866 3.2. REML estimation for precision parameters The score equations that maximize the log–REML function in (7) are then given by 1 2Tr Q−1Qi −1 2Tr (X TQX)−1X TQiX −1 2(z −Xβ) TQi(z −Xβ) = 0 1 2Tr Q−1Qi −1 2Tr (X TQX)−1X TQiX −1 2(z −Xβ) TQi(z −Xβ) = 0 for i = 1, 2 and 3. It is not diﬃcult to see that the above score equations can also be written as 1 2Tr Q−1Qi −1 2Tr (X TQX)−1X TQiX −1 2(z −Xβ) TQi(z −Xβ) = 0 for i = 1, 2 and 3. It is not diﬃcult to see that the above score equations can also be written as for i = 1, 2 and 3. It is not diﬃcult to see that the above score equations can also be written as 1 2Tr Q−1Qi −1 2Tr HQ−1Qi −1 2(z −Xβ) TQi(z −Xβ) = 0, i = 1, 2, 3, (8) where H = X(XTQX)−1XTQ denotes the ‘hat’-matrix of the linear regression model (5). Typically, Fisher’s scoring method is used to solve the score equations and to obtain REML estimates. However, this also requires computation of the second derivatives of the log REML function or the information matrix I whose (i, j)th entry is equal to I(i, j) = 1 2Tr (I −H)Q−1Qi(I −H)Q−1Qj , (9) (9) and whose inverse is used to produce estimates for dispersion of the REML estimators of the precision parameter. 3.1. Estimation of ﬁxed eﬀects and prediction of spatial eﬀects Let B denote the matrix of orthogonal contrasts formed by the last rc−1 eigen- vectors of W, and let G denote the diagonal matrix formed by the corresponding eigenvalues of W, i.e., G = diag{(d01,i + d10,i)ν, i = 2, . . . , rc} where d01,i and d10,i are the ith diagonal entries of D01 and D10 respectively. It then follows that Bψ has an rc −1 dimensional Gaussian distribution with zero mean and covariance matrix λ−1 ψ G−1. Extending Henderson (1950, 1975), Lee and Nelder (1996, 2001) and Dutta and Mondal (2015a), we represent the mixed model equations in (1)-(3) by y 0 = T F 0 B τ ψ + ϵ ζ , (5) (5) where ϵ and ζ are independent centered Gaussian distributions random vectors with covariance matrices λ−1 y I and λ−1 ψ G−1 respectively. For notational brevity, we further deﬁne z = y 0 , X = T F 0 B , β = τ ψ , η = ϵ ζ and Q = λyI 0 0 λψG . Then we can express the mixed model in (1) as a linear regression model z = Xβ + η where η ∼Nn+rc−1(0, Q−1). Under the assumption that T1 = F1 = 1, the design matrix X has one rank deﬁciency; that is there is a non-zero vector ℓ such that Xℓ= 0. For ﬁxed parameters λy, λψ and ν, we then estimate β by solving the normal equation λyTTT λyTTF λyFTT λyFTF + λψWν τ ψ = λyTTy λyFTy or in more succinct notation or in more succinct notation X TQXβ = X TQz or, Aβ = b (6) (6) X TQXβ = X TQz or, Aβ = b subject to the constraint Aℓ= 0. Let β be the estimate of β that we obtain solving the normal equation (6). Let τ and ψ be the corresponding estimates of τ and ψ, which we obtain from β. Extending the argument given in Dutta and Mondal (2015a), one can then see that the contrasts of τ coincide with the best linear unbiased estimators of the contrasts of τ. Similarly, the contrasts of ψ coincide with the best linear unbiased predictors of the contrasts of ψ. Furthermore, even if the symmetric coeﬃcient matrix A = XTQX is not sparse, we shall see that the above normal equation is solved using a novel and eﬃcient matrix-free Lanczos algorithm. 3.2. REML estimation for precision parameters Extending Lee and Nelder (1996, 2001) and Dutta and Mondal (2015a), the log-likelihood function of the residuals obtained from the regression model (5) takes the form of 2ℓr(λ, ν) = log det Q −log \|X TQX\|+ −(z −Xβ) TQ(z −Xβ). (7) (7) The derivations provided in Dutta and Mondal (2015a) further show that the traditional REML log-likelihood arising from C0y and (7) is actually same up to an additive constant that depends only on the contrast matrix C0. Thus, max- imizing (7) gives rise to REML precision parameter estimate of θ = (λy, λψ, ν). The derivations provided in Dutta and Mondal (2015a) further show that the traditional REML log-likelihood arising from C0y and (7) is actually same up to an additive constant that depends only on the contrast matrix C0. Thus, max- imizing (7) gives rise to REML precision parameter estimate of θ = (λy, λψ, ν). The derivations provided in Dutta and Mondal (2015a) further show that the traditional REML log-likelihood arising from C0y and (7) is actually same up to an additive constant that depends only on the contrast matrix C0. Thus, max- imizing (7) gives rise to REML precision parameter estimate of θ = (λy, λψ, ν). Traditional maximization of the log REML function uses score equations which is obtained by equating the gradient of ℓr to zero. Thus suppose Q1 = ∂Q/∂λy, Q2 = ∂Q/∂λψ, and Q3 = ∂Q/∂ν. The score equations that maximize the log–REML function in (7) are then given by The derivations provided in Dutta and Mondal (2015a) further show that the traditional REML log-likelihood arising from C0y and (7) is actually same up to an additive constant that depends only on the contrast matrix C0. Thus, max- imizing (7) gives rise to REML precision parameter estimate of θ = (λy, λψ, ν). Traditional maximization of the log REML function uses score equations which is obtained by equating the gradient of ℓr to zero. Thus suppose Q1 = ∂Q/∂λy, Q2 = ∂Q/∂λψ, and Q3 = ∂Q/∂ν. The score equations that maximize the log–REML function in (7) are then given by g ( ) g p p ( y, ψ, ) Traditional maximization of the log REML function uses score equations which is obtained by equating the gradient of ℓr to zero. Thus suppose Q1 = ∂Q/∂λy, Q2 = ∂Q/∂λψ, and Q3 = ∂Q/∂ν. 3.3. Nonconvexity in REML estimation Neither the log REML function (7), nor the scoring equations (8) give insight into the exact non-convex nature of the optimization problem. Here, instead of maximizing the REML function directly, we consider an alternative approach that allows us to characterize the REML optimization problem in terms of a non-linear gamma regression. This alternative approach follows the work of Lee and Nelder (1996, 2001) and Dutta and Mondal (2015a). The basic idea is as follows. Rather than using traditional REML computations that separate estimations of β and θ, we can consider an iterative approach. Thus, starting with an initial estimate β, we compute the residuals e = z −Xβ and use them REML estimation with intrinsic Mat´ern dependence 2867 to estimate the precision parameters λy, λψ and ν. Then, once estimates λy, λψ and ν are obtained, we update β by computing the normal equation from (6). The process continues until the estimates converge numerically. It is just that the ﬁnal estimates are actually REML estimates. Speciﬁcally, suppose hjj is the jth diagonal element of the hat matrix H, and qjj is the jth diagonal entry of Q. Then qjj = λyvy,j + λψ(v01,j + v10,j)ν Q qjj = λyvy,j + λψ(v01,j + v10,j)ν where where where vy,j = 1 if 1 ≤j ≤n 0 if n + 1 ≤j ≤n + rc −1 , v01,j = 0 if 1 ≤j ≤n d01,j−n+1 if n + 1 ≤j ≤n + rc −1 and v10,j = 0 if 1 ≤j ≤n d10,j−n+1 if n + 1 ≤j ≤n + rc −1. . Accordingly the score equations in (8) then can be written as n+rc−1 j=1 (1 −hjj)q(i) jj e2 j 1 −hjj −1 qjj = 0, i = 1, . . . , 3, (10) (10) where q(i) jj denote the jth diagonal entry of Qi. The above score equations co- incide with the estimating equations of a nonlinear gamma regression, where we assume that the adjusted residuals e2 j/(1 −hjj)’s are the responses vari- ables that are distributed independently as Gamma random variables, and we have an inverse link, prior weights (1 −hjj)’s and nonlinear predictors qjj = λyvy,j + λψ(v01,j + v10,j)ν. The above provides a precise characterization of the non-convex nature of REML estimation in that non-convexity arises through the estimation of the dependence parameter ν. 3.3. Nonconvexity in REML estimation In particular, when ν is ﬁxed and known, the opti- mization becomes a convex optimization in which case any local maximum is also the global maximum. 4. Matrix-free computations Typical statistical computations such as REML estimation from Mat´ern mixed models of spatial data with n irregularly distributed sampling locations require at least O(n3) computations and O(n2) storage space. The main objective here is to reduce computations for REML estimation of our mixed linear model in (1) to O(Kn(log n)2) operations, where K << n (and eﬀectively a constant) and storage to O(n) space. We do this without losing any eﬃciency properties of the REML estimators. To this end, we divide our task into three parts. First, we S. Dutta and D. Mondal 2868 discuss the two dimensional discrete cosine transformation that is essential for various matrix-free matrix-vector statistical computations for our mixed model. Second, we indicate how we can compute BLUEs and BLUPs of contrasts using a novel matrix-free Lanczos algorithm. Here we also develop a novel precon- ditioning method to reduce the order of computations. Third, we develop fast matrix-free ways to obtain REML estimates using stochastic trace approxima- tions and a trust region method. In this paper we do not pursue equation (8) for estimating the precision parameters, which would require computing the di- agonal entries of the hat matrix. In a future paper, we shall take up matrix-free computations of such quantities. Furthermore, in this section, we provide some preliminary matrix-free computations of the log REML function based on Taylor series approximations. In our limited experience, it appears that these compu- tations work better than those by Aune et al. (2014). More accurate scalable matrix-free computations of the log REML function will be a matter of future investigation. 4.1. DCT and matrix–vector multiplications For any r × c matrix E = (ei,j), its two dimensional discrete cosine transforma- tion gives rise to a r × c matrix whose (s, t)th entry is given by csc′ t r i=1 c j=1 ei,j cos(π(i −1/2)(s −1)/r) cos(π(j −1/2)(t −1)/c) where cs = 1/r if s = 1 and 2/r otherwise, and c′ t = 1/c if t = 1 and 2/c otherwise. On the contrary, the two dimensional inverse discrete cosine transformation on E produces another r × c matrix with (s, t)th entry: r i=1 c j=1 cic′ jei,j cos(π(s −1/2)(i −1)/r) cos(π(t −1/2)(j −1)/c). The above transformation is very closely related to the two dimensional fast Fourier transformation (FFT). Indeed, following the works of Cooley and Tukey (1965) and Rao and Yip (1990), one can factorize the above computations fur- ther, as done in the case of FFT. These factorizations reduce the computa- tional complexities of DCT to O(rc log(rc)). Alternatively, it is also possible to use FFT to compute the DCTs with additional O(rc) pre- and post-processing steps, as shown by Makhoul (1980). Over the decades, highly optimized algo- rithms for FFT have been developed for various machine architectures. We have found that these algorithms in conjunction with O(rc) pre- and post-processing steps are more time eﬃcient to compute DCTs than directly implementing the Cooley-Tuckey and the Rao-Yip algorithms. In this paper, we follow Frigo and Johnson (2005) that show how to eliminate the redundant operations of the FFT algorithms to compute the DCTs. The codes from Frigo and Johnson (2005) are freely available on the web: http://www.fftw.org/. 2869 REML estimation with intrinsic Mat´ern dependence In what follows, the two dimensional DCT is used in various matrix-vector multiplications. We encounter these matrix-vector multiplications in solving the normal equation (6), and in computing the REML score equations and in computing the entries of the Fisher information matrix. Essentially, these matrix-vector multiplications would require us to compute either Wνx or W−νx for certain candidate vector x. The matrix Wν and its pseudo-inverse W−ν are non sparse. However, spectral decomposition allows us to write Wν = MT(D01 + D10)−νM and W−ν = MT(D01 + D10)−νM, where (D01 + D10)−ν is obtained by inverting all the non-zero entries of (D01 + D10)ν. 4.1. DCT and matrix–vector multiplications Thus, mul- tiplying a vector with Wν or W−ν essentially reduces to multiplying a vector with M or MT. At this point, we note that obtaining Mv is same as performing a two-dimensional DCT on the r × c matrix formed using elements of v ﬁlling each column at a time and then converting the resulting matrix back into a vector by stacking the columns. Similarly computing MTv is same as obtaining a two-dimensional inverse DCT. It must be noted that, for very large arrays, one can further improve the speed of the two-dimensional DCTs using distributed computing. The main idea comes from the fact that a two-dimensional DCT consists of many one-dimensional DCTs along the columns and then along the rows. Thus, these one-dimensional DCT computations can be distributed equally or parallely among the cores of the processors. This division of computations is particularly useful on a graphical processing unit (GPU) which typically has thousands of processing cores and this division of computations can provide substantial gain in computational time. For further discussions on parallel implementation of DCT, we refer to the website: http://www.fftw.org/parallel/parallel-fftw.html. 4.2. Solving linear equations with Lanczos algorithm 4.2. Solving linear equations with Lanczos algorithm First, note that the Lanczos algorithm in Dutta and Mondal (2015a) for solving the sparse system of equations Aβ = b with ν = 1 can be extended to solve a dense system structured by general values of ν > 0 in matrix-free way. This matrix-free extension of the algorithm from sparse to the dense case is possible because all that the Lanczos algorithm uses are matrix vectors products of the form Ax which can be computed via the DCT and the inverse DCT described in the previous section. The Lanczos algorithm proceeds as follows. It computes a set of orthonormal vectors v1, v2, . . . called the Lanczos vectors, from the span of b, Ab, A2b, . . . . At the kth iteration, it then obtain an approximate tri- diagonal factorization of AVk ≈VkΔk where Vk has columns v1, . . . , vk and Δk is a k × k positive deﬁnite tridiagonal matrix. In the implemented version of the algorithm none of these matrices Vk and Δk is stored but the solution is iteratively updated on the ﬂy by progressively computing the lower bidiagonal Cholesky factorization of Δk. We refer to Dutta and Mondal (2015a) for all technical details on the algorithm including computing the norm of the resid- uals in almost no additional cost that gives a practical stopping criterion. The algorithm stores only few vectors of length m + rc and a few scalars thus re- quiring only O(rc) memory. With the use of the discrete cosine transformation, 2870 S. Dutta and D. Mondal a matrix-vector multiplication of the form Av incurs a O(rc log(rc)) computa- tional cost. Thus, if the Lanczos algorithm takes K0 iterations to converge, then the total computational cost of solving Aβ = b becomes O(K0rc log(rc)). Second, we do not solve Aβ = b directly, but rather we carefully construct a preconditioning matrix P and solve a well-conditioned system of linear equations PAP Tβ′ = Pb. We then obtain the ﬁnal solution as β = PTβ′. The idea is to reduce the number of iterations K0 as much as possible. 4.2. Solving linear equations with Lanczos algorithm The matrix P has the following requirements: 1) we want P such that the matrix-vector products Px and PTx can be computed in matrix–free way at a computational cost of O(rc log(rc)), and 2) the condition number of PAP T will be ﬁnite so that Lanczos algorithm for solving PAP Tβ′ = Pb converges geometrically fast, i.e., in O(log(rc)) iterations. To this end, we apply a block preconditioner for A where the ﬁrst block is a Cholesky factor of the small dimensional matrix λyTTT and the second block is a preconditioner for the matrix C = λyF TF + λψWν. When ν = 1 (or some other positive integer), the above matrix is sparse, in which case Dutta and Mondal (2015a) proposed using its incomplete Cholesky factor as preconditioner. For a general non-integer value of ν, we construct here a practical sparse approximation C of C by thresholding or tapering in such a way that the small eigenvalues of C stay at the same order of the small eigenvalues of C. We then apply an incomplete Cholesky factor of C to obtain an eﬀective matrix-free preconditioner of C. In order to construct the sparse approximation C of C, we use the following decomposition of the matrix W: 4.3. Stochastic trace approximation Equations (8) and (9) require computations of the trace terms Tr HQ−1Qi and Tr(I −H)Q−1 Qi(I −H)Q−1Qj, which are expensive as computing individual diagonal elements requires at least O(rc(log(rc))2) calculations even with the use of discrete cosine transformation and the Lanczos algorithm. In other words, overall trace computations using this method require at least O((rc)2(log(rc))2) operations. Here, instead we apply Hutchinson’s method that stochastically ap- proximates the trace of a symmetric matrix using a Monte Carlo average of its quadratic forms in random vectors with zero mean and identity covariance ma- trix. Thus, for a symmetric matrix E, the Hutchinson’s method approximates the trace of E by Tr E = 1 K K t=1 u T t Eut where ut’s are either i.i.d Rademacher or Gaussian random variables and K is an integer that is much smaller than the dimension of E. Throughout, we only consider i.i.d. Rademacher variables because they minimize the variance of the above estimate among all i.i.d random variables of size K from a distribution with zero mean vector and identity covariance matrix. Although further reduc- tion in variances of the trace approximations are possible if we allow dependent random variables in trace estimation, we do not pursue such stochastic approx- imations in this paper. It then follows that the REML score equations reduce to the following unbiased estimation equations where ut’s are either i.i.d Rademacher or Gaussian random variables and K is an integer that is much smaller than the dimension of E. Throughout, we only consider i.i.d. Rademacher variables because they minimize the variance of the above estimate among all i.i.d random variables of size K from a distribution with zero mean vector and identity covariance matrix. Although further reduc- tion in variances of the trace approximations are possible if we allow dependent random variables in trace estimation, we do not pursue such stochastic approx- imations in this paper. It then follows that the REML score equations reduce to the following unbiased estimation equations gi(θ) := 1 2K K t=1 u T t Q−1Qi(I −H)ut −1 2(z −Xβ) TQi(z −Xβ) = 0. (11) (11) And the (i, j)th entry of the Hessian matrix becomes 1 2K K t=1 u T t (I −H)Q−1Qi(I −H)Q−1Qj ut. W = Ic ⊗Wr + Wc ⊗Ir, W = Ic ⊗Wr + Wc ⊗Ir, where 4Wk = MT kDkMk and k is either r or c. In the above the matrix Wk is a tridiagonal matrix and Wν k is a non-sparse matrix, but its entries far away from the diagonal are very small in magnitude compared to the ones on or near the diagonal. Let Wν k be a sparse matrix approximation of Wν k obtained by suitable thresholding or tapering. Then a candidate for the matrix C is given by C = λyF TF + λψ Ic ⊗ Wν r + Wν c ⊗Ir . Thus the block diagonal preconditioner for A becomes P = diag{L−1 1 , L−1 2 } where L1 is the lower triangular Cholesky factor of λyTTT and L2 is the lower triangular incomplete Cholesky factor of C. This eﬀectively makes the condi- tion number of the matrix PAP T bounded. It can seen that, if r and c are of the same order, the overall cost of constructing this matrix P is O(rc log(rc)). Furthermore, since the inverse of P sparse, the matrix vector multiplications Pv and PTv incurs a computational cost of at most O(rc log(rc)). 2871 REML estimation with intrinsic Mat´ern dependence The above preconditioner eﬀectively brings down the computation cost of calculating the BLUEs of the contrasts of τ and the BLUPs of the contrasts of ψ to O(rc(log(rc))2) operations. Alternatively, one can obtain a preconditioner of A using Chebyshev poly- nomial approximations, see e.g., (Saad, 1985), but we did not pursue its use in this work. 4.4. Trust region method Finding a solution to the REML score equation that minimizes the negative log REML function is a non-convex optimization problem, and in such settings, the traditional Netwon-Rhapson algorithm with line search methods are often perceived to be susceptible in practice. Thus, following Powell (1970, 1984) and Nocedal and Wright (1999), we adopt here a trust-region method that can be considered as a global line search method and that allows us to obtain solutions to the REML score equations in a numerically stable way. At any iteration, the trust region method approximates the objective function by a suitable quadratic function, identiﬁes a region within which it perceives the quadratic approxima- tion to be good and then minimizes the quadratic approximation over the iden- tiﬁed region around the current value of the variable to obtain the next iterate. Understandably, this method avoids directly computing the negative log REML function ℓR(θ). Instead, it sets the objective function to be (1/2)∥∇g(θ)∥2, where ∇g(θ) denotes gradient of the function g(θ) = (g1(θ), g2(θ), g3(θ)), namely, the approximate REML score equations in (11) . Thus, the objective function is minimized at x = c if and only if ∇g(c) = 0 provided that the Hes- sian ∇2g(θ) is positive deﬁnite. Furthermore, this method uses the following the quadratic function to approximate (1/2)∥∇g(θ)∥2 around a point θk. pk(δ) = 1 2∥∇g(θk)∥2 + δ T[∇2g(θk)] T∇g(θk) + 1 2δ T[∇2∇g(θk)]2δ. It must be emphasized that the above quadratic function can computed in a matrix free way, as we can do so for computing the gradient and the Hessian using the stochastic trace approximations. Next, the trust region method picks the step size δk by minimizing pk(δ) in a suitable region. The choice of the suitable region is critical here. If the region is too large then pk(δ) could be a poor approximation to (1/2)∥∇g(θk)∥2 and minimizing pk(δ) could become unsuitable. On the other hand, if the region is too small then the algorithm will pick a tiny step size resulting in little eﬀective gain. Thus, the trust region method adaptively chooses the radius of the region based on the performance of the previous iteration. If there was a signiﬁcant gain in the previous iteration then the algorithm becomes optimistic and expands the radius of the trust region; a loss on the other hand results in shrinking the radius. 4.4. Trust region method Overall, the key steps of the algorithm are then given by: 4.3. Stochastic trace approximation Overall the computations of the trace terms, REML score equations and the information matrix require O(Krc(log(rc))2) ﬂops and O(rc) storage and allow us to pursue numeric optimization of the log REML function via the Newton- Raphson line search method or the trust region methods. S. Dutta and D. Mondal 2872 • End for. The algorithm stops when either there is no signiﬁcant change in the θk’s and the value of the objective function is suﬃciently close to zero. Sometimes the algorithm does not converge in the sense that the radius of the trust region becomes very small and yet the value of the objective function remains far from zero. In such a case the algorithm must be restarted with a diﬀerent initial value. Furthermore, this algorithm computes the Jacobian matrix of the score equa- tion, which is nothing but the negative of the observed information matrix. Thus, as a by-product of the trust region algorithm, we can obtain standard errors of the θ as the square roots of the diagonal entries of the inverse of the observed information matrix (of the stochastic score equations). ( ) Powell (1984) proves convergence to stationary points for φ0 = 0, i.e. when a step is taken whenever the value of the objective function goes down under some weak assumptions. Mor´e and Sorensen (1983) and Nocedal and Wright (1999, ch. 4) provide convergence results for φ0 ∈(0, 1/4) under stronger as- sumptions that the objective function is Lipschitz, continuously diﬀerentiable and the corresponding Hessian matrix is bounded. Trust-Region algorithm: • Set a maximum possible trust radius ρ > 0, an initial radius 0 < ρ0 < ρ and 0 < φ0 < 1/4. • Set a maximum possible trust radius ρ > 0, an initial radius 0 < ρ0 < ρ and 0 < φ0 < 1/4. It t F k ≥0 • Iterate: For k ≥0 1. Compute steepest descend direction: δ1k = ∇g(θk)T[∇2g(θk)]2∇g(θk) ∇g(θk)T[∇2g(θk)]4∇g(θk)∇2g(θk)∇g(θk) 2873 REML estimation with intrinsic Mat´ern dependence 2873 2. Compute the Newton-Raphson direction: 2. Compute the Newton-Raphson direction: 2. Compute the Newton-Raphson direction: δ2k = −[∇2g(θk)]−1∇g(θk) 3. Compute the trust region step size by: 3. Compute the trust region step size by: δk = δ1k + λ(δ2k −δ1k) where λ is the largest number in [0, 1] such that ∥δk∥≤ρk and this can be computed analytically. 4. Compute the eﬀectiveness of the step: 4. Compute the eﬀectiveness of the step: φk = ∥∇g(θk)∥2 −∥∇g(θk + δk)∥2 2(pk(0) −pk(δk)) 5. Decide whether the trust region radius shrinks, expands or stays the same: If φk < 1/4, set ρk+1 = ∥δk∥/4 If φk < 1/4, set ρk+1 = ∥δk∥/4 If φk < 1/4, set ρk+1 = ∥δk∥/4 Else if φk > 3/4 and ∥δk∥= ρk, set ρk+1 = min(2ρk, ρ0). Else set ρk+1 = ρk. Else if φk > 3/4 and ∥δk∥= ρk, set ρk+1 = min(2ρk, ρ0). Else set ρk+1 = ρk. 6. Decide whether to update the solution: 6. Decide whether to update the solution: If φk > φ0, set θk+1 = θk + δk. Else keep θk+1 = θk. • End for. 4.5. Approximate computations of log REML likelihood function Some preliminary computations of REML log-likelihood function can be ob- tained in a matrix-free way using Taylor series expansions. We indicate these 2874 S. Dutta and D. Mondal computations in the no covariate case (i.e., with T = 0). First, in order to compute the log REML function, we need to ﬁnd the log determinant of C = λyFTF+R, where R = λψWν as given in (2). Using C = (λyI+R)−λy(I−FTF) the log determinant of C can then be expressed as: log det C = rc i=1 log{λy +λψ(d01,i +d10,i)ν}+Tr log{I−(I+λ−1 y R)−1(I−F TF)}. Next, we use the Taylor series expansion and write the second term as Next, we use the Taylor series expansion and write the second term as Tr log{I−(I+λ−1 y R)−1(I−F TF)} = J j=1 (−1)j j Tr[{(I+λ−1 y R)−1(I−F TF)}j]+oJ, where oJ is a negligible term. The above is possible because the matrix (I + λ−1 y R)−1(I −FTF) has spectral radius less than 1. In particular, note that the spectral radii of each of the matrices I −FTF and (I + λ−1 y R)−1 are exactly equal to 1. However, 1 is not an eigenvalue of their product because the matrix λyFTF + R is non-singular. This also implies that the above Taylor series con- verges geometrically, a value of J can be set carefully to make the approximation suﬃciently accurate. Next we approximate the trace terms using Huchinson’s estimators. Let ˜uk’s be i.i.d Rademacher vectors. We then approximate the trace as Tr[{(I + λ−1 y R)−1(I −F TF)}j] = 1 K′ K′ k=1 ˜u T k{(I + λ−1 y R)−1(I −F TF)}j ˜uk Furthermore, using the two-dimensional DCT, we can compute all of the above quadratic forms in a matrix-free way. This allows us to approximately evaluate the REML log-likelihood function in a matrix-free way. In practice, negative log REML likelihood values can be substantially large, and can cause some numerical instability. However, in practice, we are mostly required to evaluate the diﬀerence of the log REML functions at two diﬀerent parameter values, in which case we can write the diﬀerence of the log REML function in terms of term by term diﬀerences of quadratic forms. Computing these series of term by term diﬀerences is numerically more stable and they converge faster than the individual series of quadratic forms. 5.1. Local maxima in REML estimations The non-linear gamma score equations derived in Section 3.3 characterize the non-convex nature of the REML computations. The trust region method consid- ered in Section 4.4 guarantees convergence to a local maximum, but its conver- gence to the global maximum requires a more careful investigation, as such con- vergences are often proved under strong regularity assumptions. Here we present REML estimation with intrinsic Mat´ern dependence 2875 through simulations the performance of the REML estimators, computed using matrix-free trust region method and summarized for two diﬀerent values of the dependence parameter. Results for further values of the dependence parameter and for varying proportions of missing observations are reported in the sup- plement to the paper. However, the overall conclusions of these more extensive simulation studies do not change from what we cover here. Thus the simulations summarized below are largely representative of what we expect to see for the entire range of parameter space and provide some critical understanding of the strength and weakness of the REML computations. The speciﬁc details of the simulation runs are as follows. We generate real- izations from the spatial mixed linear model (1) on 512 × 512 arrays by setting T = 0, τ = 0, λψ = 8, λy = 4 and using ν = 1.25 and 1.50. This is done in several steps. First, for a ﬁxed value of the dependence parameter ν, a random eﬀect ψ is generated on a 512 × 512 array from a Gaussian distribution with mean 0 and a precision matrix 8Wν with the sum constraint ψ T1 = 0. Then, each ψu,v value, independent of others, is removed randomly with probability p = 0.8, and a Gaussian white noise with precision λy = 4 is added to the remaining ones. This produces a random incidence matrix F and a vector of realization y with an expected sample size of n = 52428. The procedure is then repeated with diﬀerent values of ν. Tables 1 and 2 display results of REML computations done on each of the simulated dataset using our matrix-free trust region method with 9 diﬀerent starting values of the dependence parameter, namely, ν(0) = 0.8, 0.9, 1.0, . . . , 1.6. Throughout we consider K = 50 Rademacher vectors to stochastically approx- imate the traces in the score equation (11). 5.1. Local maxima in REML estimations We then pick the values of λ(0) y and λ(0) ψ by ﬁnding the solutions to the REML score equation with ﬁxed ν = ν(0). Using this triplet (ν(0), λ(0) y , λ(0) ψ ) as the starting point we next run the trust region algorithm and obtain ﬁnal solutions ν, λy and λψ to the REML score equation. In some instances, the estimate of λy grew increasingly large with successive iterations and we mark it by ‘∞’ in the tables. Thus, in a typical trust-region run ‘∞’ signiﬁes a value that is larger than 10, 000. While the ﬁrst three columns of each table record the initial values of ν(0), λ(0) y and λ(0) ψ , the last three columns of each table comprise of the ﬁnal estimated values ν, λy and λψ. Tables 1 and 2 display results of REML computations done on each of the simulated dataset using our matrix-free trust region method with 9 diﬀerent starting values of the dependence parameter, namely, ν(0) = 0.8, 0.9, 1.0, . . . , 1.6. Throughout we consider K = 50 Rademacher vectors to stochastically approx- imate the traces in the score equation (11). We then pick the values of λ(0) y and λ(0) ψ by ﬁnding the solutions to the REML score equation with ﬁxed ν = ν(0). Using this triplet (ν(0), λ(0) y , λ(0) ψ ) as the starting point we next run the trust region algorithm and obtain ﬁnal solutions ν, λy and λψ to the REML score equation. In some instances, the estimate of λy grew increasingly large with successive iterations and we mark it by ‘∞’ in the tables. Thus, in a typical trust-region run ‘∞’ signiﬁes a value that is larger than 10, 000. While the ﬁrst three columns of each table record the initial values of ν(0), λ(0) y and λ(0) ψ , the last three columns of each table comprise of the ﬁnal estimated values ν, λy and λψ. As we glean through the numbers in these Tables, several conclusions can be drawn: (a) When the initial value ν(0) is close to the true ν value, we ﬁnd that the estimates of λ(0) y and λ(0) ψ are very reasonable, and the ﬁnal estimates ν, λy and λψ are indeed very close to the true parameter values. Table 1 f Table 1 Starting values and ﬁnal REML estimates from trust region methods. True values of ν are as follows. Top: ν = 1.25; bottom: ν = 1.5. The true values of other parameters are kept at λψ = 8 and λy = 4. ν(0) λ(0) y λ(0) ψ ν λy λψ 0.8 ∞ 2.419 0.988 ∞ 2.908 0.9 ∞ 2.688 0.988 ∞ 2.908 1.0 12.038 3.549 1.253 4.032 7.941 1.1 6.181 4.876 1.253 4.032 7.941 1.2 4.511 6.696 1.253 4.032 7.941 1.3 3.729 9.209 1.253 4.032 7.941 1.4 3.281 12.708 1.253 4.032 7.941 1.5 2.993 17.611 1.253 4.032 7.941 1.6 2.794 24.528 1.253 4.032 7.941 ν(0) λ(0) y λ(0) ψ ν λy λψ 0.8 ∞ 1.542 1.229 ∞ 2.988 0.9 ∞ 1.932 1.229 ∞ 2.988 1.0 ∞ 2.287 1.229 ∞ 2.988 1.1 ∞ 2.611 1.505 4.004 8.030 1.2 17.534 3.259 1.505 4.004 8.030 1.3 7.325 4.385 1.505 4.004 8.030 1.4 5.043 5.887 1.505 4.004 8.030 1.5 4.043 7.902 1.505 4.004 8.030 1.6 3.485 10.620 1.505 4.004 8.030 run the trust region iterations, the algorithm makes subsequence adjustments to get close to the true values. (c) When the initial value of ν(0) is really far from the true ν value, the non-convex nature of the REML likelihood function takes over, and in some instances, we see that trust region algorithm converges to ﬁnal values that are far from the true parameter values. Furthermore, it is just that only the global maximum is in the interior of the parameter space and is very close to the true parameter values. However, the second local maximum, found from poor starting points, falls on the boundary of the parameter space with λy = ∞. 5.1. Local maxima in REML estimations (b) As we locally move the value of ν(0) away from the true ν value, the estimates of λ(0) y and λ(0) ψ get further away from the true parameter values, but the procedure still converges to the same ﬁnal estimates ν, λy and λψ. There is an interesting pattern here, namely, if we increase the value of ν(0) locally from the true ν value, we force a slightly smoother spatial process, and as a compensation we end up with a larger value of λ(0) ψ but a smaller value of λ(0) y . However, as we As we glean through the numbers in these Tables, several conclusions can be drawn: (a) When the initial value ν(0) is close to the true ν value, we ﬁnd that the estimates of λ(0) y and λ(0) ψ are very reasonable, and the ﬁnal estimates ν, λy and λψ are indeed very close to the true parameter values. (b) As we locally move the value of ν(0) away from the true ν value, the estimates of λ(0) y and λ(0) ψ get further away from the true parameter values, but the procedure still converges to the same ﬁnal estimates ν, λy and λψ. There is an interesting pattern here, namely, if we increase the value of ν(0) locally from the true ν value, we force a slightly smoother spatial process, and as a compensation we end up with a larger value of λ(0) ψ but a smaller value of λ(0) y . However, as we 2876 S. Dutta and D. Mondal Table 1 f 5.2. Numeric consistency towards geostatistical models The purpose of our next computer experiment is to demonstrate that using lattice-based approximations we can obtain inference for the continuum Mat´ern dependence plus the white noise structures, even when observations are made at irregular sampling locations. The basic idea is to embed the study region and irregular sampling locations into ﬁner and ﬁner lattice arrays by diminishing the lattice spacing and then check for numeric consistency (in terms of con- vergence in distribution of the estimators of dependence parameters) as we ﬁt the approximations of the continuum Mat´ern dependence with nugget eﬀect on ﬁner and ﬁner lattices. To this end, we consider the unit square (0, 1) × (0, 1) as REML estimation with intrinsic Mat´ern dependence 2877 Table 2 Estimates of the precision parameters for diﬀerent array sizes. The standard errors are shown in parenthesis. r ν λy λψ 100 1.301 (0.044) 1.391 (0.068) 3.395 (0.374) 200 1.224 (0.030) 1.040 (0.039) 3.540 (0.355) 300 1.233 (0.028) 0.987 (0.035) 4.322 (0.464) 400 1.226 (0.026) 0.973 (0.034) 4.946 (0.552) 500 1.242 (0.025) 0.956 (0.033) 5.848 (0.691) Table 2 Estimates of the precision parameters for diﬀerent array sizes. The standard errors are shown in parenthesis. the study region and pick 20, 000 points uniformly within this study region as sampling locations. At these randomly generated irregular sampling locations, we then draw a realization from the intrinsic Mat´ern process with ν = 1.25 and σ−2 = 2/(4π2). We also add a random noise to each observations by generating i.i.d. standard normal random variables. Next, we embed the unit square and the irregular sampling points in an r × r regular square array and estimate pa- rameters using methods detailed in Sections 2, 3 and 4. Table 2 provides these estimates along with their standard errors for various values of r. The results vindicate the theoretical ﬁndings in Section 2. First, the esti- mates of ν hover around the true value 1.25 as the lattice spacing decreases. Second, the sample size is ﬁxed, but with diminishing lattice spacing, stan- dard errors for ν are essentially constant, which endorse that the procedure is converging in distribution and approximating geostatistical inference. Third, as lattice spacing decreases, λy gets closer to the true value 1. 5.2. Numeric consistency towards geostatistical models The slight diﬀer- ence between the lattice-based nugget eﬀect λy and the geostatistical nugget 1 occurs because of (essentially) constant diﬀerence between the variograms of the intrinsic Mat´ern process and the fractionally diﬀerenced process. Fourth, the estimates λψ increase with the diminishing lattice spacing. But this increase and its exact nature can be explained by the approximation theory detailed in Section 2. Speciﬁcally, it follows from the scaling limit equations (3) that at lattice spacings 1/m and 1/m′ the ratio (σ2 m/σ2 m′)(m/m′)2ν−2 should be close to 1. Thus, for example, between arrays of size 400 × 400 and 500 × 500, this ratio is seen to be (4.946/5.848) × (500/400)0.5 = 0.946. Overall, the estimates and the standard errors are indicative of numeric convergences of estimators in distribution, and, it can be seen that after appropriate rescaling of parameters, we can obtain geostatistical inference from the lattice-based approximations. 5.3. Eﬃciency of matrix-free REML estimators In classical statistics, eﬃciency of an estimator is often judged based on the vari- ance of the estimator and both the Fisher information matrix and the Cram˙er– Rao lower bound play a central role in assessing the eﬃciency of an estimator. In particular, for linear mixed models, it is typical that REML estimators are asymptotically eﬃcient and achieve the Cram˙er–Rao lower bound. The ques- tion that we ask here is how eﬃcient are our matrix-free estimators. We address 2878 S. Dutta and D. Mondal Table 3 Monte Carlo standard deviation and average of the standard errors of matrix-free REML estimates when ν = 1.25. Estimate Mean Monte Carlo SD Average of SE ν 1.251 0.022 0.021 λy 3.997 0.157 0.151 λψ 8.055 0.539 0.517 Table 4 Monte Carlo standard deviation and average of the standard errors of matrix-free REML estimates when ν = 1.75. Estimate Mean Monte Carlo SD Average of SE ν 1.751 0.018 0.018 λy 3.998 0.119 0.119 λψ 8.032 0.380 0.372 Table 3 Monte Carlo standard deviation and average of the standard errors of matrix-free REML estimates when ν = 1.25. Estimate Mean Monte Carlo SD Average of SE ν 1.251 0.022 0.021 λy 3.997 0.157 0.151 λψ 8.055 0.539 0.517 Table 4 Monte Carlo standard deviation and average of the standard errors of matrix-free REML estimates when ν = 1.75. Estimate Mean Monte Carlo SD Average of SE ν 1.751 0.018 0.018 λy 3.998 0.119 0.119 λψ 8.032 0.380 0.372 Table 4 f h Table 4 Monte Carlo standard deviation and average of the standard errors of matrix-free REML estimates when ν = 1.75. Estimate Mean Monte Carlo SD Average of SE ν 1.751 0.018 0.018 Table 4 Monte Carlo standard deviation and average of the standard errors of matrix-free REML estimates when ν = 1.75. Estimate Mean Monte Carlo SD Average of SE ν 1.751 0.018 0.018 λy 3.998 0.119 0.119 λψ 8.032 0.380 0.372 this question using another set of computer experiments where we generate 1000 Monte Carlo samples from the spatial mixed linear model (1) on 256×256 arrays by setting T = 0, τ = 0, λψ = 8, λy = 4 and using ν = 1.25. As in Section 5.1, this is done in several steps. 5.3. Eﬃciency of matrix-free REML estimators First, for each Monte Carlo sample, a random eﬀect ψ is generated on a 256 × 256 array from a Gaussian distribution with mean 0 and a precision matrix 8Wν with the sum constraint ψ T1 = 0. Then, each ψu,v value, independent of others, is removed randomly with probability p = 0.6, and a Gaussian white noise with precision λy = 4 is added to the remaining ones. For each Monte Carlo sample, this produces a random incidence matrix F and a vector of realization y with an expected sample size of n = 39322. For each Monte Carlo sample, we then apply our matrix free computations and obtain the corresponding REML estimates ν, λy and λψ and their standard errors by calculating the inverse of observed Fisher information matrix in a matrix-free way. Table 3 provides the Monte Carlo summaries of these estimates and their standard errors. It can be seen that the estimates are very accurate, and the Monte Carlo standard deviations of the estimators match very well with the cor- responding Monte Carlo averages of the standard errors of estimates. (There is a slight discrepancy which occurs because only 50 Rademacher vectors are used in approximating the trace terms and this introduces a very slight additional variation in the estimates). this question using another set of computer experiments where we generate 1000 Monte Carlo samples from the spatial mixed linear model (1) on 256×256 arrays by setting T = 0, τ = 0, λψ = 8, λy = 4 and using ν = 1.25. As in Section 5.1, this is done in several steps. First, for each Monte Carlo sample, a random eﬀect ψ is generated on a 256 × 256 array from a Gaussian distribution with mean 0 and a precision matrix 8Wν with the sum constraint ψ T1 = 0. Then, each ψu,v value, independent of others, is removed randomly with probability p = 0.6, and a Gaussian white noise with precision λy = 4 is added to the remaining ones. For each Monte Carlo sample, this produces a random incidence matrix F and a vector of realization y with an expected sample size of n = 39322. Table 5 Run times in seconds of our REML method (with 30 Rademacher vectors) and INLA. The results of REML and INLA* are obtained by running INLA (version 0.0-1420281647) on a laptop with Intel R ⃝Core i7-4700MQ processor and 8GB RAM. The results for INLA** are obtained by running the same version of INLA on a workstation with two I t l R ⃝X R ⃝E5430 d 32GB RAM Th lt d d t th t d obtained by running the same version of INLA on a workstation with two Intel R ⃝Xeon R ⃝E5430 cpus and 32GB RAM. The results are rounded to the next second. Array size (r × r) REML INLA* INLA** 200 × 200 26 23 48 300 × 300 54 75 148 400 × 400 92 261 471 500 × 500 143 Out-of-memory 858 600 × 600 206 Out-of-memory 1962 700 × 700 277 Out-of-memory 3735 5.3. Eﬃciency of matrix-free REML estimators For each Monte Carlo sample, we then apply our matrix free computations and obtain the corresponding REML estimates ν, λy and λψ and their standard errors by calculating the inverse of observed Fisher information matrix in a matrix-free way. Table 3 provides the Monte Carlo summaries of these estimates and their standard errors. It can be seen that the estimates are very accurate, and the Monte Carlo standard deviations of the estimators match very well with the cor- responding Monte Carlo averages of the standard errors of estimates. (There is a slight discrepancy which occurs because only 50 Rademacher vectors are used in approximating the trace terms and this introduces a very slight additional variation in the estimates). We next repeat the above simulation experiment with ν = 1.75 and report the summaries in Table 4. Again we see that Monte Carlo standard deviations of the estimators match very well with the corresponding Monte Carlo aver- ages of the standard errors of estimates. These conﬁrm that there is little loss of statistical eﬃciency when computations are implemented in a matrix-free way. The above summaries of computer simulations along with those from Sec- tion 5.2 now suggest that we can use lattice-based fractional random ﬁelds as proxies for continuum Mat´ern random ﬁelds and when we do so we can still achieve geostatistical inference with little loss of statistical eﬃciency. REML estimation with intrinsic Mat´ern dependence REML estimation with intrinsic Mat´ern dependence 2879 6. Arsenic mapping in Bangladesh Arsenic contamination of the groundwater in Bangladesh (and in West Bengal, India) is a serious problem with approximately one in ﬁve of the wells used for drinking water currently contaminated with arsenic above the government’s drinking water standard (50 mg/L). Following an agreement with the Govern- ment of Bangladesh, the British Geological Survey (BGS) and the Department of Public Health Engineering (DPHE) conducted an extensive investigation of the arsenic problem during the period 1998 to 2001 and their website http://www. bgs.ac.uk/research/groundwater/health/arsenic/Bangladesh/data.html provides a rich treasury of groundwater arsenic concentration data. See also BGS and DPHE (2001) for a full report of the survey. The primary focus of the survey was to assess the scale of the groundwater arsenic contamination so that appropriate arsenic mitigation program could be developed. Consequently, a key component of the study was to construct an extensive geographic map of arsenic contamination. Over the years, numerous important works on the cause and damage of arsenic contamination and on the mechanism of arsenic mobi- lization came out of this study. These include Nickson et al. (2000), Chowdhury et al. (2000), McArthur et al. (2001), Smith et al. (2000), Harvey et al. (2002) and Neumann et al. (2009). Although till date the exact cause of arsenic con- tamination remained sketchy, scientists speculate that it is the supply of excess oxygen during pumping the tube wells that accelerates hydrolysis of precipitated arsenate and that releases soluble arsenous acid into groundwater. Here, using methods developed in Sections 3, 4 and 5, we explore mapping groundwater arsenic concentration data collected by BGS and DPHE during 1998 and 1999. These data are from 3534 tube wells located at irregularly dis- tributed sites at 61 out of 64 districts in Bangladesh and are measured in parts per billion (ppb which is same as micrograms per liter). The three districts in the south-eastern Bangladesh that are left out of the study are known to be arsenic safe. Overall the arsenic concentration measurements in the study re- gion have a large range with values varying from less than 0.5 ppb to 1660 ppb, and a fraction of measurements are truncated on the right at the instrumental detection limit. The left panel of Figure 2 displays these data after grouping into diﬀerent categories. 5.4. Computational times and practical gains We now demonstrate a practical gain of our matrix-free method by comparing our actual run times with those of Lindgren et al. (2011) based on iterated nested Laplacian approximations (INLA). It must be noted that the current version of INLA (numbered 0.0-1420281647) can only ﬁt a Mat´ern dependence model for the ﬁrst few integer values of the dependence parameter ν. Furthermore, INLA is a Bayesian method where the estimates of parameters can be very sensitive to the choice of prior. Thus, in order to keep the comparison fair, we only consider the run times for the case ν = 1. In a fashion identical to Section 5.1, we simulate observations on r × r arrays with ν = 1, λy = 4 and λψ = 8, and, discard 40% as missing. We then estimate these parameters using our matrix-free method and INLA. Table 5 provides the average run times in seconds of these methods. Specif- ically, the second column indicates run times for our matrix-free method, and the third and the fourth columns show run times for INLA. These are obtained respectively by running INLA on (1) a laptop with a Intel R ⃝i7-4700MQ proces- sor and 8GB of RAM on Linux operating system and (2) a workstation with two Intel R ⃝Xeon R ⃝E5430 processors and 32GB RAM on a Linux operating system. Furthermore, our matrix-free REML computations are done with some prelim- inary codes running on MATLAB 8.4 using only a single core of the laptop, while INLA is granted to use four cores on either machine. We see that the computation time of our matrix-free REML algorithm scales very well with increasing dimension and does so at an approximate rate O(n log n) as suggested by the theory in Section 4. Furthermore because RAM requirement of our matrix-free algorithms scales only linearly with the array size, we ﬁnd that these computations are possible on an ordinary laptop for moderately large array dimension. In contrast, we see that the computation times of INLA algo- rithm scales poorly with the array size. And to make things worse, INLA runs out of memory on the laptop even for a moderate array size. Only when it is run on the workstation, it can handle moderately large arrays. Thus, we conclude that we save both time and storage space. 2880 S. Dutta and D. 5.4. Computational times and practical gains Mondal Although we used only a single core to carry out the matrix-free REML optimization, our method can be made much faster with parallel implementa- tion. Furthermore, we can deal with non-integer values of ν, which the sparse computations of Lindgren et al. (2011) can not. 6. Arsenic mapping in Bangladesh Notice that, 0.5ppb is the machine detection limit, 10ppb is the WHO permissible limit, 50ppb is the Bangladesh Governtment permissible limit and 150ppb is believed to be the threshhold above which cancer mortality appears (Lamm et al., 2006). We see arsenic contamination is endemic in the middle southern part of Bangladesh. The south-west and the parts of northern Bangladesh region also have a patchy high arsenic concentration values. Over- all, about 42% wells sampled had arsenic concentration more than the World REML estimation with intrinsic Mat´ern dependence 2881 Fig 2. Plot of the raw data. Group legends are described at the top right corner. The barplot gives the number of observations in each group and the box-plot of the log-arsenic concentra- tion is shown at the bottom right panel. Fig 2. Plot of the raw data. Group legends are described at the top right corner. The barplot gives the number of observations in each group and the box-plot of the log-arsenic concentra- tion is shown at the bottom right panel. Health Organization’s permissible limit of 10 ppb and about a quarter of the wells sampled had arsenic more than the Bangladesh Government permissible limit of 50 ppb. In order to map groundwater log arsenic concentration, we then embed the study region with latitudes between 20 and 27 degrees north (approximately 778 km in length) and longitudes between 88 to 93 degrees east (approximately between 495 and 522 km in width) into a 500 × 300 array. Thus each array cell is approximately 2.64 square kilometers1 in area and we tally arsenic concen- trations in n = 3224 of these cells, (which is only about 2.15% of total array cells). Moreover, among these n = 3224 array cells, 2941 contain only one sam- pling location each, 258 contain two sampling locations each, 23 contain three sampling locations each and only 2 contain four sampling locations. Thus, due to clustering nature of the sampling locations, some array cells contained two or more sampling locations even after placing a such large array. To rectify this problem partially, we then take the average of logarithm of the arsenic concentrations over each cell and obtain our data vector y. 1Computed from http://www.nhc.noaa.gov/gccalc.shtml. 6. Arsenic mapping in Bangladesh However, since a small fraction of cells contained multiple sampling locations, in our preliminary analysis we decided against adjusting the residual or nugget vector ϵ using the number of sampling locations over which these averages are calculated. We then ﬁt the mixed model (1) with T = 0 to obtain BLUP of ψ −¯ψ1 where 1 is the rc−vector of all ones, and to obtain REML estimates of λy, λψ and ν. First, we ﬁt the data with ν = 1. This is mainly because ν = 1 corresponds to ﬁtting a Gaussian autoregression, which is often the standard practice in spatial S. Dutta and D. Mondal 2882 Fig 3. The BLUPs of ψ. Left: for ν = 1, Center: for ν = 0.858 (no nugget), Right: for ν = 1.240. Fig 3. The BLUPs of ψ. Left: for ν = 1, Center: for ν = 0.858 (no nugget), Right: for ν = 1.240. Table 6 Estimates of the precision parameters for the ground water log arsenic concentration data. Estimates of the precision parameters for the ground water log arsenic concentration d Initial value Final Solution ν(0) λ(0) y λ(0) ψ ν λy λψ 0.7 892.470 0.533 0.858 ∞ 0.660 0.9 1.449 0.983 1.240 0.596 4.607 1.1 0.715 2.399 1.240 0.596 4.607 1.3 0.567 6.143 1.240 0.596 4.607 1.5 0.508 16.688 1.240 0.596 4.607 1.7 0.477 48.634 1.240 0.596 4.607 Initial value Final Solution ν(0) λ(0) y λ(0) ψ ν λy λψ 0.7 892.470 0.533 0.858 ∞ 0.660 0.9 1.449 0.983 1.240 0.596 4.607 1.1 0.715 2.399 1.240 0.596 4.607 1.3 0.567 6.143 1.240 0.596 4.607 1.5 0.508 16.688 1.240 0.596 4.607 1.7 0.477 48.634 1.240 0.596 4.607 statistics. To this end we ﬁx a sample of 20 Rademacher variables and use the Lanczos algorithm and trust-region algorithm described in Section 3 and obtain global REML estimates λy = 0.906 (with a s.e. 0.074) and λψ = 1.527 (with a s.e. 0.106). The left panel in Figure 3 provide the image plots of the BLUP of ψ −¯ψ1 and the estimates of the residuals. These plots suggests that an intrinsic autoregression model captures the local variation of arsenic log-concentration fairly well. Overall the BLUP of ψ−¯ψ1 highlights the high arsenic concentration areas in northern Bangladesh, central and southwest Bangladesh. statistics. Table 7 Final estimates (with nugget) of the precision parameters for the arsenic data on diﬀerent resolutions. Here r denotes the number of grid cells along the latitude and c denotes the number of grid cells along the longitude. Final estimates (with nugget) of the precision parameters for the arsenic data on diﬀerent resolutions. Here r denotes the number of grid cells along the latitude and c denotes the number of grid cells along the longitude. r c ν λy λψ 350 210 1.315 (0.065) 0.595 (0.039) 4.477 (1.266) 400 240 1.143 (0.047) 0.737 (0.073) 2.392 (0.515) 450 270 1.379 (0.065) 0.547 (0.028) 7.688 (2.411) 500 300 1.240 (0.054) 0.596 (0.040) 4.607 (1.260) 550 330 1.322 (0.064) 0.606 (0.043) 5.310 (1.647) 600 360 1.329 (0.071) 0.588 (0.039) 6.661 (2.371) and right panels in Figure 3 provide corresponding image plots of the BLUP of ψ −¯ψ1 at these local maxima. Next we compute the diﬀerence of log REML function at these two local maxima and found this diﬀerence to be −49.82. This conﬁrms that second set of the estimates are the global REML estimates and they corresponds to a model that includes nugget eﬀects or residual values to capture the small scale variations. Next we reanalyze the data at diﬀerent lattice resolutions and check for nu- meric consistency of the results. In particular, we embed the study region and irregular sampling locations into ﬁner and ﬁner lattice arrays by diminishing the lattice spacing but by keeping the aspect ratio (r/c) of the arrays ﬁxed. We then ﬁt the lattice approximations of continuum Mat´ern dependence with nugget eﬀect to the observed arsenic contamination data. Table 7 provides the estimates of the precision parameters, and their standard errors for various lat- tice sizes. We ﬁnd that, at diﬀerent lattice resolutions, there are little changes in the estimates of ν and λy after accounting for their uncertainties. The estimates of λψ, however, increases with diminishing lattice spacings, but this increase can further be explained by the approximation theory laid out in Section 2. Specif- ically, the scaling limit equation (3) suggests that at lattice spacings 1/m and 1/m′ the ratio (σ2 m/σ2 m′)×(m/m′)2ν−2 should be close to 1. Thus, for example, by inserting ν = 1.322, in arrays of size 500 × 300 and 550 × 330, we ﬁnd that this ratio is equal to 0.923. 6. Arsenic mapping in Bangladesh To this end we ﬁx a sample of 20 Rademacher variables and use the Lanczos algorithm and trust-region algorithm described in Section 3 and obtain global REML estimates λy = 0.906 (with a s.e. 0.074) and λψ = 1.527 (with a s.e. 0.106). The left panel in Figure 3 provide the image plots of the BLUP of ψ −¯ψ1 and the estimates of the residuals. These plots suggests that an intrinsic autoregression model captures the local variation of arsenic log-concentration fairly well. Overall the BLUP of ψ−¯ψ1 highlights the high arsenic concentration areas in northern Bangladesh, central and southwest Bangladesh. Next we ﬁt the data with an arbitrary dependence parameter ν > 0. Here, we use the same 20 Rademacher variables, but pursue REML computations using 11 diﬀerent starting values of ν as in the simulation study. We summarize both the initial set of estimates and the ﬁnal REML estimates in Table 6. As was the case with our simulation study, we found two local maxima; one corresponds to no nugget model with λy = ∞, ν = 0.858 (with a s.e. 0.016) and λψ = 0.660 (with a s.e. 0.020); and other with nugget eﬀect where λy = 0.596 (with a s.e. 0.040), ν = 1.24 (with a s.e. 0.054) and λψ = 4.607 (with a s.e. 1.260). The center 2883 REML estimation with intrinsic Mat´ern dependence Table 7 Overall, these results are largely consistent with what saw in computer generated experiments in Section 5. and we can conclude that the inference drawn from lattice-based approximations actually mimic the inference from the usual continuum intrinsic Mat´ern dependence structures with nugget eﬀects. We now extend the arsenic mapping problem further to study various covari- ate eﬀects. It is believed that water from old and/or shallow tube wells tend to have higher arsenic concentration than water from new and deeper tube-wells. The plots of arsenic concentration against depth (in meters) and age (in years) shown in Figure 4 graphically supports this view. In other words, there is an ap- parent negative correlation between the arsenic concentration and the depth of the tube-wells and an apparent positive correlation between the arsenic concen- tration and the age of these tube-wells. Thus, we include the depth and the age of the tube-wells as covariate information into our spatial mixed linear model and make some preliminary investigation on the signiﬁcance of these covariate S. Dutta and D. Mondal 2884 Fig 4. Plot of arsenic log-concentration versus log-depth (left) and age (right) of tubewells in Bangladesh. The horizontal lines denote the permissible limits by WHO and Bangladesh Government (log 10 and log 50). Fig 4. Plot of arsenic log-concentration versus log-depth (left) and age (right) of tubewells in Bangladesh. The horizontal lines denote the permissible limits by WHO and Bangladesh Government (log 10 and log 50). Table 8 BLUEs of the covariate eﬀects and REML estimates of the precision parameters for the ground water log arsenic concentration data. f ﬀ f f ground water log arsenic concentration data. r c log-depth Age ν λy λψ 300 180 Estimate -0.620 0.019 1.177 0.719 2.675 Standard error 0.048 0.003 0.051 0.072 0.051 500 300 Estimate -0.659 0.019 1.151 0.663 3.612 Standard error 0.046 0.003 0.047 0.066 0.047 600 360 Estimate -0.646 0.019 1.134 0.787 2.683 Standard error 0.044 0.003 0.044 0.102 0.397 eﬀects and robustness of such analyses to the changes of scales. To this end, Table 8 provides the BLUEs of the age and depth eﬀects, REML estimates of the precision parameters, and the corresponding standard errors for two array sizes. These estimates are obtained by using the same sample of 20 Rademacher variables that we used before for the same array sizes to obtain our REML esti- mates for the kriging problem. As expected, we ﬁnd that these covariate eﬀects are signiﬁcant. But the most striking observation here is that the BLUEs of the covariate eﬀects appear to be robust to any changes of scales. This exact same phenomenon was observed also in Dutta and Mondal (2015a) in the context of agricultural variety trial, and, in the current context this observation reaﬃrms that even for a moderately small lattice spacings, a fractionally diﬀerenced ran- dom ﬁeld plus a white noise on a lattice grid is a very good approximation to the intrinsic Mat´ern process plus a little white noise. This oﬀers further justiﬁcations for the use of lattice-based approximations. Finally, some words should be added on why we did not ﬁt a stationary Mat´ern covariance to the arsenic concentration data and instead ﬁt a limit- ing intrinsic version. This question of stationary vs. intrinsic has dominated many discussions of spatial statistics in the past; see e.g., Beran (1992), Be- sag and Kooperberg (1995), Besag (2002), McCullagh and Cliﬀord (2006) and many others that address the inadequacy of stationary models in various spatial applications. In the arsenic example, we ﬁrst need to acknowledge that these observations are not point-wise measurements. Rather they corresponds to av- erage arsenic concentrations over water intake areas of the wells. This adds REML estimation with intrinsic Mat´ern dependence 2885 diﬃculties to ﬁtting a stationary Mat´ern covariance model. 7. Discussion We open the discussion by noting the practical beneﬁts of implementing our lattice-based approximations of continuum spatial models. As we have seen through simulations and REML analysis of arsenic contamination, there is lit- tle loss in turning a geo-statistical dataset into an areal one by embedding the study region on to a ﬁne regular grid. In this context, we must recognize that we never measure a spatial random variable at a point in space, but as an av- erage value (or an integral) over a small non-null, perhaps inﬁnitesimal, region. Thus, conceptually, there is no problem in discretizing the space, and treating the observed values as averages over discretized regions. Furthermore, in most spatial applications, the scale of sampling is neither inﬁnitesimally small nor inﬁnitely great. Thus, in a range of applications, it should not be diﬃcult to im- plement a lattice-based model at a reasonably ﬁne grid and still obtain the same inference that we would have obtained had we ﬁt the corresponding continuum geostatistical model. As statisticians our rule of computations is simple. We should pursue ex- act computations when possible. When we can not pursue exact computations directly (for example if the sample size is large), we can look for alternatives that will give us answers that are as good as exact answers. In this context, our matrix-free scalable REML computations are relevant, as it is diﬃcult to pursue exact REML computations in large datasets. Numerical experiments in Section 5.3 show that there is little loss of statistical eﬃciency due to our matrix- free scalable computations and we can obtain answers that are as good as exact answers. We believe that the fractional Laplacian diﬀerenced random ﬁelds on regular arrays (that are presented in this paper) are of interest on their own. Lattice based models, particularly Markov random ﬁelds, have played a fundamental role in the development of spatial statistics and the use of fractional diﬀerencing will widen the overall scope of lattice models. In the current paper, we did not consider any stationary models, but they deserve some attention here. Typically, for stationary models, one can pursue REML computations ﬁrst by embedding the sampling locations in a ﬁner rect- angular grid and then embedding the rectangular grid into a much larger torus lattice using block circulant embedding, as proposed in Dietrich and Newsam (1993, 1997) and Wood and Chan (1994). Table 8 Second, even if we assume that data are point-referenced, we could not estimate MLEs of the co- variance parameters as ﬁtting a stationary Mat´ern model to these data runs into boundary and other numerical problems. Similar to the computer experi- ments in Section 5.1, we tried to explore basins of attraction to local maxima by doing a grid search on few parameters. But these eﬀorts proved futile and the log-likelihood surface revealed a long ﬂat ridge. Presence of such ﬂat ridges not only complicate the numerical optimization problem but is also indicative of parameter non-interpretability and redundancy. 7. Discussion This allows for the use of station- S. Dutta and D. Mondal 2886 ary models on a torus lattice, where one can take computational advantages of fast Fourier transform (Besag and Moran, 1975) and derive matrix-free scalable REML computations within our h-likelihood framework. From a computational point of view, this h-likelihood framework will be easier to implement than di- rectly maximizing the REML function by adapting the works of Anitescu et al. (2012). Furthermore, the h-likelihood framework will allow us to characterize non-convexity in the optimization in terms of gamma non-linear models and one will be able to obtain useful circulant or other preconditioners. However, the main advantage here is that these computations will be not just matrix-free and scalable but also statistically eﬃcient (in terms of achieving Cramer–Rao lower bound) and thus will yield qualitatively better estimates than those by Fuentes (2007). In the above context, we must note that, even for stationary models, to date there is no known Whiitle likelihood method that can deal with irregularly sampled observations and produce asymptotically eﬃcient estimators. Thus, the work of Anitescu et al. (2012), Dutta and Mondal (2015a), this paper and the computations mentioned above for the stationary case are a signiﬁcant step forward. Furthermore, if, in some applications, stationary Mat´ern model is of inter- est, one can also pursue lattice-based approximations. Speciﬁcally, as in Mondal (2011), we can consider a sequence of Gaussian random ﬁelds on Z2 m with spec- tral densities ˜fm(ω, η) = σ2 m m2 1 −4βm + 4βm{sin2( 1 2mω) + sin2( 1 2mη)} ν , with ω, η ∈(−mπ, mπ]. In this case, we need to take, as m →∞, βm ↑1/4, 4m2(1 −4βm) →κ2 > 0 and mν−1σm →σ/2ν. It then follows that ˜fm(ω, η) converges to ˜f(ω, η) = σ2 (κ2 + ω2 + η2)ν , σ > 0, κ > 0, which is the spectral density formula of the continuum Gaussian stationary Mat´ern random ﬁeld. On ﬁnite rectangular lattice, we can then approximate the precision matrix by λψ{(1 −4β)I + 4βW}ν and pursue our matrix-free REML computations. Notice that this precision matrix has a ﬁnite condition number and hence the computational complexity becomes only O(n(log n)2), even with- out a preconditioner. The strength and weakness of such approximations will be a matter of future research. 7. Discussion In arsenic data example, Gaussian distribution is used as an approximation, as only a small fraction of the data is truncated to the right. If there is a concern that this right-censoring can aﬀect the Gaussianity assumption, one can further consider a non-linear model using truncated Gaussian distribution and obtain appropriate statistical estimation and inference. In this regard, it must be noted that REML estimation applies only for Gaussian models and not for truncated Gaussian models. Thus, although REML estimation can not be extended to a REML estimation with intrinsic Mat´ern dependence 2887 non-linear truncated Gaussian model, one can still derive meaningful statistical estimation and inference extending the computations we have developed in this paper. It is worthwhile to mention that there are many interesting directions in which we can take our research forward in the future. One direction is to de- velop matrix-free methods for conditional simulations of the spatial eﬀect ψ. The works of Borici (2000), Schneider and Willsky (2003), Parker and Fox (2012) and Aune et al. (2013) have paved ways for matrix-free Lanczos methods for solving equations of the form A1/2x = b, where A is a positive deﬁnite matrix that can be multiplied with a vector in matrix-free way in less than O(n2) computations. By applying these works, we can thus advance conditional simulations of ψ. Speciﬁcally note that the conditional precision matrix of ψ is λyFTF + λψWν, which can be multiplied with a vector in matrix-free way in O(n log n) computa- tions. Thus such conditional simulations will allow us to make various statistical inference about the underlying latent spatial random ﬁeld. At the same time, they would help us develop novel Bayesian computations, as such simulations can be implemented for block Gibbs updates or within Matropolis-Hasting steps or other Markov chain Monte Carlo computations. Another direction will be to combine the current work with that of Mon- dal (2013) to develop fractionally diﬀerenced conditional autoregressive spatial models. This will allow for advancement of Box–Jenkins type methodology in spatial statistics. A third direction is to develop more complex spatial models that can accom- modate anisotropy and heterogeneity. In reality, there are several reasons why substantial anisotropy and heterogeneity may be present in arsenic concentra- tion. For example, aquifers may not be homogeneous, the ages and the depths of the wells can vary from place to place. 7. Discussion Similarly, demographic variables such as population density, agricultural practices and industrial variables may also aﬀect local arsenic contamination. Furthermore, it is typical that older or deeper wells supply more oxygen to the aquifers and accelerate hydrolysis arsenates to a greater extent. It is also typical that the underground water usage is high where population density is high or where the land is used for agriculture and these factors have an adverse eﬀect on contamination. Thus it would be of in- terest to think how we can collect more covariate information and how we can develop a more elaborate spatial model that can accommodate diﬀerent sources of anisotropy and heterogeneity. Appendix: Variogram calculations when ν ≥2 We refer to the supplement of the paper for numeric comparisons of γD(s, t) and γD,m(s, t). Appendix: Variogram calculations when ν ≥2 Consider a zero mean stationary Gaussian Mat´ern process Z(x) on R2 with the covariance function {2πσ2/Γ(ν)}{∥x∥/2κ)}ν−1Kν−1(κ∥x −y∥), for some ν ≥2, κ > 0 and σ > 0. Take μ1 and μ2 to be ﬁnitely supported signed measures such that xi1 1 xi2 2 μ1(dx) = xi1 1 xi2 2 μ2(dx) = 0, i1, i2 ≥0, i1 + i2 ≤⌊ν −1⌋. S. Dutta and D. Mondal 2888 Next, deﬁne higher-order contrasts or diﬀerences by Z(μj) = Z(x)μj(dx). Consequently, we obtain cov(Z(μ1), Z(μ2)) = 2πσ2 Γ(ν) ∥x∥ 2κ ν−1 Kν−1(κ∥x −y∥)μ1(dx)μ2(dy). (12) (12) As x →0+, equations (9.6.2) and (9.6.10) of Abramowitz and Stegun (1972) give xν−1Kν−1(x) = 2ν−1π −sin(νπ) k j=0 (x/2)2j j!Γ(j + 2 −ν) + 2−νπ sin(νπ)Γ(ν)x2(ν−1) +O(x2k+2), where k = ⌊ν −1⌋, when ν is not an integer. Similarly, when ν is an integer, equation (9.6.11) of Abramowitz and Stegun (1972) give where k = ⌊ν −1⌋, when ν is not an integer. Similarly, when ν is an integer, equation (9.6.11) of Abramowitz and Stegun (1972) give xν−1Kν−1(x) = 2ν−2 ν−2 j=0 (ν −j −2)! j! (−1 2x)2j + (−1)ν−1 Ψ(1) + Ψ(ν) + log 4 2ν(ν −1)! x2(ν−1) + (−1)ν 2ν−1(ν −1)!x2(ν−1) log x + o(x2ν), as x →0, where Ψ is the digamma function. It then follows that, as κ →0, the covariance in (12) converges to 41−νπ2σ2 Γ(ν)2 sin(νπ) ∥x −y∥2ν−2μ1(dx)μ2(dy) when ν > 2 is not an integer, and to (−1)ν 41−νπσ2 Γ(ν)2 ∥x −y∥2ν−2 log ∥x −y∥2μ1(dx)μ2(dy) when ν ≥2 is a positive integer. We refer to Matheron (1973) further discussions on intrinsic spatial models. Since the limit κ →0+ corresponds to an intrinsic Mat´ern random ﬁeld with spectral density (4), we can apply the above results to compute variogram of higher-order contrasts of Z(u, v). Speciﬁcally, if 2 < ν < 4, we can exactly compute γD(s, t) = var {DZ(s, t) −DZ(0, 0)} by appropriately deﬁning two ﬁnitely supported signed measures μ1 and μ2. Furthermore, we can compute γD,m(s, t) = var {DZ(m)(s, t) −DZ(m)(0, 0)} REML estimation with intrinsic Mat´ern dependence 2889 2889 by numerically computing by numerically computing σ2 m mπ −mπ mπ −mπ sin2( 1 2ω) + sin2( 1 2η) 2 1 −cos(sω) cos(tη) 4π2m2 sin2 ω 2m + sin2 η 2m ν dωdη. Supplementary Material Supplementary to “REML estimation with intrinsic Mat´ern depen- dence in the spatial linear mixed model” (doi: 10.1214/16-EJS1125SUPP; .pdf). Supplementary to “REML estimation with intrinsic Mat´ern depen- dence in the spatial linear mixed model” (doi: 10.1214/16-EJS1125SUPP; .pdf). 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https://openalex.org/W2905388513	https://europepmc.org/articles/pmc6294048?pdf=render	English	null	Stress among on-duty firefighters: an ambulatory assessment study	PeerJ	2,018	cc-by	8,890	How to cite this article Rodrigues S, Paiva JS, Dias D, Cunha JPS. 2018. Stress among on-duty firefighters: an ambulatory assessment study. PeerJ 6:e5967 http://doi.org/10.7717/peerj.5967 ABSTRACT Background. Stress at work has been broadly acknowledged as a worldwide problem and has been the focus of concern for many researchers. Firefighting, in particular, is frequently reported as a highly stressful occupation. In order to investigate firefighters’ occupational health in terms of stress events, perceptions, symptoms, and physiological reactions under real-world conditions, an ambulatory assessment protocol was devel- oped. Methods. Seventeen firefighters’ cardiac signal was continuously monitored during an average of three shifts within a working week with medical clinically certified equipment Methods. Seventeen firefighters’ cardiac signal was continuously monitored during an average of three shifts within a working week with medical clinically certified equipment (VitalJacket R⃝), which allows for continuous electrocardiogram (ECG) and actigraphy measurement. Psychological data were collected with a software application running on smartphones, collecting potential stressful events, stress symptoms, and stress appraisal. Results. A total of 450.56 h of medical-quality ECG were collected, and heart rate variability (HRV) analysis was performed. Findings suggest that although ‘fire’ situations are more common, ‘accidents’ are more stressful. Additionally, firefighters showed high levels of physiological stress (based on AVNN and LF/HF HRV metrics) when compared to normative healthy population values that may not be diagnosed using merely self-reports. Discussion. The proposed ambulatory study seems to be useful for the monitoring of stress levels and its potential impact on health of first responders. Additionally, it could also be an important tool for the design and implementation of efficient interventions and informed management resolutions in real time. Potential applications of this research include the development of quantified occupational health (qOHealth) devices for real life monitoring of emergency personnel stress reactions. Submitted 3 July 2018 Accepted 19 October 2018 Published 11 December 2018 Corresponding author Susana Rodrigues, scpr@inescporto.pt, rodriguesusanacmp@gmail.com Academic editor Tjeerd Boonstra Additional Information and Declarations can be found on page 15 DOI 10.7717/peerj.5967 Submitted 3 July 2018 Accepted 19 October 2018 Published 11 December 2018 Corresponding author Susana Rodrigues, scpr@inescporto.pt, rodriguesusanacmp@gmail.com Academic editor Tjeerd Boonstra Additional Information and Declarations can be found on page 15 DOI 10.7717/peerj.5967 Copyright 2018 Rodrigues et al. Distributed under Creative Commons CC-BY 4.0 Subjects Bioengineering, Psychiatry and Psychology, Public Health Keywords Occupational health, Firefighters, Stress, Ambulatory assessment, Wearable devices, Work analysis Subjects Bioengineering, Psychiatry and Psychology, Public Health Keywords Occupational health, Firefighters, Stress, Ambulatory assessment, Wearable devices, Work analysis Subjects Bioengineering, Psychiatry and Psychology, Public Health Keywords Occupational health, Firefighters, Stress, Ambulatory assessment, Wearable devices, Work analysis Stress among on-duty firefighters: an ambulatory assessment study Susana Rodrigues1,2, Joana S. Paiva1,3, Duarte Dias1,2 and João Paulo S. Cunha1,2 1 Institute for Systems Engineering and Computers—Technology and Science (INESC TEC), Porto, Portugal 2 Faculty of Engineering, University of Porto, Porto, Portugal 3 Astronomy and Physics Department, Sciences Faculty, University of Porto, Porto, Portugal INTRODUCTION The existing global challenges of our society, the escalation of crime and violence contribute to the increase of critical incidents; therefore, there is a greater need for security forces and emergency services to intervene (Shaffer, 2010). Firefighting is one of the professions with strong responsibilities towards society safety and well-being, considering that firefighters are the first to respond to a critical incident. Firefighters emergency responsibilities includes not only preventing and combating fire, but also tasks such as supporting in Distributed under Creative Commons CC-BY 4.0 OPEN ACCESS major transport accidents, natural disasters, terrorist attacks, or when special technical help is required (European Agency for Safety and Health at Work, 2011). Society has strong expectations of the ability of firefighting personnel to carry out their responsibilities effectively. However, firefighters perform their typical occupational activities in very hard, unpredictable emergency circumstances, exposing them to severe stress. According to previous findings, working contexts with high levels of stress, are related with increased risks of cardiovascular problems and other work-related illnesses (Vrijkotte, Van Doornen & De Geus, 2000). Furthermore, the effects of stress among firefighters are well recognized (Kales et al., 2007; Perrin et al., 2007). Stress can be defined as a process, whereas a situation is perceived as exceeding individual’s resources or threatens the person’s well-being (Lazarus & Folkman, 1984). Despite its psychological component, stress also changes the physiological balance of the autonomic nervous system (ANS). The ANS is divided into two main divisions: the sympathetic and the parasympathetic nervous system. Both divisions operate simultaneously and balance each other dynamically in regular situations. During an acute stress event, the sympathetic system is overactive and the parasympathetic system level of activation is decreased. The sympathetic branch increases heartbeat rates, while also increasing the perspiring activity of the adrenal glands and breathing rates, preparing the body to respond, while the parasympathetic system decrease the heartbeat, sweating, and breathing rates (Clifford, Azuaje & McSharry, 2006). This process was firstly called as the ‘fight-or-flight’ response (Cannon, 1935). The chronic exposure to high levels of stress, such as experienced in working situations, can result in a chronic body activation, that can overwhelm the body systems (hormonal, cardiovascular, neural, and muscular systems). This can seriously impact health, for example, by causing long-term damages to the immune system (Taelman et al., 2009). Rodrigues et al. (2018), PeerJ, DOI 10.7717/peerj.5967 INTRODUCTION There are a wide range of physiological indicators (e.g., blood pressure, cortisol, skin conductance), however heart rate variability (HRV) has been foreseen as a reliable indicator of physiological stress reactions (Castaldo et al., 2015). HRV refers to the variations in heart rates or heartbeat intervals, which has been recognized as an instantaneous quantitative measure of the ANS activity related with stress (Zhong et al., 2005). Developments in stress diagnosis methods are crucial for the understanding of individual functioning during hazardous events and for the design of efficient prevention programs. However, despite the fact that much attention has been dedicated to the study of stress over the years, there are still some limitations with respect to its conceptualization and evaluation. First, traditional stress assessment designs in psychology are frequently retrospective and cross-sectional, only based on self-reported measures (Segerstrom & O’Connor, 2012). As a result, the collected data may be plagued by memory bias and cognitive biases. Second, and as a possible solution to overcome the limitations presented above, laboratory experiments are typically used to assess stress (Zanstra & Johnston, 2011). These controlled conditions avoid retrospective reporting problems and are more rigorous (Smyth & Stone, 2003). However, considering their artificial conditions, results may not represent real-world settings, and their ecological validity is limited. Third, there is a lack of a gold standard stress measurement in either experimental settings or the field (Hovsepian et al., 2015). Rodrigues et al. (2018), PeerJ, DOI 10.7717/peerj.5967 2/19 Finally, studies in this area frequently include clinical populations without contemplating a healthy cohort and/or reference to healthy values (Nunan, Sandercock & Brodie, 2010). In order to overcome the previous shortcomings in this area and taking into account that stress is a complex issue, interdisciplinary research approaches integrating psychophysiological dimensions of stress that are able to collect data under natural settings are necessary in order to fully investigate stress (Rodrigues, Kaiseler & Queirós, 2015). Ambulatory assessment has been recommended as a novel research method that combines self-reports, and physiological measurements under natural environments (Trull & Ebner- Priemer, 2013). In sum, ambulatory monitoring offers higher ecological validity and higher clinical reliability outcomes than laboratory studies (Houtveen & De Geus, 2009). INTRODUCTION However, stress research in the field has also been plagued by several problems, including noisy data, the influence of confounding variables, and difficulties in finding discriminative features that can detect and differentiate a stress response from similar physiological responses (Hovsepian et al., 2015). In order to overcome the previous shortcomings in this area and taking into account that stress is a complex issue, interdisciplinary research approaches integrating psychophysiological dimensions of stress that are able to collect data under natural settings are necessary in order to fully investigate stress (Rodrigues, Kaiseler & Queirós, 2015). Based on previous research recommendations, the current study developed a novel multi-method ambulatory stress approach in order to assess firefighters stress events, perceptions and symptoms during their daily work duties. Taking this into account, the current interdisciplinary method consists of a combination of self-reports with electrocardiogram (ECG) data analysis, relying on user-friendly and non-intrusive technology adapted to firefighters’ needs and requirements. According to a recent review on wearable health devices (Dias & Cunha, 2018), wearable ECG devices are of extreme importance for health monitoring because they allow to acquire valuable physiological data without interfering in users normal activities and daily routines. As opposed non-wearable devices have greater dimensions, are static and not comfortable and the awareness of being monitored can strongly interfere with subjects behavior, causing more stress. The major concern when using wearable devices is the associated noise and data quality. The device used in the current study (Vital Jacket R⃝) has the huge advantage of having medical certification, which means that the acquired data has medical quality, although it can be naturally be affected by intensive movement. This is something that has been very difficult to overcome, however the use of these kind of devices contributes for a lower amount of noise artifacts and a higher level of quality of the data. Additionally, the use of softwares that implement machine learning techniques and novel algorithms are also helpful for the removing of movement artifacts (Dias & Cunha, 2018). To the best of our knowledge, no previous studies were found internationally with this population using a similar approach. Taking this in account, we believe that this study makes a novel and original contribution to the advancement of knowledge in psychophysiological assessment in daily work conditions of firefighters. Findings will particularly increase the understanding of firefighters’ stress experience on duty. PARTICIPANTS Seventeen firefighters (15 males and 2 females) from a fire department in Portugal voluntarily participated in this study. The age range was 18–54 years (M = 29.35±8.85). Regarding the educational background, five had completed primary school, six completed secondary school, and three had a graduation level. The firefighting years of practice ranged between less than one year to 23 years (M = 9.41±7.3). Regarding the self-perception of participant’s health status, 47% reported that they have a good health, 35% an excellent health and 18% reported that they have an average health. Regarding physical activity practice, 76% of the sample reported that they practice physical activity regularly. Regarding smoking habits, 53% of the participants smokes. The exclusion criteria for participating in the study were subjects having a history of cardiovascular disease and/or taking medication that interfere with their cardiovascular function. The study was approved by the University of Porto Ethics Committee (ethical application ref: 29/CEUP/2016). After presenting the study protocol, the participants voluntarily provided written informed consent prior to the study implementation. METHODS In order to provide a standardized reporting of methodologies investigating HRV in psychiatry and behavioral sciences, we will present our research method based on the Rodrigues et al. (2018), PeerJ, DOI 10.7717/peerj.5967 3/19 Guidelines for Reporting Articles in Psychiatry and Heart Rate Variability (GRAPH) (Quintana, Alvares & Heathers, 2016). The current method fits the four domains proposed by these guidelines: participant selection, interbeat interval collection, data preparation and HRV calculation. However, small amendments were made to this checklist, considering that our study is multidisciplinary and involves other variables, such as self-report measures, besides HRV metrics. Figure 1 illustrates our method in general. Rodrigues et al. (2018), PeerJ, DOI 10.7717/peerj.5967 Rodrigues et al. (2018), PeerJ, DOI 10.7717/peerj.5967 MATERIALS A ‘kit’ was specifically developed for this study, including (a) a wearable t-shirt that acted like a wearable ECG monitor, VitalJacket R⃝(Cunha et al., 2010; Cunha, 2012) (see Fig. 2), and (b) an electronic diary based on an Android smartphone application (see Fig. 3). A ‘kit’ was specifically developed for this study, including (a) a wearable t-shirt that acted like a wearable ECG monitor, VitalJacket R⃝(Cunha et al., 2010; Cunha, 2012) (see Fig. 2), and (b) an electronic diary based on an Android smartphone application (see Fig. 3). The VitalJacket R⃝is a wearable bio-monitoring platform (in the form of a t-shirt) able to collect ECG signals in real-time, without affecting daily activities of users. It also contains a three-axis accelerometer system, allowing ECG signal correction for actigraphy profiles, and a Bluetooth transmitter that enables visualisation of the ECG signal in real time and saves all data in a SD memory card. This equipment is certified according to the MDD93/42/EEC medical device directive and holds the European Conformity mark (Biodevices, 2017). Figure 4 shows an example of an ECG trace collected using VitalJacket R⃝. The electronic diary contained an Android smartphone application that was specifically developed by biomedical engineers for this study. This tool allows to describe events and to rate stress symptoms and perceptions. Additionally, it allows data synchronisation and event marking (Fig. 3). This system pairs with VitalJacket R⃝via Bluetooth and enables the exact annotation of events in the device using ‘radiobuttons’. These events are saved in the device and synchronised with the ECG that is being acquired at that moment. The app stores all the data about the events in an SQL Light DataBase, from where a report of the event data can be generated and exported for processing and analysis, ensuring Rodrigues et al. (2018), PeerJ, DOI 10.7717/peerj.5967 4/19 Figure 1 Study method illustration based on Guidelines for Reporting articles in Psychiatry and Heart Rate Variability (Quintana, Alvares & Heathers, 2016). Full-size DOI: 10.7717/peerj.5967/fig-1 Figure 1 Study method illustration based on Guidelines for Reporting articles in Psychiatry and Heart Rate Variability (Quintana, Alvares & Heathers, 2016). Full-size DOI: 10.7717/peerj.5967/fig-1 Figure 1 Study method illustration based on Guidelines for Reporting articles in Psychiatry and Heart Rate Variability (Quintana, Alvares & Heathers, 2016). Full-size DOI: 10.7717/peerj.5967/fig-1 Figure 2 Vital Jacket R⃝equipment. F ll i DOI 10 7717/ j 5967/fi 2 Figure 2 Vital Jacket R⃝equipment. Rodrigues et al. (2018), PeerJ, DOI 10.7717/peerj.5967 Self-reports A demographic and a medical questionnaire were used in order to evaluate participants’ current health state. In order to find a measure of chronic stress, the Portuguese version (Mota Cardoso et al., 2002) of the original Perceived Stress Scale (PSS) was implemented (Cohen, Kamarck & Mermelstein, 1983). The validation studies of this scale presented a coefficient alpha reliability above 0.84 (N = 332) in Cohen’s and colleagues study and 0.86 (N = 200) in Mota Cardoso and colleagues study. In the current study, the Cronbach’s alpha for the PSS was 0.76. The PSS is a 14-item scale ranging from ‘0—never’ to ‘4—very often’. Participants were asked to indicate how often they felt or thought a certain way in the past month. Scores range from 0 to 56, with higher scores indicating more stress. MATERIALS Full-size DOI: 10.7717/peerj.5967/fig-2 Figure 2 Vital Jacket R⃝equipment. Full-size DOI: 10.7717/peerj.5967/fig-2 Figure 3 Smartphone application layout. Full-size DOI: 10.7717/peerj.5967/fig-3 Figure 2 Vital Jacket R⃝equipment. Full-size DOI: 10.7717/peerj.5967/fig-2 Full-size DOI: 10.7717/peerj.5967/fig-2 Figure 3 Smartphone application layout. Full-size DOI: 10.7717/peerj.5967/fig-3 Figure 3 Smartphone application layout. Full-size DOI: 10.7717/peerj.5967/fig-3 Figure 3 Smartphone application layout. Full-size DOI: 10.7717/peerj.5967/fig-3 Rodrigues et al. (2018), PeerJ, DOI 10.7717/peerj.5967 6/19 Figure 4 Data acquired from VitalJacket R⃝wearable medical device captured during non-movement and movement. This figure shows an example of the medical grade ECG waveform acquired and below an activity intensity from the accelerometer acquired data. This intensity scale from the accelerometer goes from yellow (no movement, zero value) to red (high movement) showing the level of each participant activity. Full-size DOI: 10.7717/peerj.5967/fig-4 the synchronisation between those events’ information and the data gathered by the VitalJacket R⃝. Figure 4 Data acquired from VitalJacket R⃝wearable medical device captured during non-movement and movement. This figure shows an example of the medical grade ECG waveform acquired and below an activity intensity from the accelerometer acquired data. This intensity scale from the accelerometer goes from yellow (no movement, zero value) to red (high movement) showing the level of each participant activity. Full-size DOI: 10.7717/peerj.5967/fig-4 h h i i b h ’ i f i d h d h d b h Figure 4 Data acquired from VitalJacket R⃝wearable medical device captured during non-movement and movement. This figure shows an example of the medical grade ECG waveform acquired and below an activity intensity from the accelerometer acquired data. This intensity scale from the accelerometer goes from yellow (no movement, zero value) to red (high movement) showing the level of each participant activity. Full-size DOI: 10.7717/peerj.5967/fig-4 Figure 4 Data acquired from VitalJacket R⃝wearable medical device captured during non-movement and movement. This figure shows an example of the medical grade ECG waveform acquired and below an activity intensity from the accelerometer acquired data. This intensity scale from the accelerometer goes from yellow (no movement, zero value) to red (high movement) showing the level of each participant activity. Full-size DOI: 10 7717/peerj 5967/fig-4 the synchronisation between those events’ information and the data gathered by the VitalJacket R⃝. the synchronisation between those events’ information and the data gathered by the VitalJacket R⃝. Rodrigues et al. (2018), PeerJ, DOI 10.7717/peerj.5967 Diary information The Android smartphone application included a stress symptoms questionnaire (Cohen & Williamson, 1988). This instrument included four questions related to physical aspects and four questions related to cognitive aspects. An example of a physical symptom question is ‘I have a stiff neck’; an example of a cognitive symptom question is ‘I lack concentration’. Participants were asked to rate each item on a free scale ranging from ‘0—not felt at all’ to ‘4—extremely felt’. These questions were answered at the beginning and end of the day, aiming to evaluate whether there were alterations in stress symptoms experienced from the beginning to the end of the day. Additionally, a Visual Analogue Scale (VAS) (Lesage, Berjot & Deschamps, 2012) was used after each event and before and after each shift. Participants were asked to rate their perceived stress levels on a 10-level scale, ranging from ‘0—None’ to ‘10—As bad as it could be’. Work stress was classified as high when VAS >6 and low when VAS <3 (Ritvanen et al., 2006). Single–item measures were chosen since they can offer useful information and present numerous benefits (e.g., reduced survey length; respondent burden); for a detailed review of these please see Fisher, Matthews & Gibbons (2016). Rodrigues et al. (2018), PeerJ, DOI 10.7717/peerj.5967 7/19 The firefighters also provided information regarding the experienced events in an open-ended event question displayed in the smartphone application. The firefighters also provided information regarding the experienced events in an open-ended event question displayed in the smartphone application. Ambulatory measures Following the guidelines presented by the task force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology (Task Force of the European Society of Cardiology, 1996) a feasible HRV time-domain parameter for stress assessment is the average time between consecutive normal-to-normal (AVNN) heartbeat time intervals. Lower values of this HRV component reflect higher stress (Castaldo et al., 2015). As for spectral domain parameters, both the low-frequency (LF) component (0.04–0.15 Hz) and the high-frequency (HF) component (0.15–0.4 Hz) of all NN intervals provide information about sympathetic nervous system (SNS) and parasympathetic nervous system (PSNS) activity. The LF/HF ratio has been frequently used as an indicator of the overall balance between the sympathetic and parasympathetic systems, respectively. Higher values reflect domination of the sympathetic system, whereas lower values refer to a higher activity from the parasympathetic system (Kaur et al., 2013). HRV values achieved with this population during entire shifts will be compared with normative values obtained by the study conducted by Voss et al. (2015). These authors conducted a study with the largest population of healthy subjects (N = 1906) and analyzed age and gender- related HRV differences. PROCEDURE A presentation meeting was organised to present the study. To assess the appropriateness of the study ‘kit’, a pilot study was conducted with two firefighters to validate the methodology’s feasibility and user-friendliness and the accuracy of the data received. Participants underwent ambulatory monitoring on about three days of the same work week (eight hours each shift). At first, all firefighters completed the demographics and medical questionnaire and the PSS. At the beginning of each shift, participants dressed in the VitalJacket R⃝, switched the smartphone on, launched the software application, and filled in the initial stress symptoms questionnaire and the stress VAS. Following this procedure, the firefighters were ready to carry out the equipment for the full working-day periods. After experiencing a stressful situation, the firefighters were required to fill in the event questionnaire on the smartphone, including a description of the event and ratings of stress appraisal, using the stress VAS. At the end of the shift, the firefighters again filled in the stress symptoms questionnaire and the stress VAS. Physiological stress data y g For the extraction of heartbeat information from the ECG recordings, the Biodevices, S.A. ECG analyser (which is the same commercialised by this company to cardiology specialists), was used. This analyser has an algorithm that detects each heartbeat in the ECG recording, detecting the ‘R’ points of the ECG waveform and the RR interval (time between two consecutives ‘R’ peaks in the ECG) was extracted. A simple verification according to the literature was implemented to verify if all the RR intervals were physiologically valid (Clifford, Azuaje & McSharry, 2006). This procedure can eliminate any possible mistake made by the RR interval detection algorithm that can occur in case of a noisy ECG signal. The RR intervals that are physiologically valid are called normal-to-normal (NN) intervals. Physiological data were collected during the entire shift and during the stressful events. For the event-based approach, AVNN and LF/HF values were computed for each 5-minute ECG segment for each subject, during the period of the event annotation made by the firefighter in the smartphone application. It is important to remind that only ECG valid data was considered after the data quality verification described above. Since the total number of 5-minute ECG blocks among all subjects was not balanced between the three categories (‘accidents’, ‘pre-hospitalar assistance’, and ‘fires’), a number of AVNN and LF/HF 5-minute block values corresponding to the minimum number of 5-minute ECG For the extraction of heartbeat information from the ECG recordings, the Biodevices, S.A. ECG analyser (which is the same commercialised by this company to cardiology specialists), was used. This analyser has an algorithm that detects each heartbeat in the ECG recording, detecting the ‘R’ points of the ECG waveform and the RR interval (time between two consecutives ‘R’ peaks in the ECG) was extracted. A simple verification according to the literature was implemented to verify if all the RR intervals were physiologically valid (Clifford, Azuaje & McSharry, 2006). This procedure can eliminate any possible mistake made by the RR interval detection algorithm that can occur in case of a noisy ECG signal. The RR intervals that are physiologically valid are called normal-to-normal (NN) intervals. Physiological data were collected during the entire shift and during the stressful events. DATA ANALYSIS AND PROCESSING Event categorisation and correspondent stress perceptions The event open-ended responses were transcribed verbatim and subjected to an inductive content analysis technique (Maykut & Morehouse, 1994). The data was coded into stressor categories by the first author and then verified by the other authors (Nicholls & Polman, 2007). Then, stressor categories generated for stressor responses were categorised into 8/19 Rodrigues et al. (2018), PeerJ, DOI 10.7717/peerj.5967 more general dimensions labelled as ‘fires’, ‘pre-hospitalar assistance’, and ‘accidents’, as recommended in the literature (Pallauf et al., 2011). The frequency and mean values of the stress VAS were calculated considering the intensity and mean intensity of each stressor. This approach is similar to previous research in the area of stress appraisal (Kaiseler, Polman & Nicholls, 2009). Kruskal–Wallis (non-parametric alternative) was used to compare if there were statistical differences across stress VAS scores for the three stress event categories. Stress symptoms and self-perceptions End-of-the-day and beginning-of-the-day stress symptoms mean scores were subtracted in order to achieve an overall mean score, symbolising accumulated stress symptoms over the shift. Internal consistency of the eight questions was calculated using Cronbach’s alphas. A study conducted by Gomes et al. (2012) with a sample of Portuguese firefighters, using the same questionnaire obtained a Cronbach’s alpha for the 8 questions of 0.93. This value provides a coefficient of reliability, and it is used as a measure of internal consistency for participants’ answers. As recommended, these values should be above 0.70 (Pallant, 2013). In the current study, the Cronbach’s alpha for the eight questions was 0.82, with an acceptable reliability (r = 0.7). Paired sample T-tests were performed to determine if there were differences between stress symptoms during the end and the beginning of the shift. For stress VAS scales implemented at the end and the beginning of the shift, mean scores were subtracted in order to achieve an overall mean score, representing the evolution of stress perceptions during the shift. Spearman rank order correlations were also used to test if there were relations between the stress symptoms and the perceived stress levels along the shift. Rodrigues et al. (2018), PeerJ, DOI 10.7717/peerj.5967 Event categorisation and correspondent stress perceptions Event categorisation and correspondent stress perceptions A total of 42 events were reported by the firefighters. The categorisation of events was divided into three main classes: ‘fires’, ‘pre-hospitalar assistance’, and ‘accidents’ (Table 1). The mean results of the VAS after each event showed that ‘accidents’ were rated as the most stressful events (M = 3.16), followed by ‘pre-hospitalar assistance’ (M = 2.27) and ‘fires’ (M = 1.95). However, results from the Kruskal–Wallis test revealed no significant statistical differences in the stress VAS across the three stress events (χ2 = 2.37, p = .31). Rodrigues et al. (2018), PeerJ, DOI 10.7717/peerj.5967 Physiological stress data For the event-based approach, AVNN and LF/HF values were computed for each 5-minute ECG segment for each subject, during the period of the event annotation made by the firefighter in the smartphone application. It is important to remind that only ECG valid data was considered after the data quality verification described above. Since the total number of 5-minute ECG blocks among all subjects was not balanced between the three categories (‘accidents’, ‘pre-hospitalar assistance’, and ‘fires’), a number of AVNN and LF/HF 5-minute block values corresponding to the minimum number of 5-minute ECG Rodrigues et al. (2018), PeerJ, DOI 10.7717/peerj.5967 9/19 blocks among the three categories was chosen randomly from the raw samples distribution of AVNN and LF/HF. Hence, a total of 30 blocks of 5-minute ECGs was considered for each condition in the statistical analysis. This allowed to perform statistical comparisons and correlations considering a balanced sample number per condition. Kruskal–Wallis was used to compare the AVNN and LF/HF mean differences for those stress event categories. For the entire shift analysis, AVNN and LF/HF mean values for each subject were subtracted from the normative values according to the age and gender of each subject. Normative values were obtained from Voss et al. (2015) study with 1,906 healthy subjects. The statistical analysis was conducted using IBM SPSS AMOS (v.22) software and ECG data analysis was performed using an interface developed in Matlab (R2015a) For the entire shift analysis, AVNN and LF/HF mean values for each subject were subtracted from the normative values according to the age and gender of each subject. Normative values were obtained from Voss et al. (2015) study with 1,906 healthy subjects. The statistical analysis was conducted using IBM SPSS AMOS (v.22) software and ECG data analysis was performed using an interface developed in Matlab (R2015a). RESULTS A total of 450.56 h were collected during shifts throughout the fire season in Portugal (July–October 2016). Considering that the combined effect of exercise and mental stress in HRV variables is still not well discriminated by the literature, some adjustments on the analysis had to be made, resulting in a total of 133 h of ECG signal considered suitable for analysis. Hence, in order to remove higher values of movement that may affect HRV changes due to stress, a selection of collected data was made according to a movement threshold measured by the wearable device accelerometer. It is also important to mention that, as stated before, a RR interval validation was made to ensure that the extracted values were physiological accepted. These adjustments enables the reduction of noise artefacts and to exclude wrong values before compute HRV measurement. The overall sum of PSS results (28 ± 4.99) was in the average (28), considering that the highest possible value is 56, and higher levels suggest more stress. The mean value obtained was 2.0 ± 1.1. Event categorisation and correspondent stress perceptions Rodrigues et al. (2018), PeerJ, DOI 10.7717/peerj.5967 Stress symptoms and self-perceptions A total of 34 entries were provided using the smartphone application. Paired sample T-test results showed statistically significant differences between the beginning and the end of the shift for all cognitive stress symptoms, with the exception of two physical stress symptoms: ‘stiff neck’ and ‘difficulty keeping the body straight’ (Table 2). There has been an increase of the symptoms from the beginning to the end of the shift, showed by the positive direction of scores between beginning and end of shift (Table 2). Spearman’s rho showed significant positive correlations concerning the difference between Visual Analogue Scales at the end and at the beginning of the shift and the stress symptoms reported, with the exception of ‘unconformable abdominal pain or stomach ache’ and ‘difficulty in controlling reactions’. Rodrigues et al. (2018), PeerJ, DOI 10.7717/peerj.5967 10/19 Table 1 Event categories, events, frequency, mean and standard deviation (SD) values of stress Visual Analogue Scales (VAS) after each event. General dimension/categories Stress events Frequency VAS Mean ± SD Accidents 6 3,16 ± 1,86 Road accidents victims 5 Accident with an infrastructure 1 Pre-hospitalar assistance 15 2,27 ± 1,29 Glycaemia alterations 12 Blood pressure changes 2 Patient transportation 1 Fires 21 1,95 ± 1,05 Forest fire 16 Transport fire 2 Fire in an abandoned house 1 Aftermath 2 Notes. Bold indicates the total frequency of each general dimension and its correspondent mean and standard deviation. Table 1 Event categories, events, frequency, mean and standard deviation (SD) values of stress Visual Analogue Scales (VAS) after each event. Notes. Bold indicates the total frequency of each general dimension and its correspondent mean and standard deviation. Table 2 Statistical analysis of differences between end and beginning of the shift for stress symptoms and their correlations with mean difference of VAS along shift (end shift minus beginning of the shift). Table 2 Statistical analysis of differences between end and beginning of the shift for stress symptoms and their correlations with mean difference of VAS along shift (end shift minus beginning of the shift). Stress symptoms and self-perceptions Stress symptoms Mean ± SDa T-Test values Spearman rho Stiff neck 0.21 ± 0.08 −1.49 0.483** Tiredness in the eyes or heavy head 0.39 ± 0.95 −2.34* 0.414* Uncomfortable abdominal pain or stomach ache 0.32 ± 0.73 −2.60* 0.339 Difficulty to keep the body straight 0.06 ± 0.89 −0,39 0.639** Lack of concentration 0.29 ± 0.84 −2.05* 0.503** Difficulty to think, and make decisions 0.44 ± 0.96 −2.69* 0.435* Anxiety 0.15 ± 0.66 −1.30* 0.365* Difficulty in controlling reactions 0.21 ± 0.54 −2.23* 0.225 Notes. aEnd shift–beginning of the shift. p < .05. p < .005. SD, standard deviation. ymptoms Mean ± SDa T-Test values Spearman rho Physiological stress data analysis Significant differences were also found for ‘‘pre-hospitalar assistance’’ (M = 1.92 ± 0.68), and ‘‘accidents’’ (M = 2.85±1.56), z = −3.29, p = .001, r = 1.1. Results from the ECG analysis conducted during the entire shifts, based on AVNN and LF/HF ratio, are presented in Fig. 5. These results were individually compared with normative values obtained by Voss et al.’s (2015) study with 1,906 healthy subjects, controlling for gender and age (Fig. 4). AVNN results suggested that 82% of participants (14 out of 17 firefighters) presented lower values of this ECG measure, and for LF/HF a total of 71% of the firefighters (12 out of 17) were above the normative values. Overall mean results from the group analysis suggested that AVNN was lower for the firefighters’ sample (M = 792.64±92.29) when compared to health subjects’ baseline (M = 930±133). Complementarily, LF/HF is higher in firefighters (M = 3.82±1.76) when compared to normative values (M = 3.33±3.47). Rodrigues et al. (2018), PeerJ, DOI 10.7717/peerj.5967 Physiological stress data analysis Physiological stress data analysis ECG data were obtained for 17 firefighters. The ECG analysis was performed during the entire shifts and during the main event categories—an event-based approach. Results for the event-based analysis suggested that ‘accidents’ were the most stressful events, considering that AVNN presented the lowest values, followed by the highest values of LF/HF, when compared to the other reported events: ‘fires’ and ‘pre-hospitalar assistance’ (Table 3). These results were in accordance with stress perceptions obtained with the VAS. Kruskal–Wallis revealed statistically significant differences only for LF/HF (χ2 = 12.54, p = .002). In order to test for significant differences in LF/HF between the groups (fires; accidents and pre-hospitalar assistance) Mann–Whitney test was used. Results showed Rodrigues et al. (2018), PeerJ, DOI 10.7717/peerj.5967 11/19 Table 3 AVNN and LF/HF mean values and SD divided into the three stress events categorizations (N = 30). AVNN (milliseconds) LF/HF Mean ± SD Mean ± SD Accidents 811.39 ± 145.65 2.85 ± 1.56 Pre-hospitalar assistance 830.24 ± 142.29 1.92 ± 0.68* Fires 823.01 ± 134.05 2.51 ± 0.98* Notes. Significant differences between conditions ‘‘accidents’’, ‘‘pre-hospital assistance’’ and ‘‘fires’’; Kruskal-Wallis, two-tailed; p < 0.01. Table 3 AVNN and LF/HF mean values and SD divided into the three stress events categorizations (N = 30). Notes. Significant differences between conditions ‘‘accidents’’, ‘‘pre-hospital assistance’’ and ‘‘fires’’; Kruskal-Wallis, two-tailed; p < 0.01. tes. *Significant differences between conditions ‘‘accidents’’, ‘‘pre-hospital assistance’’ and ‘‘fires’’; Kruskal-Wallis, two-tailed; p < 0 01 significant differences in LF/HF metric during ‘‘fires’’ (M = 2.51 ± 0.98) and ‘‘pre- hospitalar assistance’’ (M = 1.92±0.68), z = 2.74, p = .001, r = 0.9. Significant differences were also found for ‘‘pre-hospitalar assistance’’ (M = 1.92 ± 0.68), and ‘‘accidents’’ (M = 2.85±1.56), z = −3.29, p = .001, r = 1.1. significant differences in LF/HF metric during ‘‘fires’’ (M = 2.51 ± 0.98) and ‘‘pre- hospitalar assistance’’ (M = 1.92±0.68), z = 2.74, p = .001, r = 0.9. Significant differences were also found for ‘‘pre-hospitalar assistance’’ (M = 1.92 ± 0.68), and ‘‘accidents’’ (M = 2.85±1.56), z = −3.29, p = .001, r = 1.1. significant differences in LF/HF metric during ‘‘fires’’ (M = 2.51 ± 0.98) and ‘‘pre- hospitalar assistance’’ (M = 1.92±0.68), z = 2.74, p = .001, r = 0.9. Rodrigues et al. (2018), PeerJ, DOI 10.7717/peerj.5967 DISCUSSION The present study used an ambulatory multi-method approach to assess stress, particularly stress events, perceptions, symptoms, and physiological reactions, considering a combination of self-reports and ECG data. The ambulatory cardiovascular analysis during shifts suggests that firefighters experience high levels of physiological stress when compared to healthy individuals based on the study developed by Voss et al. (2015). There is a lack of studies in the literature concerning normative values of HRV in large healthy populations, not to mention that there is no study indicating these values for a firefighter population. The reduced values of AVNN during ‘accidents’ and also during shifts propose an increase in cardiac sympatho-excitation, characteristic of stress conditions (Tharion, Parthasarathy & Neelakantan, 2009). This process reflects an increased activation of the cardiovascular system, triggered in order to prepare the body to quickly respond to stress. Therefore, lower values are associated with stress responses (Taelman et al., 2009). On the other hand, increased LF/HF suggests an activation of the SNS as well as withdrawal of PNS activity (Pagani et al., 1997). Therefore, stress is related to an increase in LF/HF ratio (Kaur et al., 2013). Additionally, statistically significant differences were found for LF/HF during the reported events as opposed to AVNN, suggesting that this measure could be more sensitive for the identification of stress, considering that this parameter is directly related to Rodrigues et al. (2018), PeerJ, DOI 10.7717/peerj.5967 Figure 5 Differences between (A) AVNN and (B) LF/HF obtained during entire shifts and normative values for each subject, individually controlling against baseline HRV for age and gender (Voss et al., 2015). Full-size DOI: 10.7717/peerj.5967/fig-5 Figure 5 Differences between (A) AVNN and (B) LF/HF obtained during entire shifts and normative values for each subject, individually controlling against baseline HRV for age and gender (Voss et al., 2015). Full-size DOI: 10.7717/peerj.5967/fig-5 sympathetic activation (Traina et al., 2011). Complementarily, results obtained from PSS, used as a measure of chronic stress suggest a moderate level of this measure, potentially indicating the presence of a chronic stress state (Cohen, Kamarck & Mermelstein, 1983). When examining the differences between event self-perceptions, it is interesting to note that ‘accidents’ were reported as the most stressful (M = 2.85±1.86). However, the differences were not statistically relevant, probably due to the reduced sample used. These results were also found in a similar study with firefighters (Pallauf et al., 2011). DISCUSSION This suggests that, although ‘fire’ situations are more common, they are not the most stressful. One possible explanation for ‘accidents’ being the most stressful events could be that firefighters are usually first on the scene of accidents, which are unpredictable situations that usually involve people’s lives. However, it is important to consider that stress self-perceptions, based on the VAS, presented very low values, considering that the scale ranged from 1 to 10 and the overall mean of this measure was 2.38. The possible mismatch between self-report levels after events and their physiological responses could be explained by the fact that the reported events (mainly ‘fire’ situations) are commonly experienced by firefighters, particularly in the course of the fire season, when the data were collected. sympathetic activation (Traina et al., 2011). Complementarily, results obtained from PSS, used as a measure of chronic stress suggest a moderate level of this measure, potentially indicating the presence of a chronic stress state (Cohen, Kamarck & Mermelstein, 1983). g p When examining the differences between event self-perceptions, it is interesting to note that ‘accidents’ were reported as the most stressful (M = 2.85±1.86). However, the differences were not statistically relevant, probably due to the reduced sample used. These results were also found in a similar study with firefighters (Pallauf et al., 2011). This suggests that, although ‘fire’ situations are more common, they are not the most stressful. One possible explanation for ‘accidents’ being the most stressful events could be that firefighters are usually first on the scene of accidents, which are unpredictable situations that usually involve people’s lives. However, it is important to consider that stress self-perceptions, based on the VAS, presented very low values, considering that the scale ranged from 1 to 10 and the overall mean of this measure was 2.38. The possible mismatch between self-report levels after events and their physiological responses could be explained by the fact that the reported events (mainly ‘fire’ situations) are commonly experienced by firefighters, particularly in the course of the fire season, when the data were collected. Rodrigues et al. (2018), PeerJ, DOI 10.7717/peerj.5967 13/19 Complementarily to stress perceptions and physiology, stress symptoms were also analysed. The stress symptoms questionnaire showed an increase of stress symptomatology from the beginning of the shift to the end, with almost all stress symptoms positively correlated with perceived stress level differences based on VAS means. DISCUSSION In fact, VAS stress levels reported by participants also increased from the beginning to the end of shifts. These results are in line with previous findings obtained with this population, showing the cumulative effects of stress throughout the day (Gomes et al., 2012). Additionally, these effects are more evident for cognitive stress symptoms, considering that all of them significantly increased from the beginning of the shift to the end. Accordingly, a study conducted with 21 firefighters concluded that stress has an impact on cognitive functioning, which could explain the maladaptive responses observed during real fires (Robinson et al., 2013). These should be of great concern for firefighters’ administrators and clinicians, providing important insights for the design of tailored stress-management programs. The current study has some methodological limitations particularly: the reduced sample size that limits the generalizability of results and short time duration of the data collection procedures (only an average of three shifts were monitored). Hence, further research is required, using larger samples and different professionals for longer periods (more shifts and including days off) and comprising longitudinal designs. Regarding the wearable device used for ECG monitoring there were also some technological limitations. Firstly, the fact that the wearable t-shirts need to be washed after used, due to the sweat and transpiration. Secondly, there were two cases where the gel-electrode was displaced due to the transpiration, leading to artifacts in the ECG data. Considering these limitations, our research group has been working on the development of a new device version with a reduced skin patch form-factor (Cunha, 2016). Despite the current limitations, we do believe in the potential of this study, considering that the information we have collected from the firefighters health status could be helpful to prevent limit situations during real duties, caused by extreme stress (overexaustion; heat stress). This information could be sent in real time to fire commanders and this could help on the management of teams in the field. Rodrigues et al. (2018), PeerJ, DOI 10.7717/peerj.5967 Funding This work was supported by National Funds through the FCT Funda¸cão para a Ciência e Tecnologia (Portuguese Foundation for Science and Technology) within the project VR2Market CMUPERI/FIA/0031/2013 and FCT PhD grant PD/BD/135023/2017. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Grant Disclosures The following grant information was disclosed by the authors: The following grant information was disclosed by the authors: National Funds through the FCT Funda¸cão para a Ciência e Tecnologia (Portuguese Foundation for Science and Technology). PD/BD/135023/2017. National Funds through the FCT Funda¸cão para a Ciência e Tecnologia (Portuguese Foundation for Science and Technology). PD/BD/135023/2017. Rodrigues et al. (2018), PeerJ, DOI 10.7717/peerj.5967 CONCLUSIONS In sum, there is some evidence that stress is part of firefighters’ routines, but they may not be truly aware of their stress levels; therefore, they are unlikely to ask for help. Stress in the workplace could be problematic if not managed well (Schultz & Schultz, 2010). Therefore, further attention should be dedicated to firefighters’ occupational health by collecting not only information about what causes stress but also concerning its real impact on psychological and physical health. This study reinforces the importance of firefighter research for the monitoring of stress levels and its potential impact on health and the design of intervention plans and programmes adapted to this population’s real needs. Moreover, the multi-dimensional and complex nature of stress for first responders requires that administrators, and associated policy makers take a corresponding and comprehensive Rodrigues et al. (2018), PeerJ, DOI 10.7717/peerj.5967 14/19 method for the management of stress in these hazardous occupations (Reynolds & Wagner, 2007). Such a comprehensive approach to stress management will improve the well-being, safety, and productivity not only of the individual workers but also the communities they serve. Findings from the current study provide a novel contribute in the area of occupational health, particularly for a better understanding of stress processes. A novel and accurate methodology was developed and its feasibility was showed among hazardous professionals working under real-world environments. List of Abbreviations ANS Autonomic Nervous System AVNN Average of Normal-to-Normal Intervals ECG Electrocardiogram HF High Frequency component HRV Heart Rate Variability LF Low Frequency component LF/HF Ratio of Low Frequency and High Frequency power band PSNS Parasympathetic Nervous System PSS Perceived Stress Scale qOHealth Quantified Occupational Health SNS Sympathetic Nervous System VAS Visual Analogue Scale Supplemental Information Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj 5967#supplemental information Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.5967#supplemental-information. Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.5967#supplemental-information. peerj.5967#supplemental-information. Data Availability The following information was supplied regarding data availability: The raw data are included in the Supplemental Files. The raw data are included in the Supplemental Files. The raw data are included in the Supplemental Files. Field Study Permissions The following information was supplied relating to field study approvals (i.e., approving body and any reference numbers): The following information was supplied relating to field study approvals (i.e., approving body and any reference numbers): Field experiments were approved by the University of Porto Ethical Comission and the Chief Command of the Portuguese firefighter department. Human Ethics The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers): The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers): The Ethical Commission of the University of Porto granted ethical approval to carry out the study (Ethical Application Ref: 29/CEUP/2016). Author Contributions Author Contributions • Susana Rodrigues, Joana S. Paiva and Duarte Dias conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft. • João Paulo S. Cunha conceived and designed the experiments, analyzed the data, contributed reagents/materials/analysis tools, authored or reviewed drafts of the paper, approved the final draft. Rodrigues et al. 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https://openalex.org/W4385361088	https://ejournals.epublishing.ekt.gr/index.php/pnyx/article/download/33658/26300	Greek, Modern	null	Krinagoras and Imperial Glory	Pnyx	2,023	cc-by	16,174	Mitropoulos, G. (2023). Krinagoras and Imperial Glory: An Interplay Between Irony, Mockery, and Flattery. Pnyx: Journal of Classical Studies, 2(1), 1–19. https://doi.org/10.55760/pnyx.2023.33658 Vol 2, No 1 (2023) Vol 2, No 1 (2023) Pnyx: Journal of Classical Studies Krinagoras and Imperial Glory Giorgos Mitropoulos doi: 10.55760/pnyx.2023.33658 Copyright © 2023, Giorgos Mitropoulos This work is licensed under a Creative Commons Attribution 4.0. Keywords Krinagoras; Augustus; imperial court; court poets; Greek epigrams; irony, flattery; mockery; imperial ideology To cite this article: To cite this article: https://epublishing.ekt.gr \| e-Publisher: EKT \| Downloaded at: 24/10/2024 08:17:41 ISSN: 2754-5725 2023 \| Vol. 2\|1, 1-19 DOI: 10.55760/pnyx.2023.33658 Krinagoras and Imperial Glory: An Interplay Between Irony, Mockery, and Flattery Giorgos Mitropoulos Institute of Historical Research, National Hellenic Research Foundation \| National and Kapodistrian University of Athens gmitro@eie.gr Submitted 03/09/2022 Accepted (Internal Review) 07/11/2022 Revised (External Review) 28/12/2022 Published 30/06/2023 Krinagoras and Imperial Glory: An Interplay Between Irony, Mockery, and Flattery Giorgos Mitropoulos Institute of Historical Research, National Hellenic Research Foundation \| National and Kapodistrian University of Athens gmitro@eie.gr Submitted 03/09/2022 Accepted (Internal Review) 07/11/2022 Revised (External Review) 28/12/2022 Published 30/06/2023 Abstract This paper examines two epigrams of the Mytilenean poet Krinagoras (AP 9.562 and 9.224), traditionally interpreted as court flattery examples. Krinagoras was a poet close to the Augustan court, and many of his epigrams praised members of the imperial house. However, a closer examination of these epigrams reveals a discreet dose of irony towards the glory of Augustus, a seemingly strange choice on the part of a court poet of the Augustan circle. It seems that Krinagoras, who travelled from his native island Mytilene to the westernmost part of the Roman Empire in Tarragona as a member of the embassy to meet Augustus, used irony as a stylistic instrument to insinuate his discontent for the hardships he had suffered. The paper contributes to a growing literature that examines the ways literary works functioned as instruments of flattering the ruler but also concealed resentment or mockery, even against the Roman emperor. Krinagoras’ irony is merely one instance among many where subtle mockery and satire of the imperial family were exercised in the early Augustan period. Acknowledgements This paper expands on a reflection expressed by Kostas Buraselis during one of his always thought-provoking lectures at the University of Athens. I want to express my sincere gratitude to him, as well as to Stefanos Apostolou and Manolis Pagkalos for their precious remarks and assistance. Copyright © 2023 \| Giorgos Mitropoulos Giorgos Mitropoulos Krinagoras and Imperial Glory: An Interplay Between Irony, Mockery, and Flattery Giorgos Mitropoulos i Introduction K rinagoras, son of Kallippos, was born in Mytilene around 70 BCE to a family of high social rank, as his participation in embassies sent to Julius Caesar and Augustus indicates (see below). He was next in a line of Mytilenaean elite members and writers associated with Roman power. Before him, Theophanes was an amicus of Pompey, and Potamon was an ambassador to Caesar and Octavian and also wrote encomia for Brutus and Octavian.1 In his lifetime, Krinagoras became one of the poets supported by the domus Augusta. His works (he is attributed with the authorship of 51 epigrams in total) span a rich thematic range of themes well-represented in the Hellenistic epigrammatic tradition, including sepulchral (ἐπιτύμβια), erotic (ἐρωτικά), dedicatory (ἀναθηματικά), and epideictic (ἐπιδεικτικά) epigrams.2 However, the most characteristic feature of the poetry of Krinagoras is his inspiration from personal experiences and observation as material for his epigrams.3 K r r b B After his last embassy to Augustus in 26–25 BCE (see below), Krinagoras resided for many years in Rome. Though details of his residence are lacking, it can be presumed from many references in his epigrams that he was connected with the highest social circles in Rome and enjoyed the support of the Augustan family. Indeed, the poet praised and bestowed fame in his epigrams to Augustus and members of the imperial family, such as the future emperor Tiberius, Marcellus, Germanicus, and Antonia the Younger.4 Many of his poems celebrate fortunate events in the domus Augusta, such as the first shave of Marcellus and the forthcoming birth of a child by Antonia,5 while others glorify military victories, like those of Tiberius in Germany and Armenia.6 Epigram AP 9.224, which is discussed below, reveals Krinagoras’ knowledge of Augustus’ private habits when he travelled, while in ΑP 9.239 the poet offers an intellectual gift to Antonia. Some of these poems were probably written during his residence in Rome and offer insight into his experience of contemporary events during his stay. More specifically, these epigrams reveal his direct knowledge of events associated with the imperial family and his close contacts with many members of the 1 For Potamon as an ambassador to Caesar, together with Krinagoras in 48 BCE and 46–45 BCE (see below on Krinagoras’ embassies): RDGE, n. 26, ll. a, 3–5 and b, ll. 13, 16, respectively. Introduction To Octavian, together with Krinagoras in 26–25 BCE: IG XII, 2, 44, cf. the discussion in Arrayás Morales, 2010: 144–148. Encomia: FGH ΙΙ. B. 147. On the figure of Potamon, see Parker, 1991; Thériault, 2011. h 2 These categories were already established by Meleager, although more existed; Argentieri, 2007: 153; discussion in Ypsilanti, 2018: 3–6. Cogitore (2010: 257) adds a category she styles as ‘imperial epigrams’ (‘épigrammes impériales’) and estimates that around 40 epigrams of this kind exist in the ‘Garland’. h 2 These categories were already established by Meleager, although more existed; Argentieri, 2007: 153; discussion in Ypsilanti, 2018: 3–6. Cogitore (2010: 257) adds a category she styles as ‘imperial epigrams’ (‘épigrammes impériales’) and estimates that around 40 epigrams of this kind exist in the ‘Garland’. g 3 On an introduction to Krinagoras’ life, works and style, Ypsilanti, 2018: 1–14, cf. Cichorius, 1888: 47–61; Bowersock, 1965: 36–37; Labarre, 1996: 99. On the poets’ inspiration from personal experiences, Bowersock, 1964: 255; 1965: 36– 37; Ypsilanti, 2018: 13. g 3 On an introduction to Krinagoras’ life, works and style, Ypsilanti, 2018: 1–14, cf. Cichorius, 1888: 47–61; Bowersock, 1965: 36–37; Labarre, 1996: 99. On the poets’ inspiration from personal experiences, Bowersock, 1964: 255; 1965: 36– 37; Ypsilanti, 2018: 13. 4 APl 61 (Tiberius), AP 6.161 (Marcellus), 9.283 (Germanicus), 9.239 (Antonia). ( ), ( ), 9 3 ( ), 9 39 ( ) 5 AP 6.161 and 6.244 respectively. According to Cogitore (2010: 255, 266–268) such epigrams demonstrate Krinagoras’ closeness to certain members of the Julio-Claudian family and single him out from the other court poets by his proximity to the imperial court. The praise of rulers was rooted firmly in the Hellenistic tradition of kings’ flattery by court poets, and especially the Alexandrian one, where poets like Kallimachos flattered the Ptolemies with their works, e.g., the famous Lock of Berenike and the Deification of Arsinoe (Clayman, 2014 on Berenike; Carney, 2013 on Arsinoe, both with detailed discussions of the poems). For an examination of Krinagoras’ poetry against the backdrop of flattery to Hellenistic rulers by writers, but in new imperial contexts, Buraselis, 2020: 4–6. On the influence of Kallimachos’ Hecale in Krinagoras’ poem addressed to Marcellus (AP 9.545), Bowie, 2008: 231; Höschele, 2019: 479–482. 6 APl 61, cf. Bowie, 2008: 232. ( ), ( ), ( ), ( ) 5 AP 6.161 and 6.244 respectively. Introduction According to Cogitore (2010: 255, 266–268) such epigrams demonstrate Krinagoras’ closeness to certain members of the Julio-Claudian family and single him out from the other court poets by his proximity to the imperial court. The praise of rulers was rooted firmly in the Hellenistic tradition of kings’ flattery by court poets, and especially the Alexandrian one, where poets like Kallimachos flattered the Ptolemies with their works, e.g., the famous Lock of Berenike and the Deification of Arsinoe (Clayman, 2014 on Berenike; Carney, 2013 on Arsinoe, both with detailed discussions of the poems). For an examination of Krinagoras’ poetry against the backdrop of flattery to Hellenistic rulers by writers, but in new imperial contexts, Buraselis, 2020: 4–6. On the influence of Kallimachos’ Hecale in Krinagoras’ poem addressed to Marcellus (AP 9.545), Bowie, 2008: 231; Höschele, 2019: 479–482. 6 APl 61, cf. Bowie, 2008: 232. [2] PNYX 2023 \| Volume 2 \| Issue 1, 1-19 Giorgos Mitropoulos Krinagoras and Imperial Glory: An Interplay Between Irony, Mockery, and Flattery Giorgos Mitropoulos i Augustan house and Augustus himself. Therefore, some researchers have reasonably concluded that Krinagoras was a cliens of the imperial court.7 Krinagoras’ case can be compared to other Greek writers who stayed in Rome close to the Augustan house and other powerful Romans of the time. Presumably, he became acquainted with some of them, for example Parthenios of Nicaea, a freedman of Cinna who was also active in Rome during that time and wrote an elegy entitled Κριναγόρας (Krinagoras). As one of the most famous members of a large circle of Greek writers and poets who lived in Rome and enjoyed the benefits coming from a court that appreciated highly the various fruits of the rich Greek literary and epigrammatic tradition, Krinagoras influenced in various ways the poets Antipatros, Philip (the anthologist of the ‘Garland’), the later Leonidas of Alexandria, even the Latin epigrammatist Martial.8 However, in epigrams AP 9.562 and 9.224, Krinagoras seems to have employed a curiously ironical tone to negotiate Augustan glory. Introduction This paper examines these two epigrams and investigates the reasons behind this seemingly strange choice on the part of a ‘court poet’ of the Augustan circle and interprets Krinagoras as a talented poet who employed both irony and flattery when addressing imperial glory.9 The sense of irony in these two epigrams has been noticed but not studied by other researchers,10 but, most importantly, the interpretation of these epigrams as extremes of flattery remains prevalent.11 Therefore, a thorough analysis of these poems is necessary to challenge this view. lt My elaboration on the skillful deployment of those two instruments, that is, irony and flattery, supplements earlier and recent analytical works of others and aims at exploring the reasons behind this stylistic choice by Krinagoras.12 The present paper discusses some aspects of Krinagoras’ life and works going beyond his interpretation as merely an Augustan court poet or flatterer. The aim is to contribute to a growing literature that examines the ways literary works function as instruments of flattering the ruler that also concealed resentment or mockery, even against Augustus himself.13 Indeed, scholars now acknowledge the complexity in the interpretation of Augustan poets that was previously lost 7 Dimopoulou-Pelioune, 2015: 519; Ypsilanti, 2018: 8–9. Bowersock (1965: 36) thinks that he took residence in Rome immediately after his journey to Tarragona. Bowie (2008: 232) remarks that Krinagoras’ stay in Rome perhaps was not uninterrupted. Οn the relationship between epigram and ‘political power’ in the ancient Greek and Roman world, see the assessment of Coleman, 2019. 8 On the numerous Greek poets and writers who were active in early imperial Rome, Syme, 1978: 107; Bowie, 2008; Cogitore, 2010; Ypsilanti, 2018: 6, 8–14 (with a discussion of similarities between the poetry of Krinagoras, Archias, and Philodemos, the acquaintance of Krinagoras with Parthenios and the influence of Krinagoras on other poets). Notably, among others, was Nicolaos of Damascus, who enjoyed the patronage of Augustus; Strabo, who was protected by Aelius Gallus; Dionysios of Halicarnassos, favoured by Q. Tubero; Antipatros of Thessalonica, who was under the protection of L. Calpurnius Piso Frugi. According to Gow and Page (1968: 210), the impact of Krinagoras’ personality is marked more than that of any other epigrammatist since Kallimachos. 9 It seems that Krinagoras used poignant double entendres in his poems. For example, in an epigram dedicated to Marcellus (AP 6.161), the phrase ἄνδρα λαβεῖν [s.c. 7 Dimopoulou-Pelioune, 2015: 519; Ypsilanti, 2018: 8–9. Bowersock (1965: 36) thinks that he took residence in Rome immediately after his journey to Tarragona. Bowie (2008: 232) remarks that Krinagoras’ stay in Rome perhaps was not uninterrupted. Οn the relationship between epigram and ‘political power’ in the ancient Greek and Roman world, see the assessment of Coleman, 2019. epigrams of Krinagoras have been analysed by Gow and Page, 1968: 210–260; Bowie, 2008: 230–235; 2011: 186 Ypsilanti, 2018. Introduction ἡ Ἰταλία] means that Italy will receive Marcellus as an adult, but may also signify that the patria will take him as husband in a symbolic union between Italy and the heir-apparent of Augustus; cf. Höschele, 2019: 476–477. 9 It seems that Krinagoras used poignant double entendres in his poems. For example, in an epigram dedicated to Marcellus (AP 6.161), the phrase ἄνδρα λαβεῖν [s.c. ἡ Ἰταλία] means that Italy will receive Marcellus as an adult, but may also signify that the patria will take him as husband in a symbolic union between Italy and the heir-apparent of Augustus; cf. Höschele, 2019: 476–477. 10 Ypsilanti, 2018: 10, 243; Buraselis, 2020: 5. Likewise, Bowie (2008: 234–235; 2011: 194–195) expressed doubts over the interpretation of these poems as solely products of flattery. 10 Ypsilanti, 2018: 10, 243; Buraselis, 2020: 5. Likewise, Bowie (2008: 234–235; 2011: 194–195) expressed doubts over the interpretation of these poems as solely products of flattery. lt 11 E.g., Albiani, 2002: 943; see also Cogitore, 2010: 265, n. 43.h 13 Ahl, 1984; Pittore, 2004 (who discusses irony in the epigrams of the Anthologia Palatina, but not Krinagoras); Bertini Conidi, 2012 (on Juvenal’s third Satire); Pandey, 2018. Case-studies of irony in Greek and Latin literature are collected in Reinhold, 2009; among them, Hamm (2009) discusses the use of irony by poets in the Ptolemaic court, such as Kallimachos. In contrast to Krinagoras, Ovid is often labelled as an anti-Augustan poet, mainly because of his exile at the orders of Augustus, but modern research has revealed the intricacies of his case, Philips, 1983; Pandey, 2018. PNYX 2023 \| Volume 2 \| Issue 1, 1-19 [3] Giorgos Mitropoulos Krinagoras and Imperial Glory: An Interplay Between Irony, Mockery, and Flattery Giorgos Mitropoulos i because of simplistic dichotomies and designations as pro- or anti-Augustan poets.14 In this way, new interpretations can be ventured beyond those focusing on mere flattery or opposition to the princeps. In the next section, I discuss an important event in the life of Krinagoras, namely his embassy to Augustus in 26–25 BCE, in which he travelled from Mytilene to the other edge of the Mediterranean Sea, to the remote community of Tarragona in Spain. I argue that this mission was a significant event for Krinagoras because the reasons behind his stylistic choices in the two epigrams lie in the many hardships he had experienced. Introduction Finally, in the third section, I examine the two epigrams of Krinagoras together with AP 9.419, where irony and flattery of the Augustan glory intermingle and illustrate the intricate negotiation of imperial glory in the poems of Krinagoras, essentially an interplay between mockery, irony, and admiration. 14 For example, Philips, 1983: 780–782; Griffin, 1984; and especially Miller (2009: 5): ‘In response to the language of Augustanism, poets of the stature of Horace and Ovid not only mirror or incorporate that language but also, if to varying degrees, contest it in their private visions of the world. […] (Augustan) [p]oets both collaborate and resist’. 15 The first two poems will be commented in the present section, while the last one in the following one, cf. also Ypsilanti, 2018: 3 for the connection of these poems with the journey to Spain. 16 IG 12.2.35a. 17 IG 12.2.35b. 18 On this treaty, RDGE, n. 26; Labarre, 1996: n. 20D; Arrayás Morales, 2010: 138–140. 19 Cf. Gow and Page, 1968: 211–212; Labarre, 1996: 105; Cogitore, 2010: 255; Dimopoulou-Pelioune, 2015: 542; 2017: 407; Ypsilanti, 2018: 9. 20 Suet. Aug. 26.3, 59, 81.1; Plin. HN 25.38, 29.5; Hor. Carm. 3.14; Flor. Epit. 2.33.51; Cassius Dio 53.25.5–7. 20 Suet. Aug. 26.3, 59, 81.1; Plin. HN 25.38, 29.5; Hor. Carm. 3.14; Flor. Epit. 2.33.51; Cassius Dio 53.25.5–7. 14 For example, Philips, 1983: 780–782; Griffin, 1984; and especially Miller (2009: 5): ‘In response to the language of Augustanism, poets of the stature of Horace and Ovid not only mirror or incorporate that language but also, if to varying degrees, contest it in their private visions of the world. […] (Augustan) [p]oets both collaborate and resist’.hi Reaching for the Emperor across the Mediterranean Sea In this section, emphasis will be placed on Krinagoras’ journey to Augustus in Tarragona as a member of the Mytilenean embassy and the personal sufferings he endured on his long journey. I argue that the poet’s experiences significantly influenced his conscious decision to employ irony in two epigrams that treated Augustan glory. In contrast, otherwise and elsewhere, he had nothing but praise for the emperor and his family. Indeed, the journey to Spain served as inspiration for Krinagoras to compose several other poems, such as AP 7.376 (the death of Seleukos), 9.516 (a practice of Ligurian bandits), and 9.419 (on the Baths of Augustus at the Pyrenees).15 Arguably, his journey across the Mediterranean Sea left him a lasting impression, which is worth analysing in depth here.hi The connection of Krinagoras with Roman power began in the period of the first triumvirate when he was a member of the embassy sent by Mytilene to bestow honours upon Caesar after his victory at Pharsala (September 48 BCE).16 He also participated in a second embassy to Caesar, which succeeded in renewing φιλία and συμμαχία between Mytilene and Rome (between April 46 and January/February 45 BCE).17 However, as illustrated below, the most challenging embassy in terms of distance and hardships was undoubtedly the third one of 26–25 BCE, concerning the seal of the foedus aequum between Mytilene and Rome.18 Krinagoras and his last embassy travelled from the far eastern side of the Mediterranean Sea to the westernmost, to Tarragona—an undoubtedly long and harsh adventure.19 The ambassadors made their way there because Augustus was in Hispania at that time, personally commanding the Roman legions in the war against the Cantabri and the Astures (25 BCE); he had to withdraw to Tarragona to recuperate from a severe illness.20 In AP 9.559, Krinagoras asks for the professional help of a renowned geographer to ensure safety for his trip to Italy. The epigram reveals parts of the itinerary of the third embassy: the ambassadors sailed through the Cyclades and Corcyra (“περίπλοον”, ὅς μ’ ἐπὶ νήσους Κυκλάδας ἀρχαίην τ’ ἄξει ἐπὶ 14 For example, Philips, 1983: 780–782; Griffin, 1984; and especially Miller (2009: 5): ‘In response to the language of Augustanism, poets of the stature of Horace and Ovid not only mirror or incorporate that language but also, if to varying degrees, contest it in their private visions of the world. 15 The first two poems will be commented in the present section, while the last one in the following one, cf. also Ypsilanti, 2018: 3 for the connection of these poems with the journey to Spain. 16 IG 12 2 35a 18 On this treaty, RDGE, n. 26; Labarre, 1996: n. 20D; Arrayás Morales, 2010: 138–140. 19 Cf. Gow and Page, 1968: 211–212; Labarre, 1996: 105; Cogitore, 2010: 255; Dimopoulou-Pelioune, 2015: 542; 2017: 407; Ypsilanti, 2018: 9. 20 Suet. Aug. 26.3, 59, 81.1; Plin. HN 25.38, 29.5; Hor. Carm. 3.14; Flor. Epit. 2.33.51; Cassius Dio 53.25.5–7. 17 IG 12.2.35b. y, , ; , ; y , w and Page, 1968: 211–212; Labarre, 1996: 105; Cogitore, 2010: 255; Dimopoulou-Pelioune, 2015: 542; 2017: anti, 2018: 9. 407; Ypsilanti, 2018: 9. 20 Suet. Aug. 26.3, 59, 81.1; Plin. HN 25.38, 29.5; Hor. Carm. 3.14; Flor. Epit. 2.33.51; Cassius Dio 53.25.5–7. 21 On the connection of this epigram with the third embassy, Gow and Page, 1968: 243; Ypsilanti, 2018: 328–329. Eilers (2009) collects essays that discuss aspects of embassies to the emperor and diplomacy in the Roman world. On the sea route from the Aegean Sea to Italy via Corinth, Steinhauer, 2009: 722–723; cf. Strab. 8.6.20 on the importance of Corinth for maritime trade and communications. Bowie (2008: 233–234; 2011: 190) thinks that Krinagoras took the route Corinth – Corcyra – Brundisium. The route Corinth – Buthrotum (opposite to Corcyra) – Brundisium is depicted in the Antonine and Bordeaux Itineraries, Cuntz, 1990. 22 RDGE, n. 26, p. 155. Reaching for the Emperor across the Mediterranean Sea The entire journey, therefore, must have comprised a total of 27 to 45 days of travel time alone, excluding rest periods, dead times, and possibly a stay in Rome. Around the same period, another embassy to Augustus in Tarragona left from Asia Minor led by a person named Chaeremon. Αmbassadors from Tralleis reached the emperor to petition support in ameliorating the damages inflicted on the polis by an earthquake; the later historian Agathias stressed the difficulties of such a long journey. It is worth citing his testimony in full because it allows us a glimpse into the hardships Krinagoras may have faced on his trip to Tarragona: 2 οὕτω δὲ τοῦ ἄστεος οἰκτρότατα κειμένου, ἄγροικόν τινά φασι τούτων δὴ τῶν γεηπόνων, Χαιρήμονα τοὔνομα, σφόδρα τὴν ψυχὴν ἁλῶναι τῷ πάθει, καὶ οὖν οὐκ ἐνεγκόντα θαυμάσιόν τι ἡλίκον καὶ ἄπιστον ἔργον ἀνύσαι. 3μήτε γὰρ τῆς ὁδοῦ τὸ μῆκος ἢ τῆς πρεσβείας τὸ μέγεθος κατορρωδή σαντα μήτε ὅτι μεγίστοις, ὥσπερ εἰκός, ὁμιλήσειν ἤμελλε κινδύνοις, καὶ ταῦτα ἐπ’ ἀδήλῳ τῇ τύχῃ, μήτε τῶν οἴκοι τὴν ἐρημίαν μήτε ἄλλο τι τῶν ὁπόσα διανοούμενοι ἄνθρωποι μεταμανθάνουσι τὰ δοκηθέντα, ἀφικέσθαι μὴ ὅτι ἐν Ῥώμῃ, ἀλλὰ γὰρ καὶ ἐς τῶν Κανταβαρηνῶν τὴν χώραν ἀμφ’ αὐτὰς δή που τὰς τοῦ Ὠκεανοῦ ἠϊόνας (ἐτύγχανε γὰρ αὐτοῦ που ὁ Καῖσαρ ἐν τῷ τότε πρός τι τῶν ἐθνῶν διαμαχόμενος). 2 The story goes that, when the city [sc. Tralleis] lay in a tragic heap of ruins, a certain rustic, a tiller of the soil by the name of Chaeremon was so deeply moved by the calamity that he could bear it no longer and so set out to accomplish an incredible and extraordinary feat (θαυμάσιόν τι ἡλίκον καὶ ἄπιστον ἔργον ἀνύσαι). 3 Deterred neither by the distance involved (τῆς ὁδοῦ τὸ μῆκος), nor by the magnitude of his petition, nor by the dangers he was likely to face (ὅτι μεγίστοις, ὥσπερ εἰκός, ὁμιλήσειν ἤμελλε κινδύνοις), nor indeed by his doubtful chances of success, nor, for that matter, by the fact that he would be leaving his family to fend for themselves, nor by any of the other considerations that lead men to change their minds, he went not just to Rome but to the land of the Cantabri on the very shores of the Ocean. For Caesar was there at that time conducting a campaign against some of the local tribes.25 Agath. Hist. 2.17.2-3 (Trans. t 25 My italics for emphasis. The testimony of Agathias is partly based on the ‘traditional history’ (patria) of the city, as Jones (2011: 109) argues. 23 Dimopoulou-Pelioune, 2015: 542, cf. RDGE, n. 26, pp. 156–157; Arrayás Morales, 2010: 147–148. 24 https://orbis.stanford.edu/ Reaching for the Emperor across the Mediterranean Sea […] (Augustan) [p]oets both collaborate and resist’.hi g , p [ ] ( g ) [p] 15 The first two poems will be commented in the present section, while the last one in the following one, cf. also Ypsilanti, 2018: 3 for the connection of these poems with the journey to Spain. 16 IG 12 2 35 PNYX 2023 \| Volume 2 \| Issue 1, 1-19 [4] Giorgos Mitropoulos Krinagoras and Imperial Glory: An Interplay Between Irony, Mockery, and Flattery Giorgos Mitropoulos Σχερίην). He then must have landed at Brundisium and continued to Rome.21 The two senatus consulta connected with the treaty between Mytilene and Rome are dated between May and June 25 BCE,22 so we can assume that the Mytilenean ambassadors travelled to Rome in the late spring of 25 BCE. Then, they continued their journey to the remote Tarragona in Spain to reach the emperor, either because they decided to submit their request to Augustus himself before the decision of the Senate or to thank him for his approval.23 According to calculations on ORBIS,24 the journey from Mytilene to Rome via Corinth, Corcyra, and Brundisium in early spring could take about 20 days, and the second part of the embassy’s itinerary, from Rome to Tarragona seven days (seaborne) or 25 days (on land and by horse). The entire journey, therefore, must have comprised a total of 27 to 45 days of travel time alone, excluding rest periods, dead times, and possibly a stay in Rome. Σχερίην). He then must have landed at Brundisium and continued to Rome.21 The two senatus consulta connected with the treaty between Mytilene and Rome are dated between May and June 25 BCE,22 so we can assume that the Mytilenean ambassadors travelled to Rome in the late spring of 25 BCE. Then, they continued their journey to the remote Tarragona in Spain to reach the emperor, either because they decided to submit their request to Augustus himself before the decision of the Senate or to thank him for his approval.23 According to calculations on ORBIS,24 the journey from Mytilene to Rome via Corinth, Corcyra, and Brundisium in early spring could take about 20 days, and the second part of the embassy’s itinerary, from Rome to Tarragona seven days (seaborne) or 25 days (on land and by horse). 26 Gow and Page, 1968: 211, 241; Ypsilanti, 2018: 307. Gow and Page (1968: 241) suggest that it was a result of personal observation, while Ypsilanti finds it more likely that Krinagoras heard about this practice while travelling through Liguria. The embassy probably travelled through Marseille, as discussed below. The Ligurians are otherwise mentioned in Roman contexts as opponents in Roman campaigns (e.g., Liv. 40.27.9–13). 27 On the connection of the epigram with the third embassy, Gow and Page, 1968: 224; Labarre, 1996: 105; Dimopoulou, 2015: 542; 2017: 407. On the dangers of embassies on their way and back, Arrayás Morales, 2010: 147, n. 67. Pawlak (2020: 185) unnecessarily rejects the connection between the epigrams of Krinagoras that mention Iberia and this particular journey. It is much more likely that Seleukos passed away during this journey and not in an otherwise unknown trip to Spain. Reaching for the Emperor across the Mediterranean Sea by Gow-Page, with emendations)28 In the epigram, Seleukos, most likely one of the Mytilenean ambassadors to Tarragona,29 is buried on a coast far away from Lesbos, to the ‘outermost Iberians’ in Spain and Krinagoras vividly expresses his sorrow and pain for losing one charismatic young compatriot in this remote place, far away from their πατρίς (‘a stranger on untrodden shores’). The poem’s emphasis lies precisely on the fact that the death 26 Gow and Page, 1968: 211, 241; Ypsilanti, 2018: 307. Gow and Page (1968: 241) suggest that it was a result of personal observation, while Ypsilanti finds it more likely that Krinagoras heard about this practice while travelling through Liguria. The embassy probably travelled through Marseille, as discussed below. The Ligurians are otherwise mentioned in Roman contexts as opponents in Roman campaigns (e.g., Liv. 40.27.9–13). 27 On the connection of the epigram with the third embassy, Gow and Page, 1968: 224; Labarre, 1996: 105; Dimopoulou, 2015: 542; 2017: 407. On the dangers of embassies on their way and back, Arrayás Morales, 2010: 147, n. 67. Pawlak (2020: 185) unnecessarily rejects the connection between the epigrams of Krinagoras that mention Iberia and this particular journey. It is much more likely that Seleukos passed away during this journey and not in an otherwise unknown trip to Spain. p p 28 I chose Patton’s translation over that of Gow and Page in two cases. ‘Speech and character’ in line 3 is closer to the original Greek (μύθοισι καὶ ἤθεσι) than Gow and Page’s ‘words and ways’. The same reasoning led to the selection of ‘uncharted’ (l. 6) over ‘untrodden’, as the adjective ἀμέτρητος means ‘unmeasured’ and ‘uncharted’ conveys this on geographical terms too, since Krinagoras had an interest in geography (cf. AP 9.559). p p 28 I chose Patton’s translation over that of Gow and Page in two cases. ‘Speech and character’ in line 3 is closer to the original Greek (μύθοισι καὶ ἤθεσι) than Gow and Page’s ‘words and ways’. The same reasoning led to the selection of ‘uncharted’ (l. 6) over ‘untrodden’, as the adjective ἀμέτρητος means ‘unmeasured’ and ‘uncharted’ conveys this on geographical terms too, since Krinagoras had an interest in geography (cf. AP 9.559). g g g p y 29 Gow and Page (1968: 224) observe that the pairing of μύθοισι καὶ ἤθεσι (words and ways) in l. Reaching for the Emperor across the Mediterranean Sea by Frendo, 1975: 50) p 23 Dimopoulou-Pelioune, 2015: 542, cf. RDGE, n. 26, pp. 156–157; Arrayás Morales, 2010: 147–148. 24 https://orbis.stanford.edu/ t 25 My italics for emphasis. The testimony of Agathias is partly based on the ‘traditional history’ (patria) of the city, as Jones (2011: 109) argues. PNYX 2023 \| Volume 2 \| Issue 1, 1-19 [5] Giorgos Mitropoulos Giorgos Mitropoulos Krinagoras and Imperial Glory: An Interplay Between Irony, Mockery, and Flattery Krinagoras himself described the difficulties and dangers he went through in his quest to find Augustus. During the journey from Italy to Spain (or vice versa), the embassy made its way through Liguria and over the Alps. In AP 9.516, Krinagoras reproaches a common practice of Ligurian bandits to evade watchdogs while stealing sheep. The poet likely learned of this practice on his way to meet Augustus in Tarragona, as Liguria lay on the main land route from Italy to Spain, or at least heard about it while travelling through the land.26 Indeed, this trick of Ligurian bandits is otherwise unknown, so it can be presumed that knowledge of it was not diffused enough for Krinagoras to have known about it indirectly. By describing the ferocity of the Ligurians, whose territories he most likely traversed, the poet implicitly enhances the dangers of the trip. Moreover, in the course of the long journey to meet the emperor, Krinagoras lost one comrade, Seleukos, to whom he dedicated a moving epigram, the first of the three I discuss in detail:27 δείλαιοι, τί κεναῖσιν ἀλώμεθα θαρσήσαντες ἐλπίσιν, ἀτηροῦ ληθόμενοι θανάτου; ἦν ὅδε καὶ μύθοισι καὶ ἤθεσι πάντα Σέλευκος ἄρτιος, ἀλλ᾽ ἥβης βαιὸν ἐπαυρόμενος ὑστατίοις ἐν Ἴβηρσι, τόσον δίχα τηλόθι Λέσβου κεῖται ἀμετρήτων ξεῖνος ἐπ᾽ αἰγιαλῶν 5 5 Poor fools, why do we wander thus heartened by empty hopes, forgetful of baneful death? Once there existed Seleukos, so perfect in speech and character, yet partaking in youth’s prime for brief, among the outermost Iberians he lies, sundered so far from Lesbos, 5 a stranger on uncharted shores Poor fools, why do we wander thus heartened by empty hopes, forgetful of baneful death? Once there existed Seleukos, so perfect in speech and character, yet partaking in youth’s prime for brief, among the outermost Iberians he lies, sundered so far from Lesbos, 5 a stranger on uncharted shores AP 7.376 (Trans. by Gow-Page, with emendations)28 AP 7.376 (Trans. by Gow-Page, with emendations)28 AP 7.376 (Trans. Reaching for the Emperor across the Mediterranean Sea 3 implies the skills of Seleukos as a diplomat and likely a member of the embassy who died either on his way to meet Augustus in Tarragona or on the way back. 29 Gow and Page (1968: 224) observe that the pairing of μύθοισι καὶ ἤθεσι (words and ways) in l. 3 implies the skills of Seleukos as a diplomat and likely a member of the embassy who died either on his way to meet Augustus in Tarragona or on the way back. [6] PNYX 2023 \| Volume 2 \| Issue 1, 1-19 Giorgos Mitropoulos Krinagoras and Imperial Glory: An Interplay Between Irony, Mockery, and Flattery Giorgos Mitropoulos of the young diplomat occurred far from Lesbos.30 In my view, the first two lines of the poem, and specifically the wanderers trusting in empty hopes, refer to the embassy itself, that long wander across the Mediterranean when the hope to gain benefits from the emperor led to simply ‘let aside’ the fact that death lies everywhere in such a perilous journey. Indeed, Krinagoras uses the first plural person, referring obviously to his companions in the embassy (ἀλώμεθα, θαρσήσαντες, ληθόμενοι). In fact, it may not have been the first time Krinagoras lost a travel companion during state service. Twenty years earlier, during the second embassy of Krinagoras to Caesar at Rome, a boy named Eros, probably the servant of a fellow ambassador, also passed away and was buried on an island east of Ithaca. The poet dedicated a moving epigram to his honour (AP 7.628). Therefore, Seleukos was at least the second casualty of a Mytilenaean embassy in which Krinagoras participated, but the epigram dedicated to him has a much more personal and dramatic tone. Krinagoras does not elaborate on the circumstances of Seleukos’ death. However, the phrase κεῖται ... ἐπ’ αἰγιαλῶν is attested in sepulchral epigrams after losses in shipwrecks.31 If a shipwreck did indeed occur and Krinagoras did not simply employ a common expression, then we must presume that the embassy travelled partly by sea from Rome to Tarragona.32 The philosophical introduction in the first two lines is a common motif in funerary poems preserved in the Anthologia Palatina, and it has been interpreted as an expression of Krinagoras’ pessimistic approach to life.33 However, it is clear that this one draws on his personal experiences. Reaching for the Emperor across the Mediterranean Sea The wanderers’ futile trust in potentially empty imperial assurances, ‘forgetful of ruinous death’ is a lesson Krinagoras painfully experienced in this adventure. He employed a common topos to express his suffering, the death of Seleukos on the way to Tarragona. Indeed, the reference to personal experiences is a characteristic feature of the poetry of Krinagoras.34 The poet gave prominence to subjects deriving from his everyday experiences and often coloured his poems with his sentiments, even at the expense of traditional topoi of the genre.35 Therefore, Krinagoras effectively brings out the sadness of the death of a young Greek in a distant land that has only been introduced into Mytilenean horizons not simply by Roman conquest but by the search for the emperor himself.36 All the hardships endured by the embassy and the death of Seleukos were unfortunate events on their way to the emperor. This is an important point to bear in mind for what follows. f 32 According to Ypsilanti (2018: 187–188), ‘shores’ (ἐπ᾽ αἰγιαλῶν) stand here for ‘land’ generally, so it essentially means that Seleukos is lying in a foreign country. But this interpretation does not agree with the overall context of line 6 (ἀμετρήτων ... αἰγιαλῶν) that states clearly that Seleukos was buried ‘at the shores’, most likely on a shore of Tarragona, cf. rightly Dimopoulou-Pelioune, 2015: 542. 32 According to Ypsilanti (2018: 187–188), ‘shores’ (ἐπ᾽ αἰγιαλῶν) stand here for ‘land’ generally, so it essentially means that Seleukos is lying in a foreign country. But this interpretation does not agree with the overall context of line 6 (ἀμετρήτων ... αἰγιαλῶν) that states clearly that Seleukos was buried ‘at the shores’, most likely on a shore of Tarragona, cf. rightly Dimopoulou-Pelioune, 2015: 542. g p 33 Ypsilanti, 2018: 180–181. Similar introductions appear in AP 7.420 (Diotimos, in which the motif of ‘light hopes’ also appears), 7.519 (Kallimachos), 7.534 (Automedon). Krinagoras’ AP 9.516 also opens up with a gnome, presumably referring again to the events during the trip of the third embassy (Ypsilanti, 2018: 306). Δείλαιος and κενὴ ἐλπὶς are also attested in 9.234, l. 1, again in a personal context (Krinagoras addresses his heart). More examples in Ypsilanti, 2018: 182. 30 Krinagoras makes this clearer by calling Seleukos ξεῖνος, not the land which is more usual (κεῖται ἀμετρήτων ξεῖνος ἐπ᾽ αἰγιαλῶν). Death away from the fatherland is a common topic in sepulchral poems, as also seen in these of Krinagoras (AP 7.371, 7.628, 7.645). 31 E.g., Antipatros, AP 7.286 (l. 2), cf. Xenokritos, AP 7.291 (l. 6). On travel in the Roman period, Casson, 1974: 115–329, cf. the contributions in Niehoff, 2017 on travel in literary works of Roman-period writers. Reaching for the Emperor across the Mediterranean Sea 34 Bowersock (1964: 255) notes that most of his poems are inspired from real-life events and refers to named or nameable personalities, e.g., AP 9.559 (preparations for Krinagoras’ trip to Italy), 9.560 (an earthquake stroked his new house), 6.161 (Marcellus), 9.283 (Germanicus), 9.239 (Antonia), 9.81 (the tyrant Nikias of Kos). 35 Ypsilanti, 2018: 13. 33 Ypsilanti, 2018: 180–181. Similar introductions appear in AP 7.420 (Diotimos, in which the motif of ‘light hopes’ also appears), 7.519 (Kallimachos), 7.534 (Automedon). Krinagoras’ AP 9.516 also opens up with a gnome, presumably referring again to the events during the trip of the third embassy (Ypsilanti, 2018: 306). Δείλαιος and κενὴ ἐλπὶς are also attested in 9.234, l. 1, again in a personal context (Krinagoras addresses his heart). More examples in Ypsilanti, 2018: 182. 34 Bowersock (1964: 255) notes that most of his poems are inspired from real-life events and refers to named or nameable personalities, e.g., AP 9.559 (preparations for Krinagoras’ trip to Italy), 9.560 (an earthquake stroked his new house), 6.161 (Marcellus), 9.283 (Germanicus), 9.239 (Antonia), 9.81 (the tyrant Nikias of Kos). 35 Ypsilanti, 2018: 13. 36 Here, I adapt Bowie, 2008: 234: ‘Crinagoras effectively brings out the sadness of the death of an ephebic Greek in a distant land that has only been brought into Greek horizons by Roman conquest’. 36 Here, I adapt Bowie, 2008: 234: ‘Crinagoras effectively brings out the sadness of the death of an ephebic Greek in a distant land that has only been brought into Greek horizons by Roman conquest’. PNYX 2023 \| Volume 2 \| Issue 1, 1-19 [7] Giorgos Mitropoulos i Giorgos Mitropoulos Krinagoras and Imperial Glory: An Interplay Between Irony, Mockery, and Flattery Eventually, the foedus aequum (‘equal treaty’) between Rome and Mytilene was agreed.37 After the return of the embassy, a decree was issued, according to which divine honours were bestowed upon Augustus.38 Copies of the decree were set up at the temple of Augustus in Pergamon, Actium (the site of the emperor’s victory against Mark Antony and Kleopatra), but also in Brindisium, Marseille, and Tarragona (ll. 12–14). The only reason these copies were set up in Brindisium and Marseille could have been because the embassy travelled through these cities.39 Τhe great honours of Mytilene to the benevolent emperor were proclaimed at important stations along the route of the embassy and at its final destination. Reaching for the Emperor across the Mediterranean Sea i There can be little doubt that Krinagoras participated in the shared joy for imperial favour and divine honours. Indeed, he resided afterwards in Rome, composed many epigrams in honour of numerous members of the imperial family, and became one of the most important Greek poets in the Augustan court. How is it then explained that a pair of his poems express irony against Augustus? ( ) 39 Rightly, Dimopoulou-Pelioune, 2015: 543, n. 317. 37 IG 12.2.35c. 38 IG XII 2, 58, a.1 (ca. 25 BCE). 39 Rightly, Dimopoulou-Pelioune, 2015: 543, n. 317. 40 Texts and English translations of the poems in this section are derived from Ypsilanti, 2018, with emendations. 41 Ypsilanti, 2018: 3, 250, 254, If so, that ‘would make it comparable to the epigram on the goat’ (see immediately below on the latter), cf. Bowie, 2008: 235. The ascription to Krinagoras remains prevalent, cf. also Gow and Page, 1968: 232. Texts and English translations of the poems in this section are derived from Ypsilanti, 2018, with emendations. 41 Ypsilanti, 2018: 3, 250, 254, If so, that ‘would make it comparable to the epigram on the goat’ (see immediately below on the latter), cf. Bowie, 2008: 235. The ascription to Krinagoras remains prevalent, cf. also Gow and Page, 1968: 232. 39 Rightly, Dimopoulou-Pelioune, 2015: 543, n. 317. 40 Texts and English translations of the poems in this section are derived from Ypsilanti, 2018, with emendations. 41 Ypsilanti, 2018: 3, 250, 254, If so, that ‘would make it comparable to the epigram on the goat’ (see immediately below on the latter), cf. Bowie, 2008: 235. The ascription to Krinagoras remains prevalent, cf. also Gow and Page, 1968: 232. 38 IG XII 2, 58, a.1 (ca. 25 BCE). 37 IG 12.2.35c. Mocking Flattery: The Parrot and the She-goat40 Orpheus bid beasts obey him in the mountains; but to you, Caesar, now all birds, unbidden, squawk their addresses. 5 AP 9.562 Krinagoras narrates the adventures of a parrot with a human voice: having learnt to repeat the salutation Ave Caesar (χαῖρε Καῖσαρ), the bird escaped from its cage and taught the exact phrase to all the other birds who then repeated it enthusiastically. The parrot functions as an apostle of Augustan divinity to the natural world.42 The animal kingdom is a simile for humankind, further elevated because it participates in the glorification of Augustus.43 Except for the apparent flattery and glorification of the emperor by Krinagoras, a hint of satirical irony traverses this epigram. Firstly, the parrot is described as a person who speaks in a human voice, has studied extensively and did not forget its learnings (βροτόγηρυς; αἰεὶ δ᾽ ἐκμελετῶν; οὐδ᾽ ἀν᾽ λήθη ἤγαγεν οὐνόματος). It left its cage and escaped to the mountains but still pays allegiance to the emperor. It stands in a superior position to the rest of the birds but remains a servant. A paradoxical interplay is formed between lofty and base, human and animal, that imbues the poem with a dose of fascinating levity.44 The parrot stands in the middle between the human and the animal world and between pomposity (e.g., ἀνθοφυεῖ πτέρυγι) and servility. Furthermore, the adjectives employed for the description of the use of the parrot and its cage are unique or rare (βροτόγηρυς, λυγοτευχής, ἀνθοφυής).45 The use of flowery language for them emphasises the underlying satire. It should also be underlined that the talking parrot became a symbol of the flattering court poet in Latin literature, to describe those who merely recited whatever may benefit them.46 Therefore, another irony of Krinagoras can perhaps be traced to poets of the imperial court, possibly even to himself. Mocking Flattery: The Parrot and the She-goat40 After examining the difficulties Krinagoras experienced in his embassy to Augustus, we can proceed with the analysis of the irony and mockery against the imperial glory that manifest subtly in two Krinagorean poems, and the role the journey to Tarragona may have played in the poet’s stylistic choices. These poems stand in contrast to another epigram of Krinagoras that celebrates the glory of Augustus, also dated after the journey and making reference to Spain. The analysis reveals the literary relationship of the Mytilenean poet with Augustan glory as a complex interplay between irony and flattery. lt An elegant tone of satirical irony can be traced in the elegiac poem AP 9.562 that Ypsilanti dates after 25 BCE, during the residence of the poet in Rome (that is, after his embassy to Augustus in Tarragona), but attributes it to Philip, the anthologist of the ‘Garland’, except if humour was the purpose of the poem.41 As I argue below, humour and irony are traced in the epigram, and thus, the ascription to Krinagoras can be maintained: Ψιττακὸς ὁ βροτόγηρυς ἀφεὶς λυγοτευχέα κύρτον ἤλυθεν ἐς δρυμοὺς ἀνθοφυεῖ πτέρυγι, αἰεὶ δ᾽ ἐκμελετῶν ἀσπάσμασι Καίσαρα κλεινόν οὐδ᾽ ἀν᾽ ὄρη λήθη ἤγαγεν οὐνόματος· ἔδραμε δ᾽ ὠκυδίδακτος ἅπας οἰωνός ἐρίζων τίς φθῆναι δύναται δαίμονι ‘χαῖρ᾽’ ἐνέπειν. Ὀρφεὺς θῆρας ἔπεισεν ἐν οὔρεσιν, ἐς σὲ δέ, Καῖσαρ, νῦν ἀκέλευστος ἅπας ὄρνις ἀνακρέκεται. 5 A parrot, who speaks in human voice, left its wickerwork cage and came to the forest with flower-like wings. As he was always practising ways to embrace glorious Caesar, l g As he was always practising ways to embrace glorious Caesar, 40 Texts and English translations of the poems in this section are derived from Ypsilanti, 2018, with emendations. 41 Ypsilanti, 2018: 3, 250, 254, If so, that ‘would make it comparable to the epigram on the goat’ (see immediately below on the latter), cf. Bowie, 2008: 235. The ascription to Krinagoras remains prevalent, cf. also Gow and Page, 1968: 232. PNYX 2023 \| Volume 2 \| Issue 1, 1-19 PNYX 2023 \| Volume 2 \| Issue 1, 1-19 [8] Giorgos Mitropoulos i Giorgos Mitropoulos Krinagoras and Imperial Glory: An Interplay Between Irony, Mockery, and Flattery he did not forget them even while living in the mountains. And all birds, newcomers to this art, hurried to quarrel over who would be the first to say ‘hail’ to the god. 42 Αnalysis in Buraselis, 2020: 4–5. This is the earliest example of what later became a topos in Latin literature, the theme of birds saluting the emperor. Cf. Whitmarsh, 2013: 152, n. 49 with examples. 43 Ypsilanti, 2018: 253. Augustus is characterised as κλεινός (‘glorious’, l. 3) and in 9.419 as πουλυσέβαστος (‘most eminent’, l. 3; see below), a ἅπαξ λεγόμενον. 44 Cf. the similar remarks in Newlands, 2005: 162 on the parrot in Statius, Silvae 2.4. Similar to Krinagoras, Statius has also been characterised as mere court flatterer, but the poem Silvae 2.4 in which a parrot features again reveals a more complex poetic personality, see Newlands, 2005. 45 Βροτόγηρυς and λυγοτευχής do not appear elsewhere, while ἀνθοφυής once more in an inscription from Chalkis, IG 12, 9, 954 (l. 13). Ypsilanti, 2018: 252–253, also observes the infrequency of the adjectives. 46 Dietrich, 2002: 105–108 (for the parrot in Statius’ Silvae 2.4); Newlands, 2005: 162–165. 47 Buraselis, 2020: 9, n. 12 (comment of Massimo Nafissi); Whitmarsh (2013: 152–153), who also spots irony in the poem and finds it ‘a perfect embodiment of the conflicted approach to the poetic gift dramatised in the patronal poetry of the late Republic and early Empire’, but he did not elaborate on the causes of this stylistic choice. 48 LSJ, s.v. κρέκω, esp. AP 7.191: κίσσα κρέξασα (Archias); Aristoph. Orn. 772: βοὴν πτεροῖς κρέκοντες, cf. the verb κράζω. 46 Dietrich, 2002: 105–108 (for the parrot in Statius’ Silvae 2.4); Newlands, 2005: 162–165. 45 Βροτόγηρυς and λυγοτευχής do not appear elsewhere, while ἀνθοφυής once more in an inscription from Ch 9, 954 (l. 13). Ypsilanti, 2018: 252–253, also observes the infrequency of the adjectives. 42 Αnalysis in Buraselis, 2020: 4–5. This is the earliest example of what later became a topos in Latin literature, the birds saluting the emperor. Cf. Whitmarsh, 2013: 152, n. 49 with examples. g ti, 2018: 253. Augustus is characterised as κλεινός (‘glorious’, l. 3) and in 9.419 as πουλυσέβαστος (‘most eminent’, elow), a ἅπαξ λεγόμενον. 48 LSJ, s.v. κρέκω, esp. AP 7.191: κίσσα κρέξασα (Archias); Aristoph. Orn. 772: βοὴν πτεροῖς κρέκοντες, cf. the verb , ( p ); , 47 Buraselis, 2020: 9, n. 12 (comment of Massimo Nafissi); Whitmarsh (2013: 152–153), who also spots irony in the poem and finds it ‘a perfect embodiment of the conflicted approach to the poetic gift dramatised in the patronal poetry of the late Republic and early Empire’, but he did not elaborate on the causes of this stylistic choice. s in Buraselis, 2020: 4–5. This is the earliest example of what later became a topos in Latin literature, the theme of ting the emperor. Cf. Whitmarsh, 2013: 152, n. 49 with examples. 50 Cf. Buraselis, 2020: 5. Bowie (2008: 235; 2011: 194) traces similarities with Kallimachos’ Lock of Berenike, another famous case of court flattery, in which the lock of queen Berenike II was also catasterised and spoke in the first person (frag. 110), cf. Ypsilanti (2018: 242–243), who also takes note of the similarity. On Kallimachos’ poem, Clayman, 2011; 2014: 97–104. 49 Whitmarsh (2013: 152–153) views AP 9.562 as an allegory of patronage. The salutationes by clientes became a target of mockery in Greek and Latin writers, as for example Plut. Mor. 814D, where it is stated that provincials who sought offices in Rome ‘grew old haunting the doors of other men’s houses’ (οἱ πολλοὶ γηράσκουσι πρὸς ἀλλοτρίαις θύραις); cf. Saller, 1982: 129 for more passages. Other Latin authors sometimes described clientes as parasites, e.g., Juv. Sat. 1.139, 5.1–5; cf. Damon, 1995. 51 Bowersock (1965: 36 with n. 5) argues that Krinagoras wrote AP 9.224 and 9.419 (on the Baths of Augustus at the Pyrenees) when accompanying the emperor on his travels and Ypsilanti (2018: 9, 243) regards it highly probable for 9.224 too, accordingly; Buraselis, 2020: 5. Mocking Flattery: The Parrot and the She-goat40 Moreover, there is a clear antithesis between the parrot with the human voice (βροτόγηρυς) as described in the first line of the poem and the final en-masse cries of all birds (ἅπας ὄρνις ἀνακρέκεται) that supposedly repeated the salutation addressed to Augustus, that is, in imitation of the Roman people who performed the morning salutatio to the emperor.47 Indeed, ἀνακρέκειν, a unicum and a compound of the verb κρέκω, is used to describe the sharp noise (‘squawk’).48 That said, the word was intentionally coined to mock the rest of the birds that merely reproduced (ἀνα-κρέκειν) the initial salutation to the emperor taught to them by the parrot with the human voice. The use of this verb explicitly sets the rest of the birds in an inferior position to the parrot. Opposite the leading 43 Ypsilanti, 2018: 253. Augustus is characterised as κλεινός (‘glorious’, l. 3) and in 9.419 as πουλυσέβαστος (‘most eminent’, l. 3; see below), a ἅπαξ λεγόμενον. PNYX 2023 \| Volume 2 \| Issue 1, 1-19 [9] Giorgos Mitropoulos Krinagoras and Imperial Glory: An Interplay Between Irony, Mockery, and Flattery Giorgos Mitropoulos parrot stands a multitude of birds of unspecified species competing with each other in repeating and crying their salutations to Augustus, a mocking simile of the imperial clientes.49 Therefore, it seems that the poem is intended to satirise the flattery and flatterers of Augustus. The use of lofty language for the birds (ὠκυδίδακτος, ἀνακρέκεται) mocks those new students of high praise taught by a parrot. The poem was composed after the third embassy, in which Krinagoras travelled to Spain to reach the emperor. Arguably, Krinagoras employs irony as a stylistic instrument to insinuate his discontent with the hardships he suffered. The glory of Augustus stretched across the entire natural world, but imperial omnipresence had certain practical drawbacks, as Krinagoras had to chase Augustus to the westernmost part of the Roman Empire. An underlying sense of irony can also be discerned in AP 9.224, a poem about the favourite she- goat of Augustus: Αἶγά με τὴν εὔθηλον, ὅσων ἐκένωσεν ἀμολγεὺς οὔθατα πασάων πουλυγαλακτοτάτην, γευσάμενος μελιηδὲς ἐπεί τ᾽ ἐφράσσατο πῖαρ Καῖσαρ, κἠν νηυσὶν σύμπλοον ἠγάγετο. Ἥξω αὐτίκα που καὶ ἐς ἀστέρας· ᾧ γὰρ ἐπέσχον μαζὸν ἐμὸν, μείων οὐδ᾽ ὅσον Αἰγιόχου. 52 The she-goat compares her master with the Aegis-bearer, who is Zeus himself. Mocking Flattery: The Parrot and the She-goat40 5 Me, the goat with the heavy udders, of all whose breast the dairy-pail has drained, the richest in milk, Caesar, after tasting and praising my honey-sweet cream, took me on his ship as his shipmate. Soon I shall perhaps reach the stars; for he to whom I offered 5 my breast is no lesser to the Aegis-bearer. AP 9.224 The poem demonstrates that Krinagoras knew about the private habits of Augustus when travelling. The Mytilenean poet had personal knowledge that the emperor took his she-goat with him in his travels outside Rome, even onboard his vessel, always to enjoy her milk.50 Hence, also considering that most of Krinagoras’ poems derive from his personal experiences, it is very likely that the poet accompanied Augustus on this trip (see below for possible destinations).51 The emperor’s affection for the animal is impressive, and Krinagoras has her claiming (in the first person) that she will become a constellation at the order of her divine companion.52 Perhaps the 49 Whitmarsh (2013: 152–153) views AP 9.562 as an allegory of patronage. The salutationes by clientes became a target of mockery in Greek and Latin writers, as for example Plut. Mor. 814D, where it is stated that provincials who sought offices in Rome ‘grew old haunting the doors of other men’s houses’ (οἱ πολλοὶ γηράσκουσι πρὸς ἀλλοτρίαις θύραις); cf. Saller, 1982: 129 for more passages. Other Latin authors sometimes described clientes as parasites, e.g., Juv. Sat. 1.139, 5.1–5; cf. Damon, 1995. 51 Bowersock (1965: 36 with n. 5) argues that Krinagoras wrote AP 9.224 and 9.419 (on the Baths of Augustus at the Pyrenees) when accompanying the emperor on his travels and Ypsilanti (2018: 9, 243) regards it highly probable for 9.224 too, accordingly; Buraselis, 2020: 5.h 52 The she-goat compares her master with the Aegis-bearer, who is Zeus himself. PNYX 2023 \| Volume 2 \| Issue 1, 1-19 [10] Giorgos Mitropoulos Krinagoras and Imperial Glory: An Interplay Between Irony, Mockery, and Flattery Giorgos Mitropoulos most striking element of the epigram is that the favourite animal of the princeps will follow him in his ascension to heaven; a telling example of court flattery and homage to the emperor’s affection to the she-goat, which will provide him with milk even in the divine world.53 However, a subtle irony is also detected in this animal apotheosis.54 The image of a she-goat ‘reaching for the stars’ cannot but cause amusement to some readers of the poem, both ancient and modern. AP 9.224 Irony towards divine honours for the emperor can be detected here, and this should not confuse modern researchers, as the criticism of ancient authors against ruler worship was an already established tradition since the Hellenistic age.55 Moreover, even the later imperial-age writers and Roman emperors themselves satirised the imperial cult.56 One recalls the famous satire Apocolocynthosis of Claudius, written by Seneca, or the famous last words of Vespasian in his dying bed: Vae, puto deus fio.57 Moreover, as Ypsilanti justly remarks, the excessive boasting of the goat in different parts of the poem (e.g., ‘I am the goat with the heavy udders, the richest in milk of all whose breast the dairy- pail has drained’, ll. 1–2) ‘slyly subverts the ostensible purpose of the epigram, that is court flattery’.58 Indeed, the goat lays claims to a life parallel to that of Augustus in several ways: she will follow him to heaven; she is superior to any other (πασάων, l. 2) in terms of the richness of her milk, as Augustus excels everyone else in superiority; she is πουλυγαλακτοτάτην (l. 2), which is ironically close to Augustus πουλυσέβαστος in Krinagoras’ AP 9.419 (l. 3);59 she is a σύμπλοος (fellow shipmate) of Augustus, a term which implies equality with the emperor in an ironical tone.60 Ypsilanti notes that both poems AP 9.224 and 9.562 involve an animal whose activities are suggestive of the divinity of the emperor.61 In my view, these affinities between the poems and the interpretation offered here for the causes of Krinagoras’ irony are sufficient for the attribution of AP 9.562 to Krinagoras. This pair of epigrams constitutes an elegant embroidery of irony, mockery, and flattery, prompted by the sufferings of the poet on his long trip to Tarragona. Unfortunately, the epigram of the she-goat cannot be dated with certainty, and the voyage of Augustus mentioned in this poem cannot be identified. However, suppose Krinagoras accompanied the emperor on this trip, as I argue. In that case, it must have occurred most probably after 25 BCE, when he resided in the imperial court in Rome. Indeed, earlier scholarship identified this journey with the emperor’s visit to Greece and Asia Minor in 21–19 BCE. This would have been interesting, as Krinagoras would have returned to familiar places in this case. 57 Suet. Vesp. 23.4. 58 Ypsilanti, 2018: 243; cf. 245, 247. 56 Buraselis, 2020 on approaches to the imperial cult by Greek intellectuals. 53 Likewise, the lock of Berenike ascended to heaven in Kallimachos’ poem, as did the goat that fed Zeus, the lion of Nemea, and the golden ram that carried Phrixos and Helle, Ypsilanti, 2018: 242–243. The catheterisation of Augustus himself was described by the Augustan poets, Hor. Od. 1.2.45 (serus in caelum redeas), cf. Virg. Georg. 4.562 (viamque adfectat Olympo). 54 Buraselis (2020: 5) characteristically ended his analysis on the poem with this remark: ‘One is tempted to comment: an apotheosis of and on four legs’. 60 ‘Σύμπλοος’ as ‘fellow-voyager’ in Hdt. 2.115, 3.41; Ephor. 27J, Plut. Mor. 2.148a. described by the Augustan poets, Hor. Od. 1.2.45 (serus in caelum redeas), cf. Virg. Georg. 4.562 (viamque adfectat Olympo). 54 Buraselis (2020: 5) characteristically ended his analysis on the poem with this remark: ‘One is tempted to comment: an apotheosis of and on four legs’. and the golden ram that carried Phrixos and Helle, Ypsilanti, 2018: 242 243. The catheterisation of Augustus himself was described by the Augustan poets, Hor. Od. 1.2.45 (serus in caelum redeas), cf. Virg. Georg. 4.562 (viamque adfectat Olympo). 54 Buraselis (2020: 5) characteristically ended his analysis on the poem with this remark: ‘One is tempted to comment: an apotheosis of and on four legs’. 60 ‘Σύμπλοος’ as ‘fellow-voyager’ in Hdt. 2.115, 3.41; Ephor. 27J, Plut. Mor. 2.148a. 61 Ypsilanti, 2018: 254, n. 6. p g 55 E.g., Plut. Mor. 360D on the witty retort of king Antigonos Gonatas to claims of his divine nature from his courtier Hermodoros. Plutarch (Alex. 28) also records Lysippos’ accusation against Apelles, because he painted Alexander the Great holding a thunderbolt, an attribute of Zeus. Alexander the Great was also said to have expressed irony and concerns over his own divinity on various occasions (Ath. 6.251C). 53 Likewise, the lock of Berenike ascended to heaven in Kallimachos’ poem, as did the goat that fed Zeus, the lion of Nemea, and the golden ram that carried Phrixos and Helle, Ypsilanti, 2018: 242–243. The catheterisation of Augustus himself was described by the Augustan poets, Hor. Od. 1.2.45 (serus in caelum redeas), cf. Virg. Georg. 4.562 (viamque adfectat Olympo). 61 Ypsilanti, 2018: 254, n. 6. 62 For example, the elegant ironic tone of this poem does not preclude another function as imperial praise; the apotheosis of the she-goat can also refer to Augustus’ sign, capricornus (Αἰγόκερως) and Augustus is compared explicitly to Zeus in the last lines. 64 Suetonius (Aug. 81.1) states that Augustus tried first hot and then cold baths, most likely those mentioned in the poem; cf. Cassius Dio (53.25.7) for Augustus’ retirement in Tarragona due to an illness. 65 Buraselis, 2020: 4. AP 9.224 The she-goat would have followed him in the stars after death, while the body of Seleukos was buried on a beach in a foreign place far away from his homeland (κεῖται ἀμετρήτων ξεῖνος ἐπ᾽ αἰγιαλῶν). It should be underlined here that the ironic tone in both poems is subtle and tactful. These i d fl tt d i h f K i d h l d h ’ animal. The she-goat would have followed him in the stars after death, while the body of Seleukos was buried on a beach in a foreign place far away from his homeland (κεῖται ἀμετρήτων ξεῖνος ἐπ᾽ αἰγιαλῶν). h It should be underlined here that the ironic tone in both poems is subtle and tactful. These epigrams served to flatter and praise the patron of Krinagoras, and the poet exalted the emperor’s superiority and divinity in these literary works.62 Crucially, another poem (AP 9.419), also inspired by the journey of the third embassy (25 BCE) as the reference to the waters of the Pyrenees in Spain illustrates (Πυρήνης ὕδατα μαρτύρια), Krinagoras used nature again (the aforementioned waters) to praise Augustus:63 Κἢν μυχὸν Ὀρκυναῖον ἢ ἐς πύματον Σολόεντα ἔλθῃ καὶ Λιβυκῶν κράσπεδον Ἑσπερίδων Καῖσαρ ὁ πουλυσέβαστος, ἅμα κλέος εἶσιν ἐκείνῳ πάντῃ· Πυρήνης ὕδατα μαρτύρια. Οἷσι γὰρ οὐδὲ πέριξ δρυτόμοι ἀπεφαιδρύναντο λουτρὰ καὶ ἠπείρων ἔσσεται ἀμφοτέρων. Whether to the depths of Ercynaean nooks or to the outermost Soloeis and the fringes of Libyan Hesperides should most august Caesar travel, glory follows him everywhere; the waters of Pyrene are my witness. These, wherein not even the native woodcutters ever bathed, 5 will become baths of both continents. AP 9.419 Whether to the depths of Ercynaean nooks or to the outermost Soloeis and the fringes of Libyan Hesperides should most august Caesar travel, glory follows him everywhere; the waters of Pyrene are my witness. These, wherein not even the native woodcutters ever bathed, 5 will become baths of both continents. A The poem conveys the remoteness of the places where Octavian travelled – and of which Krinagoras had personal knowledge – as among the places mentioned are the baths in the Pyrenean mountains, close to Tarragona where Augustus withdrew to be cured from his illness.64 No sense of irony is detected in this testimony and praise of the long-reaching glory of Augustus. AP 9.224 Krinagoras may have been reminiscent of his hardships during the long journey to Tarragona that claimed the life of a young comrade, Seleukos, while the emperor enjoyed all comforts in this voyage, including the milk of his favourite 53 Likewise, the lock of Berenike ascended to heaven in Kallimachos’ poem, as did the goat that fed Zeus, the lion of Nemea, and the golden ram that carried Phrixos and Helle, Ypsilanti, 2018: 242–243. The catheterisation of Augustus himself was described by the Augustan poets, Hor. Od. 1.2.45 (serus in caelum redeas), cf. Virg. Georg. 4.562 (viamque adfectat Olympo). 54 Buraselis (2020: 5) characteristically ended his analysis on the poem with this remark: ‘One is tempted to comment: an apotheosis of and on four legs’. p g 55 E.g., Plut. Mor. 360D on the witty retort of king Antigonos Gonatas to claims of his divine nature from his courtier Hermodoros. Plutarch (Alex. 28) also records Lysippos’ accusation against Apelles, because he painted Alexander the Great holding a thunderbolt, an attribute of Zeus. Alexander the Great was also said to have expressed irony and concerns over his own divinity on various occasions (Ath. 6.251C). 56 Buraselis, 2020 on approaches to the imperial cult by Greek intellectuals. 56 Buraselis, 2020 on approaches to the imperial cult by Greek intellectuals. 58 Ypsilanti, 2018: 243; cf. 245, 247. 59 Cf. above, n. 43. [11] PNYX 2023 \| Volume 2 \| Issue 1, 1-19 Giorgos Mitropoulos Krinagoras and Imperial Glory: An Interplay Between Irony, Mockery, and Flattery Giorgos Mitropoulos i animal. The she-goat would have followed him in the stars after death, while the body of Seleukos was buried on a beach in a foreign place far away from his homeland (κεῖται ἀμετρήτων ξεῖνος ἐπ᾽ αἰγιαλῶν). It should be underlined here that the ironic tone in both poems is subtle and tactful. These epigrams served to flatter and praise the patron of Krinagoras, and the poet exalted the emperor’s superiority and divinity in these literary works.62 Crucially, another poem (AP 9.419), also inspired by the journey of the third embassy (25 BCE) as the reference to the waters of the Pyrenees in Spain illustrates (Πυρήνης ὕδατα μαρτύρια), Krinagoras used nature again (the aforementioned waters) to praise Augustus:63 animal. 63 AP 9.419, dated c. 25 BCE: Ypsilanti, 2018: 300, 303. ( )i 63 AP 9.419, dated c. 25 BCE: Ypsilanti, 2018: 300, 303. AP 9.224 Krinagoras draws again on his experiences from his journey, but this time to glorify the emperor without any shade of irony. So, how should we interpret the discreet employment of irony and humourous skepticism in Krinagoras’ poems AP 9.562 and AP 9.224 in relation to Augustan glory and divinity? The poem conveys the remoteness of the places where Octavian travelled – and of which Krinagoras had personal knowledge – as among the places mentioned are the baths in the Pyrenean mountains, close to Tarragona where Augustus withdrew to be cured from his illness.64 No sense of irony is detected in this testimony and praise of the long-reaching glory of Augustus. Krinagoras draws again on his experiences from his journey, but this time to glorify the emperor without any shade of irony. So, how should we interpret the discreet employment of irony and humourous skepticism in Krinagoras’ poems AP 9.562 and AP 9.224 in relation to Augustan glory and divinity? Κrinagoras was a skilful master of the Greek language and knew well how to express complex notions elegantly. Accordingly, he employed his poetic talents to praise the imperial house, including Augustus, ‘without becoming cheap in his praise’.65 But at the same time, deeply embodied in the Greek cultural tradition, Krinagoras did not hesitate to compose poems expressing an almost anti- Roman spirit, melancholy, and nostalgic glory of Greek independence. In AP 9.284, Krinagoras expresses sorrow for the settlement of Italian libertini (παλίμπρητοι, ‘slaves’) in Corinth, the ancient city of the Bacchiadai. Elsewhere (AP 9.235, ll. 5–6), hope is expressed that the kingdom of Cleopatra 62 For example, the elegant ironic tone of this poem does not preclude another function as imperial praise; the apotheosis of the she-goat can also refer to Augustus’ sign, capricornus (Αἰγόκερως) and Augustus is compared explicitly to Zeus in the last lines. 6 , p , , 64 Suetonius (Aug. 81.1) states that Augustus tried first hot and then cold baths, most likely those mentioned in the poem; cf. Cassius Dio (53.25.7) for Augustus’ retirement in Tarragona due to an illness. 65 Buraselis 2020: 4 [12] PNYX 2023 \| Volume 2 \| Issue 1, 1-19 Giorgos Mitropoulos Krinagoras and Imperial Glory: An Interplay Between Irony, Mockery, and Flattery Giorgos Mitropoulos i will rise again through the adverb πάλι (again);66 resentment against the Romans through the adjective ὀθνεῖοι (foreign) for them (AP 7.645, l. AP 9.224 5); and there is also the ironic tone in AP 9.224 and 9.562 analysed here. The poetic world of Krinagoras is decisively Greek, as his poems are full of references to the geography, myth, and history of Greece and his topics are derived mainly from the Greek cultural tradition, for example, the Eleusinian Mysteries (AP 11.42); the poems of Anakreon that Krinagoras offers as a gift to Antonia (AP 9.239); or Kallimachos’ Hecale that he offers to Marcellus (AP 9.545).67i ftf However, in the two epigrams discussed here that subtly mock specifically the Augustan glory and divinity, except for whatever role the poet’s ‘patriotic’ sentiments may have played (if any) and the wish to display an independent artistic spirit, the hardships Krinagoras personally suffered in the long and dangerous journey to Tarragona and the loss of a young countryman while wandering ‘κεναῖσιν ἐλπίσιν’ could also be an important factor for his choice to adopt this seemingly strange style. The influence of sufferings when travelling for long, from one side of the Mediterranean Sea to the other, and through unknown and perilous lands in search of the emperor should not be underestimated. A talented poet like Krinagoras, who largely anchored his writings on personal experience, would take advantage of topics, such as the praise of Augustan glory even in the animal world (AP 9.562) and the luxuries Augustus indulged during his travel – in which Krinagoras probably accompanied him (AP 9.224) –, to formulate a distinct, yet subtle ironic tone. Surely, then, it is not a coincidence that both epigrams seem to have been composed after the events of 25 BCE. t We must keep in mind that being a keen supporter of Augustus, as Krinagoras was, did not mean obsequious flattery (adulatio): praise does not necessarily imply servility, and irony does not equal opposition or to an ‘anti-Augustan’ stance.68 The poet could praise the emperor, but he could also subtly, skilfully, and carefully insinuate irony for his glory; epigrams were useful for both purposes, especially scoptic epigrams.69 Indirect irony and wordplay could express complaints and bitterness elegantly. Indeed, many passages of Latin poets, especially Ovid, on the Augustan monuments and symbols (such as the sidus Iulium, the Danaids of the portico on the Palatine Hill, and the triumph) are ambiguous and often on the knife’s edge between flattery and criticism (including irony as a technique of criticism). 68 Clearly, flattery served to the glorification of Augustus and his achievements, as is the case, more straightforwardly, with Horace’s Carmen Saeculare, excerpts of which were even inscribed on marble. However, open adulatio was viewed negatively by contemporaries (Griffin, 1984: 205–213). Ahl (1984: 187–208) discusses figured speech as a way to safely and effectively express veiled criticism of tyrants in terms that rely on reader conclusions, e.g., through double-edged discourse (cf. Pandey, 2018: 28 with a collection of ancient sources). Ahl discusses figured speech in Roman poetry and oratory, although with a different focus (mainly Quintilian against the ‘tyrant’ Domitian). In his opinion, figured speech is an intermediate stage between direct criticism and flattery. There were surely many more intermediate stages, such as the elegant combination of irony and flattery in these epigrams of Krinagoras. 67 For the Greek poetic world of Krinagoras, Bowie, 2011.lti 66 AP 9.235, ll. 5–6: Ἐκ πατέρων εἴη παισὶν πάλι τοῖσιν ἀνάκτων \| ἔμπεδον ἠπείροις σκῆπτρον ἐπ᾽ ἀμφοτέραις (trans. Let the children of kings hold from their fathers a strong rule over both lands again). g 69 Coleman, 2019: 65–69. 67 For the Greek poetic world of Krinagoras, Bowie, 2011.lti 66 AP 9.235, ll. 5–6: Ἐκ πατέρων εἴη παισὶν πάλι τοῖσιν ἀνάκτων \| ἔμπεδον ἠπείροις σκῆπτρον ἐπ᾽ ἀμφοτέραις (tran children of kings hold from their fathers a strong rule over both lands again). , 70 Pandey, 2018: 27–29. Pandey supports that Ovid encouraged his audience to read his texts and search for veiled meanings that criticized Augustus, Pandey, 2018: 24–26, with analysis of specific Ovidian passages. 71 AP 9.224 In this eloquent way, the poets elicited sympathy from like-minded readers while avoiding retaliations from the imperial house.70 Moreover, Augustan court poets encouraged their readers to apply critical thinking to symbols linked to Augustus to allow some discussion and reflection on aspects that the fear of retaliation, or excessive respect for Augustan authority, might otherwise exclude.71 For example, the paradox, which we have already pinpointed in Krinagoras’ epigrams (the talking parrot or the she-goat ascending to the heavens), was consciously used by poets 70 Pandey, 2018: 27–29. Pandey supports that Ovid encouraged his audience to read his texts and search for veiled meanings that criticized Augustus, Pandey, 2018: 24–26, with analysis of specific Ovidian passages. i Contemporary audiences, both Greek and Roman, were educated in the critical study of literature, Konstan, 2006. PNYX 2023 \| Volume 2 \| Issue 1, 1-19 [13] Giorgos Mitropoulos Krinagoras and Imperial Glory: An Interplay Between Irony, Mockery, and Flattery Giorgos Mitropoulos i as a tool to engage the readers actively with the poem and encourage them to interpret it as they wished.72 Roman writers were keen to use satire and irony in their works; one example is Horace in his Satires and Ode 2.4.73 Ovid is also a typical case. Many passages can equally be read as flattering or ironic. For example, it has been argued that Ars Amatoria 1 parodies the Augustan militaristic values espoused by the Forum Augustum, and that Ovid gently satirises Augustan legislation on morality in Amores 2.2. Moreover, it is stated that the description of the deification of Caesar in Metamorphoses 15.745–851 almost parodied Augustus’ hegemony.74 In many passages of the Metamorphoses, Ovid compares Augustus to Jupiter and insists that he is a ‘god on earth’, presumably intending to flatter the emperor.75 Yet, some of the very ideas satirised by Ovid continued to be circulated, precisely because these passages could equally be understood as ‘pro-Augustan’ as well as ironic. Therefore, the epigrams of Krinagoras contain a long-lasting literary topos, where animals provide a humourous, subtle, and safe way to convey social and political criticism.76 By using animals and their relationship to the emperor, their undisputable master, Krinagoras comments indirectly and carefully on the omnipresent imperial glory and the unequal power relations between emperor and subjects, matters that surely dramatically came into his mind during the embassy to Tarragona and the death of Seleukos. 72 Cf. Pittore (2004: 15, 33–35, esp. 35), who argues that the world created by writers distorts reality in ways that correspond and react to the existing social system. Among literary texts, the ironic one possesses most this characteristic attribute of systematic re-invention of facts and values. For example, it assigns people and objects functions that are not normally theirs. In the fictitious world of the ironic text, the atypical, the unexpected, the unorthodox, appears as normal. On their part, readers can decide whether and how much they will accept of the new image of reality the text offers. 73 Hejduk, 2018 and Pelliccia, 2018, respectively. j , , , p y 74 Pandey, 2018: 31, 116 and 74–75, respectively. Cf. also Pandey, 2018: 77–78 on the treatment of the Julian paternity of Augustus by Ovid. 75 Feeney (1991: 219–224) collects and discusses the relevant passages of Metamorphoses, and comments that the comparisons between Augustus and Jupiter elegantly criticised the absolute power of Augustus (Feeney, 1991: 222–223). 76 Newlands, 2005: 153–157 on animal fable, among which features Aesopos in a prominent position. 72 Cf. Pittore (2004: 15, 33–35, esp. 35), who argues that the world created by writers distorts reality in ways that correspond and react to the existing social system. Among literary texts, the ironic one possesses most this characteristic attribute of systematic re-invention of facts and values. For example, it assigns people and objects functions that are not normally theirs. In the fictitious world of the ironic text, the atypical, the unexpected, the unorthodox, appears as normal. On their part, readers can decide whether and how much they will accept of the new image of reality the text offers. 73 Hejduk, 2018 and Pelliccia, 2018, respectively. 74 Pandey, 2018: 31, 116 and 74–75, respectively. Cf. also Pandey, 2018: 77–78 on the treatment of the Julian paternity of Augustus by Ovid. 75 Feeney (1991: 219–224) collects and discusses the relevant passages of Metamorphoses, and comments that the comparisons between Augustus and Jupiter elegantly criticised the absolute power of Augustus (Feeney, 1991: 222–223). 76 Newlands, 2005: 153–157 on animal fable, among which features Aesopos in a prominent position. 77 As Pittore (2004: 14) puts it, the ironical discourse ‘è così costruito per essere ambiguo’. Pittore (2004: 50–52) discusses the role of ambiguity in ironical discourse and argues that ambiguous irony can be more effective than direct language. In this regard, characteristic is the flattery of the fisherman who suggested to Domitian that the fish he caught purposefully swam in his net to honour the emperor’s table, an obvious irony to the rest of us (Juv. 4.69–71; Ahl, 1984: 197–198). 78 See for example the assessment of the so-called Demetrius on the treatment of Aeschines on Telauges as being poised ambiguously between praise and irony, Demetr. Eloc. 291: πᾶσα γὰρ σχεδὸν ἡ περὶ τὸν Τηλαυγῆ διήγησις ἀπορίαν παρέχοι, εἴτε θαυμασμὸς εἴτε χλευασμός ἐστι. Τὸ δὲ τοιοῦτον εἶδος ἀμφίβολον, καίτοι εἰρωνεία οὐκ ὄν, ἔχει τινὰ ὅμως καὶ εἰρωνείας ἔμφασιν. According to the author, this ambiguity cannot be defined exactly as irony, but it possesses a clear affinity to it. 79 Sources collected in Yavetz, 1990: 34–35, as for example Augustus’ advice to Tiberius to tolerate criticism in Suet. Aug. 51.3. According to Pandey (2018: 27–28), Augustus allowed a certain degree of freedom of speech to writers that ultimately confirmed his domination (what Herbert Marcuse termed ‘repressive tolerance’). 80 E.g., Suet. Aug. 85.2; Macrob. Sat. 2.4.1,14; and characteristically Quint. Inst. 6.3.77 on his reaction for the neglect of his cult in Tarragona (cf. Yavetz, 1990: 36–38 for a discussion of Augustus’ sense of humour). Augustus by Ovid. 75 Feeney (1991: 219–224) collects and discusses the relevant passages of Metamorphoses, and comments that the comparisons between Augustus and Jupiter elegantly criticised the absolute power of Augustus (Feeney, 1991: 222–223). 76 Newlands, 2005: 153–157 on animal fable, among which features Aesopos in a prominent position. 77 As Pittore (2004: 14) puts it, the ironical discourse ‘è così costruito per essere ambiguo’. Pittore (2004: 50–52) discusses the role of ambiguity in ironical discourse and argues that ambiguous irony can be more effective than direct language. In this regard, characteristic is the flattery of the fisherman who suggested to Domitian that the fish he caught purposefully swam in his net to honour the emperor’s table, an obvious irony to the rest of us (Juv. 4.69–71; Ahl, 1984: 197–198). 78 See for example the assessment of the so-called Demetrius on the treatment of Aeschines on Telauges as being poised ambiguously between praise and irony, Demetr. Eloc. 291: πᾶσα γὰρ σχεδὸν ἡ περὶ τὸν Τηλαυγῆ διήγησις ἀπορίαν παρέχοι, εἴτε θαυμασμὸς εἴτε χλευασμός ἐστι. Τὸ δὲ τοιοῦτον εἶδος ἀμφίβολον, καίτοι εἰρωνεία οὐκ ὄν, ἔχει τινὰ ὅμως καὶ εἰρωνείας ἔμφασιν. According to the author, this ambiguity cannot be defined exactly as irony, but it possesses a clear affinity to it. 79 Sources collected in Yavetz, 1990: 34–35, as for example Augustus’ advice to Tiberius to tolerate criticism in Suet. Aug. 51.3. According to Pandey (2018: 27–28), Augustus allowed a certain degree of freedom of speech to writers that ultimately confirmed his domination (what Herbert Marcuse termed ‘repressive tolerance’). 80 E.g., Suet. Aug. 85.2; Macrob. Sat. 2.4.1,14; and characteristically Quint. Inst. 6.3.77 on his reaction for the neglect of his l ( f f d f ’ f h ) p 73 Hejduk, 2018 and Pelliccia, 2018, respectively. AP 9.224 Krinagoras, like many other poets and orators of the imperial age, could tweak imperial noses as much as they liked so long as what was said could be interpreted in another, safer way.77 Indeed, the line between flattery and irony is sometimes very thin, and this explains sufficiently why the epigrams treated are still interpreted as ‘extremes of flattery’. After all, the line between flattery and irony sometimes confused even ancient writers.78 On his part, Augustus generally demonstrated tolerance against verbal criticism coming from his political opponents so that he would have accepted the indirect and well-hidden irony in a pair of epigrams of one of his court poets.79 More than that, many anecdotes illustrate the emperor’s good sense of humour, even his ability and tendency for self-mockery.80 After all, Augustus himself does See for example the assessment of the so-called Demetrius on the treatment of Aeschines on Telauges as being poised ambiguously between praise and irony, Demetr. Eloc. 291: πᾶσα γὰρ σχεδὸν ἡ περὶ τὸν Τηλαυγῆ διήγησις ἀπορίαν παρέχοι, εἴτε θαυμασμὸς εἴτε χλευασμός ἐστι. Τὸ δὲ τοιοῦτον εἶδος ἀμφίβολον, καίτοι εἰρωνεία οὐκ ὄν, ἔχει τινὰ ὅμως καὶ εἰρωνείας ἔμφασιν. According to the author, this ambiguity cannot be defined exactly as irony, but it possesses a clear affinity to it. 79 Sources collected in Yavetz, 1990: 34–35, as for example Augustus’ advice to Tiberius to tolerate criticism in Suet. Aug. 51.3. According to Pandey (2018: 27–28), Augustus allowed a certain degree of freedom of speech to writers that ultimately confirmed his domination (what Herbert Marcuse termed ‘repressive tolerance’). i 80 E.g., Suet. Aug. 85.2; Macrob. Sat. 2.4.1,14; and characteristically Quint. Inst. 6.3.77 on his reaction for the neglect of his cult in Tarragona (cf. Yavetz, 1990: 36–38 for a discussion of Augustus’ sense of humour). 80 E.g., Suet. Aug. 85.2; Macrob. Sat. 2.4.1,14; and characteristically Quint. Inst. 6.3.77 on his reaction for the neglect of his cult in Tarragona (cf. Yavetz, 1990: 36–38 for a discussion of Augustus’ sense of humour). [14] PNYX 2023 \| Volume 2 \| Issue 1, 1-19 Giorgos Mitropoulos Krinagoras and Imperial Glory: An Interplay Between Irony, Mockery, and Flattery Giorgos Mitropoulos not play an active role in these poems. He is praised in the epigram of the parrot, and in the she-goat epigram he merely boards the she-goat on his vessel to enjoy her milk while travelling. Conclusions In this paper, I argue that Krinagoras drew inspiration from and anchored his epigrams on his personal experiences. The long and hazardous trip from his native home in Mytilene to Tarragona and the death of a young compatriot along the way would have profoundly impacted him. Something evident in numerous epigrams that can reasonably be associated with this journey and convey the sufferings Krinagoras and the other ambassadors endured. Having established this as a starting point, I interpret the distinct but delicate tone of irony in two epigrams of Krinagoras addressing Augustan glory as a poetic way of conveying criticism. In this regard, it is important to note that the epigrams of the parrot and the she-goat were written after the embassy, and the latter perhaps when the poet accompanied Augustus in one of his travels. The parrot and the she-goat represent flatterers of the emperor, animal symbols that revere Augustus willingly and disseminate his glory. At the same time, both poems undoubtedly praise the emperor. Especially Krinagoras’ epigram of the talking parrot seems to be the earliest exemplar of a tradition of talking birds praising emperors, an eager poetic attempt to flatter them via nature’s indisputable acknowledgement of and submission to their greatness. However, Krinagoras differentiated his work from courtly and slavish speech. The two epigrams provide a form of ‘safe speech’ in which Krinagoras could subtly satirise the excessive efforts of others to flatter Augustus. Though light-touch and discreet, there is an underlying tone of irony in these epigrams; not everyone was meant to understand it fully, and this probably offered sufficient self-protection. Krinagoras took the opportunity to make a personal, not overly positive, comment on the omnipresent Augustan glory and divinity, contemplating and acknowledging his hardships during the long and calamitous journey to the remote Tarragona and the loss of a young compatriot in his task to meet the emperor. There is no reason to interpret these epigrams exclusively as flattery or as covert irony – both apply, as they do in other passages of Augustan poets. Therefore, the paper offers new perspectives on the differentiated interpretations of Augustan glory and the interplay between irony and flattery in the approach of imperial grandeur by a renowned Greek epigrammatist who lived in the imperial court. AP 9.224 He is a distant figure that appears to be glorified and revered by the natural world. Therefore, also considering his intensive contact with Greek and Latin writers and poets and his affinity with poetry,81 he might have enjoyed the elegantly satirical pun in those epigrams of Krinagoras.82 The fact that Augustus took no issue with that ambiguity of flattery and irony adds him to the list of Hellenistic rulers who oscillated between indulgence and realisation of absurdity – but they did indulge in those praises nonetheless. 81 Suet. Aug. 85.2 (on the composition of poems by Augustus), 89.1 (on his affinity with Greek poetry), cf. Griffin, 1984: 204 on the emperor’s affinity with poetry. 82 Cf. the remark in Hamm (2009: 103–104) that irony in the right mixture and dose in literary works gave the ruler a certain freedom to react and ensured that he could still laugh, and maybe he even had to laugh (‘der Herrscher trotzdem lachen konnte, ja vielleicht lachen musste’). Conclusions From this aspect, it would be interesting to examine how far and in which contexts Greek writers of the imperial period employed irony when they referred to Roman principes. Krinagoras’ irony is merely one instance among many where subtle mockery and satire of the imperial family were exercised in the early Augustan period. PNYX 2023 \| Volume 2 \| Issue 1, 1-19 [15] Giorgos Mitropoulos Krinagoras and Imperial Glory: An Interplay Between Irony, Mockery, and Flattery Giorgos Mitropoulos i d Bibliography Ahl, F. 1984. The Art of Safe Criticism in Greece and Rome. The American Journal of Philology, 105(2), 174–208. ni, M.-G. 2002. Krinagoras. In: Cancik, H., Schneider, H., and Lanfester, M. (eds.) Der Neue Pauly, 3, 94 Albiani, M.-G. 2002. Krinagoras. In: Cancik, H., Schneider, H., and Lanfester, M. (eds.) Der Neue Pauly, 3, 943. Argentieri, L. 2007. Meleager and Philip as Epigram Collectors. In: Bing, P. and Bruss, J. S. (eds.) Brill’s Companion to Hellenistic Epigram, Leiden: Brill, 147–164. Argentieri, L. 2007. Meleager and Philip as Epigram Collectors. In: Bing, P. and Bruss, J. S. (eds.) Brill’s Companion to Hellenistic Epigram, Leiden: Brill, 147–164. Arrayás Morales, I. 2010. Diplomacy in the Greek Poleis of Asia Minor: Mytilene’s Embassy to Tarraco. Classica & Medievalia, 61, 127–149. Bertini Conidi, R. 2012. Giovenale: un intellettuale nella Roma imperiale: introduzione, traduzione e commento della Satira. Milano: Prometheus. Bowersock, G. 1964. Anth. Pal. VII 638 (Crinagoras). Hermes, 92, 255–256. Bowersock, G. 1965. Augustus and the Greek World. Oxford: Clarendon Press. Bowie, E. 2008. Luxury Cruisers? Philip’s Epigrammatists between Greece and Rome. Aevum Antiquum, 8, 223–258. Bowie, E. 2011. Men from Mytilene. In: Schmitz, T. and Wiater, N. (eds.) The Struggle for Identity: Greeks and their Past in the First Century BCE, Stuttgart: Franz Steiner Verlag, 181–195. Buraselis, K. 2020. On Greek Intellectuals and the Roman Emperor Cult. Mythos, 14, 1–12. Carney, E. 2013. Arsinoe of Egypt and Macedon: A Royal Life. Oxford: Oxford University Press. Casson, L. 1974. Travel in the Ancient World. Toronto, ON: Hakkert. Casson, L. 1974. Travel in the Ancient World. Toronto, ON: Hakkert. Cichorius, C. 1888. Rom und Mytilene. Leipzig: B. G. Teubner. Clayman, D. 2011. Berenice and her Lock. Transactions of the American Philological Association, 141(2), 229–246. Clayman, D. 2014. Berenice II and the Golden Age of Ptolemaic Egypt. Oxford: Oxford University Press. Cogitore, I. 2010. Crinagoras et les poètes de la couronne de Philippe: la cour impériale romaine dans les yeux des Grecs. In: Savalli-Lestrade, Ι. and Cogitore, I. (eds.) Des Rois au Prince. Pratiques du pouvoir monarchique dans l’Orient hellénistique et romain (Ive siècle avant J.-C.-Iie siècle après J.-C.), Grenoble: ELLUG, Université Stendhal, 253–269. Coleman, K. 2019. Epigram, Society, and Political Power. In: Henriksén, C. (ed.) A Companion to Ancient Epigram, Hoboken, NJ: Wiley, 59–75. Cuntz, O. 1990. Itineraria Romana, vol. Bibliography I: Itineraria Antonini Augusti et Burdigalense; accedit tabula geographica; conspectum librorum recentiorum adiecit Gerhard Wirth, Stuttgart: Teubner. Damon, C. 1995. Greek Parasites and Roman Patronage. Harvard Studies in Classical Philo Damon, C. 1995. Greek Parasites and Roman Patronage. Harvard Studies in Classical Philology, 97, 181–195. Dietrich, J. 2002. Dead Parrots Society. The American Journal of Philology, 123(1), 95–110. Damon, C. 1995. Greek Parasites and Roman Patronage. Harvard Studies in Classical Philology, 97, 181–195. h d Th l f h l l ( ) Dietrich, J. 2002. Dead Parrots Society. The American Journal of Philology, 123(1), 95–110. h Dimopoulou-Pelioune, Α. 2015. Λεσβίων πολιτείαι. Πολίτευμα, θεσμοί και δίκαιο των πόλεων της Λέσβου. Athens: Eurasia. opoulou-Pelioune, A. 2017. Lesbos sous Auguste. Du renouveau des traités à l’apothéose. In: L. Cavalier, L Dimopoulou-Pelioune, A. 2017. Lesbos sous Auguste. Du renouveau des traités à l’apothéose. In: L. Cavalier, L., Ferriès, M. C. and Delrieux, F. (eds.) Auguste et l’Asie Mineure, Bordeaux: Ausonius, 399–412. Ferriès, M. C. and Delrieux, F. (eds.) Auguste et lAsie Mineure, Bordeaux: Ausonius lers, C. (ed.) 2009. Diplomats and Diplomacy in the Roman World. Leiden: Brill. Eilers, C. (ed.) 2009. Diplomats and Diplomacy in the Roman World. Leiden: Brill. Feeney, D. 1991. The Gods in Epic: Poets and Critics of the Classical Tradition. Oxford: Clarendon Press.h Feeney, D. 1991. The Gods in Epic: Poets and Critics of the Classical Tradition. Oxford: Clarendon Press. Frendo, J. 1975. Agathias: The Histories, Corpus Fontium Historiae Byzantinae – Series Berolinensis 2A. Feeney, D. 1991. The Gods in Epic: Poets and Critics of the Classical Tradition. Oxford: Clarendon Press. Frendo, J. 1975. Agathias: The Histories, Corpus Fontium Historiae Byzantinae – Series Berolinensis 2A. Berlin: De Gruyter. J. 1975. Agathias: The Histories, Corpus Fontium Historiae Byzantinae – Series Berolinensis 2A. n: De Gruyter. Gow, A. S. F. and Page, D. L. 1968. The Greek Anthology: The Garland of Philip, and Some Contemporary Epigrams. Cambridge: Cambridge University Press. [16] PNYX 2023 \| Volume 2 \| Issue 1, 1-19 Giorgos Mitropoulos Krinagoras and Imperial Glory: An Interplay Between Irony, Mockery, and Flattery Giorgos Mitropoulos i d Griffin, J. 1984. Augustus and the Poets: ‘Caesar qui cogere posset’. In: Millar, F. and Segal, E. (eds.) Caesar Augustus: Seven Aspects, Oxford: Clarendon Press, 189–218. Griffin, J. 1984. Augustus and the Poets: ‘Caesar qui cogere posset’. In: Millar, F. and Segal, E. (eds.) Caesar Augustus: Seven Aspects, Oxford: Clarendon Press, 189–218. Hamm, U. Bibliography 2009. Zum Phänomen der Ironie in höfischer Dichtung oder: Ironie ist, wenn der Herrscher trotzdem lacht. In: Reinhold, G. F. (ed.) Ironie. Griechische und lateinische Fallstudien, Bochumer Altertumswissenschaftliches Colloquium 80, Trier: Wissenschaftlicher Verlag Trier, 77–105. tt Hejduk, J. 2018. Saepe stilum uertas: Moral and Metrical Missteps in Horace’s Satires. In: Knox, P. E., Pelliccia, H. and Sens, A. (eds.) They Keep it All Hid: Augustan Poetry, its Antecedents and Reception, Berlin: De Gruyter, 63–73. Höschele, R. 2019. Greek Epigram in Rome in the First Century CE. In: Henriksén, C. (ed.) A Companion to Ancient Epigram, Hoboken, NJ: Wiley, 475–490. hele, R. 2019. Greek Epigram in Rome in the First Century CE. In: Henriksén, C. (ed.) A Companion t Ancient Epigram, Hoboken, NJ: Wiley, 475–490. Jones, C. 2011. An Inscription Seen by Agathias. Zeitschrift für Papyrologie und Epigraphik, 179, 107–115. Jones, C. 2011. An Inscription Seen by Agathias. Zeitschrift für Papyrologie und Epigraphik, 179, 107–115.h Konstan, D. 2006. The Active Reader in Classical Antiquity. Argos, 30, 7–18. h Labarre, G. 1996. Les cités de Lesbos aux époques hellénistique et impériale. Paris: de Boccard. Miller, J. 2009. Apollo, Augustus, and the Poets. Cambridge: Cambridge University Press. Newlands, C. 2005. Animal Claquers: Statius Silv. 2.4 and 2.5. In: Batstone, W. W. and Tissol, G. (eds.) Defining Genre and Gender in Latin Literature: Essays Presented to William S. Anderson on His Seventy-Fifth Birthday, Lang Classical Studies 15, New York: Lang, 151–173. Birthday, Lang Classical Studies 15, New York: Lang, 151–173. Niehoff, M. R. (ed.) 2017. Journeys in the Roman East: Imagined and Real. Tübingen: Mohr Siebeck Pandey, N. 2018. The Poetics of Power in Augustan Rome: Latin Poetic Responses to Early Imperial Iconography. Cambridge: Cambridge University Press.t Parker, R. W. 1991. Potamon of Mytilene and his Family. Zeitschrift für Papyrologie und Epigraphik, 85, 115–130. Parker, R. W. 1991. Potamon of Mytilene and his Family. Zeitschrift für Papyrologie und Epigraphik, 85, 115–130. Pawlak, M. N. 2020. Theophanes, Potamon and Mytilene’s Freedom. Electrum, 27, 173–188. 115–130. Pawlak, M. N. 2020. Theophanes, Potamon and Mytilene’s Freedom. Electrum, 27, 173–188. Pawlak, M. N. 2020. Theophanes, Potamon and Mytilene’s Freedom. Electrum, 27, 173–188. h Pelliccia, H. 2018. The Reception of Horace Odes 2.4 in Horace Odes 2.5. In: Knox, P. E., Pelliccia, H. and Sens, A. (eds.) They Keep it All Hid: Augustan Poetry, its Antecedents and Reception, Berlin: De Gruyter, 75–88. Philips, C. R. 1983. Rethinking Augustan Poetry. Ο Κριναγόρας και η αυτοκρατορική δόξα: Μια αλληλεπίδραση μεταξύ ειρωνείας, σάτιρας και κολακείας Ο Κριναγόρας ήταν ένας Έλληνας ποιητής που καταγόταν από την Μυτιλήνη (γεννήθηκε γύρω στο 70 π.Χ.). Έζησε επομένως κατά την Αυγούστεια περίοδο και ήταν μέλος επιφανούς οικογένειας. Του έχουν αποδοθεί συνολικά 51 επιγράμματα που χωρίζονται σε διάφορες κατηγορίες, όπως «ἐπιτύμβια», «ἐρωτικά», «ἀναθηματικά» και «ἐπιδεικτικά». Όμως, το πιο χαρακτηριστικό στοιχείο της ποίησης του Κριναγόρα είναι η έμπνευσή του από προσωπικές εμπειρίες και παρατηρήσεις, που συχνά χρησιμοποιεί ως υλικό για τα επιγράμματά του. Ο Κριναγόρας συμμετείχε ενεργά στα δημόσια θέματα που απασχολούσαν τη Μυτιλήνη. Χαρακτηριστικά, πήρε μέρος σε συνολικά τρεις πρεσβείες προς Ρωμαίους πολιτικούς ηγέτες: δύο στον Καίσαρα (κατά τα έτη 48 και 46/5 π.Χ.) και μία στον Οκταβιανό – Αύγουστο το 26/5 π.Χ. προκειμένου να επισφραγιστεί το foedus aequum μεταξύ της Μυτιλήνης και της Ρώμης. Μετά από την τρίτη πρεσβεία, ο Κριναγόρας έμεινε στη Ρώμη για πολλά χρόνια, όπου συνδέθηκε στενά με τον αυτοκρατορικό κύκλο και ίσως έγινε ποιητής της αυλής του Αυγούστου. Πράγματι, σώζονται πολλά επιγράμματά του, τα οποία είναι αφιερωμένα σε μέλη της αυτοκρατορικής οικογένειας, όπως στον Τιβέριο, τον Μαρκέλλο και την Αντωνία. Σε αυτό το άρθρο, επιχειρώ να ερμηνεύσω ένα παράδοξο φαινόμενο. Ενώ τα περισσότερα επιγράμματα του Κριναγόρα που απευθύνονται ή σχετίζονται με τον Αύγουστο και την οικογένειά του χαρακτηρίζονται προφανώς από έπαινο και κολακεία των αναφερόμενων προσώπων, δύο επιγράμματα φαίνεται να διέπονται από έναν διακριτικό και λεπτό τόνο ειρωνείας. Πιστεύω πως αυτή η ειρωνική χροιά ερμηνεύεται βάσει των δυσχερειών που αντιμετώπισε ο Κριναγόρας κατά την πρεσβεία του προς τον Οκταβιανό, όταν ταξίδεψε από τη Μυτιλήνη στην άλλη άκρη της Μεσογείου, στην Ταρραγόνα της Ισπανίας, όπου βρισκόταν ο αυτοκράτορας. Στη διάρκεια αυτού του μακρινού και δύσκολου ταξιδιού, ο Κριναγόρας έχασε έναν συμπατριώτη του, τον Σέλευκο, τον οποίο θρήνησε σε ένα επίγραμμα. Επομένως, αντλώντας έμπνευση από τις δύσκολες συνθήκες του ταξιδιού, ο Κριναγόρας προχώρησε στη συγγραφή δύο επιγραμμάτων στα οποία στοιχεία ειρωνείας και κολακείας της αυτοκρατορικής δόξας και θειότητας συμπλέκονται στενά. Στο πρώτο μέρος του άρθρου εξετάζω το ταξίδι της πρεσβείας από τη Μυτιλήνη στην Ταρραγόνα. Γνωρίζουμε από επιγράμματα του Κριναγόρα πως η αποστολή διέσχισε τις Κυκλάδες και τη «βάρβαρη» περιοχή της Λιγουρίας, όπου ο Κριναγόρας συνάντησε ή πληροφορήθηκε για μια πρακτική των ντόπιων ληστών, την οποία επίσης περιέγραψε σε ένα επίγραμμα. Κατά τη διάρκεια του ταξιδιού ή στην επιστροφή, πέθανε ο Σέλευκος, την απώλεια του οποίου θρήνησε ο Κριναγόρας. Bibliography Latomus, 42, 780–817. Pittore, M. 2004. L’ironia negli epigrammi dell’Anthologia palatina: tra manipolazione linguistica e allusività. Alessandria: Edizioni dell’Orso. hold, G. (ed.) 2009. Ironie: griechische und lateinische Fallstudien. Trier: Wissenschaftlicher Verlag Trie Reinhold, G. (ed.) 2009. Ironie: griechische und lateinische Fallstudien. Trier: Wissenschaftlicher Verlag Trier. Saller, R. 1982. Personal Patronage Under the Early Empire. Cambridge: Cambridge University Press. gt g Saller, R. 1982. Personal Patronage Under the Early Empire. Cambridge: Cambridge University Press. einhauer, G. 2009. Ιστορική γεωγραφία του αρχαίου κόσμου: Ελλάδα – Ρώμη. Athens: Papadimas. Syme, R. 1978. History in Ovid. Oxford: Clarendon Press. Syme, R. 1978. History in Ovid. Oxford: Clarendon Press. Thériault, G. 2011. Honneurs cultuels et évergétisme: le cas de Potamôn de Mytilène. In: Daviault, Α. (ed.) Mélanges offerts à Pierre Senay, Cahiers des études anciennes Supplément 1, Trois Rivières: Société des études anciennes du Québec, 55–64. Whitmarsh, T. 2013. Greek Poets and Roman Patrons in the Late Republic and Early Empire. In: Whitmarsh, T. (ed.) Beyond the Second Sophistic: Adventures in Greek Postclassicism, Berkeley, CA: University of California Press, 137–153. Yavetz, Z. 1990. The Personality of Augustus: Reflections on Syme’s Roman Revolution. In: Raaflaub, K. A., and Toher, M. (eds.) Between Republic and Empire: Interpretations of Augustus and His Principate, Berkeley, CA: University of California Press, 21–41. h Ypsilanti, M. 2018. The Epigrams of Crinagoras of Mytilene: Introduction, Text, Commentary. Oxford: Oxford University Press. Oxford University Press. [17] PNYX 2023 \| Volume 2 \| Issue 1, 1-19 Giorgos Mitropoulos i d Giorgos Mitropoulos Krinagoras and Imperial Glory: An Interplay Between Irony, Mockery, and Flattery Ο Κριναγόρας και η αυτοκρατορική δόξα: Μια αλληλεπίδραση μεταξύ ειρωνείας, σάτιρας και κολακείας Σύμφωνα με το κείμενο, ο νεαρός άντρας τάφηκε σε μια παραλία της Ισπανίας, «πολύ μακριά από τη Λέσβο» (τόσον δίχα τηλόθι Λέσβου). Έτσι, παίρνουμε μια εικόνα των κινδύνων και των δυσχερειών που αντιμετώπισε η πρεσβεία. Μάλιστα, γνωρίζουμε ότι περίπου την ίδια περίοδο μια πρεσβεία από τις Τράλλεις στάλθηκε στην Ταρραγόνα και ο ιστορικός Αγαθίας (Ἱστορίαι 2.17.2–3) επίσης ανέλυσε τις δυσκολίες ενός τέτοιου ταξιδιού προς αναζήτηση του αυτοκράτορα. Στο δεύτερο μέρος του άρθρου αναλύω τα επιγράμματα AP 9.562 και 9.224 του Κριναγόρα. Το πρώτο επίγραμμα περιγράφει έναν παπαγάλο που αφήνει το κλουβί του και διδάσκει στα πουλιά του δάσους πώς να απευθύνουν χαιρετισμό στον Αύγουστο, ενώ στο δεύτερο επίγραμμα η αγαπημένη κατσίκα του Αυγούστου αναφέρει περήφανα ότι αυτή προμηθεύει τον αυτοκράτορα με γάλα ακόμα PNYX 2023 \| Volume 2 \| Issue 1, 1-19 [18] Giorgos Mitropoulos Krinagoras and Imperial Glory: An Interplay Between Irony, Mockery, and Flattery Giorgos Mitropoulos i και κατά τη διάρκεια των ταξιδιών του και εκφράζει την ελπίδα της ότι σύντομα θα αποθεωθεί, ακολουθώντας τον στον ουρανό. Τα δύο επιγράμματα χρονολογούνται μάλλον μετά από το ταξίδι της τρίτης πρεσβείας (25 π.Χ) και έχει υποστηριχθεί ότι ο Κριναγόρας μπορεί να συνόδευε τον Αύγουστο στο ταξίδι που περιγράφει η κατσίκα του επιγράμματος AP 9.224. και κατά τη διάρκεια των ταξιδιών του και εκφράζει την ελπίδα της ότι σύντομα θα αποθεωθεί, ακολουθώντας τον στον ουρανό. Τα δύο επιγράμματα χρονολογούνται μάλλον μετά από το ταξίδι της τρίτης πρεσβείας (25 π.Χ) και έχει υποστηριχθεί ότι ο Κριναγόρας μπορεί να συνόδευε τον Αύγουστο στο ταξίδι που περιγράφει η κατσίκα του επιγράμματος AP 9.224. Τα δύο επιγράμματα έχουν χαρακτηριστεί ως «αποκορύφωμα της κολακείας», αλλά υπάρχουν ευδιάκριτα, αν και λεπτά, ίχνη ειρωνείας. Ειδικότερα, δεν σατιρίζεται άμεσα ο ίδιος ο Αύγουστος, αλλά ο Κριναγόρας χρησιμοποιεί δύο ζώα για σχολιάσει με ασφάλεια την πανταχού παρουσία και δόξα του Αυγούστου. Αυτό το γεγονός δεν πρέπει να μας παραξενεύει. Υπάρχουν και άλλοι ποιητές, όπως ο Οράτιος και ο Οβίδιος που χρησιμοποίησαν έντεχνους τρόπους για να ειρωνευτούν πτυχές της ιδεολογίας του Αυγούστου χωρίς να θέσουν σε κίνδυνο τόσο το έργο τους όσο και τους ίδιους. Επιπλέον, γνωρίζουμε πως ο ίδιος ο Αύγουστος ήταν γενικά ανεκτικός με την σάτιρα και πολλές ανεκδοτολογικές αφηγήσεις μαρτυρούν την αίσθηση χιούμορ που τον διακατείχε, ακόμα και τον αυτοσαρκασμό του. Επομένως, δεν θα πρέπει να ερμηνευτούν τα επιγράμματα AP 9.562 και 9.224 είτε ως απόπειρες κολακείας του Αυγούστου, είτε ως προσεκτική ειρωνεία. Ο Κριναγόρας και η αυτοκρατορική δόξα: Μια αλληλεπίδραση μεταξύ ειρωνείας, σάτιρας και κολακείας Ισχύουν και τα δύο ταυτόχρονα, όπως συμβαίνει και σε άλλα έργα Αυγούστειων ποιητών. Συνεπώς, το άρθρο αυτό προσφέρει νέες οπτικές πάνω στην διαφοροποιημένη ερμηνεία της Αυγούστειας δόξας και την αλληλεπίδραση μεταξύ ειρωνείας και κολακείας στην προσέγγιση του αυτοκρατορικού μεγαλείου από έναν φημισμένο Έλληνα ποιητή που έζησε στην αυτοκρατορική αυλή. Η ειρωνεία του Κριναγόρα αποτελεί μόνο ένα παράδειγμα μεταξύ πολλών όπου χρησιμοποιούνταν ο διακριτικός σαρκασμός και η σάτιρα της αυτοκρατορικής οικογένειας σε έργα της πρώιμης Αυγούστειας περιόδου. [19] PNYX 2023 \| Volume 2 \| Issue 1, 1-19
https://openalex.org/W2736307986	https://vbn.aau.dk/ws/files/267285766/Framing_Design_to_support_Social_Innovation_The_Open4Citizens_Project.pdf	English	null	Framing Design to support Social Innovation: The Open4Citizens Project	The design journal	2,017	cc-by	6,799	Aalborg Universitet Framing Design to support Social Innovation The Open4Citizens Project Morelli, Nicola; Aguilar, Marc; Concilio, Grazia; Götzen, Amalia De; Mulder, Ingrid; Pedersen, Janice; Torntoft, Louise Klitgaard Published in: Design for Next DOI (link to publication from Publisher): 10.1080/14606925.2017.1352823 Creative Commons License CC BY 4.0 Publication date: 2017 Document Version Publisher's PDF, also known as Version of record Link to publication from Aalborg University Citation for published version (APA): Morelli, N., Aguilar, M., Concilio, G., Götzen, A. D., Mulder, I., Pedersen, J., & Torntoft, L. K. (2017). Framing Design to support Social Innovation: The Open4Citizens Project. In L. Di Lucchio, L. Imbesi, & P. Atkinson (Eds.), Design for Next: Proceedings of the 12th European Academy of Design Conference (pp. 171-184). Routledge. https://doi.org/10.1080/14606925.2017.1352823 Citation for published version (APA): Morelli, N., Aguilar, M., Concilio, G., Götzen, A. D., Mulder, I., Pedersen, J., & Torntoft, L. K. (2017). Framing Design to support Social Innovation: The Open4Citizens Project. In L. Di Lucchio, L. Imbesi, & P. Atkinson (Eds.), Design for Next: Proceedings of the 12th European Academy of Design Conference (pp. 171-184). 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Take down policy If you believe that this document breaches copyright please contact us at vbn@aub.aau.dk providing details, and we will remove access to the work immediately and investigate your claim. Aalborg Universitet Aalborg Universitet Citation for published version (APA): Morelli, N., Aguilar, M., Concilio, G., Götzen, A. D., Mulder, I., Pedersen, J., & Torntoft, L. K. (2017). Framing Design to support Social Innovation: The Open4Citizens Project. In L. Di Lucchio, L. Imbesi, & P. Atkinson (Eds.), Design for Next: Proceedings of the 12th European Academy of Design Conference (pp. 171-184). Routledge. https://doi.org/10.1080/14606925.2017.1352823 The Design Journal An International Journal for All Aspects of Design SSN: 1460-6925 (Print) 1756-3062 (Online) Journal homepage: http://www.tandfonline.com/loi/rfdj20 Date: 09 January 2018, At: 23:55 Downloaded from vbn.aau.dk on: October 24, 2024 Downloaded from vbn.aau.dk on: October 24, 2024 The Design Journal Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=rfdj20 Framing Design to support Social Innovation: The Open4Citizens Project Morelli, Nicolaa; Aguilar, Marcb, Concilio, Graziac, De Götzen, Amaliad; Mulder, Ingride Pedersen, Janicef, Klitgaard Torntoft, Louiseg; Organisation(s): aAalborg University, Denmark; bI2Cat, Barcelona, Spain; cPolitecnico di Milano Italy; dAalborg University, Denmark; eTUD Delft, The Netherlands; fAntropologerne Aps, Denmark; gAalborg University, Denmark Corresponding author e-mail: nmor@create.aau.dk Abstract: In the recent years, new forms of organization have emerged, that have a disruptive power over the existing social and economic system. This phenomenon is challenging the traditional design approach, based on the idea that designers could design services for citizens and public administrations. In the new processes designers and service provider are simply mediating the process of co-creation and supporting the ecosystem for the value creation process. This paper will propose a logical framework for the design action, according to a multi-level structure that includes the value-creation level, in which design is a prerogative of the stakeholders participating in the value-creation action; the level of infrastructuring in which designers use their expert knowledge to support the interaction in the value-creation phase; and the level of governance, in which designers must figure out the structure of the ecosystem in which the value-creation process can be adequately organized and possibly scaled-up. Keywords: Design for Services, Service Dominant Logic, Open4Citizen, open data. Nicola Morelli, Marc Aguilar, Grazia Concilio, Amalia De Götzen, Ingrid Mulder, Janice Pedersen & Louise Klitgaard Torntoft To cite this article: Nicola Morelli, Marc Aguilar, Grazia Concilio, Amalia De Götzen, Ingrid Mulder, Janice Pedersen & Louise Klitgaard Torntoft (2017) Framing Design to support Social Innovation: The Open4Citizens Project, The Design Journal, 20:sup1, S3171-S3184, DOI: 10.1080/14606925.2017.1352823 To link to this article: https://doi.org/10.1080/14606925.2017.1352823 © 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group Published online: 06 Sep 2017. Submit your article to this journal Article views: 91 View related articles View Crossmark data Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=rfdj20 Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=rfdj20 Download by: [Aalborg University Library] Design for Next 12th EAD Conference Sapienza University of Rome 12-14 April 2017 Design for Next 12th EAD Conference Sapienza University of Rome 12-14 April 2017 Design for Next 12th EAD Conference Sapienza University of Rome 12-14 April 2017 © 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. doi: 10.1080/14606925.2017.1352823 MORELLI, NICOLA; DE GÖTZEN, AMALIA; MULDER, INGRID; KLITGAARD TORNTOFT, LOUISE; AGUILAR, MARC; CONCILIO, GRAZIA This type of initiatives enables citizens to organise new forms of value creation in a very independent way, in order to solve everyday problems or to find new forms of social cohesion in a context of growing atomisation of society. Citizens’ problem solving attitude has been recognised as a form of diffuse design (Manzini 2015) that has always existed, but is now becoming highly visible because of the emergence of new tools to communicate, collaborate, interact, exchange and co-create value along peer to peer social structures. This means that both focus and direction of the value creation process for solving several everyday problems are changing; this in turn is challenging the existing value creation mechanisms. From a designer’s perspective this shift is critical, because it questions the essence of the designer’s professional profile: why should designers have a role in those mechanisms of change, and how can they best play that role? The first question could be addressed with the consideration that a large part of the new forms of social interaction and innovation are using technologies, which are the result of a design action. Beside few cases of innovation in limited local contexts, the activation of new mechanisms of social innovation is often based on remote interaction and require citizens’ engagement to understand the technical or service mechanisms they are going to use or change. Several new social initiatives are based on citizens’ capability to use an aggregation of technical resources and infrastructure, which are not necessarily designed for the aims they are used for. Such resources need to be adapted to new forms of interaction, or new platforms and infrastructure could be generated, that support this emerging phenomenon. Hence the need for a design activity or design tools to address this new demand. The second question, concerning the modalities of design interventions in such a context, requires a perspective change from a logic inspired by the industrialisation mechanisms of the last century to a new logic. The industrial logic was based on a linear sequence of value creation actions that ended at the point of sale. The consumer was using (consuming) the value created until the point of sale (Ramirez 1999). The consumer was therefore external to the value creation process. The new logic is instead moving the value creation process towards the user, by giving him/her a role and clear responsibility in value co-production. 1. Introduction New emerging phenomena in the last few decades are posing new challenges in the way we live, use technology and organise our society. The balance between the various components of our society are challenging, new forms of inequality, large migration flows, new ways of organising our cities, new ways of managing our healthcare systems are the emerging results of the rapid change brought together by the convergence of different political social and technological occurrences. The different directions that can be observed when looking at future perspectives of the present situation are often the result of unplanned interactions between different components of our society: local communities are finding new ways to use existing online social networking systems to create offline local communities, as in the case of the Social Streets (http://www.socialstreet.it/). S3171 MORELLI, NICOLA; DE GÖTZEN, AMALIA; MULDER, INGRID; KLITGAARD TORNTOFT, LOUISE; AGUILAR, MARC; CONCILIO, GRAZIA MORELLI, NICOLA; DE GÖTZEN, AMALIA; MULDER, INGRID; KLITGAARD TORNTOFT, LOUISE; AGUILAR, MARC; CONCILIO, GRAZIA MORELLI, NICOLA; DE GÖTZEN, AMALIA; MULDER, INGRID; KLITGAARD TORNTOFT, LOUISE; AGUILAR, MARC; CONCILIO, GRAZIA The perspective shift is therefore from the value chain of the industrial logic to the value constellation (Normann and Ramirez 1994). Goods, which were the end result of a process of value production fully controlled by the producer, are now replaced by a bundle of material and immaterial components that support users to define and co-create the value. In an economic and social system in which the exchange of material goods is becoming less relevant than the exchange of information, knowledge and social value, enterprises can no longer deliver value independently. They can rather offer an aggregation of resources for collaboratively and interactively creating value (Vargo and Lusch 2008). This changes the framework for design action, which is no longer focused on the final good, but rather on the interaction in a value production process. Together with the shift of the locus of value creation towards the user (or customer or citizen), the role of design and production are also shifting. This is particularly evident in the most recent services, such as Uber, Airbnb or even social networks. In those services the basic unit of value is produced by users: Uber’s trips are operated by the drivers as well as Airbnb accommodations are provided by the platform customers; finally, the content of the most diffuse social networks (information, text, videos, photographs) are provided and exchanged among users. The control of the value production process is shifted from producers to users through the creation of infrastructures, that support the users’ interaction (Figure 1). S3172 S3172 Framing Design to support Social Innovation: The Open4Citizens Project Figure 1 Shifting control over value creation processes Product Dominant Fast food IKEA Education AirB&B Social neworks EXPERT DESIGN DIFFUSE DESIGN Service Dominant Producer (Designer) Producer/user User Control Education Downloaded by [Aalborg University Library] at 23:55 09 January 2018 Service Dominant Figure 1 Shifting control over value creation processes Although the value creation depends on the users’ problem solving and design capabilities, expert design does not become irrelevant, but it is rather articulated into different levels: designers can become the trigger of facilitators of the interaction in the value creation process, but they are also responsible for the creation of the infrastructure that would support and trigger interaction. The platforms on which the interaction is happening needs indeed to be accurately designed, in order to make sure that the right process, the most adequate methods, the right people and the right technological infrastructures are provided to support the value creation process. Moreover, for the value creation process to be scalable and reproducible in different and larger contexts, the design of the infrastructure must be configured as an ecosystem in which relevant roles, actors and competences are aggregated and identified. The case proposed in this paper gives a good overview of the articulation of the design action over the different levels of the framework outlined in this section. The following sections will therefore outline the project and propose a clearer framework for design action. Framing Design to support Social Innovation: The Open4Citizens Project 2. A Case: Open4Citizens The Open4Citizens (O4C) project is a valuable ground for exploring this new approach to design. O4C is a project funded by the EU under the H2020 program. The starting point for this project is the increasing availability of large amounts of open data; the project’s aim is to ensure broad citizens recognition of the potential of this type of data as a resource, thus enabling citizens to make meaningful use of open data. In order to achieve this aim, the project is articulated in a number of hackathons, that are open not just to programmers, but also to citizens, interest groups, organisation and public authorities. The O4C team include partners with different competences, including IT expert, anthropologists, Service Designers, Urban Designers, Researchers and Public Servants. Although not all of them would MORELLI, NICOLA; DE GÖTZEN, AMALIA; MULDER, INGRID; KLITGAARD TORNTOFT, LOUISE; AGUILAR, MARC; CONCILIO, GRAZIA MORELLI, NICOLA; DE GÖTZEN, AMALIA; MULDER, INGRID; KLITGAARD TORNTOFT, LOUISE; AGUILAR, MARC; CONCILIO, GRAZIA ORELLI, NICOLA; DE GÖTZEN, AMALIA; MULDER, INGRID; KLITGAARD TORNTOFT, LOUISE; AGUIL ARC; CONCILIO, GRAZIA define themselves as designers, their activity in the project can be considered as a design activity, therefore the paper will refer to them as the designers or the design team. define themselves as designers, their activity in the project can be considered as a design activity, therefore the paper will refer to them as the designers or the design team. define themselves as designers, their activity in the project can be considered as a design activity, therefore the paper will refer to them as the designers or the design team. The design activity in this project is organised around hackathons, i.e. co-design sessions in which citizens can co-design new services, together with IT experts and any other relevant stakeholders. The design team is working to support this co-creation process, by framing the hackathon event in a cycle, including also pre-hackathon meetings, and post-hackathon development and test phases (Figure 2). 2. A Case: Open4Citizens Figure 2 The O4C hackathon cycle Downloaded by [Aalborg Univers Figure 2 The O4C hackathon cycle Figure 2 The O4C hackathon cycle The activity of each of the OpenDataLab is articulated around a cycle of hackathons on specific The thematic focus for the O4C hackathon activities is chosen on the basis of three dimensions: a) the most relevant challenges in the local context, b) the possibility to aggregate an ecosystem of relevant people and c) the availability of data. Starting from the pre-hackathon phase the challenges are explored in detail, the relevant actors are identified and invited to participate and the most relevant datasets are collected, to be used in the hackathon event. While the pre- and post-hack phases consist of different activities, including meetings, workshops, interviews, service jams, coaching and facilitation, the hackathon event in itself is a 2-3 days long co- creation workshop, including citizens, coders, data owners, representatives of interest groups and public institutions. 2.1. The Pilots and the OpenDataLabs The hackathons are organised in 5 different EU pilot locations: Barcelona, Copenhagen, Karlstad, Milan and Rotterdam. The activities in these 5 cities are focusing on different challenges. In Copenhagen team works on how services could better integrate newcomers into the Danish society. S3174 Framing Design to support Social Innovation: The Open4Citizens Project The team in Rotterdam works closely with communities around the self-management of urban park areas. Karlstad zooms-in on healthcare services. In Barcelona the team works to improve the cultural opportunities in the city, and the Milan team is working on transparency in public works in the city. The team in Rotterdam works closely with communities around the self-management of urban park areas. Karlstad zooms-in on healthcare services. In Barcelona the team works to improve the cultural opportunities in the city, and the Milan team is working on transparency in public works in the city. The project vision is that that the activities in the project result in the establishment of a network of OpenDataLabs; i.e. physical and digital meeting places for joint exploration of the value of data and how this makes up an actual resource for residents and interest groups. Ideally the OpenDataLab-platform will enable connections between bottom-up initiatives and top- down policies: Supporting various parties to make better use of open data, through dialogue and co- creation. The primary focus for the various OpenDataLabs, is to raise awareness of data and increasing data literacy, as well as organizing the wider support for innovative ideas and for joint implementation. The scope of OpenDataLabs will not be only about developing apps, but might have a wider scope of economic and social value, contributing to new initiatives, public services, and to new businesses. At the time of the submission of this paper the first hackathon cycle has been hosted in almost all the cities. This gives us the opportunity for some considerations, concerning the process of co-design with data, the organisation of the hackathon cycle, with the tools and methods proposed and the strategic definition of the function of the OpenDataLab. 2.2. Co-designing with data The hackathons of the O4C project are different from most common hackathons. The latter are quite unstructured events in which a group of technical experts with a very high coding capability work together in a full-immersion 2-3 days’ event, to generate new applications. The solutions resulting from such events might be interesting, but most often a large part of the resulting ideas are not implemented, because of the scarce relevance on social contexts they refer to. The O4C hackathon instead, brings together a broader and more varied group of include other participants, such as citizens, interest groups or public authorities and all those who could be seen as the problem holders. Therefore, most of the participants are not familiar with coding, but may instead have a deep knowledge of specific problems or needs. A more structured hackathon process has also been developed, in order to enable full and fruitful participation of such varied and diverse groups of participants. The new process consists of co-design a sequence of phases, from the need definition to the data validation. The Hackathon Starter Kit includes tools to apply in each phase. S3175 S3175 S3176 Framing Design to support Social Innovation: The Open4Citizens Project d use cases Figure 4 Overview of the concepts developed in the "Integration hack" in Copenhagen Figure 4 Overview of the concepts developed in the "Integration hack" in Copenhagen S3175 MORELLI, NICOLA; DE GÖTZEN, AMALIA; MULDER, INGRID; KLITGAARD TORNTOFT, LOUISE; AGUILAR, MARC; CONCILIO, GRAZIA Figure 3 The hackathon starter kit Downloaded by [Aalborg University Library] at 23:55 09 January 2018 Figure 3 The hackathon starter kit Figure 3 The hackathon starter kit As outlined in Figure 3 the O4C hackathon process is articulated in three main phases: Inspiration, Ideation and Implementation. As outlined in Figure 3 the O4C hackathon process is articulated in three main phases: Inspiration, Ideation and Implementation. In the initial phase inspiration cards are used to illustrate well known cases based on open data. This phase, together to the need definition phase have been developed in pre-hack meetings or in service jams, in order to gather information about the datasets that could be used during the hackathon event. Hackathon events are co-design session organised along a sequence of phases, from the need definition to the data validation, in some cases the participants have been able to develop initial prototype to be tested in a post-hackathon phases. The hackathon event should be supported by an online platform including relevant datasets, links to other external datasets and tools to analyse, represent and manage data. The ideation phase included a session of brainstorming and a data validation phase, in which the participants could look into the platform or search for external data sources. Another specific feature of the O4C hackathon concerns the access and use of open data. Although a large number of open datasets is now available, the retrieval of relevant data for the proposed challenges is not an easy exercise, due to the lack of specific data and the different formats used for the publication of the available data. The O4C team is developing and experimenting with an online platform including processual and digital tools to integrate data into the design activity. The platform as such includes links to relevant datasets, links to other external datasets and various selected tools to analyse, represent, visualize and manage data. Depending on the quality and quantity of datasets available in each pilot, the participants were able to work in the implementation phase of the hackathon by developing concepts (Figure 4), graphical interface prototypes or even partly functioning apps. 2.3. Design in the co-production phase In the hackathon event the value creation activity was in the hands of the participants, who worked in groups that included people with different competences. The results of this activity depended very much on the composition of the design group and the competences of the participants. The presence of public authority representative in Rotterdam, for instance, opened a dialogue between the various city-makers and enabled better ways of articulating data requests, which seem to be crucial to ensure meaningful reuse of open data. The presence of the data owners in the Barcelona, instead was determinant to orient the outcome towards challenges that could better use the available datasets. The role of designers in this process was to support, stimulate, inspire and trigger interaction and co- creation in the group. The inspiration cards (Figure 5), used in the early phase, were useful to introduce the discussion about open data. The cards included description of commonly used services, such as public transport apps, which opened the citizens’ eyes about the potential of open data as a new resource. S3177 S3177 MORELLI, NICOLA; DE GÖTZEN, AMALIA; MULDER, INGRID; KLITGAARD TORNTOFT, LOUISE; AGUILAR, MARC; CONCILIO, GRAZIA MORELLI, NICOLA; DE GÖTZEN, AMALIA; MULDER, INGRID; KLITGAARD TORNTOFT, LOUISE; AGUILAR, MARC; CONCILIO, GRAZIA MORELLI, NICOLA; DE GÖTZEN, AMALIA; MULDER, INGRID; KLITGAARD TORNTOFT, LOUISE; AGUILAR, MARC; CONCILIO, GRAZIA Figure 5 Example of an inspiration card Figure 5 Example of an inspiration card Figure 5 Example of an inspiration card Figure 5 Example of an inspiration card 2.4. Infrastructuring the co-design process Although the design team did not have direct control in the value co-production process, they had a critical role in the creation of the infrastructure that facilitated co-production. Beside the above mentioned hackathon starter kit, the preparation of the hackathon also consisted in the definition of an online repository of data and the aggregation of a relevant ecosystem of stakeholders in the pre-hack phase and the facilitation of prototype development in the post-hack phase. The digital OpenDataLab platform includes relevant datasets, links to external datasets, digital collaboration tools, or app development tools, that facilitate data visualisation and coding (Figure 6). g p p g tools, or app development tools, that facilitate data visualisation and coding (Figure 6). Figure 6 The home page of the OpenDataLab online platform (www.opendatalab.eu The process of infrastructuring innovation in the O4C process consisted in aggregating resources to support the co-production process, including: a) relevant technological infrastructures, such as data, visualisation tools, links to relevant design tools, b) an ecosystem of relevant stakeholders, who are able to contribute to the co-design process from different perspectives, with different competences Figure 6 The home page of the OpenDataLab online platform (www.opendatalab.eu gure 6 The home page of the OpenDataLab online platform (www.opendatalab.eu Figure 6 The home page of the OpenDataLab online platform (www.opendatalab.eu The process of infrastructuring innovation in the O4C process consisted in aggregating resources to support the co-production process, including: a) relevant technological infrastructures, such as data, visualisation tools, links to relevant design tools, b) an ecosystem of relevant stakeholders, who are able to contribute to the co-design process from different perspectives, with different competences The process of infrastructuring innovation in the O4C process consisted in aggregating resources to support the co-production process, including: a) relevant technological infrastructures, such as data, visualisation tools, links to relevant design tools, b) an ecosystem of relevant stakeholders, who are able to contribute to the co-design process from different perspectives, with different competences S3178 S3178 Framing Design to support Social Innovation: The Open4Citizens Project and with different motivations to develop and implement innovation and c) organisational tools, such as toolkits, information and communication tools, that support the interaction among stakeholders and technological elements in the ecosystem. 2.5. Setting the framework for innovation The role of the design team in the O4C project is not limited to the facilitation of innovation along the hackathon process, it also defines the framework for a permanent physical or virtual infrastructure that will provide knowledge and services to facilitate innovation and design with open data. The OpenDataLab (ODL) is the most tangible instance of this framework. The vision of it is inspired to the concept of FabLabs, which are physical places where materials are turned into products. Fablabs are the places where common citizens, usually unfamiliar with the coding language of 3D printers, can meet IT experts and exchange knowledge, create partnerships and co-create new solutions. Like Fablabs, the ODL will represent an opportunity for citizens to concretely develop their ideas on new services based on open data. It is envisioned to be places where different stakeholders, knowledges and motivations come together to design and develop new services - making meaningful use of data. A key activity of the ODL platform will be to work as a local advocate to push the demand for new datasets that could be created either by opening existing sources, or by transforming existing data into more suitable format or by collecting new crowdsourced data. An ODL consists of a) a physical location, that represents a point of reference for innovation based on open data, b) the body of expert knowledge capable of bringing citizens, coders and other relevant stakeholders together, supporting the design process, the organisation and facilitation of events and assisting start-ups with funding opportunities, c) an online platform that includes links to existing datasets, a repository of open datasets generated for previous projects, as well as a curated/recommended collection of tools to support data-driven design. Overall, the physical and online instances of the ODLs are supporting different scenarios of interaction between their users (Figure 7): S3179 MORELLI, NICOLA; DE GÖTZEN, AMALIA; MULDER, INGRID; KLITGAARD TORNTOFT, LOUISE; AGUILAR, MARC; CONCILIO, GRAZIA Figure 7 Different scenarios of interaction among users in the OpenDataLab Downloaded by [Aalborg University Library] at 23:55 09 January 2018 Figure 7 Different scenarios of interaction among users in the OpenDataLab The online ODL platform will work as a repository of available datasets (lower-left quadrant), or a portal to access to other external datasets; in this case the online ODL platform will support more conventional hackathons, i.e. 2.5. Setting the framework for innovation hackathons in which coders and citizens will be able to develop new apps. The ODL platform is possibly providing plug’n play tools that facilitate visualisation and data elaboration. The online ODL platform will transform existing datasets that have been published in unsuitable format and will also support and organise the collection of crowdsourced data related to specific themes (higher-left quadrant) The physical instance of the OpenDataLab will be a place where design activities are organised, for creating new service solutions based on open data; such activities include Open Data jams, organisation of data crowdsourcing and crowdfunding. In this sense the ODL will work as an incubator of new ideas and will reinforce citizens’ awareness of the potential of open data (upper- right quadrant). Finally, the ODL will be a physical place where citizens, hackers and other relevant stakeholders can meet to develop ideas based on existing datasets. In this sense the physical ODL will have the same function of hub for innovation as the Fablabs (lower-right quadrant). When setting up the ODL as an innovation framework, the design team contributes by providing strategic design knowledge, to suggest concrete perspectives, to represent a map of possibilities and to aggregate an ecosystem of relevant stakeholders. 3. Discussion The role of designers in facilitating participatory processes and social innovation has been discussed for a long time and in several occasions. Sanders and Stappers (2008) provide a complete overview of the way the inclusion of users has been discussed for several decades, in the discipline of design software design, in participatory processes and Co-design. Sanders and Stappers observe how the S3180 Framing Design to support Social Innovation: The Open4Citizens Project maturation of this approach from a user centred approach to a more active involvement of users into the design process has challenged both the ‘expert’ mindset and the power structures of companies, by relinquishing control to potential customers, users and consumers. This is clearly a hard challenge, which has often focused the discussion on how to capture and integrate users’ knowledge into the design process (von Hippel 1994, von Hippel 1998), how to trigger and support users’ design capability (Kimbell 2013) and how to provoke users’ own design capabilities with different forms of prototyping (Hillgren, Seravalli et al. 2011). Inspired by Vargo and Lush(2004), Kimbell (2011) observes that in a new service dominant logic the value production activity, and consequently the control of the design process, is passed on to users, and the designer can no longer design services but rather design for services, that means aggregating resources to support value creation in use. This perspective has also animated the debate on design for social innovation. Manzini (2015) reflects on the role of designers, when the activity of design is no longer fully in control of the designers but is passed on to users. When new solutions come from the spontaneous initiatives of citizens and from a diffuse design capability, the role of the ‘expert’ designer need to be more clearly defined. Downloaded by [Aalborg University Library] at 23:55 09 January 2018 At the same time Bjorgvinsson et al (2010) and Hillgren et al (2011) are looking beyond the traditional project based approach, towards an activity of infrastructuring, to build framework conditions, such as mutual trust relationships, that could extend the design action over time and consolidate social innovation processes. Downloaded by [Aalborg University Library] at 23:55 0 The lesson learned in the O4C project may cast more light on this debate, as it represents a design action distributed on three logical levels, a) the value co-production level, b) the level of ‘infrastructuring’ the value co-production and c) the level of governance of the ecosystem (Morelli and De Götzen 2016). a) A first, visible level is the value co-production process, during the hackathon. This is the process of interaction among citizens, IT experts, public authorities and other stakeholders for the creation of new solutions. S3180 The interaction at this level is depends on the stakeholders’ diffuse design capability, generated by the negotiation among different problem solving attitudes and strategies of the participants. The designers’ problem solving capabilities are just one of the components of this negotiation. At this level the designers’ contribution consists in the use of inspiration cards, prototypes, visual representations and any other tool that can support the design process. The outcome of the collaborative design activity at this level are the design solutions generated by the heterogeneous competences involved in the project. b) The second logical level includes the generation of the infrastructure to support diffuse design, that includes the definition of blueprints, templates, modular structures and working frameworks (such as the hackathons themselves). This level makes large use of expert design, that means the various competences included in the design team, from the technical expert to the anthropologist, and, of course, the designers. The outcome of the design activity at this level consists in the organisation of the hackathon cycle and the body of knowledge for a systematic application of models, tools and IT infrastructures (the O4C online platform) that can be used in the hackathon. MORELLI, NICOLA; DE GÖTZEN, AMALIA; MULDER, INGRID; KLITGAARD TORNTOFT, LOUISE; AGUILAR, MARC; CONCILIO, GRAZIA c) The third level consists of the definition of the ecosystem of actors, technical infrastructures and conditions to make the design action scalable and reproducible in different contexts. In the O4C project this logical level includes the definition of the OpenDataLab, its scenarios, conditions of use and the stakeholders that are crucial for its definition (Figure 7). The design knowledge needed at this level refers to the capability to understand the socio-technical implications of the proposed transformations into the ecosystem around the OpenDataLAb: Therefore it implies a wider, transdisciplinary and systemic perspective of a specific problem area and the relevant ecosystem. Beside the OpenDataLab, the outcome of the design activity at this level are frameworks for action and policies (Table 1). c) The third level consists of the definition of the ecosystem of actors, technical infrastructures and conditions to make the design action scalable and reproducible in different contexts. In the O4C project this logical level includes the definition of the OpenDataLab, its scenarios, conditions of use and the stakeholders that are crucial for its definition (Figure 7). The design knowledge needed at this level refers to the capability to understand the socio-technical implications of the proposed transformations into the ecosystem around the OpenDataLAb: Therefore it implies a wider, transdisciplinary and systemic perspective of a specific problem area and the relevant ecosystem. Beside the OpenDataLab, the outcome of the design activity at this level are frameworks for action and policies (Table 1). Table 1 The levels of design activities in O4C Who designs? Design knowledge Outcome Value in use Users, citizens, IT experts, public servants Diffuse design: Citizens’ problem solving capabilities Apps and new services Citizens’ awareness and empowerment Infrastructuring The design team Expert design: based on disciplinary professional knowledge Online O4C platforms Hackathon starter kit Hackathon cycles Ecosystem The design team Transdisciplinary and Strategic design: design team’s collaborative understanding of the socio-technical ecosystem around the opendatalab OpenDataLab Framework for action, policies Downloaded by [Aalborg Univers S3181 S3181 MORELLI, NICOLA; DE GÖTZEN, AMALIA; MULDER, INGRID; KLITGAARD TORNTOFT, LOUISE; AGUILAR, MARC; CONCILIO, GRAZIA MORELLI, NICOLA; DE GÖTZEN, AMALIA; MULDER, INGRID; KLITGAARD TORNTOFT, LOUISE; AGUILAR, MARC; CONCILIO, GRAZIA 4. Conclusions The present socio-technical systems are bringing about radical changes in the mechanisms of value production and call for a substantial revision of the role of the stakeholders in the production system. Users are no longer passive receivers of the offerings of enterprises. Enterprises are no longer in full control of the value production process and the emerging design practices are no longer aimed at designing well defined outcomes (products or services) but rather at designing for a purpose, i.e. designing for services or for supporting value production. In the debate about this new direction of design practice it is certainly important to develop tools and new practices that activate, empower and support users in the process of value production. Several contributions already focus on how to ensure the interaction of the user in processes of value co-production. At the same time several authors have also invited reflection about a broader view of the nature and role of design activities and their capability to support, codify, organise the value co-production process and possibly to scale it up beyond the single interaction instance. The case illustrated in this paper is a contribution to the broader view, providing a logical framework for the interpretation and qualification of design practices at different levels and related to different S3182 Framing Design to support Social Innovation: The Open4Citizens Project design knowledges: diffuse design, expert design and strategic design. Such framework can be a valuable starting point for refining and making explicit the possible impact offered by design and designers in shaping the next society..). design knowledges: diffuse design, expert design and strategic design. Such framework can be a valuable starting point for refining and making explicit the possible impact offered by design and designers in shaping the next society..). Bibliography Björgvinsson, E., P. Ehn and P.-A. Hillgren (2010). Participatory design and “democratizing innovation”. PDC2010, Sydney, Australia. Björgvinsson, E., P. Ehn and P.-A. Hillgren (2010). Participatory design and “democratizing innovation”. PDC2010, Sydney, Australia. Bollier, D. (2016). "Making Networked Sharing Socially Beneficial, Not Just Predatory and Profitable." Common Transition http://blog.p2pfoundation.net/making-networked-sharing-socially-beneficial- not-just-predatory-and-profitable/2016/01/28 2016. Bødker, S., K. Grønbæk and M. Kyng (1993). Cooperative Design, Techniques and Experiences from the Scandinavian Scene. Participatory Design. Principles and Practices. D. Shuler and A. Namioka. Hillsdale, LEA: 157-175. Cross, N. and D. R. Society (1972). Design participation: proceedings of the Design Research Society's conference, Manchester, September 1971, Academy Editions. Ehn, P. (1988). Work-Oriented Design of Computer Artifacts. Stockholm, Arbetslivcentrum. Hillgren, P.-A., A. Seravalli and A. Emilson (2011). "Prototyping and infrastructuring in design for social innovation." CoDesign 7(3-4): 169-183. Kimbell, L. (2011). "Designing for Service as One Way of Designing Services." International Journal of Design 5(2): 41-52. Kimbell, L. (2013). The Social Design Methods Menu, Fieldstudio. Manzini, E. (2015). Design, when Everybody Designs. Cambridge, Massachusetts, London, England, MIT Press. Morelli, N. and A. De Götzen (2016). Service Dominant Logic. Changing perspective, revising the toolbox. ServDes2016. N. Morelli and A. De Götzen. Copenhagen, Denmark, Linköping University Electronic Press. Normann, R. and R. Ramirez (1994). Desiging Interactive Strategy. From Value Chain to Value Constellation. New York, John Wiley and Sons. Ramirez, R. (1999). "Value Co-Production: Intellectual Origins and Implications for Practice and Research." Strategic Management Journal 20: 49-65. Sanders, E. (2008). "Co-creation and the new landscapes of design." Co-Design 1(1). Vargo, S. and R. Lusch (2004). "Evolving to a new dominant logic for marketing." Journal of Marketing 68: 1-17. Vargo, S. L. and R. F. Lusch (2008). "Service-Dominant Logic: Continuing the Evolution." Journal of the Academy of Marketing Science 36: 1-10. von Hippel, E. (1994). ""Sticky Information" and the Locus of Problem Solving: Implications for Innovation." Management Science 40(4): 429-439. von Hippel, E. (1998). "Economics of Product Development by Users: The Impact of “Sticky” Local Information”." Management Science 44(5): 629-644. S3183 MORELLI, NICOLA; DE GÖTZEN, AMALIA; MULDER, INGRID; KLITGAARD TORNTOFT, LOUISE; AGUILAR, MARC; CONCILIO, GRAZIA MORELLI, NICOLA; DE GÖTZEN, AMALIA; MULDER, INGRID; KLITGAARD TORNTOFT, LOUISE; AGUILAR, MARC; CONCILIO, GRAZIA MORELLI, NICOLA; DE GÖTZEN, AMALIA; MULDER, INGRID; KLITGAARD TORNTOFT, LOUISE; AGUILAR, MARC; CONCILIO, GRAZIA About the Authors: Nicola Morelli Professor with specific responsibilities at Aalborg University. He collaborates to the master in Service Systems Design at Aalborg University Copenhagen His teaching and research work focus on service design methods and strategies. He coordinates the O4C project Marc Aguillar is Researcher and head of European projects at the Living Labs & Digital Social Innovation Unit of the i2CAT Foundation. His current work is focused on open innovation models, citizen laboratories, and co-creation methodologies for open data Grazia Concilio is an Associate Professor in Urban Planning at Politecnico di Milano and has (Italy) and has been involved in several European Projects integrating Smart Cities and Participatory Service Design approaches in a Living Lab perspective Amalia de Götzen Associate Professor at Aalborg University Copenhagen, where she coordinates the master in Service Systems Design. Her research and teaching focus are on Service Design and Interaction Design. Ingrid Mulder, PhD is an Associate Professor of Design Techniques, Faculty of Industrial Design Engineering, Delft University of Technology. Her expertise is in transformative and social design, using strategic design and co-creation to empower people driving social change. Janice S. Pedersen is an anthropologist and project leader at Antropologerne Aps, a design-anthropological consultancy in Copenhagen, Denmark. Janice is interested in improving the evaluation of complex, innovative interventions, in mediating capacity- building across societal sectors, and in evidence-based policymaking Louise Klitgaard Torntoft, is a research assistant at the Department of Architecture, Design & Media Technology, Aalborg University/Copenhagen. Louise has undertaken both ethnographic research and design-anthropological consultancy work on various instances of technological innovation. Acknowledgements: This paper is the result of the joint collaboration of some of the members of the consortium of the Open Open4Citizens project. The project is a EU funded project under the H2020 – 2015 funding program, (ICT10 – 2015, proposal number 687818). S3184
https://openalex.org/W4301398399	https://www.frontiersin.org/articles/10.3389/fphar.2022.955401/pdf	English	null	Advances in stromal cell therapy for management of Alzheimer’s disease	Frontiers in pharmacology	2,022	cc-by	8,471	TYPE Review PUBLISHED 04 October 2022 DOI 10.3389/fphar.2022.955401 TYPE Review PUBLISHED 04 October 2022 DOI 10.3389/fphar.2022.955401 cellular therapy, mesenchymal stromal cell, Alzheimer’s, clinical trial, management cellular therapy, mesenchymal stromal cell, Alzheimer’s, clinical trial, management Advances in stromal cell therapy for management of Alzheimer’s disease OPEN ACCESS EDITED BY Mythily Srinivasan, Indiana University, Purdue University Indianapolis, United States REVIEWED BY Androniki Kretsovali, Foundation for Research and Technology Hellas (FORTH), Greece Vincent Pons, Jackson Laboratory, United States Longyang Liu, Southern Medical University, China CORRESPONDENCE Saurabh Kumar Jha, saurabh.jha@sharda.ac.in Zhe-Sheng Chen, chenz@stjohns.edu †These authors have contributed equally to this work and share ﬁrst authorship SPECIALTY SECTION This article was submitted to Experimental Pharmacology and Drug Discovery, a section of the journal Frontiers in Pharmacology RECEIVED 28 May 2022 ACCEPTED 08 September 2022 PUBLISHED 04 October 2022 OPEN ACCESS EDITED BY Mythily Srinivasan, Indiana University, Purdue University Indianapolis, United States Rashi Srivastava1†, Aidong Li2†, Tirtharaj Datta 3, Niraj Kumar Jha 3, Salehikram Talukder4, Saurabh Kumar Jha 3,5,6 and Zhe-Sheng Chen 4,7* REVIEWED BY Androniki Kretsovali, Foundation for Research and Technology Hellas (FORTH), Greece Vincent Pons, Jackson Laboratory, United States Longyang Liu, Southern Medical University, China 1Chemical and Biochemical Engineering, Indian Institute of Technology, Patna, India, 2Department of Rehabilitation, The Second People’s Hospital of Shenzhen, Shenzhen, China, 3Department of Biotechnology, School of Engineering and Technology, Sharda University, Greater Noida, India, 4Institute for Biotechnology, St. John’s University, New York City, NY, United States, 5Department of Biotechnology, School of Applied and Life Sciences, Uttaranchal University, Dehradun, India, 6Department of Biotechnology Engineering and Food Technology, Chandigarh University, Mohali, India, 7Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John’s University, New York City, NY, United States Deposition of misfolded proteins and synaptic failure affects the brain in Alzheimer’s disease (AD). Its progression results in amnesia and cognitive impairment. Absence of treatment is due to excessive loss of neurons in the patients and the delayed effects of drugs. The enhanced pluripotency, proliferation, differentiation, and recombination characteristics of stromal cells into nerve cells and glial cells present them as a potential treatment for AD. Successful evidence of action in animal models along with positive results in preclinical studies further encourage its utilization for AD treatment. With regard to humans, cell replacement therapy involving mesenchymal stromal cells, induced-pluripotent stromal cells, human embryonic stromal cells, and neural stems show promising results in clinical trials. However, further research is required prior to its use as stromal cell therapy in AD related disorders. The current review deals with the mechanism of development of anomalies such as Alzheimer’s and the prospective applications of stromal cells for treatment. CITATION CITATION Srivastava R, Li A, Datta T, Jha NK, Talukder S, Jha SK and Chen Z-S (2022), Advances in stromal cell therapy for management of Alzheimer’s disease. Front. Pharmacol. 13:955401. doi: 10.3389/fphar.2022.955401 COPYRIGHT © 2022 Srivastava, Li, Datta, Jha, Talukder, Jha and Chen. This is an open- access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Nervous stimulation of stromal cells The adult stromal cells are stimulated for differentiation via numerous pathways as shown in Figure 1. Signiﬁcance of stromal cells in Alzheimer’s disease Currently, the world lacks any proven standard therapy for the treatment of AD. Pharmacological management of AD temporarily improves cognitive symptoms. The targeted research in AD is directed towards 1) anti-oxidation, 2) removal of Aβ accumulation from the brain, and 3) regulation of the tau protein phosphorylation. Due to the advancements in regenerative and translational medicine, the usage of cells for treatment has become the limelight for producing disease- modifying strategies in the treatment of AD. Stromal cells are blank, unspecialized cells that are the result of the replenishment of lost neuronal cells through the differentiation and proliferation of neural cells upon activation via signaling pathways. They essentially restore neuroplasticity and neuromodulation (Ottoboni et al., 2020). In the recent decade, there are evidence to validate the cell-based neuroreplacement treatments for the treatment of AD in pre-clinical and clinical trials. The strategies of stromal cell therapy in AD are depicted in Figure 2. Evidence of the results from successful stromal cell therapy in animal models for AD is tabulated in Table 1. Introduction However, “disease-modifying” treatments focusing on averting or curing the speciﬁc clinical observations and biomarkers related to AD still remain under investigation (Cummings and Fox, 2017). As per the complexities of Alzheimer’s pathophysiology, a multidisciplinary treatment approach is recommended composing of pharmacological therapy, behavioural treatment, and the stimulation of endogenous or exogenous neurogenesis and synaptogenesis (Doody et al., 2008; Liperoti et al., 2008; Radad et al., 2017). The present literature anticipates the current research advancements in stromal cell treatments of AD and discusses the challenges that still need to be addressed. mechanisms, protein misfolding, and amyloid cascades. The loss of neural cells evident by temporal, parietal, frontal lobe atrophy, along with inﬂammation, increase in free radicals and the accretion of amyloid peptides and hyperphosphorylated tau protein in the form of plaques and neuro-ﬁbrillary tangles (NFTs), respectively characterize Alzheimer’s disease (Valotassiou et al., 2010). An aggregation of misfolded proteins is the primary cause of this nervous system disorder. Two of these proteins are plaques (β- amyloid) and hyperphosphorylated tau proteins, which are created by the accumulation of Aβ proteins and affect cell-to-cell communication. These proteins also correspond to the formation of intracellular NFTs, which disrupt synaptic networks and reduce neurotransmitter production, respectively. Tangles, which are made of hyperphosphorylated tau protein, obstruct the neuronal internalised transport system that carries vital nutrients to the brain. The remaining 1% of the elderly population has been shown to have presenilin 2 (PS2) and presenilin 1 (PS1) genetic changes, which are responsible for reducing -secretase activity by boosting the synthesis of self-accumulating Aβ42 peptide. The diagnostic criteria for AD is given by the National Institute of Neurological and Communicative Disorders and Stroke (NINCDS) are: 1) the occurrence of disabilities related to cognition (language, orientation, learning and memory, perception behaviour), 2) the neuropsychological testing in case of symptoms concerning dementia syndrome conﬁrmed by neuropsychological testing. There are no standard treatments presented in the literature (Loewenstein et al., 2001). However, the main strategy of treatment in AD reversal relies upon the reduction of Aβ levels. Various researchers across the globe have performed an extensive research to understand the pathogenesis, natural progression, diagnosis, and regenerative principles in the management of AD. However, “disease-modifying” treatments focusing on averting or curing the speciﬁc clinical observations and biomarkers related to AD still remain under investigation (Cummings and Fox, 2017). Introduction As per the complexities of Alzheimer’s pathophysiology, a multidisciplinary treatment approach is recommended composing of pharmacological therapy, behavioural treatment, and the stimulation of endogenous or exogenous neurogenesis and synaptogenesis (Doody et al., 2008; Liperoti et al., 2008; Radad et al., 2017). The present literature anticipates the current research advancements in stromal cell treatments of AD and discusses the challenges that still need to be addressed. of beneﬁcial and developmental medicine along with pre- clinical examination has progressed. Regenerative medicine aims to rejuvenate at cellular and tissue levels while also achieving restored regulation in the regional milieu (Sharma et al., 2022). To regenerate the tissue of concern, regenerative medicine employs the recombination of the extracellular matrix, growth factors, and most importantly stromal cells. The notion of “Cellular Therapy” (CT) or “Cytotherapy” is based on the use of paracrine signaling by biomacromolecules to direct stromal cells which are in a state of quiescence for induction or augmentation. CT attempts to induce a curative impact in the concerned area through the transplantation of autologous, allogeneic, or modiﬁed cells that are unaccompanied by changes in their biological properties. Recombinant cell-induced pluripotent cells, hematopoietic cells, genetically-modiﬁed cells, stromal cells, genetically designed cells, or an amalgamation of all of these cells can be used to create cellular therapeutic products (Kaufman, 2009; Nianias and Themeli 2019; Sharma et al., 2022). The use of human-made medical items to treat neurological problems is still in its early stages. For efﬁcient regeneration of these cells, numerous combinations have to be researched and explored. Introduction The epidemiological spread of communicable to non-communicable (not transfer from one living beings to another) diseases has raised the overall burden on healthcare workers worldwide. Neurological illnesses are serious issues that concern both medical professionals and patients when considering non-communicable diseases. Alzheimer’s disease (AD) accounts for 50%–70% of dementia along with it being the top ﬁfth leading cause of death worldwide. By 2050, the worldwide population affected by AD is expected to rise to 152 million (GBD et al., 2019 Dementia Forecasting Collaborators, 2022). The degeneration of neural cells and synaptic connections in certain subcortical regions and cerebral cortex marks the presence of Alzheimer’s disease (AD). Scientists hypothesize that AD is based on cholinergic 01 Frontiers in Pharmacology Frontiers in Pharmacology frontiersin.org 10.3389/fphar.2022.955401 10.3389/fphar.2022.955401 Srivastava et al. mechanisms, protein misfolding, and amyloid cascades. The loss of neural cells evident by temporal, parietal, frontal lobe atrophy, along with inﬂammation, increase in free radicals and the accretion of amyloid peptides and hyperphosphorylated tau protein in the form of plaques and neuro-ﬁbrillary tangles (NFTs), respectively characterize Alzheimer’s disease (Valotassiou et al., 2010). An aggregation of misfolded proteins is the primary cause of this nervous system disorder. Two of these proteins are plaques (β- amyloid) and hyperphosphorylated tau proteins, which are created by the accumulation of Aβ proteins and affect cell-to-cell communication. These proteins also correspond to the formation of intracellular NFTs, which disrupt synaptic networks and reduce neurotransmitter production, respectively. Tangles, which are made of hyperphosphorylated tau protein, obstruct the neuronal internalised transport system that carries vital nutrients to the brain. The remaining 1% of the elderly population has been shown to have presenilin 2 (PS2) and presenilin 1 (PS1) genetic changes, which are responsible for reducing -secretase activity by boosting the synthesis of self-accumulating Aβ42 peptide. The diagnostic criteria for AD is given by the National Institute of Neurological and Communicative Disorders and Stroke (NINCDS) are: 1) the occurrence of disabilities related to cognition (language, orientation, learning and memory, perception behaviour), 2) the neuropsychological testing in case of symptoms concerning dementia syndrome conﬁrmed by neuropsychological testing. There are no standard treatments presented in the literature (Loewenstein et al., 2001). However, the main strategy of treatment in AD reversal relies upon the reduction of Aβ levels. Various researchers across the globe have performed an extensive research to understand the pathogenesis, natural progression, diagnosis, and regenerative principles in the management of AD. Cellular therapy in Alzheimer’s disease In 2004, the World Health Organization (WHO) introduced the idea of medical products of human origin (MPHO), which serve as a platform for the attainment and dispersal of biological therapeutic goods to treat a variety of diseases (Martin, 2017). The use of MPHO to treat diseases has become increasingly important as research in the realms Frontiers in Pharmacology 02 frontiersin.org Srivastava et al. 10.3389/fphar.2022.955401 FIGURE 1 Mechanism of neurogenic signaling of stromal cells (MBD1: Methyl-CpG-binding domain protein one; NGF: Nerve Growth Factor; NT-3: Neurotrophin three; MeCP2: Methyl-CpG-binding protein two; BDNF: Brain-derived neurotrophic factor; IGF-1: Insulin-like growth factor; NT-4/5: Neurotrophin 4/5; VEGF: Vascular endothelial growth factor; MeCP2: Methyl-CpG-binding protein two; FGF: Fibroblast growth factor; Mll1: Mixed- lineage leukemia 1). FIGURE 1 Mechanism of neurogenic signaling of stromal cells (MBD1: Methyl-CpG-binding domain protein one; NGF: Nerve Growth Factor; NT-3: Neurotrophin three; MeCP2: Methyl-CpG-binding protein two; BDNF: Brain-derived neurotrophic factor; IGF-1: Insulin-like growth factor; NT-4/5: Neurotrophin 4/5; VEGF: Vascular endothelial growth factor; MeCP2: Methyl-CpG-binding protein two; FGF: Fibroblast growth factor; Mll1: Mixed- lineage leukemia 1). Neurogenic precursors or neural stromal cells exist in a few restricted areas of the adult brain which shows continued neurogenesis and neuromodulation. Growth and the divergence of the cell into neurons and synapses are regulated by transcription factors and signaling pathways which involve biomolecules that act on neuronal progenitor cells, referred to as neurotransmitters. The genetic modiﬁcation of neural stromal cells increases migratory efﬁciency. As a result, it allows to successfully employ them for the enhancement of gene expression and for the delivery of neurotrophic factors which subsequently modify the progression of AD. FIGURE 2 Strategies of stromal cell utilization in AD therapies. Embryonic stromal cells Stromal cells Animal model used NSCs Transgenic mice with triple mutation (3xTg-AD) expressing PS-1, APP, and tau NPCs Focal cerebral ischemia in rat model UC-MSC Transgenic mice with double mutation of APP and PS1 Transdifferentiated human Wharton’s jelly MSCs into neuron-like cells Transgenic mice with double mutation of AβPP/PS1 MSCs Aβ treated mice BM-MSC APP/PS1 transgenic mice Human olfactory bulb NSCs Ibotenic acid induced AD rat model P-MSCs Aβ1-42 peptide infused in mouse model VEGF overexpressing BM-MSCs Transgenic mice with double mutation of APPswe/PS1dE9 Choline acetyltransferase expressing hNSC AF64A-cholinotoxin induced learning deﬁcit rat model ENCSCs Aβ1-40 administration in rat UC-MSC APP/PS1 transgenic mice model NSC, neural stromal cells; HNSC, human neural stromal cells; NPC, neural precursor cells; MSC, mesenchy ENCSC epidermal neural crest stromal cell; UC-MSC umbilical cord-derived mesenchymal stromal cells Results/Outcome NSCs Transgenic mice with triple mutation (3xTg-AD) expressing PS-1, APP, and tau Reduced memory deprivation associated with position, location and learning along with increase in synaptic density NPCs Focal cerebral ischemia in rat model Interaction between transplanted and endogenous cells was focussed; Enhanced intrinsic neural growth UC-MSC Transgenic mice with double mutation of APP and PS1 Microglial activation improves spatial learning and memory; Diminished accumulation of Aβ Transdifferentiated human Wharton’s jelly MSCs into neuron-like cells Transgenic mice with double mutation of AβPP/PS1 Development of perceptual observation Decrease in Aβ through expression of neprilysin and enzymes and activation of microglial cells MSCs Aβ treated mice Enhanced neural tissue formation in hippocampus; Formation of neurons via Wnt signalling pathway from neural progenitor cells BM-MSC APP/PS1 transgenic mice Decrease in accumulated plaques Escalated expression DeltaNp73 protein Improvement in spatial learning Human olfactory bulb NSCs Ibotenic acid induced AD rat model Transcription of nerve growth factors Improved psychological abilities P-MSCs Aβ1-42 peptide infused in mouse model Improved psychological abilities Increases neural cell development VEGF overexpressing BM-MSCs Transgenic mice with double mutation of APPswe/PS1dE9 Improvement in behaviour associated symptoms Decrease in amyloid plaque build-up Improvement in abnormal blood vessels formation Choline acetyltransferase expressing hNSC AF64A-cholinotoxin induced learning deﬁcit rat model Translocation of implanted cells to the damaged area; differentiation of neural stromal cells ENCSCs Aβ1-40 administration in rat Increased granule cells in hippocampus; Expression of neuronal markers by implanted cells UC-MSC APP/PS1 transgenic mice model Enhanced intrinsic expression of neprilysin Decrease in Aβ plaques NSC, neural stromal cells; HNSC, human neural stromal cells; NPC, neural precursor cells; MSC, mesenchymal stromal cells; BM-MSC, bone marrow derived mesenchymal stromal cell; ENCSC, epidermal neural crest stromal cell; UC-MSC, umbilical cord-derived mesenchymal stromal cells; P-MSCs, placenta derived mesenchymal stromal cells. Embryonic stromal cells Reduced memory deprivation associated with position, location and learning along with increase in synaptic density Interaction between transplanted and endogenous cells was focussed; Enhanced intrinsic neural growth Microglial activation improves spatial learning and memory; Diminished accumulation of Aβ Development of perceptual observation Decrease in Aβ through expression of neprilysin and enzymes and activation of microglial cells Enhanced neural tissue formation in hippocampus; Formation of neurons via Wnt signalling pathway from neural progenitor cells Decrease in accumulated plaques Escalated expression DeltaNp73 protein Improvement in spatial learning Transcription of nerve growth factors Improved psychological abilities Improved psychological abilities Increases neural cell development Improvement in behaviour associated symptoms Decrease in amyloid plaque build-up Improvement in abnormal blood vessels formation Translocation of implanted cells to the damaged area; differentiation of neural stromal cells Increased granule cells in hippocampus; Expression of neuronal markers by implanted cells Enhanced intrinsic expression of neprilysin Decrease in Aβ plaques mal stromal cells; BM-MSC, bone marrow derived mesenchymal stromal cell; P-MSCs, placenta derived mesenchymal stromal cells. Decrease in Aβ plaques NSC, neural stromal cells; HNSC, human neural stromal cells; NPC, neural precursor cells; MSC, mesenchymal stromal cells; BM-MSC, bone marrow derived mesenchymal stromal cell; ENCSC, epidermal neural crest stromal cell; UC-MSC, umbilical cord-derived mesenchymal stromal cells; P-MSCs, placenta derived mesenchymal stromal cells. astrocytes which enhances memory and learning performance in AD animal models (J et al., 2008). However, certain ethical concerns are associated with hESCs (Human ESCs) in FDA- approved clinical trials (Liras, 2010; Fouad, 2019). Estimating the effectiveness of transplantation-based therapy is crucial from an ethical standpoint, as is lowering the possibility of therapeutic misunderstanding, minimizing discomfort, and emphasizing the signiﬁcance of informed permission. Actually, the ethical discussion around stem cell-based therapy demonstrated the challenging balance between the need for prudence and the advancement of clinical studies. For instance, the inﬁnite and unwanted differentiation potential of iPSCs increases the possibility of cancer, human cloning, and the unethical creation of genetically engineered human embryos. Similar safety concerns arise with MSC implantation due to their propensity to promote tumour development and metastasis. As a result, iPSCs are viewed as having higher moral standing than hESCs. However, the greatest safety concern with iPSC- based treatment is the possibility of teratoma development because of the unregulated differentiation. Additionally, the astrocytes which enhances memory and learning performance in AD animal models (J et al., 2008). Embryonic stromal cells FIGURE 2 Strategies of stromal cell utilization in AD therapies. FIGURE 2 Strategies of stromal cell utilization in AD therapies. FIGURE 2 Strategies of stromal cell utilization in AD therapies. Embryonic stromal cells (ESCs) are totipotent cells that have a self-renewing ability and differentiation capacity. ESCs are 03 Frontiers in Pharmacology frontiersin.org frontiersin.org 10.3389/fphar.2022.955401 Srivastava et al. TABLE 1 Laboratory trials of stromal cells in the treatment of AD. Neural stromal cells The multipotent nature of neural stromal cells (NSCs) allows them to differentiate into oligodendrocytes, astrocytes, and neurons after transplantation which is why they are an ideal cell-based substitution therapy in degenerative neurological disorders. The sources of NSCs are from primary tissues namely fetus, iPSCs, and ESCs. However, the required NSCs have to be customized for a particular patient to avoid immune rejection. Moreover, the isolation procedure is extremely risky as well. As contrary to other stem cells like ESCs, foetal NSCs, and MSCs, adult neural stem cells (aNSCs) are localised to certain regions of the central nervous system (CNS) and have a low proliferative potential. As a result, the initial isolation and steady in vitro growth of aNSCs are signiﬁcant technological challenges that must be overcome before aNSCs may be used. Adult CNS surgical samples are often comparatively small (1–2 ml). Since there aren’t many resident aNSCs in the tissue, isolation methods have been improved to increase the likelihood that aNSCs will be successfully isolated in the ﬁrst place. CNS tissues are manually chopped and enzymatically degraded into single cells to get aNSCs. Enzymatic digestion is one among them, and it is important because it directly impacts aNSC survival. Different researchers use different dissociating enzyme compositions and incubation periods. Trypsin, collagenase, and papain have often been utilised, and in some studies, papain dissociation was recommended as the most effective method for the ﬁrst separation of aNSCs. In the mammalian brain, NSCs exhibit neural homeostasis, repair, and regeneration, and demonstrate neural pleiotropism (Boese et al., 2020). The administration of bone marrow-derived MSCs (BM- MSCs) in the intracerebroventricular region ameliorates memory loss in AD model mice by accentuating astrocytic inﬂammation as well as synaptogenesis. BM-MSCs treated AD model of rodents expressed increased levels of miR-146 exosomes in the hippocampus which ultimately induces synaptogenesis and neurogenesis. Along with that, neuroplasticity was restored and cognitive impairment improved (Nakano et al., 2020). Intravenous injection of BM-MSCs was detected in brain parenchyma within an hour post-injection in the rat AD model which ultimately released growth factors and cytokines to recover neurobehavioral functions and stimulate endogenous regeneration (Harach et al., 2017). Administration of both stromal cells and neurotrophic peptides, stimulates differentiation of the neural stromal cells via paracrine signaling. The mentioned peptides increase neural formation in zones of the brain that concern memory such as the subgranular and subventricular zone. Embryonic stromal cells However, certain ethical concerns are associated with hESCs (Human ESCs) in FDA- approved clinical trials (Liras, 2010; Fouad, 2019). Estimating the effectiveness of transplantation-based therapy is crucial from an ethical standpoint, as is lowering the possibility of therapeutic misunderstanding, minimizing discomfort, and emphasizing the signiﬁcance of informed permission. Actually, the ethical discussion around stem cell-based therapy demonstrated the challenging balance between the need for prudence and the advancement of clinical studies. For instance, the inﬁnite and unwanted differentiation potential of iPSCs increases the possibility of cancer, human cloning, and the unethical creation of genetically engineered human embryos. Similar safety concerns arise with MSC implantation due to their propensity to promote tumour development and metastasis. As a result, iPSCs are viewed as having higher moral standing than hESCs. However, the greatest safety concern with iPSC- based treatment is the possibility of teratoma development because of the unregulated differentiation. Additionally, the obtained from blastocysts, speciﬁcally the inner cell mass. Due to the pluripotent nature of ESCs, tumorigenesis, uncontrolled cellular explosion, and immunogenic rejection may occur. Tang et al. suggested that hippocampal ESC transplantation can be performed without any potential risks to restore cognitive function in Aβ peptide injured rats (J et al., 2008). In this rodent model of AD, ESC-derived neural progenitor cell (NPC) transplantation following commitment to a cholinergic cell phenotype can enhance behavioral and cognitive recovery with the regeneration of cholinergic neurons (Fh et al., 2009). Transplantation in the mouse with ESC-derived NPCs to the meynert basal nucleus resulted in enhanced memory and learning capacity (Liu et al., 2020). Along with that, the enhancement of neurocognitive recovery was also observed when mouse and human ESC-derived basal forebrain cholinergic neurons were transplanted into the transgenic AD mice model (Yue and Jing, 2015). ESCs derived from humans were able to translate into dopaminergic neurons, spinal motor neurons, and astroglial cells (Lee et al., 2007). Neural progenitor cells derived from ESC develop into neuron-like cells and Frontiers in Pharmacology Frontiers in Pharmacology 04 frontiersin.org 10.3389/fphar.2022.955401 Srivastava et al. 10.3389/fphar.2022.955401 et al., 2013). PD-MSCs decrease the cytokines with pro- inﬂammatory action and increase the concentration of anti- inﬂammatory cytokines in the brain. They promote neuronal differentiation and proliferation in the hippocampus of the AD mice model. MSC transplantation in aged AD rats restores motor and cognitive activity (Liu et al., 2020). There are encouraging results in the preclinical trials of MSCs in AD. Embryonic stromal cells In one human trial, composed of nine patients, were administered MSCs in AD and have completed phase I which conﬁrms the potential, efﬁcacy, and non-toxicity of MSC injection. proliferative and differentiation capacity is decreased due to the host cells’ niche differing from that of the in vitro cultivated cells. Mesenchymal stromal cells MSCs play a signiﬁcant role in controlling the symptoms of AD through 1) immunoregulation, 2) neurotrophic, 3) neuroplasticity, and 4) the reduction of Aβ plaque burden which restores the cognitive function (Babaei et al., 2012; Qin et al., 2020). Both in vitro and in vivo, MSCs show a remarkable capacity to trigger the quick clearance of Aβ aggregates. In terms of mechanism, the interaction between microglial cells and MSCs was primarily responsible for the removal of Aβ deposits (Fouad, 2019). Additionally, by expressing CCL5, MSCs can draw more microglial cells from the bone marrow (Fouad, 2019). In this approach, MSCs can inhibit deposition of Aβ plaques and perhaps halt the progression of the illness. Intriguingly, these investigations also showed a decrease in intracellular NFTs. Various preclinical studies have demonstrated the reversal of pathological changes in AD animal models. These studies veriﬁed neuroprotection, neuroplasticity, neuromodulation, and the neurogenic effects of MSCs through the neurogenic signaling pathway activation (Fumagalli et al., 2006). These grafted MSCs upregulate the expression of the anti-apoptotic factor, accentuate the AD symptoms, and halt the progression of the AD (Ruzicka et al., 2016). MSC-induced cognition restoration is due to the suppression of microglial activation and reduced levels of Aβ plaques in the brain (Woodruff-Pak, 2008). Induced pluripotent stromal cells In 2011, Yahata et al. and Yagi et al. were the ﬁrst researchers to introduce iPSCs therapy for AD (Yagi et al., 2011; Yahata et al., 2011). Yahata et al. (2011) demonstrated the production of Aβ plaques in neurons through the transplantation of iPSCs from forebrain neurons. Yagi et al. (2011) demonstrated the enhanced Aβ42 secretion by attenuating the dysfunctional condition of familial AD with PS1 (A246E) and PS2 (N141I) mutations. Transplanted NSCs in AD animal models possess the ability to replace the destructed neural circuit through neurotrophic factors that counteract the levels of Aβ plaques. NSC transplantation improve the cholinergic neuron numbers and enhance learning and memory abilities (Hayashi et al., 2020). It is unclear whether the observed behavioral rescue, enhanced memory, and learning abilities are due to NSC differentiation or neurotrophic factors in AD models (Hayashi et al., 2020). However, transplanted grafts of Neural SCs increases neurotrophic factor levels that are derived from the brain and enhance the behavioral rescue without altering concentration concerning Aβ or tau in a mutant rodent model of AD with overexpressed hAPP (Marsh and Blurton-Jones, 2017). Induced pluripotent cells can transform into neurospheres, neuronal progenitor cells and, neural cells which is evident in the cortex of rodent. The incorporation of these cells in diseased rodents resulted in increased concentration of Aβ-degrading protease neprilysin (Han et al., 2019). Enhanced synaptic activity and the connection between excitatory and inhibitory neurons have been demonstrated by iPSC-derived neurons (Birey et al., 2017; Xiang et al., 2017). Pure, immature neuronal cells, after incubation, differentiated into mature nerves exhibiting the inhibitory GABAergic and excitatory glutamatergic synapses along with the potentiation of action potentials. Human iPSC derived cortical neuronal cells attenuate the deposition of Aβ plaques and tau protein complexes in the forebrain (Nieweg et al., 2015). In APP/PS1 transgenic mice AD model, intranasal incorporation of human NSCs that differentiate into cholinergic neurons decrease β-amyloid accumulation by increasing neprilysin expression along with enzymes that can degrade β-amyloid. They also downregulate neuroinﬂammation, synaptic and pericytic loss ultimately rescuing the cognitive function. Intranasal administration of human NSCs possess greatest potential as a noninvasive tool in treating AD (Lu et al., 2021). NSCs ameliorates Aβ levels in AD mouse models by degrading plasmin, cathepsin B, and insulin-degrading enzymes. Treatment of AD models by AD-iPSCs derived from neuronal cells consequently suppressed p-tau protein. Neural stromal cells These neural stromal cells differentiate into neuronal cells and glial cells (Shohayeb et al., 2018). Genetic engineering of these cells ampliﬁes the release of amyloid plaque degrading enzymes which increases synaptic density by removing Aβ aggregates (Hayashi et al., 2020). The immune protection of MSCs can be provided by the capacity to regulate immune activities thus providing neural protection from Endovenous introduction of MSCs derived from adipose tissue (AD-MSCs) in rodent-model with AD improved neural functioning. It was accompanied by an increase in the formation of neurons along with differentiation of cells resembling neurons and astrocytes. Advancement in cognitive abilities was evident due to the decrease in accumulation of amyloid plaque and the increase in neural cells. The cells were then observed 12 days after injection (Tian et al., 2012). Intracerebroventricular transplantation of placenta-derived MSCs (PD-MSCs) demonstrated the inhibitory effect of neuronal apoptosis and memory impairment in Aβ1–42-infused mice (Yun Frontiers in Pharmacology 05 frontiersin.org 10.3389/fphar.2022.955401 Srivastava et al. inﬂammation. Aside from that, mi-RNAs and si-RNAs released from these MSCs act to degrade accumulated amyloid plaques. cognition. In phase one of the clinical trial, genetically modiﬁed ﬁbroblasts coded with NGF gene transplantation into forebrain found to be safe (NGF-gene therapy) for AD (Tuszynski et al., 2005). These evidence provide a right path and new horizon in the management of AD in humans. Transplantation of neural SCs in the lateral ventricle resulted in the formation of neuronal progenitor cells and glial cells. The cells had the capability of migration and regeneration which reduced Aβ deposition and tau phosphorylation and increased neural cell and synapse formation (Hayashi et al., 2020). Findings were proven through the incorporation of ﬂuorescent protein labeled BM-MSCs in the hippocampus of AD diagnosed models where the same results were witnessed (Duncan and Valenzuela, 2017). Human MSCs were able to restore neuromuscular, cognition, and memory function after administration in aged rat models. Induced pluripotent stromal cells AD- iPSC derived neuronal cells were able to upregulate AD associated genes which was evident by increased transcription (Hossini et al., 2015). In 5XFAD transgenic AD mouse model, differentiation of protein-iPSCs into glial cells, attenuation of Aβ plaque depositions, and improved cognitive ability were observed. In proteomic analysis, upregulation of oligodendrocyte-related genes in brain when protein-iPSCs are injected were observed (Cha et al., 2017). Zhang et al. (2019) transplanted human induced neural progenitor cells (iNPCs) into the hippocampal region of brain in wild type immunodeﬁcient AD mice. This study demonstrated the long-term survival, graft-host synaptic connections, neuronal circuits, and regenerated neural network of host hippocampus. AD mice exhibited neuroplasticity, neuromodulation and synaptogenesis with enhanced cognitive and behavioral abilities. Three dimensional spheroids from human iPSCs for AD model were differentiated into neuronal cell lines that led to the reduction of both Aβ40 and Aβ42 thereby potentiating the cell lines to regenerate into neuronal cells (Lee et al., 2016). Three dimensional cerebral organoids recapitulated the human brain architecture including cerebral progenitors in the subventricular and subgranular zone of hippocampus (Machairaki, 2020). Human iPSCs yielded 16 times more extracellular vehicles, exosomes, than MSCs. These exosomes have more potential to attenuate the deposition of Aβ plaques in the brain and demonstrate an increased Aβ42/Aβ40 ratio in AD animal models (Machairaki, 2020). In APP/PS1 murine model of AD, NSC inﬁltration into ﬁmbria and fornix junction show signiﬁcant improvement in cognition and memory abilities. NSCs induce microglia activation and phagocytosis of amyloid plaques (McGinley et al., 2018). Without priming the microenvironment with neurotrophic factors, the transplantation of NSCs to the hippocampus would not produce any desired results in AD. In aged animals, the infusion of NGF demonstrated improved Frontiers in Pharmacology 06 frontiersin.org 10.3389/fphar.2022.955401 Srivastava et al. TABLE 2 Clinical trials of Alzheimer’s disease incorporating stromal cells. S.No. Clinical trials Similarly, vascular endothelial cells, neuronal cells, and glial cells are needed for therapy which aims to treat AD and traumatic brain injuries. Considering this, there is a need of speciﬁcally differentiated cell lines. Known mechanisms today act through paracrine signalling which involves neurotrophic factors that regulate immune functions. Another potential mechanism could be inducing neural cell formation (Gnecchi et al., 2008). Altogether, it is critical to comprehend these effector processes before using them to treat neurological illnesses. Induced pluripotent stromal cells Nct number Title Status Interventions Locations 1 NCT02833792 Allogeneic Human Mesenchymal Stromal cells for Alzheimer’s Disease Recruiting BMMSCs United States 2 NCT02600130 Allogeneic Human Mesenchymal Stromal cell Infusion Versus Placebo in Patients With Alzheimer’s Disease Active, not recruiting BMMSCs United States 3 NCT04040348 Alzheimer’s Disease Stromal cells Multiple Infusions Recruiting BMMSCs United States 4 NCT03117738 A Study to Evaluate the Safety and Efﬁcacy of AstroStem in Treatment of Alzheimer’s Disease Completed ADMSCs United States 5 NCT02672306 Safety and Exploratory Efﬁcacy Study of UCMSCs in Patients With Alzheimer’s Disease Unknown status UCMSCs China 6 NCT04855955 Autologous Human Adipose-Derived Mesenchymal Stromal cells in Alzheimer’s Disease Available ADMSCs United States 7 NCT03724136 Alzheimer’s Autism and Cognitive Impairment Stromal cell Treatment Study Recruiting BMMSCs UAE 8 NCT01696591 The Long-Term Safety and Efﬁcacy Follow-Up Study of Subjects Who Completed the Phase I Clinical Trial of NeurostemÂ®-AD Unknown status ADMSCs Korea 9 NCT01547689 Safety and Efﬁciency of Umbilical Cord-derived Mesenchymal Stromal cells (UC- MSC) in Patients With Alzheimer’s Disease Unknown status UCMSCs China 10 NCT02054208 Safety and Exploratory Efﬁcacy Study of NEUROSTEMÂ® Versus Placebo in Patients With Alzheimer’s Disease Completed UCMSCs Korea 11 NCT04482413 Study to Evaluate the Safety and Efﬁcacy of AstroStem in Treatment of Alzheimer’s Disease Not yet recruiting ADMSCs United States 12 NCT01297218 The Safety and The Efﬁcacy Evaluation of NEUROSTEMÂ®-AD in Patients With Alzheimer’s Disease Completed UCMSCs Korea 13 NCT04954534 Exploratory Efﬁcacy Study of NEUROSTEMÂ® in Subjects Who Control Group of NEUROSTEMÂ® Not yet recruiting UCMSCs Korea 14 NCT04684602 Mesenchymal Stromal cells for the Treatment of Various Chronic and Acute Conditions Recruiting ADMSCs United States 15 NCT03172117 Follow-up Study of Safety and Efﬁcacy in Subjects Who Completed NEUROSTEMÂ® Phase-I/IIa Clinical Trial Recruiting UCMSCs Korea 16 NCT03297177 Autologous Stem/Stromal Cells in Neurological Disorders and Disease Not yet recruiting ADMSCs United States 17 NCT00874783 Development of iPS From Donated Somatic Cells of Patients With Neurological Diseases Recruiting iPSCs Israel Status Interventions Locations Challenges posed by stromal cell treatment in Alzheimer’s disease As a result, substantial research into the tumour formation of stromal cells and the derivatives of stromal cells is critical. In order to avoid risk of such terrible incidents and their associated consequences for patients, researchers and healthcare specialists must work together. Furthermore, treatments must be effectively better in terms of safety and effectiveness when compared to conventional therapeutic interventions (Sato et al., 2019). of SCs, iPSCs, MSCs, ESCs and NSCs, can be incorporated invasively and non-invasively. This presents researchers with a wide range of possibilities for the development of customized cellular therapies. The promising results observed in pre-clinical and clinical trials were evident of the decrease in causative agents of AD which encourages its use in humans. Nevertheless, enormous amounts of research are requirement especially in the context of genetic variation due in the occurrence of AD and the inability to conclude negative consequences due to lack of replicable translation models. Issues like accuracy of treatment, mechanistic methodology, and ethical issues also need to be addressed prior to its common medical use. Stromal cell treatments are linked to moral and ethical concerns along with the possibility of transplant rejection. With these cellular therapies, the cost of customized treatment escalates as a result of poor efﬁciency in the elicitation of iPSCs from the patient’s diploid cells. In order to increase the effectiveness of these cellular therapies while minimizing cost, technical and methodological improvements are required. The use of SCs in AD treatment raises a lot of unresolved issues. There are no data on which sort of differentiated or undifferentiated pluripotent SCs would be the most efﬁcient for this treatment. Likewise, details on cell concentration, the quantity of effective dosages, and the length of the course of treatment are lacking. It is unclear if a conﬁned (using stereotactic injections) or peripheral transplant should be performed, as well as how cell migration to the afﬂicted part of the brain happens. There is no proofthatSCstreatmentworksinhumans.ItisalsounknownhowSCs can get rid of the inﬂammatory and toxic environment caused by the Aβ peptide, how healthy cells connect to one another, or how the cell repopulation will inﬂuence other metabolic pathways. It is yet unclear if the transplantation of SCs will stop the transmission of pathogenic tau protein between neurons or communication between damaged cells. Challenges posed by stromal cell treatment in Alzheimer’s disease Challenges posed by stromal cell treatment in Alzheimer’s disease The emerging application of clinical therapy by medical experts and researchers in neurological disorders is based on the proof of potential in the preclinical research. The methods discussed have proven to be efﬁcient and safe for the treatment of neural medical conditions. Following preclinical trials, clinical trials (Table 2) have begun to speciﬁcally target AD (McGinley et al., 2016). Although certain challenges still obstruct the routine use of cellular therapy in the medical practice, it is important to identify the correct cell types that are used along with understanding the mechanism of action of those cells for a maximum advantage in treatment. For instance, the therapy for treatment of PD requires particular cells such as the dopaminergic substantia nigra neurons (Freed et al., 2001). Results expressing affectivity of stromal cells in animal models are not completely reliable due to numerous reasons. The incomplete aspects and inaccuracy may have more harmful outcomes than beneﬁcial. First being, using rodent models are far from the accurate representation of the physiological state of sickness in humans. Second, the age variation between the two remains at an excessive gap. Third, animal models are still inadequate to show that clinically relevant functional impairments improve. Finally, the animal models may be incapable of indicating the negative consequences of CT (Van der Worp et al., 2010). Frontiers in Pharmacology 07 frontiersin.org Srivastava et al. 10.3389/fphar.2022.955401 Srivastava et al. of SCs, iPSCs, MSCs, ESCs and NSCs, can be incorporated invasively and non-invasively. This presents researchers with a wide range of possibilities for the development of customized cellular therapies. The promising results observed in pre-clinical and clinical trials were evident of the decrease in causative agents of AD which encourages its use in humans. Nevertheless, enormous amounts of research are requirement especially in the context of genetic variation due in the occurrence of AD and the inability to conclude negative consequences due to lack of replicable translation models. Issues like accuracy of treatment, mechanistic methodology, and ethical issues also need to be addressed prior to its common medical use. The characterization of potential tumour generation remains the next issue in CT. For the treatment of neurological disorders, a healthy average life span and the tumorigenicity potential is important. In this circumstance, even a remote potential of tumour growth through CT would be unbearable. Author contributions SJ has prepared the backbone of the manuscript. RS and AL wrote the original draft of the manuscript with TD and ST. SJ, ST, and Z-SC reﬁned the ﬁrst draft. Illustrations, NJ. Z-SC and SJ critically revised the manuscript for intellectually correct content. AL and Z-SC supported the project. All authors approved the submitted version. Challenges posed by stromal cell treatment in Alzheimer’s disease Finally, since it has been unable to show the safety of this form of therapy for AD, ongoing patient monitoring would be necessary to prevent any negative side effects, both immediate and long-term. Furthermore, there are notable changes in the brain’s structure and microenvironment between AD patients and animal models, making it challenging to precisely characterise the positive effects of stem cells in human AD. For the familial form of AD, transgenic animal models have been created, although human AD pathogenesis primarily comprises sporadic instances. This restricts our understanding of how patient-speciﬁc stem cell treatment might operate. These issues require future research. Future preclinical studies examining various stem cell sources, kinds, dosages, long- term safety, effectiveness, and exact mechanisms of action are necessary. Using recently developed in vivo biomarkers in clinical research is anticipated to give researchers more control and insight into patient selection and quantity efﬁcacy than was previously feasible using previously known marker kinds. When working with the immune system, neuro factors, enzymes, proteins, and gene therapy, to mention a few examples of applications, among many more, cell extraction and injection enhancements may be tackled from numerous viewpoints. Additionally, as part of the entire development process for the technology, it is crucial to evaluate ethical issues at each stage of building a stem cell- based technology. Many SCs treatments are currently in the testing phase. Although SCs products may have a huge potential to cure a wide range of medical conditions, there is not enough scientiﬁc proof to guarantee that their usage is safe and has any positive health effects. 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https://openalex.org/W4200067443	https://www.researchsquare.com/article/rs-1116554/latest.pdf	English	null	Recurrence and Prognosis in Intrahepatic Cholangiocarcinoma Patients with Different Etiology After Radical Resection: A Multi-Institutional Study	Research Square (Research Square)	2,021	cc-by	6,224	Recurrence and Prognosis in Intrahepatic Cholangiocarcinoma Patients with Different Etiology After Radical Resection: A Multi-Institutional Study Different Etiology After Radical Re Qi Li The First Affiliated Hospital of Xi'an Jiaotong University Chen Chen The First Affiliated Hospital of Xi'an Jiaotong University Jingbo Su The First Affiliated Hospital of Xi'an Jiaotong University Yinghe Qiu Oriental Hepatobiliary Hospital Affiliated to Naval Medical University Hong Wu West China Hospital of Sichuan University Tianqiang Song Tianjin Medical University Cancer Hospital Xianhai Mao Hunan Provincial People’s Hospital Yu He The First Hospital Affiliated to Army Medical University Zhangjun Cheng Zhongda Hospital of Southeast University Jingdong Li Affiliated Hospital of North Sichuan Medical College Wenlong Zhai the First Affiliated Hospital of Zhengzhou University Dong Zhang The First Affiliated Hospital of Xi'an Jiaotong University Zhimin Geng (  gengzhimin@mail.xjtu.edu.cn ) The First Affiliated Hospital of Xi'an Jiaotong University Zhaohui Tang Shanghai Jiaotong University School of Medicine Research Article Keywords: intrahepatic cholangiocarcinoma, hepatitis B virus, hepatolithiasis, recurrence, prognosis DOI: https://doi.org/10.21203/rs.3.rs-1116554/v1 DOI: https://doi.org/10.21203/rs.3.rs-1116554/v1 cense:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full Lic Version of Record: A version of this preprint was published at BMC Cancer on March 26th, 2022. See the published version at https://doi.org/10.1186/s12885- 022-09448-w. Page 1/13 Page 1/13 Page 1/13 Page 1/13 Abstract Objective We aimed to evaluate the prognosis and adjuvant chemotherapy (ACT) in intrahepatic cholangiocarcinoma (ICC) patients with different etiology after radical resection. We aimed to evaluate the prognosis and adjuvant chemotherapy (ACT) in intrahepatic cholangiocarcinoma (ICC resection. Results Univariate and multivariate analysis showed that different etiology was a prognostic factor for overall survival and relapse-free survival, and different etiology was an independent risk factor for overall survival in ICC patients, respectively (P<0.05). In addition, there was a statistical difference for overall survival in early recurrence patients among the three etiological subtypes (P<0.05). After PSM, the overall survival of patients with Stone-ICC was worse than those of Con-ICC and HBV-ICC subtypes (P<0.05), while the relapse-free survival of patients with Stone-ICC was equivalent to patients with Con-ICC and HBV-ICC (P>0.05). In Stone-ICC patients, the median overall survival was 16.0 months and 29.7 months, and the median relapse-free survival was 9.0 months and 20.0 months for non-ACT and ACT patients, respectively (P<0.05). Methods A total of 448 patients with ICC who underwent radical resection between 2010 and 2018 at ten Chinese tertiary hospitals were analyzed in the study. These patients were divided into conventional ICC (Con-ICC, n=261, 58.2%), hepatitis B virus ICC (HBV-ICC, n=102, 22.8%) and hepatolithiasis (Stone-ICC, n=85,19.0%) subtypes according to different etiology. Propensity score matching (PSM) was conducted to mitigate the baseline differences between related two subtypes. Introduction Intrahepatic cholangiocarcinoma (ICC), originating above the secondary branches of the bile duct, is the second most common biliary malignancy and accounts for about 10–15% of primary liver carcinoma [1, 2]. In recent years, the incidence of ICC has shown a significant upward trend worldwide [3, 4]. Surgical resection is the only potentially curative treatment option for ICC patients, but only 20% of the first-diagnosed patients are eligible for radical resection [5]. In addition, the prognosis of ICC patients is very poor, and the median overall survival (OS) and relapse-free survival (RFS) are about 30 months and 20 months, and the 5-year survival rate is approximately 30%, respectively [6, 7]. Besides, the benefits of postoperative adjuvant chemotherapy (ACT) are still unclear for ICC patients and need further research [8, 9]. Currently, the pathogenic factors of ICC are geographically different, mainly including primary sclerosing cholangitis (PSC), Caroli disease, cirrhosis, non- alcoholic fatty liver disease (NLFLD), hepatitis B virus (HBV) infection, intrahepatic bile duct stones (hepatolithiasis), and liver flukes [10–13]. According to the epidemiological data from China, hepatolithiasis and HBV infection are the most important risk factors for the occurrence of ICC, account for 15~30% of ICC patients with hepatolithiasis and 30~50% with HBV infection, respectively [14–17]. Different etiological subtypes have variable pathological characteristics, leading to the differences on prognosis of ICC patients. However, the differences on OS and RFS, as well as the benefits of ACT on prognosis of the three etiological subtypes for ICC patients are still unclear. The study aimed to evaluate the prognosis and the benefits of ACT in ICC patients with different etiology after radical resection. Conclusion The prognosis of Stone-ICC patients was significantly worse than those of Con-ICC and HBV-ICC patients. Interestingly, postoperative adjuvant chemotherapy can improve the prognosis of Stone-ICC patients effectively. The prognosis of Stone-ICC patients was significantly worse than those of Con-ICC and HBV-ICC patients. Intere can improve the prognosis of Stone-ICC patients effectively. Follow-up All patients included in the study were followed up after surgery. Routine follow-up was performed in outpatient and telephone. Liver function, tumor biomarkers (CEA, CA19-9, CA125), and ultrasound, contrast-enhanced CT or MRI examination were reviewed every 2 to 3 months within 1 year after surgery, and follow-up was conducted once every 3-6 months for more than 1 year after surgery. Recurrence referred to the discovery of new lesions by two or more imaging examinations. OS and RFS were calculated from the date of radical resection until the date of the most recent follow-up or death of the patients, and clinical evidence of tumor recurrence, respectively. Comparison of recurrence and survival By comparing overall recurrence, early recurrence (RFS≤1 year after surgery, ), and OS for the different etiological subtypes in a proportion of ICC patients, the results showed that there was no statistical difference in the proportion of patients with overall recurrence and early recurrence (Fig 3-A and B, P>0.05). By further comparing the difference of OS≤1 year, OS with 1~3 year and OS >3 years in early recurrence patients, there was a statistical difference with the three etiological subtypes, and Stone-ICC tended to have a worse prognosis (Fig 3-C, P<0.05), while there was no statistical difference in non-early recurrence patients (Fig 3-D, P>0.05). Therefore, the survival difference of the three etiological subtypes was mainly for OS in patients with early recurrence. Comparison of clinicopathological characteristic for different etiological subtypes in ICC Patients A total of 448 patients undergoing radical resection for histologically confirmed ICC between 2010 and 2018 were considered for inclusion. The comparison of clinicopathological characteristic of Con-ICC (no identifiable cause, n=261, 58.2%), HBV-ICC (n=102, 22.8%) and Stone-ICC (n=85, 19.0%) was summarized in Table 1. The three etiological subtypes in ICC patients had a certain correlation with clinicopathological characteristics of sex, age (year), obstructive jaundice, CA19-9, Child-Pugh grade, tumor location, morphologic grape, perineural invasion, liver capsule involvement, and AJCC 8th edition N stage (P<0.05). In addition, Stone-ICC had a higher proportion of CA19-9 >39.0 U/ml, presence of perineural invasion, and morphologic grape with periductal infiltrating and intraductal growth compared with HBV-ICC (P<0.05). Survival analysis on OS and RFS in the whole cohort The 1-, 3-, and 5-year OS rates of ICC patients were 84.9%, 42.4%, and 20.0%, and 1-, 3-, and 5-year RFS rates of ICC patients were 56.5%, 20.6%, and 10.3%, respectively. Median OS and RFS were 28.0 and 14.9 months, respectively. Univariate analysis showed that different etiology was a prognostic factor for OS and RFS of patients with ICC after radical resection, respectively (Supplement Fig 1, P<0.05). Multivariate analysis showed that different etiology was an independent risk factor for OS. Detailed results of the univariate and multivariate analysis are shown in Table 2. To eliminate the differences and be comparable of baseline clinicopathological characteristics between related two subtypes, a 1:1 PSM was utilized to identify 102 pairs of patients with Con-ICC and HBV-ICC, 70 pairs of patients with Con-ICC and Stone-ICC, 37 pairs of patients with HBV-ICC and Stone-ICC. Before and after PSM, the OS and RFS of patients with Con-ICC and HBV-ICC were not statistically significant (Fig 1, 2-A and B, P>0.05); however, the OS of patients with Stone-ICC was worse than those of Con-ICC and HBV-ICC subtypes (Fig 1, 2-C and E, P<0.05), while the RFS of patients with Stone-ICC was equivalent to patients with Con-ICC and HBV-ICC (Fig 2- D and F, P>0.05). Therefore, the results showed that the prognosis of Stone-ICC subtype was significantly worse than those of Con-ICC and HBV-ICC subtypes. Statistical analysis All statistical analyses were performed using SPSS version 25. Categorical variables were examined using c2 test. The Kaplan-Meier method and Log-rank test were conducted for univariate analysis, and Cox proportional hazard regression model was conducted for multivariate analysis. The Kaplan-Meier curves and histograms were conducted by GraphPad Prism (version 8.0, San Diego, California, USA). Variables with P<0.05 were considered as statistically significant. Propensity score matching Propensity score matching (PSM), as a very practical, novel, and creative statistical method for evaluating intervention effects using non-randomized controlled data, was conducted to mitigate the baseline differences between related two subtypes affecting long-term outcomes [18], which was conducted by SPSS version 25 (IBM Corp., Armonk, NY, USA). Propensity score analysis with 1:1 matching was performed within a range of 0.02 of standard deviation. Statistical analysis Patients and Design All patients undergoing curative resection for histologically confirmed ICC between 2010 and 2018 at ten tertiary hospitals in China (Oriental Hepatobiliary Hospital Affiliated to Naval Medical University; West China Hospital of Sichuan University; Tianjin Medical University Cancer Institute and Hospital; Hunan Provincial People’s Hospital; The First Hospital Affiliated to Army Medical University; Zhongda Hospital of Southeast University; The First Affiliated Hospital of Xi'an Jiaotong University; Affiliated Hospital of North Sichuan Medical College; Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine; The First Affiliated Hospital of Zhengzhou University) were considered for inclusion. The inclusion criteria were as follows: (1) patients underwent radical resection and margin status recorded microscopically negative (R0); (2) patients with HBV infection (HBsAg (+) and /or HBcAb (+)); (3) patients with bile duct stones in histology; (4) the data of clinicopathological characteristics and follow-up data were all available. The exclusion criteria were as follows: (1) the history of HBV infection and hepatolithiasis was not recorded in detail; (2) patients combined with HCV infection or HCV+HBV infection or HBV+ hepatolithiasis or HCV+ hepatolithiasis before surgery; (3) patients combined with PSC or Caroli disease in histology; (4) patients died within 30 days after surgery. Finally, a total of 448 patients were included in the study after the strict inclusion and exclusion criteria. All included patients were evaluated according to the 8th edition AJCC staging system and were followed up through December 2020. Page 2/13 Page 2/13 The regimens of postoperative ACT In this study, patients with postoperative ACT were strictly performed as follows. The regimens of included gemcitabine (1000mg/m2 on days 1 and 8) + capecitabine (1250 mg/m2 twice daily on days 1-14) of a three-week cycle; gemcitabine (1000mg/m2 on days 1 and 8) + cisplatin (30 mg/m2 on days 1 and 8) of a three-week cycle; gemcitabine (1000mg/m2 on days 1 and 8) + oxaliplatin (100 mg/m2 on day 1) of a three-week cycle; gemcitabine (1000mg/m2 on days 1 and 8) + tegafur (40~60 mg twice daily on days 1-14) of a three-week cycle. Discussion [24] have found that the patients with HBV-ICC can activate the immune memory produced by HBV infection previously, thereby enhancing anti-tumor immunity, and Iida et al. [25] have also revealed that HBV-ICC conferred a low risk of lymph node metastasis for postoperative recurrence, which may be the reason that HBV-ICC has a relatively better prognosis than the other two etiological subtypes. Many studies [20, 26, 27] also found that HBV infection was a favorable prognostic factor for ICC after surgery, which was consistent with our results. Unfortunately, patients with Stone-ICC had the worst prognosis in the study. Wang et al.[20] revealed that patients with Stone-ICC were often difficult to differentiate with benign biliary strictures, resulting in patients who were mostly diagnosed in the advanced stage. Due to the presence of hepatolithiasis, it can lead to long-term chronic inflammation, followed by dysplasia and multiple tumors. In addition, the proportion of Stone-ICC with elevated CA19-9, multiple tumors, and lymph node metastasis was significantly higher than the other two etiological types [20, 28, 29]. In this study, Stone-ICC had a higher proportion of CA19-9>39.0 U/ml, presence of perineural invasion, morphologic grape with periductal infiltrating and intraductal growth, and N1 stage compared with HBV- ICC and Con-ICC, which could explain the reason why Stone-ICC had a poor prognosis. Thus, we suggest a more aggressive preventive surgery for those patients with a long term history of hepatolithiasis to avoid the presence of ICC. At present, due to limited prospective data on the benefits of systemic ACT for ICC patients after radical resection [9], whether postoperative ACT could improve the prognosis of ICC patients is still controversial [30, 31]. In this study, we found that postoperative ACT could improve the OS and RFS effectively, and non-ACT was identified as an independent risk factor for ICC patients (Table 2). Recently, some studies [32, 33] proved that postoperative ACT was beneficial to the prognosis for ICC after surgery. By analyzing the prognostic improvement value of ACT for different etiological subtypes in ICC patients, the results showed that postoperative ACT was a benefit to Stone-ICC patients on OS and RFS. However, ACT did not significantly improve the prognosis for Con- ICC and HBV-ICC patients. Altman et al. [34] and Ke et al. [35] reported that ACT improving OS was related to more patients with lymph node-positive or T3/T4 stage. Discussion Hepatolithiasis and HBV infection as the two most common risk factors for ICC in China, and ICC without a clear cause (conventional-ICC) were the mainly specific pathogenic factors in present studies [14–17, 19]. Therefore, we divided the etiology of ICC patients into three subtypes, Con-ICC (no identifiable cause), HBV-ICC, and Stone-ICC. Survival analysis showed that the OS and RFS were statistically significant among the three etiological subtypes, and the etiology of ICC was identified as an independent risk factor for OS. To further compare the survival differences between related two etiological subtypes, PSM was conducted to eliminate differences in baseline clinicopathological characteristics. After PSM, the OS of patients with Stone-ICC was worse than those of Con-ICC and HBV-ICC subtypes, while the RFS of patients with Stone-ICC was equivalent to patients with Con-ICC and HBV-ICC. Wang et al. [20] conducted a single institutional study and found that Stone-ICC had a worse prognosis compared to HBV-ICC after PSM. Similarly, Zhang et al. [19] conducted an international multi-institutional study and also found that Stone-ICC had a worse prognosis than Con-ICC and HBV-ICC subtypes, and they further found that Stone-ICC and Con-ICC had statistical differences in OS and RFS, while Stone-ICC and HBV-ICC had no difference on prognosis after PSM. In this study, we also found that there was no statistical difference in the proportion of patients with overall recurrence, early recurrence and OS in non-early recurrence patients with the three etiological subtypes, while there was a statistical difference of OS ≤1 year, OS with 1~3 year and OS >3 years in early recurrence patients. So, the survival difference of ICC with the different etiological subtypes was mainly in OS, especially in patients with early recurrence. To further explore the reasons for the differences in the prognosis of ICC with different etiology, we analyzed the differences in clinicopathological characteristics of ICC with the three etiological subtypes. Based on the above results, we considered that the prognostic differences of ICC with different etiology had a strong correlation with its clinicopathological characteristics. Widespread epidemics with HBV infection in China increase integration of HBV gene fragments into liver cells, which contributes to ICC and also causes the HBV-ICC of China with highest distribution in the world [21–23]. In this study, the results showed that the prognosis of patients with HBV-ICC was better than those of Con-ICC and Stone-ICC subtypes. Ding et al. Comparison of OS and RFS in ACT To determine whether the ACT regimens affected the prognosis of patients, we first analyzed the prognosis differences among the four regimens for patients with postoperative ACT, and the results showed that there was no difference in OS and RFS among different chemotherapy regimens (P > 0.05). By analyzing the prognostic improvement value of ACT for different etiological subtypes in ICC patients, the results showed that in Con-ICC patients, the median OS was Page 3/13 Page 3/13 30.2 months and 30.2 months, and the median RFS was 14.9 months and 19.0 months for non-ACT and ACT patients, respectively (Fig 4-A and B, P > 0.05); in HBV-ICC patients, the median OS was 38.0 months and 44.5 months, and the median RFS was 13.0 months and 15.3 months for non-ACT and ACT patients, respectively (Fig 4-C and D, P > 0.05); in Stone-ICC patients, the median OS was 16.0 months and 29.7 months, and the median RFS was 9.0 months and 20.0 months for non-ACT and ACT patients, respectively (Fig 4-E and F, P < 0.05). Therefore, postoperative ACT can improve the OS and RFS of Stone-ICC patients effectively. 30.2 months and 30.2 months, and the median RFS was 14.9 months and 19.0 months for non-ACT and ACT patients, respectively (Fig 4-A and B, P > 0.05); in HBV-ICC patients, the median OS was 38.0 months and 44.5 months, and the median RFS was 13.0 months and 15.3 months for non-ACT and ACT patients, respectively (Fig 4-C and D, P > 0.05); in Stone-ICC patients, the median OS was 16.0 months and 29.7 months, and the median RFS was 9.0 months and 20.0 months for non-ACT and ACT patients, respectively (Fig 4-E and F, P < 0.05). Therefore, postoperative ACT can improve the OS and RFS of Stone-ICC patients effectively. Discussion We considered that ACT improving the prognosis of Stone-ICC was related to its high proportion of elevated CA19-9, perineural invasion, N1 stage patients. Of course, whether ACT can improve the prognosis of Con-ICC and HBV-ICC patients still needs further research. However, there were several limitations in our study. The study included 448 ICC patients after radical resection from 10 medical centers in China, which effectively increased the universality of the study, while the sample size was still relatively small. Besides, the study did not include ICC patients from Western countries, because the role of hepatolithiasis and HBV infection was not as important as those patients in China. In addition, the preoperative inflammatory indicators were not available, so the differences of the three etiological subtypes of inflammatory response were not further compared. Accordingly, we should clarify the molecular mechanisms and prognostic monitoring indicators for ICC with different etiology in the future, so as to provide references and decision support for the individualized diagnosis, treatment and prevention for ICC patients. Conclusion In conclusion, this study retrospectively analyzed 448 ICC patients with different etiology after radical resection, the survival difference of ICC with different etiological subtypes was mainly in OS, especially in patients with early recurrence, but there was no difference in RFS. Importantly, different etiology was identified as an independent risk factor for OS in patients with ICC after radical resection. The prognosis of Stone-ICC patients was significantly worse than those of Con-ICC and HBV-ICC patients. Interestingly, postoperative ACT can improve the prognosis of Stone-ICC patients. Page 4/13 Page 4/13 Availability of data and materials This manuscript contains all associated data. This manuscript contains all associated data. Acknowledgments No further acknowledgments. Consent for publication Not applicable. Not applicable. Competing interests Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. Abbreviations ICC: Intrahepatic cholangiocarcinoma; ACT: Adjuvant chemotherapy; PSM: Propensity score matching; PSC: Primary sclerosing cholangitis; NLFLD: Non- alcoholic fatty liver disease; HBV: Hepatitis B virus; OS: overall survival; RFS: Relapse-free survival Ethics approval and consent to participate The study was approved by the ethics committee of Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine (No: XHEC-JDYXY-2018- 002), Shanghai, China, as well as from the ethics committees of the other study centers. Written informed consent was obtained from all included patients and their families before study enrollment. Author contributions Z. Geng, and Z. Tang conceived and designed the experiments. Q. Li and J. Su performed the experiments. Y Qiu, H. Wu, T. Song, X. Mao, Y. He, Z. Cheng, J. Li, and W. Zhai collected and offered the data. Q. Li, C. Chen, and D. Zhang conducted statistical analysis. 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Table 2 Univariate and multivariate analysis of prognosis for ICC after radical resection Tables Table 1 Comparison of clinicopathological characteristic for ICC with different etiological subtypes after radical resection Table 1 Comparison of clinicopathological characteristic for ICC with different etiological subtypes after radical resection Page 6/13 Page 6/13 Page 6/13 ICC with different etiological subtypes c2 P value Con-ICC (%) HBV-ICC (%) Stone-ICC (%) Sex Male 127(48.7) 63(61.8) 33(38.8) 10.073 0.006 Female 134(51.3) 39(38.2) 52(61.2) Age (year) ≤55 110(42.1) 51(50.0) 26(30.6) 7.226 0.027 >55 151(57.9) 51(50.0) 59(69.4 Obstructive jaundice No 246(94.3) 100(98.0) 69(81.2) 21.723 <0.001 Yes 15(5.7) 2(2.0) 16(18.8) AFP (ng/ml) ≤7.0 203(78.8) 77(75.5) 63(74.1) 0.563 0.755 >7.0 58(22.2) 25(24.5) 22(25.9) CEA (ng/ml) ≤5.0 200(76.6) 77(75.5) 60(70.6) 1.260 0.533 >5.0 61(23.4) 25(24.5) 25(29.4) CA19-9(U/ml) ≤39.0 121(46.1) 48(47.1) 25(29.4) 8.260 0.016 >39.0 140(53.6) 54(52.9) 60(70.6) CA125(U/ml) ≤35.0 161(61.7) 59(57.8) 51(60.0) 0.464 0.793 >35.0 100(38.3) 43(42.2) 34(40.0) Child-Pugh Grade Grade A 247(94.6) 99(97.1) 74(87.1) 8.750 0.013 Grade B 14(5.4) 3(2.9) 11(12.9) Type of resection Wedge resection 108(41.4) 49(48.0) 28(32.9) 8.722 0.068 Minor hepatectomy 109(41.8) 43(42.2) 36(42.4) Major hepatectomy 44(16.9) 10(9.8) 21(24.7) Tumor differentiation Well 17(6.5) 4(3.9) 7(8.2) 3.227 0.521 Moderate 164(62.8) 59(57.8) 49(57.6) Poor 80(30.7) 39(38.2) 29(34.1) Tumor location Left 128(49.0) 36(35.3) 56(65.9) 18.423 0.001 Right 99(37.9) 53(52.0) 23(27.1) Left and right 34(13.0) 13(12.7) 6(7.1) Morphologic grape Mass-forming 221(84.7) 88(86.3) 51(60.0) 28.811 <0.001 Periductal infiltrating 24(9.2) 8(7.8) 17(20.0) Intraductal growth 16(6.1) 6(5.9) 17(20.0) Tumor size (cm) 126(48.3) 50(49.0) 35(41.2) >5.0 126(48.3) 50(49.0) 35(41.2) Major vascular invasion No 217(83.1) 91(89.2) 69(81.2) 2.725 0.256 Yes 44(16.9) 11(10.8) 16(18.8) Microvascular invasion No 235(90.0) 87(85.3) 71(83.5) 3.246 0.197 Yes 26(10.0) 15(14.7) 14(16.5) Perineural invasion No 223(85.4) 96(94.1) 66(77.6) 10.535 0.005 Yes 38(14.6) 6(5.9) 19(22.4) Liver capsule involvement No 180(69.0) 64(62.7) 69(81.2) 7.722 0.021 Yes 81(31.0) 38(37.3) 16(18.8) AJCC 8th edition T stage T1a/T1b 63(24.1) 27(26.5) 26(30.6) 3.817 0.431 T2 132(50.6) 45(44.1) 43(50.6) T3/T4 66(25.3) 30(29.4) 16(18.8) AJCC 8th edition N stage N0 193(73.9) 82(80.4) 61(71.8) 2.211 0.031 N1 68(26.1) 20(19.6) 24(28.2) AJCC 8th edition TNM stage IA/IB 119(45.6) 42(41.2) 45(52.9) 3.540 0.472 II 40(15.3) 20(19.6) 10(11.8) IIIA/IIIB/IV 102(39.1) 40(39.2) 30(35.3) >5.0 Page 8/13 Page 8/13 Page 8/13 OS RFS Univariate analysis Multivariate analysis Univariate analysis Multivariate analysis HR (95%CI) P HR (95%CI) P HR (95%CI) P HR (95%CI) P Sex Female vs Male 0.935(0.721~1.212) 0.610 0.891(0.717~1.109) 0.302 Age (year) >55 vs≤55 1.242(0.955~1.615) 0.105 1.046(0.838~1.305) 0.693 Obstructive jaundice Yes vs No 1.172(0.872~2.150) 0.175 1.090(0.729~1.628) 0.675 Etiology HBV-ICC vs Con- ICC 0.801(0.567~1.130) 0.206 0.862(0.608~1.223) 0.405 1.143(0.873~1.498) 0.331 Stone-ICC vs Con- ICC 2.185(1.597~2.989) <0.001 2.009(1.447~2.789) <0.001 1.563(1.181~2.068) 0.002 AFP (ng/ml) >7.0 vs ≤7.0 1.137(0.834~1.550) 0.416 1.041(0.804~1.348) 0.761 CEA (ng/ml) >5.0 vs ≤5.0 1.439(1.076~1.923) 0.014 1.348(1.054~1.725) 0.017 CA19-9(U/ml) >39.0 vs ≤39.0 1.378(1.056~1.798) 0.018 1.370(1.097~1.711) 0.006 1.252(1.010~1.568) 0.030 CA125(U/ml) >35.0 vs ≤35.0 1.604(1.218~2.111) 0.001 1.308(1.046~1.635) 0.019 Child-Pugh Grade Grade B vs A 1.150(0.709~1.866) 0.570 1.006(0.662~1.526) 0.979 Type of resection Minor hepatectomy vs Wedge resection 1.718(1.284~2.297) <0.001 1.157(1.127~2.043) 0.006 1.684(1.318~2.151) <0.001 1.515(1.183~1.941) 0.001 Major hepatectomy vs Wedge resection 1.962(1.344~2.864) <0.001 1.405(1.149~2.080) 0.019 1.829(1.324~2.528) <0.001 1.733(1.245~2.411) 0.001 Lymphadenectomy Yes vs No 0.909(0.697~1.185) 0.479 1.048(0.837~1.312) 0.683 Tumor differentiation Moderate vs Well 1.318(0.739~2.352) 0.350 1.002(0.644~1.560) 0.993 Poor vs Well 2.243(1.228~4.095) 0.009 1.597(1.108~2.302) 0.012 Tumor location Right vs Left 0.689(0.518~1.129) 0.069 0.890(0.704~1.127) 0.334 Left and right vs Left 0.857(0.556~1.321) 0.485 0.976(0.685~1.391) 0.895 Morphologic grape Periductal infiltrating vs Mass- forming 0.871(0.559~1.358) 0.543 0.997(0.700~1.420) 0.986 Intraductal 0 825(0 502~1 357) 0 448 0 789(0 530~1 174) 0 789 Page 9/13 >5.0 vs≤5.0 1.517(1.148~2.003) 0.003 1.352(1.034~1.797) 0.028 1.164(0.936~1.149) 0.173 Major vascular invasion Yes vs No 1.716(1.249~2.357) 0.001 1.606(1.217~2.121) 0.001 Microvascular invasion Yes vs No 1.506(1.009~2.248) 0.045 1.748(1.388~2.202) <0.001 1.788(1.400~2.257) <0.001 Perineural invasion Yes vs No 2.068(1.421~3.008) <0.001 1.331(0.964~1.838) 0.083 Liver capsule involvement Yes vs No 1.136(0.857~1.507) 0.375 1.262(0.894~1.783) 0.186 AJCC 8th edition T stage T2 vs T1a/T1b 1.317(0.947~1.831) 0.101 1.164(0.885~1.532) 0.277 T3/T4 vs T1a/T1b 1.559(1.080~2.250) 0.018 1.713(1.273~2.305) <0.001 AJCC 8th edition N stage N1 vs N0 1.949(1.459~2.603) <0.001 1.791(1.334~2.406) <0.001 1.648(1.289~2.106) <0.001 1.378(1.070~1.776) 0.013 AJCC 8th edition TNM stage II vs IA/IB 1.362(0.947~1.961) 0.096 1.258(0.907~1.746) 0.170 IIIA/IIIB/IV vs IA/IB 1.521(1.138~2.033) 0.005 1.732(1.365~2.198) <0.001 Adjuvant chemotherapy Yes vs No 0.685(0.520~0.902) 0.007 0.632(0.469~0.851) 0.003 0.715(0.568~0.901) 0.005 0.655(0.517~0.831) <0.001 Page 10/13 ( ) ( ) ( ) Major vascular invasion Yes vs No 1.716(1.249~2.357) 0.001 1.606(1.217~2.121) 0.001 Microvascular invasion Yes vs No 1.506(1.009~2.248) 0.045 1.748(1.388~2.202) <0.001 1.788(1.400~2.257) <0.001 Perineural invasion Yes vs No 2.068(1.421~3.008) <0.001 1.331(0.964~1.838) 0.083 Liver capsule involvement Yes vs No 1.136(0.857~1.507) 0.375 1.262(0.894~1.783) 0.186 AJCC 8th edition T stage T2 vs T1a/T1b 1.317(0.947~1.831) 0.101 1.164(0.885~1.532) 0.277 T3/T4 vs T1a/T1b 1.559(1.080~2.250) 0.018 1.713(1.273~2.305) <0.001 AJCC 8th edition N stage N1 vs N0 1.949(1.459~2.603) <0.001 1.791(1.334~2.406) <0.001 1.648(1.289~2.106) <0.001 1.378(1.070~1.776) 0.013 AJCC 8th edition TNM stage II vs IA/IB 1.362(0.947~1.961) 0.096 1.258(0.907~1.746) 0.170 IIIA/IIIB/IV vs IA/IB 1.521(1.138~2.033) 0.005 1.732(1.365~2.198) <0.001 Adjuvant chemotherapy Yes vs No 0.685(0.520~0.902) 0.007 0.632(0.469~0.851) 0.003 0.715(0.568~0.901) 0.005 0.655(0.517~0.831) <0.001 Figures Page 10/13 Microvascular invasion Yes vs No 1.506(1.009~2.248) 0.045 1.748(1.388~2.202) <0.001 1.788(1.400~2.257) <0.001 Perineural invasion Yes vs No 2.068(1.421~3.008) <0.001 1.331(0.964~1.838) 0.083 Liver capsule involvement Yes vs No 1.136(0.857~1.507) 0.375 1.262(0.894~1.783) 0.186 AJCC 8th edition T stage T2 vs T1a/T1b 1.317(0.947~1.831) 0.101 1.164(0.885~1.532) 0.277 T3/T4 vs T1a/T1b 1.559(1.080~2.250) 0.018 1.713(1.273~2.305) <0.001 AJCC 8th edition N stage N1 vs N0 1.949(1.459~2.603) <0.001 1.791(1.334~2.406) <0.001 1.648(1.289~2.106) <0.001 1.378(1.070~1.776) 0.013 AJCC 8th edition TNM stage II vs IA/IB 1.362(0.947~1.961) 0.096 1.258(0.907~1.746) 0.170 IIIA/IIIB/IV vs IA/IB 1.521(1.138~2.033) 0.005 1.732(1.365~2.198) <0.001 Adjuvant chemotherapy Yes vs No 0.685(0.520~0.902) 0.007 0.632(0.469~0.851) 0.003 0.715(0.568~0.901) 0.005 0.655(0.517~0.831) <0.001 Figures Page 10/13 Figures Page 10/13 Figure 1 Comparison of overall survival and relapse-free survival after radical resection between conventional ICC and HBV-ICC (A and B), between conventional ICC and Stone-ICC (C and D), and between HBV-ICC and Stone-ICC (E and F) before PSM. Comparison of overall survival and relapse-free survival after radical resection between conventional ICC and HBV-ICC (A and B), between conventional ICC and Stone-ICC (C and D), and between HBV-ICC and Stone-ICC (E and F) before PSM. Figure 2 Comparison of overall survival and relapse-free survival after radical resection between conventional ICC and HBV-ICC (A and B), between conventional ICC and Stone-ICC (C and D), and between HBV-ICC and Stone-ICC (E and F) after PSM. Figure 2 Figure 4 Comparison of overall survival and relapse-free survival in adjuvant chemotherapy after radical resection between conventional ICC and HBV-ICC (A and B), between conventional ICC and Stone-ICC (C and D), and between HBV-ICC and Stone-ICC (E and F). Figure 2 Comparison of overall survival and relapse-free survival after radical resection between conventional ICC and HBV-ICC (A and B), between conventional ICC and Stone-ICC (C and D), and between HBV-ICC and Stone-ICC (E and F) after PSM. Page 11/13 Figure 3 Comparison of overall recurrence ratio (A), recurrence time ≤1 year and >1 year (B), and overall survival time with recurrence time ≤1 year (C) and overall Figure 3 Comparison of overall recurrence ratio (A), recurrence time ≤1 year and >1 year (B), and overall survival time with recurrence time ≤1 year (C) and overall survival time with recurrence time >1 year after radical resection for conventional ICC, HBV-ICC and Stone-ICC. Figure 3 Comparison of overall recurrence ratio (A), recurrence time ≤1 year and >1 year (B), and overall survival time with recurrence time ≤1 year (C) and overall survival time with recurrence time >1 year after radical resection for conventional ICC, HBV-ICC and Stone-ICC. Page 12/13 Page 12/13 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. SupplementFig1.tif SupplementFig1.tif Supplementaryfigurelegend.docx Supplementaryfigurelegend.docx Page 13/13
https://openalex.org/W4375930734	https://gjmasuok.com/index.php/gjmas/article/download/151/99	English	null	DYNAMICS OF MONEY LAUNDERING AND TERRORIST FINANCING: A CHALLENGE TO PAKISTAN	Global journal for management and administrative sciences	2,022	cc-by	11,155	17 17 Global Journal for Management and Administrative Sciences ISSN (Print): 2788-4821 ISSN (Online): 2788-483X Volume 3, Issue 1, Page 17-40, June 30, 2022 Abstract This article throws light on organised crime which is chameleonic in nature and due to its dynamics; it represents and adjusts according to promulgated laws. This has prospered and overlapped with the criminal activities of organised criminal groups. In the same way, money laundering is a predicate offense and by default, it is linked to other organised crimes. The study discussed the impact of 9/11 which was a culmination of a lethal combination of money laundering and terrorist financing. This same nexus is currently being experienced by Pakistan. The study critically dissects the dissipation of organised criminal activities and money laundering in Pakistan along with transnational stakeholders’ involvement with counter measures. Various methodologies adopted by criminal networks are reviewed in the article. Criminal networks exploit the pervasiveness of technological advancement more efficiently than the government. Government should recognize that sovereignty is daily compromised at the hands of stateless organised criminal networks. These are involved in the laundering of money, financing of terrorism and other linked organised crimes. The active presence of terrorist networks like Taherik-e.-Taliban, other non-state actors, porous borders with Afghanistan, and the cash-based and undocumented economy in the presence of corruption pose serious threats to the regime and require immediate counter measures. Moreover, all countries in the region require beefing up efforts against money laundering and terrorist financing at local, regional, and international levels. Pakistan has shown its international commitment to global stakeholders by adopting multi-prong strategies. But there is a long way to go in countering money laundering and terrorist financing. Counter-terrorist operations, namely ‘Zarb-e-Azb’ (means sharp and cutting strike) in North Waziristan along the Pakistan-Afghanistan border in 2014 & Radd-ul-Fasaad (means the elimination of strife) across the country in 2017 were carried out by Pakistan army as a part of state efforts to curb local and cross border terrorism. Keywords: Money laundering, Organised crime, Terrorist financing DYNAMICS OF MONEY LAUNDERING AND TERRORIST FINANCING: A CHALLENGE TO PAKISTAN Dr. Tahseen Ahmed Shaikh Advisor Anti Money Laundering and Terrorist Financing Risk International Commercial Bank Dr. Ammad Zafar Assistant Professor Department of Management Sciences Usman Institute Technology University, Karachi, Pakistan This work is licensed under a Creative Commons Attribution 4.0 International License. Introduction Organized crime is sometime transnational, sophisticated, complex and pervasive in nature. It is multifaceted. Due to these dynamics; perpetrators tuned their criminal activities by considering enforcement regime’s counter measures. In this article, Dynamics of money laundering and terrorist financing: a challenge to Pakistan 18 more emphasis is given on nexus of money laundering and terrorist financing, since money laundering itself is a predicate offence. Thus, it is directly linked with organised crime. When we study current scenario in Pakistan; we find existence of multi-faceted organised crime. The situation further aggravates when cross border stake holders firm their nexus in organised criminal activities. Since this South Asian region has been vulnerable to various cross border interference, swayed by superpowers during the cold war and post-cold war era as well. Taking an opportunity of fragile peace in the region, in particular to Afghanistan situation, non-state holders discreetly became contributing actors in organised criminal groups. Declared boundaries of the countries, in particular to Pakistan and Afghanistan; didn’t offer any serious challenges to their cross border organised criminal activities. Thus, serious threats of smuggling and trafficking of narco- drugs, arms and ammunition found its transit passage through Pakistan to European, non- European markets. These existing organised criminal activities further proliferated when it found nexus with cross border terrorist activities. Certainly, there is no denial to the fact, fragile peace, dwindling democracy; weak functioning of state organs; poor government spending on human development; deteriorating economy; and nonexistence of pragmatic long-term policies have proved to be pushing factors in the region in general and Pakistan in particular. After happening of 9/11; it compelled national, regional and international anti money laundering and terrorist financing regime to revisit their existing counter policies and measures. Nation states witnessed influence and inter-transnational nexus of criminal groups involved in money laundering and terrorist financing. Global regime required new governance structures for countering challenges globally. Due to compelling threat of money laundering, the international counter regime sought to assist and encourage efforts and take initiatives to enhance the role of countering money laundering and terrorist financing regime globally. This led to awareness efforts, revitalise investigation, enforcement, regulatory and criminal justice system which could enforce and implement the stringent promulgated laws passed by countries. Introduction Counter terrorist operations, namely ‘Zarb-e-Azb’ (means sharp and cutting strike) in North Waziristan along the Pakistan-Afghanistan border in 2014 & Radd-ul-Fasaad (means elimination of strife) across the country 2017 were carried out by Pakistan army as a part of serious efforts at state level to curb local and cross border terrorism. Realizing the gravity of money laundering crime in Pakistan, the researcher reviewed the existing features of organised crimes in Pakistan which depict their modus operandi. The fundamental changes have given the criminal organised networks a new intensity, since the last decade, which is likely to persist. Rationale of Research The study tries to review various dynamics of money laundering. By reviewing international literature and data on Pakistan, the researcher finds no specific money laundering activities are addressed based on criminological aspects. This may involve how the organised groups commit criminal activities of money laundering in Pakistan and involvement of transnational methodologies. Since it has porous borders with politically disturbed and terrorist-ridden Afghanistan; and provides ideal passage for trafficking narco-drugs to and from Iran and China in its North & West. After happening of 9/11, the number of terrorist activities has seriously increased. Thus, to carry out research work on the subject is need of hour in Pakistan. Scope of Research Threat of terrorism is very much related to terrorist financing which finds its channels through money laundering. Pakistan, being victim to terrorism, has been countering these existing challenges of organized crimes through its counter regime measures. How so far these efforts have been effective and what further counter measures to be taken; these dynamics are examined and reviewed in this research work. Introduction Realising the gravity of nexus between money laundering and terrorist financing during 9/11; Financial Action Task Force (FATF), which is one of the counter regimes of money laundering, also clubbed its counter measures of money laundering with terrorist financing. This step was a serious effort by an international regime to keep abreast the world of new tactics used by the criminal groups and networks by changing their methods and trends according to emerging innovations and technologies. In the list of members, those countries were included, which were reluctant or not interested to implement FATF Recommendations in their respective countries. United Nations Office for Drug Control and Crime is also active for effective working on the global programme countering money-laundering activities. It involved cooperation and technical assistance to the international, regional institutions and organizations which were responsible in countering laundering of money and drug trafficking. The strategy of Pakistan has a regional and international impact, since Pakistan is strategically located in the Asia region. That is why, the USA State Department stated in its report that Pakistan is strategically important for being priority region in counterfeiting financing of terrorism. Pakistan being signatory to international compliance regime; it also beefed up its efforts against laundering money and financing Global Journal for Management and Administrative Sciences ISSN (Print): 2788-4821 ISSN (Online): 2788-483X Volume 3, Issue 1, Page 27-40, June 30, 2022 Global Journal for Management and Administrative Sciences ISSN (Print): 2788-4821 ISSN (Online): 2788-483X Volume 3, Issue 1, Page 27-40, June 30, 2022 19 of terrorism regimes at the local, regional, and international level (INL, 2010). Root causes and existing weaknesses of non-effective anti-money laundering and terrorist financing measures by Pakistan regime are discussed and analysed in the article. The main objective is to critically dissect the dissipation of organised crime and money laundering in Pakistan along with transnational stake holders’ involvement with counter measures. This existing threat is responsible for the state where sovereignty is everyday compromised at the hands of non-state actors. Counter measures are taken by Pakistan. But there are still internal challenges of combating a qualitative new phenomenon with obsolete tools, weak enforced laws, corrupt bureaucratic hierarchy and fragile strategies. Pakistan has showed its international commitment to the global stakeholders for adopting multi-prong strategies. But there is a long way to go in countering money laundering and terrorist financing. Research Questions Q1. What are elements of Organised Crime, in reference to money laundering and terrorist financing? Q2. What are the challenges of money laundering and terrorist financing? Q2. What are the challenges of money laundering and terrorist financing? 20 Dynamics of money laundering and terrorist financing: a challenge to Pakistan Dynamics of money laundering and terrorist financing: a challenge to Pakistan Q3. What are Pakistan’s Counter Regime measures against money laundering and terrorist financing? Theoretical Framework The framework of this research study examines anti-money laundering regime’s counter measures in the country particularly and globally in general. The contemporary socio-economic and psychological factors with cultural influence related to organised crime with reference to laundering of money are also reviewed. The study would lead the future research works to introduce new avenues in countering money laundering and terrorist financing. Research Objective This study is mainly focused on money laundering and terrorist financing. It dissects various elements of organised crime, in reference to the focus of this study. Various impediments are discussed, to understand challenges being faced by the counter regime in Pakistan. Significance of Research This research work is a deliberate approach to critically review and draw results from the collected literature and data of various theorists. Through various methods, unnecessary data is thrashed out from collected research works. The researcher believes that the selected research work has limited theories in social sciences and criminology. However, various dynamics of organised crime in reference to money laundering and terrorist financing is analyzed. Literature Review Elements of Organised Crime Related to Money Laundering and Terrorist Financing Article analyses various elements of organised crime and to know the outcome of their nexus in proliferating more organised crime in the society. But more emphasis is given on nexus of laundering money and financing of terrorism specific to Pakistan. Organised crime involves group organisation, and this group is controlled through hierarchical links or personal relatives. The design is to have ill-gotten earnings, controlling territories or capturing markets with the use of threats, suppression, corruption; laundering money earned through illicit proceeds and make it legal. Sometime, organised crime is transnational and crosses national borders for their activity and engages organised transnational criminal groups (Adamoli, 1998). When we discuss of organised crime; we cannot depart organised criminal group which plays a pivotal role in perpetrating any organised crime. The UN’s Palermo Convention gave the concept of ‘organised criminal group’ in its definition. As pointed out above, it is hierarchical in nature and consists of three or more actors and operating over a period. It acts with intention of perpetrating serious crimes as listed in Palermo Convention and achieve financial or other material benefits (UNTOC, 2000). According to UN Convention “organised criminal group consists of three or more individuals who exist for a certain period and acts with the aim to execute one or more serious crimes determined by this Convention, in order to obtain direct or indirect financial benefit or other kind of material benefit” (UNTOC, 2000). Gilmore also discusses organised criminal groups and to them- these vary in shape, size, specializations and skills. This extends from highly layered organizations to more fluid and dynamic mechanism (Gilmore, 2004). On the other hand, Gottschalk reviewed the characteristics and elements of organised criminal groups which involve average size of criminal organizations. Half of its members were of same racial background, while the others were not from same ethnic linkages. To him, some criminal networks had political sway at local and regional level, and also some existence at the national level in the country of intervention (Gottschalk, 2009). Limitation of Research This selected research work has also inherited limitations due to its own dynamics in Pakistan. Absence of consolidated data on the system related to money laundering and financing of terrorism is another obstacle to analyze the data. The researcher finds very limited international data and literature available on Pakistan which is an impediment in a way to review the selected research topic. Despite the fact, organised criminal networks are mainly involved in criminal activities of laundering of money in Pakistan, as well as it forms transnational criminal activity. The researcher believes that the research work in hand would contribute alot in scratching the surface for future works in the subject of criminological aspects of laundering of money and financing of terrorism along with organised crimes in the country. Global Journal for Management and Administrative Sciences ISSN (Print): 2788-4821 ISSN (Online): 2788-483X Volume 3, Issue 1, Page 27-40, June 30, 2022 Global Journal for Management and Administrative Sciences ISSN (Print): 2788-4821 ISSN (Online): 2788-483X Volume 3, Issue 1, Page 27-40, June 30, 2022 21 Literature Review Challenges of Money Laundering and Terrorist Financing With the passage of time, the criminal enterprises have changed their fabric. Earlier these were monolithic and rigidly hierarchical in structures, which were helpful to law enforcement agencies to burst criminal organizations. Now this rigid structure has been replaced with flexible and decentralized trend, which is not also easy task for the law enforcement agencies to locate. The larger size structures are substituted with small organizations also and this provides them insulation from law enforcement agencies if any single organization group is located or traced by the agencies (Southerland and Potter, 1993); (Schelling, 1976). 22 Dynamics of money laundering and terrorist financing: a challenge to Pakistan Dynamics of money laundering and terrorist financing: a challenge to Pakistan This new fabric provides an opportunity to resurrect, re-associate and re- organise their criminal activities, if these were earlier disrupted due to some reasons. This has provided them an opportunity to adept according to the national, regional and global counter regime framework (Hage and Aiken, 1970). It is observed that commission of offences by the organised criminal groups is interdependent. It involves chain of various types of crimes until the finality offence to be committed by these groups. Vertical interdependence among crimes rises till the final crime is perpetrated. However, more intermediate, or instrumental offences are committed by these groups to achieve their finality. This can be understood, in the case of fraud, corruption and money laundering crimes. These crimes functionally related to each other. One of them is the finality and other two are intermediate or instrumental towards the end. In the case of corruption, fraud and money laundering are the means to launder these proceeds and legitimacy. To criminalize the vertical interdependency among crimes; law enforcement should bring them into account collectively rather than tackling them in isolation (Savona, 1997). These trends have revolutionized organised criminal groups. They are entrepreneurial and free from impediments like-legislation, borders, morality or technology. They are strategic, dominant, monopolist, profiteering and influential. They are adaptive and goal oriented which is profit-making. They are well calculated and anticipatory of law enforcement move. They are collaborative to each other for mutual benefit and swiftly disperse and reorganise themselves in other markets if disrupted. Organised crime is not separate from legitimate businesses but operates within it and based on maximize profits and minimize risks. Pakistan’s Counter Regime Measures against Money Laundering and Terrorist Financing The researcher analysed various provisions of Anti Money Laundering Act 2010 and finds appended key impediments: • Lack of understanding amongst investigating agencies of the scope and use of the money laundering offence. • Lack of capacity to conduct financial investigation at an early stage of the investigative process. • The agencies face extreme difficulty in proving before the court of law that the money is proceeds of predicate crime. • The agencies face extreme difficulty in proving before the court of law that the money is proceeds of predicate crime. In peer review report by the Global Forum Transparency and Exchange of Information for Tax Purposes (OECD, 2022), it reviewed various aspects of Pakistan’s counter regime’s efforts under AML/CFT framework. It is observed that the counter regime framework relies on Anti-Money Laundering Act 2010 (the AMLA 2010) and various sector based regulations. During the earlier review, absence of supervisory bodies was realised in sector based designated non-financial businesses and professions (DNFBPs). Later, effective counter regime efforts were taken by the country in appointing sector based supervisory authorities in DNFBPs; in particular for accountants and lawyers. However, the modalities which are required under the Income Tax Ordinance to provide information on beneficial ownership to Federal Board of Revenue (FBR) are not yet operationalized. After various peer periodic reviews by FATF; Pakistan has taken drastic measures in counter regime framework. Already existing supervisory bodies State Bank of Pakistan (SBP) and Security Exchange Commission of Pakistan (SECP) are supported by other regulatory bodies like the FBR, the Institute of Chartered Accountants of Pakistan (ICAP), the Institute of Cost and Management Accountants of Pakistan (ICMAP), the Supervisory Board for National Savings Schemes and the Supervisory Board for Pakistan Post. Challenges of Money Laundering and Terrorist Financing Since this is strategic in its operations, it plans aggressive marketing and hires the expertise of professionalists and executors as a part of risk mitigation strategies. Complex networks are involved in illegitimate transnational transactions for making it legitimate through laundering money as earned from proceeds of narcotic drugs, cybercrime, human trafficking and other organised crimes. Organised crime may seem to be far distant threat, since it is far from people’s lives. But in real; its social, economic, moral and financial harms, are caused by illicit narcotic drugs, use of violence and intimidation, terrorist financing and money laundering. These have serious irreparable consequences on the whole society. In the non-developed countries, various forms of institutional organised crime influence state and its people. Institutionalized organised crime involves corrupted politics steered by the transnational countries. This is also facilitated through the sophistication by perfidy and most clandestine interference at the transnational level. Interference may end in the coup détente and use of violence and force against innocent civilians and ushers a reign of terror against the humanity (Coleman, 2001), (Nikolovska and Sundać, 2001). Institutionalized organised crime is not an open crime (Labović and Nikolovski, 2010). Organised criminal activities, sometime includes activities committed across borders and involves many other same activities. It is not possible globally to assess Global Journal for Management and Administrative Sciences ISSN (Print): 2788-4821 ISSN (Online): 2788-483X Volume 3, Issue 1, Page 27-40, June 30, 2022 23 accurate scope of organised criminal activities due to the conceal nature. The list is broad, and it changes between regions and always continues to increase. accurate scope of organised criminal activities due to the conceal nature. The list is broad, and it changes between regions and always continues to increase. Pakistan’s Counter Regime Measures against Money Laundering and Terrorist Financing Discussion Researcher tries to describe in brief a few of major following criminal activities, linked with organised crime I Pakistan, before proceeding to nexus of money laundering and terrorist financing: Threat of corruption is getting serious where controls are non-existing or weak with less enforcement and implementation. There is close link between corruption and transnational organised crime. It is more often, successful transnational criminal activities depend on corruption (Velkova and Georgievski, 2004). The Palermo Convention demands states to criminalise corruption. UN resolution 55/61 emphasises other independent conventions for effective international legal instruments against corruption. Corruption was particularly described in tough terms by the Secretary-General in 2003 that corruption was an insidious plague which had a wide range of corrosive effects on societies. It undermined democracy and the rule of law, which led to violations of human rights, distortion markets, erosion the quality of life, and allowed organised crime, terrorism and other threats to human security to flourish (UNODC, 2003). To sustain stability in long term, the most eminent scholars believe that corruption is one of the channels that contribute organised crime (World Bank, 2000); (Georgievski and Stanković, 2008). Corruption is considered to be an essential characteristic of institutional organised crime. To speak about the institutional organised crime, the abuse of the public authority is common practice. It involves successful operation of organised crime and it is inevitable component (Fijnaut and Paoli, 2004); (Labović, 2006). Abusing of legal institutional hierarchy and pretext of implementing a legitimate policy; are some of common traits in commission of corruption. This causes social, political and economic consequences; without using of violence and force (Albanese, 2010), (Zoutendijk, 2010), (Velkova, and Georgievski, 2004); (Van, 2004). Pakistan has general and special laws; later is particular for specific organised act; like, NAB Act (for anti-corruption) and other special laws. Punitive punishments are also already extended for individuals and groups involved in commission of corruption. Former Pakistan’s Prime Minister Nawaz Sharif was found guilty of corruption and convicted of 10 years imprisonment (The Washington Post, 2018). Pakistan is the 140 least corrupt nation out of 180 countries, according to the 2021 Corruption Perceptions Index reported by Transparency International (CPI, 2021). The Russian invasion of Afghanistan in 1979 confronted Pakistan with manifold problems, including introduction of arms smuggling, human trafficking, narco- drug/heroin cultures. These had engulfed the whole country and the nation as hostage to terrorism. Research Methodology There is no set systematic data and sharing of information at the central level in the country related to money laundering, terrorist financing and other related organised crimes. Financial institutions have limited scope of reporting suspicious transactions to Financial Monitoring Unit-FMU; which is only formal source of reporting suspicious activities. Since the financial institutions have restrictions to disclose the information other than legal obligations. Thus, it is very difficult to analyze complete methodologies involved in organised crimes in Pakistan from qualitative perspective. Such limitations have been major compelling factors which led the researcher to opt qualitative methods in the pioneered research work. Secondary information was collected under qualitative 24 Dynamics of money laundering and terrorist financing: a challenge to Pakistan methodology. Obtained information was collected, collated, analyzed and interpreted according to various forms of theories. Besides that, during the qualitative review, researcher’s own experience and exposure contributes greatly in extracting quality information under various forms. Discussion Prior to invasion, arms and narco culture were unknown to this country and the nation. The continuous and uninterrupted influx and absence of data of afghan refugees facilitated drug barons and arms smugglers to export these menaces. This also involved Global Journal for Management and Administrative Sciences ISSN (Print): 2788-4821 ISSN (Online): 2788-483X Volume 3, Issue 1, Page 27-40, June 30, 2022 25 Pakistani organised criminal groups to use Pakistan as a route for trafficking of narcotics from Afghanistan to global markets to meet their demand. Pakistan has 2,430km-long porous borders with politically disturbed and terrorist-ridden Afghanistan; and provides ideal passage for trafficking narco-drugs (UNODC, 2008). One of the most preferable crimes by the organised criminal groups is illicit narco-business, since it attracts extremely profitable business to them. Palermo Convention has classified drug trafficking as one of the transnational organised criminal activities. However, the intensity and extent of elements involved in this organised crime could vary (UNODC, 2007). During 2007, Afghanistan is figured as the largest producer of the world‘s opium production, i.e. 92%. Bolivia, Colombia, and Peru dominated coca cultivation for shipment to the world. Developed countries particularly the US and Europe have major demand of drugs. The cross-border demand of narco-drugs gives rise to illicit drug trade as transnational organised crime. In this trade, the produced narco- drugs must reach at its destination where the demand is high, after trafficking through multiple countries, where its demand is very high with final products (INCB, 2008). These factors are more likely to involve individuals and groups in narco-drug smuggling and transportation when there is an opportunity of having porous borders with the country which is producing the highest quantity of opium in the world (UNODC, 2018). After 9/11, the number of terrorist activities has seriously increased with flourishing of terrorism in the country and the cross border. Involvement of drug money in these activities cannot be denied. In order to put rigorous restrains; Pakistan promulgated special law of Control of Narcotics Substance Act with special punishments was introduced against the perpetrators of narco-drug crime. This was an effort to create deterrence of punishment in the society for creating self-restraint. Palermo Convention mentions trafficking of person implies onboarding, transfer, transportation, harbouring by means of threat, giving and receiving of payments (UNTOC, 2000). After alarming rise of narco trade; this crime has drawn a worldwide attention. Discussion Global terrorism has witnessed co-relationship of these transnational organised crimes; and has met condemnation. There is some difference between victims of human trafficking and smuggled migrants. Usually consent of migrants is involved; while in the former, there is absence of consent by the trafficked victims. Moreover, migrant smuggling is always transnational; and trafficking victims may be trafficked through their own country. The ongoing relationship between human traffickers and their victims is often based on abuse and exploitation with the motivation of profit making for the traffickers. This always results in the victimization of smuggled migrants. Human traffickers may opt same route of trafficking for their operations at transnational level. Thus, people from deprived socio-economic environment of subculture become more vulnerable to delinquency and deviance in this social and cultural set up. In sub-culture society, individuals face negative ethnicity, discrimination and morality issues and people from such societies engage in organised crime. Formation of mafias or cartels by them on the basis of racism, ethnic or with other common factors, are the outcome of ‘social disorganization’ for financial gains (UNODC, 2018). According to UNODC report; men, women, and children are trafficked from Pakistan for the purpose of forced labour and sexual exploitation and using it for transit, Dynamics of money laundering and terrorist financing: a challenge to Pakistan 26 source and destination country. Forced labour trafficking is probably more widespread through Pakistan. Presence of organised international smuggling networks, entrenched poverty, Afghan refugees and porous borders contribute as favorable factors for human traffickers in Pakistan. Victims are trafficked mostly to the Middle East. Every year in Pakistan, thousands of Afghan and Pakistani migrants are also smuggled in or out for the sake of work (UNODC, 2018). Baluchistan is one of the provinces of Pakistan, with its vast contiguous borders with Iran and Afghanistan, has hosted huge human smuggling and trafficking rackets since decades. This practice took on an organised form in the 1980s. The migrants are now largely Afghans and Pakistanis. The route from Quetta to Chaghi,via Naukundi is very notorious and preferable for the human traffickers and smugglers. According to local reports, the town of Naukundi has hardly 20,000 population and their source of livelihood is human trafficking. It is significant to note here, mineral-rich mountains lie adjacent to Naukundi and it is located in Chaghi which is the largest district of Pakistan. Discussion It 27 Global Journal for Management and Administrative Sciences ISSN (Print): 2788-4821 ISSN (Online): 2788-483X Volume 3, Issue 1, Page 27-40, June 30, 2022 27 empowers law enforcement agencies of Pakistan to effectively prosecute the organised criminal groups (UNODC, 2018). empowers law enforcement agencies of Pakistan to effectively prosecute the organised criminal groups (UNODC, 2018). The annual illegal arms trade is estimated around $US 1 billion. The increasing demand of arms and its supply at transnational level is posing a serious threat to curb national and international terrorism. UN declared a Protocol of arms trafficking. It encourages, assists and strengthens cooperation among the states. This would help to restrain, combat and eliminate the illicit production and transportation of firearms, its arms and ammunition. Organised crime is multifaceted. It involves traditional types of criminal organizations, transnational criminal networks with flexible structures and quick mobility, transformation. These may also be controlled from different locations as well. The illicit arms smuggling, and trafficking is intrinsically linked to these criminal organizations and networks. This is fundamentally considered to be useful in violent crimes, stirs armed conflicts, and cerates insecurity. Usually, various types of criminality are intertwined with trafficking of firearms, such as humans, drugs trafficking, and terrorism. No region in the world is exempted from the use of firearms and serious security challenges. To support states in establishing effective criminal justice system and countering challenges posed by organised criminal networks and organizations specifically related to trafficking in firearms and its parts; UNODC Global Firearms Programme was introduced (UNODC, 2018). Pakistan is also facing this menace. Defense expert Brigadier (R) Mahmood Shah in his interview mentioned that neighbouring Afghanistan is the main supply passage of illegal weapons into Pakistan. Khyber Pakhtunkhwa (K-P) and Baluchistan have porous borders with Afghanistan. Anti-tank shells, grenades, anti-aircraft guns and Rockets are smuggled from across the border. These are mostly Russian-made and copies of which are also built in central Asian states. According to senior police officials, roughly 20 million illegal arms in Pakistan are estimated. These include prohibited firepower which is frequently used by criminals and terrorists during their nefarious designs (The Express Tribune, 2012). According to one estimate, the generation old illicit arms trade boomed in the early 1980s in Pakistan. The warlords in Afghanistan particularly and adjacent Pakistan borders generally started buying illegal weapons during ongoing war against Soviet- union. Discussion Gold and copper mines, namely Reko Diq and Saindak are situated in the same vicinity. There are no employment opportunities for the locals despite presence of the multi- million dollar proceeds. Thus, they are left with no option to involve in human smuggling and trafficking (Butt and Notezai, 2018). In absence of government amenities, development of isolated lower-class culture is natural. It lacks conventional social opportunities and racial/ethnic discrimination is common. Therefore, this sub-culture causes increase of deviant youth with justifications against the social norms. This also holds the symptoms of crime tendency, deviance and earning of money through illicit means. The situation is further aggravated when educated youth belonging to lower-class and passed through various deprivations to get an opportunity of white-collar job. Such circumstances mostly cause of nourishing culture of committing organised crime, like human trafficking and smuggling, money laundering, fraud and other unlawful organised activities. According to one estimate, every year, between 30, 000 to 40,000, Pakistanis illegally attempt to opt route to Europe through Turkey, Russia, and the Middle Eastern countries from Balochistan and by air. Human smuggling does not involve the element of force, and it is always trans-national. An individual has consent with a smuggler to get facilitations for illegal entry into a foreign country. It is not always economic restrains that the dream chasers opt illegal passages to cross border European countries. There are many factors which contribute in increasing trend of migration from Pakistan. Financial restrains or religio-ethnic violence also cause of impetus for dream chasers to incur grave risks with the hope of finding greener pastures in foreign lands as perpetrated against the Shia-Hazaras community in Balochistan, which proved to be pushing factor to them. The current civil war in Syria also swayed both Shia and Sunni militants to opt migration from Pakistan. According to local reporters, when Bashar al-Assad started losing control in the civil war in 2013, Shia-Hazaras from Baluchistan were also smuggled into Iran to fight alongside the Syrian army. When Iran raised 15-ft high wall along its border with Afghanistan’s Nimruz province, Afghans also got smuggled out from Afghanistan via Baluchistan. New special acts of "The Prevention of Trafficking in Persons Act, 2018" and "The Prevention of Smuggling of Migrants Acts, 2018" are passed in Pakistan. These laws give guaranty to the victims of human trafficking and smuggled migrants. Discussion Further, existing porous borders contributed a lot in proliferation of this organised crime. Darra Adamkhel, the town is not far from the Afghan border, was a hub of criminal activity for decades. This area comes under semi-autonomous tribal areas joining porous borders with Afghanistan where ethnic Pashtuns live. Later, the town came under the direct influence of the Pakistani Taliban. Now the town is cleaned after Pak army operation (Dawn, 2016). Thus, to the researcher, in continuity to above discussed organise crimes in Pakistan; money laundering cannot be studied in isolation. Since it involves predicate offence and linkage of organised crimes with money laundering is natural. Particularly the terrible rise of terrorism in Pakistan and terrorist activities in the region in general, demands to review the collected information on these dynamics of organised crime which 28 Dynamics of money laundering and terrorist financing: a challenge to Pakistan is requirement of the time. Another critical aspect of these crimes cannot be ignored that these are transnational in nature. is requirement of the time. Another critical aspect of these crimes cannot be ignored that these are transnational in nature. Overlapping of criminal activities among the organised criminal groups has prospered. This change happened during post-cold war era, when earlier the terrorism was state sponsored. After cold war, those terrorist elements formed groups and found alternative sources through organised crimes. This culmination was witnessed after happening of 9/11, which was lethal combination of money laundering and terrorist financing. 9/11 terrorism drew special attention of the international regime. The General Assembly resolution 55/25 of 15 November 2000, that adopted the Palermo Convention, “...with deep concern” a growing nexus of transnational organised crime and terrorist groups (UNODC, 2000). Like organised crime, terrorism has also been difficult to define in concrete terms which could be acceptable to all. When we concentrate elements of criminal organised group, we find that these are non-ideological. Criminal organizations find it feasible to jeopardize smooth functioning of the contemporary governments. This may lead them to support corruption, kidnapping or violence (Finckenauer and Albanese, 2005). This perpetration is witnessed amongst drug trafficking cartels where use of intimidation, force and extortion against prosecutors, judges, public representatives and law enforcement officials. The object of these actions has been to disrupt and divert attention of legitimate government from drug enforcement. These criminal activities, during the 1980s, led to emergence of the term narco-terrorism (Mylonakia, 2002). Discussion Terrorism and organised crime are interrelated, and it is very difficult to distinguish both on their characteristics basis. Infact organised criminal networks prefer use of force to achieve their designs and also support other terrorist groups. This is the reason, why terrorist networks are always tended to work with organised criminal networks (Mylonakia, 2002). Despite this blur characteristics, and perhaps complementary activities; the motivating factor is the major comparative feature. For organised criminal groups, motivation emanates from profit, while terrorist groups get primary motivation from a particular so-called ideology or struggle. Due to fundamental difference in motivation, strategies to combat terrorism and transnational organised crime also vary substantially. For instance, a strategy to combat trafficking offences needs to consider those factors which give rise to demand for trafficking like drugs, arms and others. That is why, terrorism is not made part of definition of ‘organised crime’ during the Palermo Convention. Infact, there are many specific conventions and resolutions which directly and solely aim at terrorism. Criminologists consider deprivation as a major cause of social movements and deviance, which in extreme situations ends in riots, terrorism, civil disobedience and crime. It can be cited here, various political or religious organizations in Pakistan which exploit such deprivations in the name of ethnicity or religious feelings that the contemporary feelings of deprivation are the outcome of injustice and exploitation. These cheeky slogan mongering provide them an opportunity to justify their civil disobedience, terrorist activities and other unlawful activities against the existing socio-poetical and economic system. These politico or ethno or religio-based parties found their place where Global Journal for Management and Administrative Sciences ISSN (Print): 2788-4821 ISSN (Online): 2788-483X Volume 3, Issue 1, Page 27-40, June 30, 2022 Global Journal for Management and Administrative Sciences ISSN (Print): 2788-4821 ISSN (Online): 2788-483X Volume 3, Issue 1, Page 27-40, June 30, 2022 29 the societies or communities are living, where social norms or values are in conflict with the norms or values of haves. the societies or communities are living, where social norms or values are in conflict with the norms or values of haves. the societies or communities are living, where social norms or values are in conflict with the norms or values of haves. The study deduce that terrorism in Pakistan, when the researcher studies contemporary norms of Pakistani society, it is more prone to achieve ‘goals’ of accumulating wealth. Discussion Since there is more depression in the economy, more tendencies towards achieving it increases. Here the passage becomes narrower; when majority involves in achieving it; while resources are less. This leads to deviance behavior, particularly at the lower middle class. This rises to criminal behavior and traits of corruption, extortion and fraudulent means become daily norm. A novel anomic situation appears in Pakistan, where politicians, bureaucrats, and others from elite class of ‘haves’ become more active to achieve ‘goals ’of accumulating wealth at the cost of deprived. This has resulted in expansion of increasing gap between haves and have-nots. Their means of tax evasion, laundering of money, corruption and frauds have become talk of the day in Pakistan. It also triggers a race of having more and more financial security to the illicit funds. An unannounced conflict of haves and have-nots exists. Thus, commission of organised crimes, like frauds, corruption, smuggling and other organised crimes find supportive environment. These favorable factors lead to laundering of funds and existing fragile enforcement region further provides opportunity to offenders to extract maximum from the system to meet their criminal designs. Since money laundering is the predicate offence and it is naturally linked to other organised crimes. The latest scenario of the country depicts an increase in overlapping of organised criminal activities among the criminal groups. This also applies in organised narco-drug and terrorist networks in Pakistan, where narco-money crosses borders of Afghanistan and inside Pakistan for the import of terrorist activities in the country. These criminal activities are transnational in nature. In such scenario, the criminal cartels need to transfer illicit funds from one country to another by various means, whether it involves financial institutions through money laundering and informal channels of Hawala. Raufer defines financing of terrorism in these words, “financing of terrorism can be divided into two general types: financing from above, in which large scale financial support is aggregated centrally by states, companies, charities or permissive financial institutions; and financing from below, in which terrorists fund raising is small-scale and dispersed, from example based on self-financing by the terrorists themselves using employment or welfare payments” (Raufer, 2006). To the researcher, this same combination of money laundering and terrorist financing is also currently experienced by Pakistan which is the foremost threat to the security now. Discussion Various terrorist groups are involved with direct and indirect support of cross border financial channels through money laundering in the country. Tahrik-e-Taliban Pakistan (TTP) is one of the examples. These two organised crimes are different in nature and involve different modus operandi when various techniques are analysed. Different measures and controls are set for countering against laundering money and financing terrorism, all businesses consider these two simultaneously for meeting their end. There are no set principles which could operationally distinguish these two. The basic difference between them is the origin of the funds. Dynamics of money laundering and terrorist financing: a challenge to Pakistan Dynamics of money laundering and terrorist financing: a challenge to Pakistan Dynamics of money laundering and terrorist financing: a challenge to Pakistan 30 Terrorist Financing does not always involve ill-gotten funds. These funds could be from legitimate sources but always will be used for illegal objects. But money laundering always involves illicit funds earned through criminal proceeds. These funds could be used for legal purposes, but its source of funds is illegal. Thus, its main object is to disguise its origin from the trail of record. So, in the case of financing of terrorism, it is very difficult to prove legal funds to be used or used for financing of terrorism. In this case, the predicate offence is necessary to prove initially before proving laundering of money. Thus, controls and measures against financing of terrorism would be possible when evidence of predicate offences is available and collected. In the case of available legitimate funds for terrorist financing, there is no need to launder it. But problem arises to hide the purpose of legal funds involved for financing of terrorism and its linkage with terrorists. To disguise this linkage, the terrorists perform the same techniques like cash smuggling, involving of remittances, layering and integration of legitimate and illegitimate funds. The use of informal banking channels like Hawala or Hundi which have been favorite for the terrorists since it does not require identity and non-availability of trail record for the investigators. USA State Department stated in its report that after 9/11, the active presence of Al Qaida and other terrorist groups, cash-based economy and porous borders with Afghanistan encourages the use of informal financial networks, like Hawala (INL, 2010). Discussion To the researcher; in the presence of cash-based economy and informal banking channels like Hundi/Hawala in Pakistan; the serious vulnerability of money laundering and terrorist financing exists. To make their networking more effective, some terrorist groups or organizations adopt complicit charities and entrepreneurs. These networks are so organized and form justifications to their terrorist activities. The terrorist groups also influence public opinion through mass media and propagandize their acts of violence based on some ideology which could sway masses. Kohlmann describes in his work, “Groups that use terrorism as a primary means to pursue their objectives can also utilise affiliated charities as a source of financing that may be diverted to fund terrorist attacks and terrorist recruitment by providing a veil of legitimacy over an organization based on terrorism” (Kohlmann, 2006). In actual, collected charity funds may be susceptible to financing of terrorism. Mostly, these funds are collected in cash, which is also the highest risk factor. It is very difficult to find the trail record of originators of the cash deposits and their source of funds. The situation further aggravates when these organizations are globally based, and funds are deposited through different instruments in their accounts maintained in various jurisdictions. So, these pouring of funds may lead to placement and layering of transactions activities by these charity or non-profit organizations. Weak regulatory measures also create encouraging environment for these entities to meet their malafide objective of financing terrorism. To have checks over charity or non-profit organizations; there is need of taking stringent regulatory measures related to the maintaining and operation of accounts. Organizations should maintain transparency in collection of funds and its usage. Internal and external independent audit should be conducted which would confirm the proper Global Journal for Management and Administrative Sciences ISSN (Print): 2788-4821 ISSN (Online): 2788-483X Volume 3, Issue 1, Page 27-40, June 30, 2022 31 utilization of donations. Entities should be compelled to maintain their formal accounts and carry out transactions through these formal channels, so that their transaction activities could be brought in the ambit of regulatory measures. During the past decade, the world at large has witnessed rising menace of laundering of money; forcing most governments to initiate a process of containing this commission and curbing financing of terrorism alongside. However, emerging poorly regulated markets remain vulnerable to money launderers. Discussion This is largely due to strict action being taken by developed countries to check this activity and forcing money launderers to switch their activities to emerging weak regulated markets. Money laundering is a threat to the world’s major financial institutions and offshore financial centers. Every country is integrated into the international banks system and at risk since trading activities between nations, and the banking transactions relating to them, have integrated all financial networks. With the emergence of open market economies, money launderers find it an opportunity as a lucrative target. Strategy of Pakistan has regional and international impact, since Pakistan is strategically located in the Asia region. That is why, the USA State Department stated in its report that Pakistan is strategically important for being priority region in counterfeiting terrorist financing. Since the active presence of Al Qaida and other terrorist groups, porous borders with Afghanistan, and economy is cash based which involves informal financial networks, like as Hawala. Thus, all countries in the region require beefing up efforts against laundering money and financing of terrorism at the local, regional and international level (INL, 2010). Counter strategy in fighting against money laundering and terrorist financing in Pakistan relies on the measures of cooperation by the financial and non-financial institutions with enforcement and regulatory agencies and authorities. It demands curbing of actual and perceived threats of money laundering and terrorist financing. Pakistan has showed its international commitment to the global stakeholders for adopting best preventive measures against curbing money laundering and terrorist financing (Malik, 2003). Once Ishrat Hussain, the then Governor State Bank of Pakistan in 2003 stated that keeping in view the contemporary systems and measures to curb laundering money and financing of terrorism; Pakistan needs to formulate multi prong strategy. This demands various strategies. Promulgation of laws, assignment of related tasks to different institutions, beef up regulatory and supervisory competence, enforcement network, training and expertise, compliance and implementation of international standards, technical support and abiding the UN resolutions (Ishrat, 2005). Pakistan is an active and effective signatory of Asia pacific regional group of on anti-money laundering regime. Pakistan, as a part of its strategy, measures to curb and has adopted cooperative policy towards FATF objectives and adopted various legal and administrative deter money laundering. Laws & regulations are enforced by the government of Pakistan and involved other controlling authorities for getting maximum results. Discussion A number of strict counter measures form part of its strategy. Pakistan was 32 Dynamics of money laundering and terrorist financing: a challenge to Pakistan officially placed on a terrorism financing watchdog’s ‘grey list’ by the Financial Action Task Force (FATF) in June 2018 after its plenary meeting (Iqbal, 2018). This is an international regime’s effort to counter the country's financial support to militant groups (Khaleej Time, 2018). In the country, coordination among the law enforcement agencies takes place, when the high-profile cases are involved. The provisions of mutual legal assistance are also included in AML ACT and CNSA; which involve: a) exercising coercive measures to facilitate foreign investigations. b) freezing and confiscating of assets in connection with foreign legal evidence b) freezing and confiscating of assets in connection with foreign legal evidence. Under the NAO 1999, laundering of money is not an offence, unless it is sufficient suspicion that the act is committed for disguising proceeds earned through Global Journal for Management and Administrative Sciences ISSN (Print): 2788-4821 ISSN (Online): 2788-483X Volume 3, Issue 1, Page 27-40, June 30, 2022 33 corrupt practices in violation of laws. The action is taken on the “Principle of Reciprocity” (Gazette of Pakistan, 2010). corrupt practices in violation of laws. The action is taken on the “Principle of Reciprocity” (Gazette of Pakistan, 2010). Section 65 of the CNSA allows the federal government may enter into agreement for asset sharing with the foreign government. Under S. 56, Pakistan may approach to the other foreign state with request for assistance related to the narcotics offence. In the same way, Pakistan can also provide assistance to the requesting state. Since money laundering is criminalised under the S. 12 of CNSA; which requires mutual legal assistance-MLA related to laundering the proceeds of narcotics crime. So, the predicate offence should be there, and the offence is criminalised under the CNSA. Request of the foreign country could be declined if it is found against the security, sovereignty, or national interest of Pakistan. The authorities can freeze or forfeit assets of a person who has committed offence in Pakistan and holding such assets in foreign country. This rule is also applicable vice versa. By summing up; under the CNSA; Pakistan can carry out searches, provide records, transfer detained person, freeze or forfeit assets, even in the absence of relationships with the requesting state. But this is done under the requirement of double criminality (Gazette of Pakistan, 1997). The country’s regulatory bodies have authority of coordination and cooperation with other foreign supervisory or regulatory authorities. This may involve information sharing, as per legal requirements. International cooperation also involves regulator’s active membership of various international organizations and also signs MoUs with other foreign countries. However, information is shared with international counter parts on an ongoing basis. The purpose of MoUs is to bring improvement for cooperation in supervisory functions, laundering money and financing terrorism measures and other related. Information is shared with counterparts under the obligation of signed Memorandum of Understanding (MoU) that released information will not be shared with third parties without concurrence of the requested authority. Confidentiality to the sharing of information must be maintained. b) freezing and confiscating of assets in connection with foreign legal evidence. Under S.26 of the AML ACT, Pakistan can provide assistance in investigation of laundering of money to foreign countries. This involves sharing of information, transferring of assets related to the offence committed. Under S.27, the requesting state makes request through the letter of request. This request is channeled through the Ministry of Foreign Affairs. Requests can be entertained so long as these are not in violation of security, sovereignty or national interests of the country. Under S.30 (2) of AML ACT, the Federal Government can forward the letter of request by the other state, asking for freezing and confiscation. This letter can be forwarded to NAB, FIA or ANF. It may include freezing or forfeituring of assets generated directly or otherwise from proceeds of committed crime (Gazette of Pakistan, 2010). Pakistan in compliance to UN Resolutions has taken steps of freezing terrorist assets under UNSCR 1267 and 1373. Regulator can also send circulars to financial institutions holding the list of wanted persons and entities which are part of the UN 1267 Sanctions Committee’s list. Under the AML ACT, Pakistan can provide assistance in freezing or forfeiting of proceeds of money laundering transactions. But its scope is limited to this offence and cannot assist, if proceeds do not involve money laundering transactions. Section 26 of AML Act also authorizes the federal government to come into agreement with foreign government for assets sharing. It requires a letter of provision of information from court of law of a contracting State. Under the section 30 of AML ACT, request is received from contracting State for forfeiture of assets in Pakistan which is directly or otherwise derived by a person after commission of offence under AML Act. The federal government receives such request and may send the request “as it thinks fit” to “investigation agency” which could be FIA, ANF or NAB according to AML ACT. Under S.28 of AML ACT, FIA, ANF or NAB execute request which is not in violation of Law of Pakistan and prejudicial to the national interest, security and sovereignty of the country. These steps may involve intelligence, investigation of tracing assets, accounts in the banks and documents to testify. These broader powers are exercised by FIA and ANF (Gazette of Pakistan, 2010). b) freezing and confiscating of assets in connection with foreign legal evidence. But confidentiality should not prevent it from disclosing necessary information to the law enforcement agencies and other related authorities. It is also agreed that the information shared with other counterparts should be exclusively used for lawful supervisory purposes. Pakistan could not extradite cases related to financing of terrorism offences before October 2008. In October 2008, the Offence of “financing for terrorism” was made part of the extraditable offence list as given in Schedule of the Extradition Act. The offences, which are part of the Schedule, will be considered related to aiding, abetting, counselling, procuring, attempting or conspiring. The second part considers dual criminality (Gazette of Pakistan, 1972). Laundering of money was also made part of the Schedule of the Extradition Act in March 2009. On the other hand, the S. 66 of CNSA considers offences which come under the Chapter II of CNSA 2007 are “extraditable offences”. However, CNSA encompasses narcotics related offences and its laundering of proceeds under S. 12. Law enforcement authorities share information with other counterparts in the foreign countries according to their standard operating procedures. This is relies upon “Principle of Reciprocity” related to mutual legal assistance among them (Gazette of Pakistan, 1997). 34 Dynamics of money laundering and terrorist financing: a challenge to Pakistan Money Laundering (ML) and Terrorism Financing (TF) are criminalised by Pakistan. The law enforcement agencies find it difficult to collect evidence in ML for getting conviction in the predicate offence, which is prerequisite. However, the range of predicate offences in contemporary laws of Pakistan is limited. There is no one formal platform, where various agencies meet together and chalk out strategy. However, interagency coordination (provincial police, FIA, IB, and ISI) is limited in scope by involving only combating terrorism issues in Pakistan. It does not involve financing of terrorism issues, FMU and other stakeholders. The term ‘investigating agency’ in AML Act is limited to NAB, ANF, and the FIA under S. 2 (k). Thus, the investigation of information received through financial institutions is very limited to only some federal enforcement agencies. It does not consider provincially controlled police department, customs or the tax authority. Since NGOs have proved to be a serious channel of layering in laundering of money. There is need of coordination among Social Welfare Ministry and FMU for information sharing. SECP and FBR can play critical role in this regard. b) freezing and confiscating of assets in connection with foreign legal evidence. However, Ministry Social Welfare shares information on financing of terrorism issues with Ministry of Interior. Involvement of Police at the provincial level and multi federal agencies in curbing ML, CFT and predicate offences has resulted in fragmentation and complexity of work (UNODC, 2018). Due to complexities of international financial system, criminal networks are taking full advantage of it. It is now mandatory part of investigation to trace overseas proceeds which involve multiple jurisdictions and bank accounts, if any investigation is initiated holding organised criminal activities. This is slow shift of law enforcement agencies from individual criminals to criminal networks related to financial gains. This involves enforcement operations with support of legislation providing forfeiture of proceeds from criminal activities. It is also combined with prosecutorial and other instruments in fight against organised transnational crime. These instruments should also be further improved with witness protection schemes, electronic monitoring, computerised information systems, funding for forensic expertise, international agreements, asset-management/asset-sharing mechanisms, and financial and business co- operation. The acceptability of many of these methods, such as electronic surveillance, should be bound by strict observance of legal requirements under the principles of criminal procedure. Conclusion Pakistan has not been able to cope up with the global anti money laundering and terrorist financing regime’s requirements. It requires reviewing of factors which compel Pakistan from following global requirements in letter and spirit. So that anti organised crime regime in Pakistan with particular reference to countering money laundering and terrorist financing could firm its footing against these serious rising challenges in the country. To the researcher, the tacit acceptance of corruption in the society is another rising organised criminal trend in the country, which needs more attention by the counter regime in Pakistan. Despite experiencing limitations, the researcher believes this work in Global Journal for Management and Administrative Sciences ISSN (Print): 2788-4821 ISSN (Online): 2788-483X Volume 3, Issue 1, Page 27-40, June 30, 2022 35 hand would contribute a lot in scratching the surface of criminological aspects of laundering money and financing of terrorism along with organised crimes in the country. If timely action is not taken; it would facilitate money launderers and terrorist financers to be more organised at larger scale in the country. This would also distort social and moral values and pave the way for waning respect of rule of law as whole. Thus, Pakistan has also to cope up with the global anti-money laundering and terrorist financing regime’s requirements. Recommendations During the qualitative study, weak anti-money laundering and terrorist financing regime’s measures found. In psycho-social terms, human deviance in the form of rising corruption also contributes to deteriorating regime’s performance in the country. Money laundering and terrorist financing have serious socio-politico and economic consequences. Weak money laundering regime is the main cause of fragile financial system and reputational risk. This is not limited to country but has broader impacts on global economy. Strong anti-money laundering and terrorist financing regime is need of hour which is responsible for integral working of financial institutions with lower reputational risks. It is found that offender is motivated by various gains after laundering illicit generated proceeds through various layering tactics of concealment. So that, it should look legal in the country and may contribute to financing terrorism through legal means with malafide designs. Another aggravating factor, which motivates criminals in commission of crime, is non-impressive enforcement or operations, lacunae in investigations, weak prosecution, and inherited mapping loopholes in the legal system for countering laundering money and financing of terrorism. Since in each country; the structures, techniques, methods and participants used in combating organised crime are in some cases radically different. The key element in regulating their interaction at the national and international level is co-operation among law enforcement agencies in investigating, apprehending, and prosecuting international criminals. It is important to establish lines of co-operation and communication and shared understanding of common goals throughout the world. Effective help in tracing organised criminal networks is provided by investigative co-operation between law enforcement agencies and special units because this permits exchange of information so essential to neutralize the activities and movements of criminal networks. Pakistan still has to do more in meeting international standards of AML and CFT, especially synchronizing local laws and provisions with FATF Recommendations. This is expected in particular to FATAF’s decision in June 2018 by placing Pakistan in ‘grey list’. Now, there is compelling need to take stringent reforms through organised crime legislation in line with international measures. The country needs non-traditional anti-organised crime measures. This requires a community-based engagement in countering money laundering and terrorist financing. This involves creating awareness among youth against criminal commission by organised criminal networks in the society. Recommendations Thus, through legal assurances, the private sector of the country should be engaged to Dynamics of money laundering and terrorist financing: a challenge to Pakistan 36 minimize opportunities for organised criminal groups to limit their available channels for laundering ill-gotten funds. The organised crimes, particularly money laundering and terrorist financing are penetrating in social fabrics and demanding legislative bodies to promulgate stricter legislative measures with stringent law enforcement. The Protection of Pakistan Bill 2014 adopted by the legislature of Pakistan was one of the precedents of making efforts to counter extra ordinary challenges in the extra ordinary terrorist activities in the country. The bill bestows more authority to enforcement agencies against the arrests, investigation and collection of evidence. The use of force against the crushing of terrorists’ networks in border areas is also welcome action by Pakistan army. It declared formal operation namely ‘Zarb-e-Azab’ and “Radd-ul-Fasaad” against terrorists and their sanctuaries inside Pakistan and near border region. Such type of combat action is also recommended, when anti-money laundering steps are taken in the country on war footing. Thus, under this research, there is need to assess the actual participation of organised crime groups in the criminal markets of Pakistan. It demands to analyze and throw more light on the modus operandi of organised criminal groups. This may specifically require multiple empirical methodologies. This would lead to know, how organised criminal groups launder their ill-gotten money and systematically infiltrate in available legitimate channels. Earlier this aspect was taken up by the various researchers from law enforcement point of view. 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https://openalex.org/W3134775266	https://www.researchsquare.com/article/rs-244360/latest.pdf	English	null	Oscillatory visual mechanisms revealed by random temporal sampling	Scientific reports	2,021	cc-by	5,704	Arguin et al. Visual oscillations Oscillatory visual mechanisms revealed by random temporal sampling Martin Arguin1, Roxanne Ferrandez2 & Justine Massé2 1- Centre interdisciplinaire de recherche sur le cerveau et l’apprentissage (CIRCA), Département de psychologie, Université de Montréal and Centre de recherche, Institut Universitaire de Gériatrie de Montréal 2- Département de psychologie, Université de Montréal Address correspondence to: Martin Arguin (ORCID ID: 0000-0002-1036-2932) Département de psychologie Université de Montréal C.P. 6128, Succ. Centre-ville Montréal, Qc H3C 3J7, Canada email : martin.arguin@umontreal.ca Classification: Biological sciences – Psychological and Cognitive Sciences Arguin et al. Arguin et al. Visual oscillations Visual oscillations Research Article Keywords: Vision, visual recognition, neural oscillations, visual processing Posted Date: March 1st, 2021 DOI: https://doi.org/10.21203/rs.3.rs-244360/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Oscillatory visual mechanisms revealed by random temporal sampling Martin Arguin1, Roxanne Ferrandez2 & Justine Massé2 1 y by random temporal sampling Martin Arguin1, Roxanne Ferrandez2 & Justine Massé2 1- Centre interdisciplinaire de recherche sur le cerveau et l’apprentissage (CIRCA), Département de psychologie, Université de Montréal and Centre de recherche, Institut Universitaire de Gériatrie de Montréal 2- Département de psychologie, Université de Montréal *Address correspondence to: Martin Arguin (ORCID ID: 0000-0002-1036-2932) Département de psychologie Université de Montréal C.P. 6128, Succ. Centre-ville Montréal, Qc H3C 3J7, Canada email : martin.arguin@umontreal.ca Classification: Biological sciences – Psychological and Cognitive Sciences Keywords : Vision, visual recognition, neural oscillations, visual processing 1 1 Visual oscillations Arguin et al. Arguin et al. ABSTRACT It is increasingly apparent that functionally significant neural activity is oscillatory in nature. Demonstrating the implications of this mode of operation for perceptual/cognitive function remains somewhat elusive. This report describes the technique of random temporal sampling for the investigation of visual oscillatory mechanisms. The technique is applied in visual recognition experiments using different stimulus classes (words, familiar objects, novel objects, and faces). Classification images reveal variations of perceptual effectiveness according to the temporal features of stimulus visibility. These classification images are also decomposed into their power and phase spectra. Stimulus classes lead to distinct outcomes and the power spectra of classification images are highly generalizable across individuals. Moreover, stimulus class can be reliably decoded from the power spectrum of individual classification images. These findings and other aspects of the results validate random temporal sampling as a promising new method to study oscillatory visual mechanisms. 2 2 Visual oscillations Arguin et al. Arguin et al. Visual oscillations experiments described below also reveal crucial features of the oscillatory mechanisms involved. Some of these features are largely shared among observers and are diagnostic of the stimulus class or specific task participants perform. Adult neurologically intact human observers carried out visual recognition tasks using words, familiar or novel objects, or faces. Throughout their exposure duration (200 ms), the stimuli were sampled by a manipulation of target visiblity (controlled by signal-to-noise ratio), which oscillated according to a random function. Classification images [13] of visual processing efficiency were then constructed as a function of different coding formats for the temporal features of stimulus visibility. Processing efficiency refers to the capacity of participants to use the stimulus information available to perform their task correctly. INTRODUCTION There is growing evidence suggesting that functionally relevant neural communication takes the form of periodic synchronized firing by groups of neurons [1-4]. At a macroscopic level, this takes the form of oscillatory activity which can be recorded in humans by electroencephalography (EEG) or magnetoencephalography (MEG; [5]). If brain oscillations underlie faculties such as perception and cognition, this should be manifest in behavior. For instance, perceptual effectiveness should oscillate rapidly through time, in contrast to subjective experience which rather suggests a stable capacity. Empirical evidence in support of perceptual oscillations has been reported based on the periodicity of performance measures in humans carrying out various perceptual tasks [6- 10]. The capacity of this approach to characterize putative oscillatory perceptual mechanisms remains limited, however, and a source for more potent evidence is clearly desirable. Initially proposed by [11; see also 12], we have enhanced the test and analysis procedures for the technique of random temporal sampling to make it a powerful tool to investigate oscillatory visual mechanisms. In addition to demonstrating temporal inhomogeneity in processing effectiveness, the 3 3 Arguin et al. RESULTS The classification images (Fig. 1) show the evolution of processing efficiency according to the temporal features analysed. All differences in processing efficiency illustrated are significant (2-way Pixel test, α = .05; [14]). 4 Visual oscillations Visual oscillations Insert Fig. 2 near here. Visual oscillations Visual oscillations Arguin et al. Arguin et al. Arguin et al. Insert Fig. 1 near here. Insert Fig. 1 near here. In the temporal domain, the variation of processing efficiency is markedly different according to stimulus class (Fig. 1a-d). Processing efficiency was also analysed according to a time-frequency representation of the variations of stimulus visibility through time (Fig. 1e- h). Efficiency oscillates through time and the pattern of these changes is affected by sampling frequency, i.e. the temporal variations of target visibility. These sampling frequency x time patterns are unique for each stimulus class. Classification images of static frequencies of stimulus oscillation based on the Fourier descriptors of the temporal profiles of stimulus sampling were also calculated. None of these classification images showed any significant effect; i.e. they were all perfectly uniform. The consistency of classification images across participants carrying out the same task served to assess their generalizability. This was used to index the degree to which the mechanisms captured by the classification images are fundamental, or essential, for the visual system. Between-subject 5 Arguin et al. Arguin et al. individual classification images to stimulus class with a leave-one out validation procedure. Out of four possibilities, stimulus class was predicted with 75% accuracy from the power spectra of the time domain classification images and with 52% accuracy using the power spectra of the time- frequency classification images. These classification rates are well above chance (time domain: χ2 (9) = 70.0; p < .001; time-frequency domain: χ2(9) = 31.0; p < .001). Arguin et al. consistency for each stimulus class was measured by the average of all possible pairwise correlations between the classification images of individual participants. The mean across stimulus classes is very low for both time (mean r = .17) and time-frequency (mean r = .08) classification images. Classification images however, contain generalizable information which may be revealed when properly extracted. Fourier analyses were carried out on individual time domain classification images as well as separately for each stimulus sampling frequency of individual time-frequency classification images. They were thus decomposed into their power and phase spectra, which were then submitted to an assessment of between-subject consistency. Consistency was very low for the phase spectra (mean r = .18 across stimulus classes for the time domain; .03 for the time-frequency domain). In contrast, the power spectra (Fig. 2) were remarkably consistent (mean r across stimulus classes = .74 for the time domain; .80 for the time- frequency domain). The power spectra of individual classification images also comprise signature properties that are diagnostic of stimulus class. This was determined with support vector machines (SVMs; [15]) trained to map 6 DISCUSSION Four visual recognition experiments using the technique of random temporal sampling were carried out in normal adult observers with different stimulus classes. The classification images constructed from the relation between response accuracy and temporal features of stimulus sampling demonstrate that visual processing efficiency varies markedly throughout stimulus exposure in conjunction with stimulus oscillation frequencies. The classification images as well as the capacity of their power spectra to discriminate reliably between stimulus classes indicate that the oscillatory mechanisms revealed are different or are engaged differently across experiments. This is congruent with the existence of specialized cortical mechanisms for visual word, object, and face recognition [16]. In turn, since 7 Arguin et al. Arguin et al. Visual oscillations functional specialization for stimulus classes only emerges at relatively high- levels in the cortical hierarchy, this suggests that random temporal sampling may tap neural mechanisms that are several synapses away from receptors. The results also indicate that temporal and time-frequency classification images are highly variable across individuals. A decomposition of these classification images into their power and phase components however, yields a more substantial picture. It demonstrates highly consistent power spectra among participants tested with the same stimulus class and shows that between-subject classification image variability largely rests on their phase spectra. This finding indicates that observers engage to the same degree a number of oscillatory mechanisms which are necessary to perform a given task. However, the temporal relation between these oscillations and stimulus events is what varies substantially across observers. It is the latter which appears to be responsible for the poor between-subject consistency of raw classification images. Across all tasks, the oscillation frequencies that seem the most engaged range from 5 Hz to about 25 Hz, as can be seen from the frequencies that carry the most power in Fig. 2. However, the particular pattern of power per frequency is rather unique for each task, except perhaps for the two object recognition tasks (familiar and novel), which demonstrate highly similar 8 Visual oscillations Visual oscillations A noteworthy finding from the present study is that classification images of processing effectiveness as a function of the static temporal frequencies present in the sampling functions revealed no significant effect. This finding demonstrates the incapacity of static temporal frequencies of stimulus visibility oscillations to modulate visual recognition performance. This contrasts with the time-frequency classification images (Fig. 1e-h), which show the large impact of transient stimulus oscillations. These observations appear relevant to the issue of whether the neural oscillations that may be captured in humans by EEG/MEG reflect sustained or transient-burst events [20]. The incapacity of static frequencies to account for processing effectiveness is predicted by the transient-burst theory whereas it is incompatible with sustained oscillations of stable amplitude. Arguin et al. patterns. From these power spectra of classification images, it was possible to determine the stimulus class they originate from with significant accuracy. This observation strongly reinforces the idea that random temporal sampling taps fundamental visual mechanisms that are largely shared among neurologically intact adults. What appears critical for the ability of random temporal sampling to deliver relevant observations is for perceptual mechanisms to synchronize precisely with stimulus events. Indeed, without synchrony, the timing relations between the state of the visual system and stimulus events would be random. This would prevent classification images, which are based on large collections of trials, from showing variations of processing efficiency according to the timing of stimulus events since this relation would be entirely blurred. To prevent such problems, great care was taken in designing the experiments to present preparatory signals that would permit participants to precisely anticipate the moment of target onset and to maximize their level of alertness at that instant [17, 18]. Also, the features of the auditory tone preceding this event were selected based on a prior demonstration of their capacity to reset neural oscillations in the visual system [19]. Future studies will be required to assess the actual value of these preparatory signals. 9 9 Arguin et al. Participants All 44 participants, 29 women and 15 men, were French speaking neurologically intact undergraduate students at Université de Montréal aged between 19 and 35 years old. All add normal or corrected vision. Eight participants took part in the word recognition experiment, whereas distinct groups of 12 participants took part in each of the others, which were conducted later. The recruitment of participants was initiated only after approval of the studies by the relevant ethics committee of the Université de Montréal (Comité d’éthique de la recherche en education et psychologie). All participants gave their informed consent to participate and all procedures were carried out in accordance with the relevant guidelines and regulations. Note added in proof: We have recently completed analyses for a new experiment involving one condition that was identical to the word recognition task reported here. These new results closely replicate those reported here in every respect. 10 Visual oscillations Arguin et al. Arguin et al. Arguin et al. Arguin et al. Materials and stimuli All experiments were run on an HPZ230 computer equipped with an NVIDIA GeForce GTX970 videocard and an ASUS VG248QR HD monitor with maximum luminance of 200 cd/m2 and a 120 Hz refresh rate. All stimuli 11 Visual oscillations were achromatic and all manipulations of intensity were linear. Experiments were programmed in Matlab [Mathworks Inc.] and made the use of the Psychophysics toolbox [21]. Observation distance was 57 cm, which was stabilized by having participants rest their head on a chin rest. For the word recognition experiment, stimuli were 600 five-letter French common words with a mean frequency of 157 per million [22]. Words were printed in Tahoma (x-height of 0.76 deg) in black letters over a grey (half of maximum intensity) background. For the familiar object recognition experiment, stimuli were the colour photographs of 300 objects shown in front of a white background from the Bank of Standardized Stimuli (BOSS; [23]) which were transformed into greyscale images. The items used were selected based on the published performance statistics for BOSS images so that it would be easy for participants to recognize them and find their name. The maximum horizontal extent of the stimuli was 17.4 deg of visual angle and their maximal vertical extent was 17.1 deg. The stimuli used for the novel object recognition task were simple randomly generated 3-D shapes with two interconnected medial axes around each of which a cylindrical shape of variable diameter was built (Fig. 3; this 12 Visual oscillations Insert Fig. 3 near here. Arguin et al. described above, overlaid by a white noise field with a contrast level which was adjusted on a trial by trial basis, as described below, in an attempt to maintain response accuracy to 50% correct. The noise was made from a second, independent white noise field of maximal contrast. Throughout the stimulus duration, the signal-to-noise ratio (SNR), that is the ratio of the weights attributed to the signal and the noise for the construction of the stimulus to be displayed, varied according to a random function constructed by the integration of sine waves with frequencies ranging between 5 and 55 Hz in steps of 5 Hz with random amplitudes and phases. A new, independent SNR function was generated on each trial. The range of the SNR was normalized between 0 and .5 and its sum across the sequence of 24 image frames making up the 200 ms stimulus duration (at the refresh rate of 120 Hz) was matched across trials. The overall luminance and contrast of the stimuli were matched across image frames and across trials. Visual oscillations Visual oscillations Arguin et al. Arguin et al. stimulus class has been used previously by [24]. The stimuli displayed in the experiment were the 2D renderings of these items, lighted from above, covered in a rich achromatic texture and presented in one of four viewpoints, all of which revealed the major features characterizing the shape of the object. Items were shown over a black background and their maximum horizontal and spatial extent was of 9.5 deg. Insert Fig. 3 near here. Insert Fig. 3 near here. Insert Fig. 3 near here. One hundred and thirty greyscale photographs of famous actors and actresses from the face bank of [25] were used for the face recognition experiment. The items selected were those which appeared as most likely to be recognized by participants. Each face appeared in frontal view and its expression was either neutral or happy. These photographs were displayed over a white background and their horizontal and vertical spatial extent was of 7.0 deg. In all experiments, the duration of the target stimulus was of 200 ms. Target stimuli were made from the linear summation of two components, the signal and the noise. The signal was made from the image of the target, as 13 Procedure In all experiments, participants completed a total of 1200 trials over two test sessions of 600 trials each. In all cases, the participant’s task was to identify the target without time pressure. With word stimuli, participants had to read the word aloud. With familiar objects, they were required to identify the item using a name that was sufficiently specific to demonstrate accurate 14 Arguin et al. Visual oscillations recognition. With novel objects, they had to indicate orally the digit assigned to it during the learning phase, which is described below. With famous faces, participants were asked to either give the name of the actor/actress, the name of his/her character in a particular movie or his/her specific role in that movie. In the sequence of trials in the word, familiar object, or faces recognition experiments, no item could be repeated before the complete stimulus set had been presented for the same number of trials. In the novel object recognition task, the order of presentation of items was random, with the constraint that each object be presented an equal number of times. For the novel object recognition experiment, participants first had to be trained with the objects and the name they were assigned, which was a digit ranging from 1 to 6. This began with a familiarization phase during which participants were exposed to a sheet of paper on which the six objects were printed in all four possible viewpoints along with the digit associated with each object. Once the participant felt having studied the sheet sufficiently, the practice phase followed during which each of the possible 24 stimulus instances were presented eight times in a random trial sequence presenting one non-degraded item at a time until the participant named it. A correct response was followed by a 1000 Hz (50 dB pure tone) auditory feedback 15 Visual oscillations Visual oscillations contrast (i.e. null) mask and the highest being made of black and white (i.e. minimum vs. maximum luminance) elements. The first experimental block began with a white noise contrast exactly at the middle of the available range for the first 10 trials. Starting at trial 11, the average response accuracy for the 10 preceding trials was determined. If accuracy was exactly 50%, white noise contrast remained the same for the following trial. If accuracy was below 50%, white noise amplitude was reduced by one step (see below) whereas it was increased by one step if accuracy was above 50%. Initial step size was of 16 contrast levels and this value was halved every time the direction of adjustment was reversed, down to a minimum of 1. The state of the algorithm adjusting this white noise contrast was maintained across consecutive blocks of trials. Arguin et al. Visual oscillations whereas errors were followed by a 300 Hz tone. Following each response, whether correct or not, the correct digit identity of the item was displayed on the screen for 500 ms. To pass to the experimental phase, participants had to obtain at least 90% correct responses in a practice block. In all experiments, the time course of each trial was as follows. A square white noise mask of 18° to a side centred on the middle of the display monitor was first presented for 1250 ms. A white fixation cross was then added at the centre of the monitor for 250 ms, which then disappeared. This offset was followed 150 ms later by a 900 Hz-60 dB – 14 ms pure tone announcing the onset of the target stimulus 100 ms later. The target stimulus was presented in the middle of the screen for a duration of 200 ms during which the SNR varied according to a random function, as described above. Following its offset, only the background white noise mask remained visible until the participant’s response. A 500 ms delay then followed prior to the beginning of the following trial. Response accuracy on experimental trials was maintained to about 50% correct using a staircase procedure which automatically adjusted the contrast of the white noise superimposed on the target image as part of the “signal” portion of the stimulus to be displayed (see above). The available range of white noise contrast had 128 levels, the lowest producing a zero- 16 Arguin et al. Arguin et al. Data analysis Response accuracy was averaged across participants separately for each experiment. The average percentages of correct responses are; words: 60.5%; familiar objects: 49.2%; novel objects: 56.0%; and faces: 30.1%. As indicated above, the contrast of the white noise mask that was part of the “signal” portion of the stimuli displayed was adjusted to maintain response accuracy to about 50% correct. The means of noise levels for each 17 Arguin et al. Visual oscillations experiment are; words: 111.3; familiar objects: 119.3; novel objects: 119.9; and faces: 11.0, over a possible maximum of 128. The main analyses pertained to the construction of classification images representing how response accuracy was affected by various temporal features of the sampling functions of the target stimuli. The temporal features which were analysed are SNR amplitude as a function of time from target onset, the Fourier descriptors for these functions, and time-frequency representations of the same functions. The Fourier descriptors are the amplitude and phase of the temporal frequencies (5-55 Hz in 5 Hz steps) used to build the SNR time functions (see above). The time-frequency representations of the SNR functions were calculated with a wavelet analysis using three-cycle complex Morlet wavelets varying in temporal frequency from 5 to 55 Hz in 5 Hz steps [26]. For each mode of coding the SNR time functions, classification images were obtained for each individual participant. This was done by the weighted subtraction of the temporal features of the sampling functions associated to errors from those associated to correct responses. These raw classification images were then transformed in Z scores by a bootstrapping operation whereby the sampling functions were randomly assigned to response accuracies while allowing for repetition, and from which classification images 18 Arguin et al. Visual oscillations were constructed. The mean and standard deviation of 1000 such random classification images for an individual participant served as reference to transform the values from his/her raw classification image into Z scores. Once transformed to a common scale, the individual classification images were averaged and then submitted to a two-way Pixel test [14] with α = .05 to determine the adjacent points in classification images which differed significantly. All classification images reported are based on group means and all the Z score differences they depict are significant. Nonsignificant differences between consecutive values were averaged. Analyses of the consistency of classification images across participants having taken part in the same experiment were carried out by performing all possible pairwise correlations between the raw classification images of individual participants, which were then averaged. The same procedure was followed to analyse the consistency among participants of the power and phase spectra of their classification images. The analysis of raw individual classification images into their power and phase components was performed by one-dimensional fast Fourier transforms applied to either the time domain classification images or to Z 19 Arguin et al. Arguin et al. score amplitude variations through time separately for each temporal frequency represented in time-frequency classification images. score amplitude variations through time separately for each temporal frequency represented in time-frequency classification images. Stimulus class was decoded from the power spectra of individual time domain and time-frequency domain classification images using linear support vector machines (SVMs; [15]) and a leave-one-out cross-validation procedure. 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Arguin et al. Arguin et al. Arguin et al. [24] Yamane, Y., Carlson, E. T., Bowman, K. C., Wang, Z., & Connor, C. E. (2008). A neural code for three-dimensional object shape in macaque inferotemporal cortex. Nature Neuroscience 11(11), 1352-1360. doi:10.1038/nn.2202 [25] Butler, S., Blais, C., Gosselin, F., Bub, D. & Fiset, D. (2010). Recognizing famous people. Attention, Perception, & Psychophysics 72(6), 1444 1449. doi:10.3758/APP.72.6.1444 [25] Butler, S., Blais, C., Gosselin, F., Bub, D. & Fiset, D. (2010). [26] Cohen, M. X. (2014). Analyzing Neural Time Series Data: Theory and Practice. Massachusetts Institute of Technology. 25 25 Visual oscillations ACKNOWLEDGEMENTS Supported by grants from the Fonds de Recherche Québec – Nature et Technologie and the Natural Sciences and Engineering Research Council of Canada (NSERC) to Martin Arguin and a summer research scholarship to Roxanne Ferrandez from NSERC. We are grateful to Prof. Charles E. Connor for the software for the generation of novel objects. 26 26 Visual oscillations Arguin et al. AUTHOR CONTRIBUTIONS Martin Arguin was responsible for conceiving and designing the work reported, programming the software for conducting the experiments and analysing the data, data collection for the word recognition experiment, advanced data analyses for all experiments and preparing the manuscript. Roxanne Ferrandez prepared the stimulus sets and performed the data collection for the familiar object and the face recognition tasks as well as the preliminary data analyses for these experiments. Justine Massé prepared the stimulus set and performed the data collection for the novel object recognition experiment and the preliminary data analyses. 27 27 Arguin et al. ADDITIONAL INFORMATION No author has any competing interest to declare. No author has any competing interest to declare. 28 28 Visual oscillations Arguin et al. Arguin et al. Arguin et al. 32 Fig. 3 – Arguin et al. FIGURE CAPTIONS Fig. 1. Classification images of encoding effectiveness (in Z scores) as a function of SOA (panels a-e) and as a joint function of the frequency content of stimulus oscillations and SOA (panels e-h). Stimuli are: words (a and e), familiar objects (b and f), novel objects (c and d), and faces (d and h). Fig. 2. Power spectra of the classification images of encoding effectiveness in the time domain (panels a-e) and in the time-frequency domain (panels e- h). Stimuli are: words (a and e), familiar objects (b and f), novel objects (c and d), and faces (d and h). Fig. 3. Illustration of a subset of the stimuli used in the novel object recognition task. The figure shows one instance of each object of the set. In the experiment, each object could be shown from one of four different viewpoints. 29 Visual oscillations Arguin et al. FIGURES Fig. 1 – Arguin et al. FIGURES FIGURES Fig. 1 – Arguin et al. 30 Visual oscillations Arguin et al. 31 Fig. 2 – Arguin et al. Fig. 2 – Arguin et al. 31 Visual oscillations Arguin et al. Arguin et al. Fig. 3 – Arguin et al. 32 32 Figures Figures Figure 1 Arguin et al. Classi¦cation images of encoding effectiveness (in Z scores) a e) and as a joint function of the frequency content of stimulus oscillations are: words (a and e), familiar objects (b and f), novel objects (c and d), and Figure 1 Figure 1 Arguin et al. Classi¦cation images of encoding effectiveness (in Z scores) as a function of SOA (panels a- e) and as a joint function of the frequency content of stimulus oscillations and SOA (panels e-h). Stimuli are: words (a and e), familiar objects (b and f), novel objects (c and d), and faces (d and h). Figure 2 Arguin et al. Power spectra of the classi¦cation images of encoding effective (panels a-e) and in the time-frequency domain (panels e-h). Stimuli are: word (b and f) novel objects (c and d) and faces (d and h) Figure 2 Arguin et al. Power spectra of the classi¦cation images of encoding effectiveness in the time domain (panels a-e) and in the time-frequency domain (panels e-h). Stimuli are: words (a and e), familiar objects (b and f), novel objects (c and d), and faces (d and h). Figure 2 Arguin et al. Power spectra of the classi¦cation images of encoding effectiveness in the time domain (panels a-e) and in the time-frequency domain (panels e-h). Stimuli are: words (a and e), familiar objects (b and f), novel objects (c and d), and faces (d and h). Figure 3 Arguin et al. Illustration of a subset of the stimuli used in the novel object recognition task. The ¦gure shows one instance of each object of the set. In the experiment, each object could be shown from one o four different viewpoints. Figure 3 Arguin et al. Illustration of a subset of the stimuli used in the novel object recognition task. The ¦gure shows one instance of each object of the set. In the experiment, each object could be shown from one of four different viewpoints.
https://openalex.org/W2122505441	https://link.springer.com/content/pdf/10.1007%2Fs40200-019-00440-z.pdf	English	null	Patient centred care in diabetology: an Islamic perspective from Iran	Journal of diabetes and metabolic disorders	2,013	cc-by	436	Khojasteh Joharchi1 & Moosareza Memari1 & Eznollah Azargashb2 & Navid Saadat3 # The Author(s) 2019 Published online: 13 2019 September # The Author(s) 2019 Published online: 13 2019 September Correction to: Journal of Diabetes & Metabolic Disorders https://doi.org/10.1007/s40200-019-00427-w https://doi.org/10.1007/s40200-019-00440-z Journal of Diabetes & Metabolic Disorders (2019) 18:583 https://doi.org/10.1007/s40200-019-00440-z Journal of Diabetes & Metabolic Disorders (2019) 18:583 CORRECTION CORRECTION Correction to: Journal of Diabetes & Metabolic Disorders https://doi.org/10.1007/s40200-019-00427-w by/4.0/), which permits use, duplication, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made. The article Efficacy and safety of duloxetine and Pregabalin in Iranian patients with diabetic peripheral neuropathic pain: a double-blind, randomized clinical trial, written by Khojasteh Joharchi, Moosareza Memari, Eznollah Azargashb, and Navid Saadat, was originally published electronically on the pub- lisher’s internet portal (currently SpringerLink) on 13 August 2019 without open access. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appro- priate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. With the author(s)’ decision to opt for Open Choice the copyright of the article changed on 12 September 2019 to © The Author(s) 2019 and the article is forthwith distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/ Publisher’s note Springer Nature remains neutral with regard to jurisdic- tional claims in published maps and institutional affiliations. The online version of the original article can be found at https://doi.org/ 10.1007/s40200-019-00427-w The online version of the original article can be found at https://doi.org/ 10.1007/s40200-019-00427-w The online version of the original article can be found at https://doi.org/ 10.1007/s40200-019-00427-w * Khojasteh Joharchi joharchi-kh@sbmu.ac.ir * Moosareza Memari reza_memari@sbmu.ac.ir * Moosareza Memari reza_memari@sbmu.ac.ir 1 Department of Pharmacology, School of Medicine, Shahid Beheshti University of Medical Sciences (SBUMS), Tehran, Iran 2 Department of Social Medicine, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran 3 Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran 1 Department of Pharmacology, School of Medicine, Shahid Beheshti University of Medical Sciences (SBUMS), Tehran, Iran 2 Department of Social Medicine, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran 3 Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran 1 Department of Pharmacology, School of Medicine, Shahid Beheshti University of Medical Sciences (SBUMS), Tehran, Iran
https://openalex.org/W4382631205	https://discovery.ucl.ac.uk/10172885/1/s41434-023-00409-x.pdf	English	null	Immunobiology of a rationally-designed AAV2 capsid following intravitreal delivery in mice	Gene therapy	2,023	cc-by	11,025	INTRODUCTION Ad i d AAV2 transduction potential via capsid mutagenesis, that is, substituting wild-type residues for those with desirable properties [6]. One example of this involves the mutation of phosphodegron residues that are thought to be phosphorylated by cytosolic protein kinase enzymes, thus facilitating ubiquitin-dependent degradation of AAV2 virions. Switching these residues to amino acids that are refractory to phosphorylation has been shown to increase target cell transduction, possibly by augmenting nuclear transfer of vector genomes [7, 8]. The reduction in cytosolic degradation of AAV2 has also been studied in the context of CD8 T-cell responses to infected cells. The authors demonstrated that a reduction in the presentation of AAV2 capsid antigen on major histocompatibility complex class I (MHC c. I) correlated with attenuated cytolytic killing of infected hepatocytes by adoptively transferred CD8 T-cells following systemic vector administration in a rag KO mouse (which exhibited abrogated endogenous B- and T-cell function) [9]. Boye et al. also showed that triple, quadruple and quintuple phosphodegron mutant capsids demonstrate attenuated binding afﬁnity to AAV2’s primary attachment receptor, heparan sulphate proteoglycan (HSPG), which may explain their capacity to permeate deeper into retinal tissue, possibly due to reduced sequestration in the inner limiting membrane (ILM) that demarcates the vitreo-retinal interface [1]. Adeno-associated virus serotype 2 (AAV2) is a gene therapy vector that can be used to deliver therapeutic genes to diseased cells in the retina [1]. The platform is favoured for its relatively low immunogenicity and capacity to induce long-term therapeutic transduction in non-dividing neural tissue [2]. This was evidenced in 2017 by the regulatory approval of Luxturna (voretigene neparvovec-rzyl), an AAV2-based gene therapy that delivers a functional copy of the RPE65 gene to retinal pigment epithelium (RPE) cells in the outer retina via subretinal injection (SRT) [3]. More recently in 2020, scientists and clinicians investigating GS010 (lenadogene nolparvovec) reported improvements in visual acuity after intravitreal injection (IVT) of AAV2.ND4 to patients diagnosed with Leber hereditary optic neuropathy (LHON), a mitochondrial genetic eye disorder caused by the m.11778 G > A point mutation in the MT-ND4 gene, which is characterised by rapid loss of retinal ganglion cell (RGC) function [4]. g g Despite these recent successes, researchers have sought to improve AAV2 vectors across multiple parameters, including their transduction capacity, ability to evade pre-existing neutralising antibodies (NAbs), genome packaging capacity, and functional viral titres during manufacturing at scale for in-human trials [5]. Gene Therapy Gene Therapy www.nature.com/gt Received: 23 August 2022 Revised: 22 May 2023 Accepted: 12 June 2023 1John Van Geest Centre for Brain Repair, Department of Clinical Neuroscience, University of Cambridge, Cambridge, UK. 2Division of Cardiovascular Medicine, University of Cambridge, Addenbrooke’s Hospital, Hills Road, Cambridge, UK. 3UCL Institute of Ophthalmology, London, UK. 4Paediatric Respiratory Medicine, Primary Ciliary Dyskinesia Centre, Guy’s and St Thomas’ NHS Foundation Trust, London, UK. 5MRC Mitochondrial Biology Unit, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK. 6NIHR Biomedical Research Centre at Moorﬁelds Eye Hospital and UCL Institute of Ophthalmology, London, UK. 7Wellcome Trust-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK. 8Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, Melbourne, VIC, Australia. 9Ophthalmology, Department of Surgery, University of Melbourne, Melbourne, VIC, Australia. ✉email: michael1993whitehead@gmail.com ARTICLE OPEN Immunobiology of a rationally-designed AAV2 capsid following intravitreal delivery in mice Andrew Sage2, Tom Burgoyne3,4, Andrew Osborne1, Patrick Yu-Wai-Man1,5,6 and Keith R. Martin 1,7,8 Michael Whitehead 1✉, Andrew Sage2, Tom Burgoyne3,4, Andrew Osborne1, Patrick Yu-Wai-Man1,5,6 and Keith R. Martin 1,7,8,9 © The Author(s) 2023 © The Author(s) 2023 © The Author(s) 2023 Adeno-associated virus serotype 2 (AAV2) is a viral vector that can be used to deliver therapeutic genes to diseased cells in the retina. One strategy for altering AAV2 vectors involves the mutation of phosphodegron residues, which are thought to be phosphorylated/ubiquitinated in the cytosol, facilitating degradation of the vector and the inhibition of transduction. As such, mutation of phosphodegron residues have been correlated with increased transduction of target cells, however, an assessment of the immunobiology of wild-type and phosphodegron mutant AAV2 vectors following intravitreal (IVT) delivery to immunocompetent animals is lacking in the current literature. In this study, we show that IVT of a triple phosphodegron mutant AAV2 capsid is associated with higher levels of humoral immune activation, inﬁltration of CD4 and CD8 T-cells into the retina, generation of splenic germinal centre reactions, activation of conventional dendritic cell subsets, and elevated retinal gliosis compared to wild-type AAV2 capsids. However, we did not detect signiﬁcant changes in electroretinography arising after vector administration. We also demonstrate that the triple AAV2 mutant capsid is less susceptible to neutralisation by soluble heparan sulphate and anti-AAV2 neutralising antibodies, highlighting a possible utility for the vector in terms of circumventing pre-existing humoral immunity. In summary, the present study highlights novel aspects of rationally-designed vector immunobiology, which may be relevant to their application in preclinical and clinical settings. Gene Therapy; https://doi.org/10.1038/s41434-023-00409-x Gene Therapy; https://doi.org/10.1038/s41434-023-00409-x Received: 23 August 2022 Revised: 22 May 2023 Accepted: 12 June 2023 Use of animals Eyes were enucleated and immersed in ice-cold 4% paraformaldehyde (PFA) for 24 h. Under a dissecting microscope, the cornea and lens were removed and the retina was dissociated from the eye cup using forceps. Four incisions were then made from the peripheral retina to within 0.5 mm of the optic nerve head to allow the retina to ﬂatten. Retinas were washed three times in PBS, then blocked with 3% normal goat serum (NGS), 1% bovine serum albumin (BSA), and 0.3% Triton X-100 diluted in PBS for 1 h at room temperature. Primary antibodies were diluted to the concentra- tions stated below (Table 1) in blocking solution and incubated with retinas overnight at 4 °C. Samples were washed three times in PBS. Alexaﬂour-conjugated secondary antibodies and 2-(4-amidinophenyl)-1H- indole-6-carboxamidine (DAPI) were diluted in PBS 1:500 and 1:5,000 respectively and incubated with retinas for 2 h at room temperature. Imaging and analysis of GFP was performed without antibody-mediated signal ampliﬁcation. Retinas were washed three times in PBS and mounted onto Superfrost Plus slides (VWR) with RGCs facing upwards, using FluoSave (Merck Millipore) to preserve the ﬂuorescent signal. To obtain tilescan images of the whole ﬂatmounted retina, a Leica DMi8 microscope was used and set to a 20x objective. An SPE confocal microscope equipped with a 40x magniﬁcation (Leica Microsystems) was used to obtain high magniﬁcation representative images. For quantiﬁcation of RBPMS and GFP co-localisation, eight images per retina were taken using a 20x objective All procedures performed on animals were approved by the UK Home Ofﬁce in accordance with the UK Animals (Scientiﬁc Procedures) Act, and undertaken in accordance with the Association for Research in Vision and Ophthalmology’s (ARVO) Statement for the Use of Animals in Ophthalmic and Visual Research. The sample size used in each experiment is stipulated in the ﬁgure legends. In vivo study design We performed bilateral intravitreal injections of 2E8 genome copies (GC)/ eye AAV2, 2E8 GC/eye phosphodegron mutant AAV2, or PBS vehicle into C57BL6/J adult male mice. After a three week incubation period, electroretinography was performed. Blood samples were also taken by cardiac puncture for serological testing, before eyes were enucleated and ﬁxed for immunohistochemical analysis. The spleens of mice were also extracted for analysis via ﬂow cytometry. A summary of the in vivo study design is given in Fig S1 (Supplementary Materials). Vector production p Mutagenesis of rep/cap gene plasmids was performed at Vector Biolabs (Vector Biolabs 293 Great Valley Parkway, Malvern, PA 19355, USA). The correct substitutions were conﬁrmed by Sanger Sequencing. AAV2 vectors were manufactured at ViGene Biosciences (9430 Key West Avenue, Suite 105, Rockville, MD 20850, USA). For AAV2 manufacturing, a standard triple plasmid transfection protocol was used (pAAV-CAG-GFP, pHelper, pRep2/ Cap2). Vectors were extracted from HEK-293T cells via repeated freeze- thawing, puriﬁed using iodixanol gradient ultracentrifugation and sus- pended in phosphate-buffered saline (PBS). All vector preps were titred by ViGene via qPCR with primers speciﬁc to the AAV ITR regions and the SYBR Green detection method. Viral preps were treated with a DNAse I followed by digestion with Proteinase K prior to qPCR analysis. All vectors were aliquoted upon arrival and stored at –80 °C until further use. INTRODUCTION Ad i d A comprehensive literature now exists detailing efforts to improve M. Whitehead et al. 2 These ﬁndings therefore demonstrated that the in vivo properties of phosphodegron mutant AAV2 may be explained by factors in addition to reducing cytosolic degradation. Lomb) was also used as a local anaesthetic and pupils were dilated with 1% tropicamide to visualise correct placement of the needle in the vitreous. Vectors were diluted in sterile PBS to obtain the desired vector concentration in 2 μL solution. For bilateral IVTs, a 5μL Hamilton Syringe (#65RN; Needle: 33 G, 8 mm, point style 2, Hamilton Co) was used to puncture the sclera approximately 1 mm posterior to the superior- temporal limbus and inject the vector solution into the vitreous. Solution was injected slowly over a 30 s period to prevent sudden increases in intraocular pressure. The cornea was then punctured with a 30 G needle to limit reﬂux of the injection solution from the site of injection. Animals were recovered in a warm cabinet and monitored every 15 min until fully recovered. To collect tissues, mice were sacriﬁced using the Schedule 1 method of cervical dislocation. Eyes would be excluded from analysis if (i) the injection score was graded ‘poor’ e.g. due to signiﬁcant reﬂux of injected solution, or (ii) a cataract developed. g y g Some literature reports have described the eye as partially immune-privileged due to physical barriers like the blood-retinal- barrier (BRB) and an apparent lack of efferent lymphatics, and expression of anti-inﬂammatory factors [10]. However, AAV2 delivery via IVT has been associated with the inﬁltration of mononuclear cell inﬁltrates into the eye [11], in addition to a possible role for dendritic cell (DC) and macrophage inﬁltration, and the proliferation of microglia cells [12]. Another publication observed AAV8 genomes in blood, splenic and lymphatic samples post-IVT, and suggested a possible role for vector shedding in facilitating systemic immune responses to vector protein via the IVT route of administration [13]. Collectively, these ﬁndings question the role of ocular immune privilege in AAV-mediated gene therapy studies and suggest investigations to better understand the nature and clinical relevance of the immune response to wild-type and mutant AAV capsids are warranted. NAb assays y HEK-293T cells were seeded at 10,000 cells per well in white-walled, clear- bottomed 96-well plates (Sigma Aldrich) and used in the neutralising antibody (NAb) assay when 80% conﬂuent. Blood sera samples were collected via saphenous vein bleeds or cardiac puncture, allowed to clot overnight at 4 °C, then centrifuged at 300 RCF. The resulting supernatant was designated sera and stored at –20 °C until further use. Two- or three- fold serial dilutions of serum samples were prepared in DMEM in a 96-well plate. AAV2.CMV.Luciferase was added to a ﬁnal concentration of 1E9 GC/ mL, and incubated for 1 h to allow the anti-AAV NAbs to bind to the vectors. Sera-AAV mixtures were then transferred to the HEK-293T cultures and incubated for 24 h. Luminescent signal was detected by aspirating the media and adding 25 µL of BrightGlo assay substrate (ProMega), covering the plates with foil and incubating for 5 min. Luminescence was recorded using a FLUOstar Omega plate reader, and IC50 (dilution of sera that yielded a 50% reduction in the luminescent signal) and area-under-the- curve (AUC) was also calculated in GraphPad Prism. Here, we demonstrate that a triple phosphodegron mutant AAV2 capsid is associated with increased humoral immune activation, retinal T-cell inﬁltration, splenic germinal centre reactions and DC activation, and retinal gliosis when compared to wild-type AAV2 particles. However, we did not detect electrophysiological perturba- tions after vector administration. We also show that the triple mutant capsid is more resistant to neutralisation by soluble heparan sulphate (HS) and anti-AAV2 NAbs compared with wild-type AAV2, highlighting a possible utility for phosphodegron mutant AAV2 vectors in circumventing pre-existing NAb responses. In summary, the present study highlights novel aspects of phosphodegron mutant AAV2 immunobiology relevant to both preclinical and clinical application. Total antigen binding antibody assays g g y y To assess total binding antibody (TAb) levels, an enzyme-linked immunosorbent assay (ELISA) was used. 96-well plates were coated with 50 µL 1E10 GC/mL AAV2.CAG.GFP for 2 h at room temperature. AAV was aspirated and plates were blocked with a 5% milk powder 0.2% tween-20 solution for 2 h at room temperature. Serum samples were diluted 1:2,000 in blocking solution and added to the wells in a 100 µL volume. Plates were incubated overnight at 4 °C, followed by one wash in blocking solution and two washes with tris-buffered saline (TBS). Horseradish peroxidase (HRP)- conjugated secondary antibodies were diluted to a ﬁnal concentration of 1:50,000 in TBS, added to the wells and incubated for 2 h at room temperature. Plates were washed three times with TBS, followed by addition of 50 µL 3,3’,5,5’-tetramethylbenzidine (TMB) substrate, and a 30 min incubation at room temperature. Signal detection was performed on a FLUOstar Omega plate reader, measuring absorbance at 650 nm. Flow cytometry Spleens were extracted and processed into a single cell suspension by passing through a 70 µm cell sieve using a syringe plunger. Red blood cells (RBCs) were ﬁrst lysed with ammonium chloride RBC lysis buffer, and then 1-2E6 cells were stained with a cocktail of ﬂuorescently-tagged antibodies and a live-dead stain (Zombie aqua, Biolegend). After washing, cells were ﬁxed with formaldehyde solution (BD Cellﬁx) and ﬁltered through a 30 µm ﬁlter before analysis on a LSRII Fortessa ﬂow cytometer (BD). Prior to antibody gating, live single cells are gated based on FSC, SSC and negative live-dead staining. A list of the antibodies used for these analyses is given below (Table 3). Intravitreal (IVT) vector injection j For anaesthesia, 50 mg/kg ketamine and 10 mg/kg xylazine were delivered through intraperitoneal (IP) injection. 1% tetracaine solution (Bausch & Gene Therapy M. Whitehead et al. 3 Table 1. List of primary antibodies used in immunohistochemical analysis of ﬂatmounted retina tissue. Target Species Dilution Vendor Catalogue number RBPMS Guinea pig 1:500 PhosphoSolutions 1832-RBPMS GFAP Rabbit 1:500 Dako Z0334 Tuj1 Mouse 1:500 Biolegend MMS-435P Iba1 Guinea pig 1:250 Synaptic Systems 234 004 Table 1. List of primary antibodies used in immunohistochemical analysis of ﬂatmounted retina tissue. Table 2. List of primary antibodies used in immunohistochemical analysis of cryosectioned retina tissue. Table 2. List of primary antibodies used in immunohistochemical analysis of cryosectioned retina tissue. Target Species Dilution Vendor Catalogue number Antigen retrieval RBPMS Rabbit 1:500 Abcam ab152101 No GFAP Rabbit 1:500 Dako Z0334 No Iba1 Guinea pig 1:250 Synaptic systems 234 004 No CD4 Rabbit 1:250 Abcam ab183685 Yes CD8 Rabbit 1:1000 Abcam ab217344 Yes and assessed using Volocity Software. Assessment of astrocyte dendritic arbour complexity was performed in ImageJ using the Skeletonise/Analyse Skeleton functions. Table 3. List of antibodies used for ﬂow cyt ble 3. List of antibodies used for ﬂow cytometry analysis y y y Target Fluorophore Clone Vendor CD19 BV650 6D5 Biolegend IgM APC II/41 ThermoFisher IgD PerCPVio700 Miltenyi CD95 PE-Cy7 Jo2 BD Biosciences GL7 eF450 Ly-77 Biolegend MHC c. II APCVio770 Miltenyi CD3 AF488 145-2C11 Biolegend CD4 AF700 RM4-5 Biolegend CD8 PerCP 53-6.7 Biolegend CXCR5 PE L138D7 Biolegend PD-1 PE-Cy7 4B12 Biolegend CD44 BV605 1M7 Biolegend CD62L PacBlue MEL-14 Biolegend CD25 APC PC61 Biolegend CD11c PE-Cy7 N418 Biolegend CD8a PerCP 53-6.7 Biolegend CD11b AF488 M1/70 Biolegend XCR1 APC MPC-11 Biolegend SiglecH PE Miltenyi Retinal cryosections A Radial Interpretation of Viral Electron Density Map (RIVEM) was also produced. This programme reads atomic coordinates from Protein Data Bank ﬁles, and converts these into a stereographic projection. residues, PyMol’s internal mutagenesis wizard was used. The residue and the appropriate substitute residue was selected, then the correct rotameric conformation of the mutated amino acid was chosen in accordance with PyMol’s prediction. Mutated residues were coloured Y444F, red; K556E, green; S662V, yellow. Heparan binding domains (HBDs; R484, R487, K532, R585, and R588) were coloured in blue. AAV receptor binding domains (AAVR BDs; R471, D528, Q589, T592, S262, Q263, G265, A266, S267, N268, H271, N382 and Q385) were highlighted in orange. A Radial Interpretation of Viral Electron Density Map (RIVEM) was also produced. This programme reads atomic coordinates from Protein Data Bank ﬁles, and converts these into a stereographic projection. Assessment of in vitro transduction efﬁciency and neutralisation by HS and anti-AAV2 NAbs y HEK-293T and ARPE-19 cells were cultured on poly-L-lysine (10 µg/mL; Sigma Aldrich) coated plates. Cells were incubated in Dulbecco’s Modiﬁed Eagles Medium (DMEM) supplemented with 10% foetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) until 80% conﬂuent. For transduction with vectors, cells were washed with PBS, then incubated with vectors diluted to stated concentrations in serum-free DMEM. Cells were passaged Retinal cryosections y Following enucleation, eyes were immersed in 4% PFA for 24 h at 4 °C, dehydrated in 30% sucrose for 24 h at 4 °C, and embedded in optimal cutting temperature compound (OCT; Sakura Finetek). 13μm tissue sections (through the dorsal-ventral/superior-inferior axis of the retina) were prepared using a Bright OTF 5000 cryostat (Bright Instruments) and Superfrost Plus slides (VWR). For some staining protocols (see Table 2 below), citrate buffer- mediated antigen retrieval was used. Slides were incubated in 90 °C sodium citrate buffer (Abcam) for 30 mins, allowed to cool to room temperature, then washed twice in PBS. Tissue sections were blocked and permeabilised with 10% NGS, 0.5% Triton X-100 and 0.5% BSA diluted in PBS for 1 h at room temperature. Primary antibodies were diluted in 5% NGS, 0.5% Triton X-100 and 0.5% BSA to the concentrations stated below and incubated overnight at 4 °C. Table 2 outlines all the primary antibodies used for immunohistochem- ical analysis of retinal cryosections. Samples were washed three times with PBS for 10 mins, then incubated with secondary antibodies and DAPI diluted 1:1,000 and 1:5,000 respectively for 2 h at room temperature. Samples were washed three times in PBS and a glass coverslip was mounted with FluoSave reagent (Merck Millipore). For imaging, a Leica DM6000 epiﬂuorescence microscope was used to obtain 8–12 images per sample for quantiﬁcation of ﬂuorescence intensity. Quantiﬁcation of ﬂuorescence intensity per image was performed in ImageJ. Images were thresholded and measurements of integrated density were taken per ﬁeld-of-view (FOV) and for each region-of- interest (ROI). For glial ﬁbrillary acidic protein (GFAP) analysis, Single Neurite Tracer, an ImageJ plugin, was used. Each observable GFAP+ ﬁbril per FOV was traced manually on the software, which then calculated the integrated density of ﬂuorescence and length of each ﬁbril. Counting of CD4 and CD8 T-cells was performed using the Analyse Particles function. residues, PyMol’s internal mutagenesis wizard was used. The residue and the appropriate substitute residue was selected, then the correct rotameric conformation of the mutated amino acid was chosen in accordance with PyMol’s prediction. Mutated residues were coloured Y444F, red; K556E, green; S662V, yellow. Heparan binding domains (HBDs; R484, R487, K532, R585, and R588) were coloured in blue. AAV receptor binding domains (AAVR BDs; R471, D528, Q589, T592, S262, Q263, G265, A266, S267, N268, H271, N382 and Q385) were highlighted in orange. Splenic lymphocyte populations are modulated by IVT of AAV2 (TM) vs. wild-type AAV2 vectors Splenic lymphocyte populations are modulated by IVT of AAV2 (TM) vs. wild-type AAV2 vectors Recent reports have suggested that IVT of AAV8 may be associated with the presence of vector genomes in the blood and spleen, suggesting that vector shedding from the ocular cavity may be responsible for humoral and cellular adaptive immune activation [13]. As such, we asked whether injection of phosphodegron mutant and/or wild-type AAV2 via IVT could be associated with changes in splenic lymphocyte populations pertinent to the generation of NAbs and retina inﬁltrating T-cells. First, spleens were extracted and cells gated by CD3 and CD4/CD8 for analysis of T-cell populations (Fig. 3a). No changes in the levels of effector memory CD4 (Fig. 3b) or CD8 (Fig. 3c) T-cell populations between AAV2 (TM), AAV2 (Y444F) and wild-type AAV2 groups were observed, however, an increase in follicular helper T-cell (Tfh) levels was evident when comparing AAV2 (TM) and wild-type AAV2 injected mice (Fig. 3d), a ﬁnding that may support the NAb and TAb data described above. Next, splenic samples were gated for CD19 to analyse B-cell populations (Fig. 3e). Germinal centre B-cell levels were assessed ﬁrst as B-cells are known to proliferate and migrate to germinal centres when activated by antigen [14]. An increase in germinal centre B-cell levels was observed when comparing mice in the AAV2 (TM) group vs. the wild-type AAV2 group (Fig. 3f). Expression of major histocompatibility complex class II (MHC c. II) by germinal centre B-cells was then assessed to determine whether increased B-cell activation had been induced by administration of AAV2 (TM), and elevated MHC c. II immunoﬂuorescence was evident in this group compared to wild-type AAV2 controls (Fig. 3g). Next, germinal centre B-cell class-switching was analysed, and an increase in ‘switched’ B-cells was seen in the AAV2 (TM) group compared to wild-type AAV2 injected mice, an observation that may corrobo- rate the TAb data described above (Fig. 3h). Finally, conventional DC subsets, cDC1 & 2, and plasmacytoid DCs (pDCs) were investigated in terms of the level of MHC c. II immunoﬂuores- cence, a marker of DC activation [15]. First, DC subsets were gated according to MHC c. II, CD11c, CD11b, XCR1 and SiglecH expression (Fig. 3i). Analysis of MHC c. II immunoﬂuorescence on In silico modelling of selected mutations To visualise VP3 AAV2 capsid monomer and oligomers in silico, 6ih9, a 2.8 Å resolution cryoelectron microscopy-derived structure, was down- loaded from the Protein Data Bank. Both monomeric and oligomeric forms of 6ih9 were visualised using PyMol software. To depict the mutated capsid Gene Therapy M. Whitehead et al. 4 Increased humoral immune activation and T-cell inﬁltration into the retina following IVT of AAV2 (TM) vs. wild-type AAV2 First, the levels of NAbs were assessed in serum samples extracted 3wk post-IVT. Higher levels of neutralising activity were detected in mice who received AAV2 (Y444F) and AAV2 (TM) injections compared to wild-type AAV2 controls (Fig. 2a). These data were converted into IC50 values (dilution of serum that yields a 50% decrease in remaining infectivity), and this demonstrated increases in NAb levels following AAV2 (TM) compared with wild-type AAV2 IVTs (Fig. 2b). Next, TAb ELISA assays were performed to establish a correlation between the neutralising activity identiﬁed in the NAb assays and the presence of immunoglobulin isotypes known to participate in the anti-virus humoral immune response. Here, elevations in IgG, IgG2b and IgG2c titres were seen when comparing mice who received AAV2 (TM) and wild-type AAV2 IVTs, however, no changes in IgG1 and IgM isotype levels could be detected in this experiment (Fig. 2c). In summary, the results of the NAb and TAb assays suggested a higher level of humoral immune activation in AAV2 (TM) vs. wild- type AAV2 injected mice. in T75 ﬂasks in a 37 °C 5% CO2 incubator. Media was replaced every two days and cells were split 1:10-1:20 when they had reached 80–90% conﬂuency. Cells were counted using haemocytometry with trypan blue live dead staining, and cultures were only used for experiments if viability exceeded 90%. To determine the efﬁciency of cellular transduction, cells were detached with TrypLE (Thermo Fisher) for 5 min in the incubator. TrypLE was deactivated by addition of DMEM + 10% FBS + 1% P/S, and cells were centrifuged at 300 RCF for 5 min. The supernatant was aspirated and the remaining pellet was ﬁxed in 150 µL 4% PFA. To assess the number of transduced GFP+ cells, an Acurri C6 ﬂow cytometer was used. Forward (FSC) and side scatter (SSC) gating was used to identify an appropriate population for analysis. In silico modelling of selected mutations An FSC/FITC-A gate was used to identify GFP+ cells, and the population with a FITC-A ﬂuorescence intensity of >10^4 RFUs was deemed to be expressing GFP. To determine the neutralisation of wild-type and mutant AAV2 capsids in the presence of HS, 4000 AAV2 GC/ng HS was mixed and incubated at room temperature for 1 h before addition to HEK293T cell cultures. To study the neutralisation of wild-type and mutant AAV2 capsids by anti- AAV2 NAbs, wild-type and mutant AAV2 were incubated with anti-AAV2 NAb containing sera for 1 h before addition to HEK293T cell cultures. To assess differences between groups, the remining infectivity (I/I0) was calculated by normalising the values for neutralised wild-type and mutant AAV2 to no-sera or no-HS control groups. In the next set of experiments, eyes were extracted and processed into cryosections to assess whether the inﬁltration of CD4+ and CD8 + T-cells would be induced by IVT of wild-type and mutant AAV2. Increases in CD4 + T-cells were observed in the murine retina after AAV2 (TM) IVTs compared to mice who received wild-type AAV2 controls (Fig. 2d, e). Similar trends were evident in the CD8 + T-cell dataset, where elevated levels of inﬁltration were seen in AAV2 (TM) compared with wild-type AAV2 groups (Fig. 2f, g). Statistical analysis y Normality of distribution was assessed with a Shapiro Wilk test. If the data did not meet this assumption, non-parametric Kruskal-Wallis and Dunn’s posthoc tests to assess more than two groups, or a Mann–Whitney test to compare two groups. The homogeneity of variance was also assessed, and for data that was normally distributed but exhibited heteroskedasticity, a Brown-Forsythe ANOVA and Dunnett’s T3 posthoc test was used to compare more than two groups, and a Welch’s t-test to compare two groups. If datasets were normally distributed and exhibited homogeneity of variance, ordinary one-way ANOVA and Tukey or Dunnett’s posthoc tests (more than two groups) or a Student t test (two groups; two-tailed, unpaired) was used. Electroretinography g p y Electroretinography (ERG) was performed using a Diagnosys ColorDome LabCradle machine. Mice were dark adapted in a closed cabinet for 18–24 h before the ERG procedure. For anaesthesia, 50 mg/kg ketamine and 10 mg/kg xylazine were delivered via IP. An eyedrop of 1% tetracaine solution was used as a local anaesthetic and pupils were dilated with 1% tropicamide and 2.5% phenylephrine hydrochloride. Before placement of electrodes, eyes were checked for cataracts or absence of a red reﬂex, which would result in exclusion of that particular eye. A grounding electrode was placed into the tail, and the reference electrodes were placed around the eyes. The recording electrodes were then placed very gently onto the apex of the cornea. A small drop of lacrilube was then applied to the cornea using a 1 mL syringe/30 g needle to couple to the recording electrodes to the eye. The protocol used had 17 steps, including 7 positive scotopic threshold responses (pSTRs; measure of retinal ganglion cell function), 4 B-waves (rod bipolar cell function) and 4 A-waves (rod photoreceptor function). Transduction proﬁles of selected phosphodegron mutations in vitro and in vivo Three phosphodegron mutations, Y444F, K556E and S662V, were selected for analysis in accordance with previous reports [6, 7]. First, the transduction potential of each vector was assessed in vivo via IVT to C57BL6/J mice. Each vector was found to induce higher levels of GFP expression in RBPMS+ RGCs compared with wild-type AAV2 (Fig. 1a–c). Next, the three mutant residues were incorporated into a single capsid, hereafter referred to as AAV2 (TM). AAV2 (TM) induced higher levels of GFP expression in HEK- 293T and APRE-19 cell cultures when assessed with ﬂow cytometry in vitro (Supplementary Fig. 1a–c). These observations translated into an in vivo setting, where AAV2 (TM) IVTs were associated with higher levels of transduction in the murine retina than wild-type AAV2 and AAV2 (Y444F) injections (Fig. 1d–f). Gene Therapy Gene Therapy M. Whitehead et al. 5 n of three phosphodegron mutations leads to a synergistic increase in transduction of the really and tissue was taken for analysis after three weeks. All data is presented as a column gra p ±SEM All statistical analyses are vs the AAV2 group a Representative images of retinal w Fig. 1 A combination of three phosphodegron mutations leads to a synergistic increase in transduction of the murine retina. Vectors were injected intravitreally and tissue was taken for analysis after three weeks. All data is presented as a column graphs showing the mean value for each group ±SEM. All statistical analyses are vs. the AAV2 group. a Representative images of retinal wholemounts depicting increased levels of GFP in the phosphodegron mutant groups compared to AAV2 WT control three weeks after bilateral intravitreal injection of 2E8 GC/eye. Quantiﬁcation of GFP expression levels in wholemounted retina samples was performed in Volocity. b Percentage of GFP + RBPMS+ cells, and (c) mean GFP immunoﬂuorescence/RBPMS + ROI/FOV was calculated. p < 0.05, p < 0.01, *p < 0.001, parametric ANOVAs and Dunnett’s posthoc tests, n = 6–8. d Representative 40x epiﬂuorescent microscope images of retinal sections transduced with AAV2, or phosphodegron mutant capsid AAV2. RGC retinal ganglion cell layer, INL inner nuclear layer, ONL outer nuclear layer. Quantiﬁcation was performed in ImageJ. Here, (e) mean GFP ﬂuorescence levels/FOV was calculated. p < 0.05, p < 0.01, *p < 0.0001, Kruskal-Wallis and Dunn’s posthoc tests, n = 6–8. f Representative tilescan images showing wholemounted retinas from each group. Fig. Transduction proﬁles of selected phosphodegron mutations in vitro and in vivo wild-type AAV2 groups could be seen (Fig. 3k). Finally, no changes in MHC c. II immunoﬂuorescence was Transduction proﬁles of selected phosphodegron mutations in vitro and in vivo 1 A combination of three phosphodegron mutations leads to a synergistic increase in transduction of the murine retina. Vectors were injected intravitreally and tissue was taken for analysis after three weeks. All data is presented as a column graphs showing the mean value for each group ±SEM. All statistical analyses are vs. the AAV2 group. a Representative images of retinal wholemounts depicting increased levels of GFP in the phosphodegron mutant groups compared to AAV2 WT control three weeks after bilateral intravitreal injection of 2E8 GC/eye. Quantiﬁcation of GFP expression levels in wholemounted retina samples was performed in Volocity. b Percentage of GFP + RBPMS+ cells, and (c) mean GFP immunoﬂuorescence/RBPMS + ROI/FOV was calculated. p < 0.05, p < 0.01, p < 0.001, parametric ANOVAs and Dunnett’s posthoc tests, n = 6–8. d Representative 40x epiﬂuorescent microscope images of retinal sections transduced with AAV2, or phosphodegron mutant capsid AAV2. RGC retinal ganglion cell layer, INL inner nuclear layer, ONL outer nuclear layer. Quantiﬁcation was performed in ImageJ. Here, (e) mean GFP ﬂuorescence levels/FOV was calculated. p < 0.05, p < 0.01, p < 0.0001, Kruskal-Wallis and Dunn’s posthoc tests, n = 6–8. f Representative tilescan images showing wholemounted retinas from each group. Gene Therapy M. Whitehead et al. 6 cDC1 revealed elevated expression of this activation marker when comparing AAV2 (TM) and wild-type AAV2 mice (Fig. 3j). This trend was recapitulated in the cDC2 subset in which elevation of MHC c. II in the AAV2 (TM) vs. wild-type AAV2 groups could be seen (Fig. 3k). Finally, no changes in MHC c. II immunoﬂuorescence was evident between any of the three groups analysed in the pD subset (Fig. 3l). To summarise, analysis of splenic lymphocyt populations revealed changes in cells associated with th generation of systemic immunity to AAV2 capsids in the AAV (TM) groups compared with wild-type AAV2 injected mice, whic evident between any of the three groups analysed in the pDC subset (Fig. 3l). To summarise, analysis of splenic lymphocyte populations revealed changes in cells associated with the generation of systemic immunity to AAV2 capsids in the AAV2 (TM) groups compared with wild-type AAV2 injected mice, which cDC1 revealed elevated expression of this activation marker when comparing AAV2 (TM) and wild-type AAV2 mice (Fig. 3j). This trend was recapitulated in the cDC2 subset in which elevation of MHC c. II in the AAV2 (TM) vs. IVT of wild-type and phosphodegron mutant AAV2 vectors are not associated with detectable electrophysiological perturbations or loss of RGCs in the retina To further understand how the mutation of AAV vectors affects the induction of the immune response, future investigations may seek to better understand how the attenuation of binding afﬁnity to HSPG impacts vector shedding to the spleen and lymph nodes. Considering recent reports that HSPG binding afﬁnity may affect extravasation of systemically administered vector into the retina [19], it is possible that AAV2 (TM) was better able to enter the circulation than wild-type AAV2. This model would be consistent with the elevation of cDC1 & 2 activation and increase in germinal centre reactions (B-cell levels, MHC c. II expression and class- switching) observed in this study, which naturally would lead to higher NAb and TAb titres. If increased vector shedding to the spleen and cDC1 activation with AAV2 (TM) did occur, then this may also explain the CD4+ and CD8 + T-cell inﬁltration dataset, as cDC1s are thought to participate in priming these immune cells [20]. The results outlined above demonstrate that AAV2 (TM) may elicit higher systemic and local immune responses than wild-type AAV2 vectors when injected into the vitreous cavity. To assess whether these immune responses could be associated with electrophysio- logical changes in the murine retina, ERG was performed and ﬂatmounted retina tissue was stained with Tuj1 antibodies to identify possible loss of RGCs 3wk after vector administration. Assessment of ERG datasets showed no detectable changes between any of the vector IVTs across positive scotopic threshold (pSTR; RGC function), B-wave (rod bipolar cell function) and A-wave (rod photoreceptor cell function) recordings (Fig. 5a, b). Furthermore, there were no detectable changes in the number of Tuj1+ RGCs in the ﬂatmounted retina tissue (Fig. 5c). b IC50 values for each group were calculated using non-linear regression (variable slope, four parameters) in GraphPad p < 0.01, Brown-Forsythe ANOVA and Dunnett’s post hoc tests, n = 6. c Immunoglobulin ELISA assays were performed to assess the antibody subtypes responsible for the neutralising effect. p < 0.01, *p < 0.0001, two-way AVOVA and Dunnett’s posthoc tests, n = 6. d Representative 40x magniﬁcation epiﬂuorescent microscope images showing that intravitreal injection of triple phosphodegron mutant AAV2 is associated with the presence of CD4+ T-cells in the retina. e Quantiﬁcation of dataset was undertaken via manual counting of the number of CD4+ ROIs/FOV. p < 0.05, *p < 0.01, Kruskal-Wallis and Dunn’s posthoc tests, n = 6–10. f Representative 40x magniﬁcation epiﬂuorescence microscope images showing that intravitreal injection of triple phosphodegron mutant AAV2 is associated with the presence of CD8+ T-cells in the retina. g Quantiﬁcation of dataset was undertaken via manual counting of CD8+ ROIs/FOV. p < 0.05, p < 0.01, Kruskal-Wallis and Dunn’s posthoc, n = 4–8. may support the NAb/TAb and T-cell inﬁltration data outlined above in which immune activation was higher in the AAV2 (TM) group. afﬁnity to the vector’s primary receptor, HSPG [1]. We hypothe- sised that phosphodegron mutant AAV2 vectors may also be more resistant to anti-AAV2 NAbs, possibly due to changes in NAb epitopes induced by conformational changes. Positioning of the three mutations was visualised in PyMol at the level of capsid monomers (Fig. 6a) and 60mer capsid oligomers (Fig. 6b). A RIVEM plot was used to determine whether the mutated residues lay proximal or distal to canonical HSPG binding residues, with AAV receptor binding amino acids included for reference (Fig. 6c). Rotameric conformation of the mutated residues was predicted in PyMol and this may have identiﬁed some slight variances with wild-type counterpart structures (Fig. 6d). First, we corroborated previous reports and demonstrated that mutants Y444F, S662V and TM exhibited attenuated neutralisation by soluble HS, which could suggest reduced binding afﬁnity to HSPG (Fig. 6e). Next, we showed that remaining infectivity was increased in all single mutant capsid and AAV2 (TM) groups compared to wild-type AAV2, suggesting that the mutant vectors were more resistant to neutralisation by anti-AAV2 NAbs (Fig. 6f). DISCUSSION I hi d In this study, we examined the immune response arising from IVT of wild-type AAV2 and a triple phosphodegron mutant capsid, AAV2 (TM). Increased humoral immune activation and retinal T-cell inﬁltration were observed with the mutant capsid, in addition to changes in splenic germinal centre reactions and cDC1 & 2 activation. Elevated retinal gliosis was also increased in the AAV2 (TM) group, however, the immune responses identiﬁed were not associated with detectable electroretinography changes in the murine retina. Interestingly, AAV2 (TM) appeared to be less susceptible to neutralisation by soluble HS and anti-AAV2 NAbs. Gene Therapy M. Whitehead et al. 7 Fig. 2 Intravitreal injection of a triple phosphodegron mutant AAV2 induces humoral and cellular adaptive immune responses. Vectors were injected and blood and tissue samples were taken three weeks later for analysis. All data is presented as a bar graph showing the mean value for each group ±SEM. All statistical analyses are vs. the AAV2 group. a Injection of phosphodegron mutant AAV2 increases murine sera neutralising antibody (NAb) levels. NAb titres were assessed using a HEK-293T/1E9 VP/mL AAV2.CMV.Luciferase system. Luminescence was measured across a range of dilutions of sera, and remaining infectivity was deﬁned as the luminescent signal in each well divided by ‘Control’ values. b IC50 values for each group were calculated using non-linear regression (variable slope, four parameters) in GraphPad p < 0.01, Brown-Forsythe ANOVA and Dunnett’s post hoc tests, n = 6. c Immunoglobulin ELISA assays were performed to assess the antibody subtypes responsible for the neutralising effect. p < 0.01, *p < 0.0001, two-way AVOVA and Dunnett’s posthoc tests, n = 6. d Representative 40x magniﬁcation epiﬂuorescent microscope images showing that intravitreal injection of triple phosphodegron mutant AAV2 is associated with the presence of CD4+ T-cells in the retina. e Quantiﬁcation of dataset was undertaken via manual counting of the number of CD4+ ROIs/FOV. p < 0.05, *p < 0.01, Kruskal-Wallis and Dunn’s posthoc tests, n = 6–10. f Representative 40x magniﬁcation epiﬂuorescence microscope images showing that intravitreal injection of triple phosphodegron mutant AAV2 is associated with the presence of CD8+ T-cells in the retina. g Quantiﬁcation of dataset was undertaken via manual counting of CD8+ ROIs/FOV. p < 0.05, p < 0.01, Kruskal-Wallis and Dunn’s posthoc, n = 4–8. Fig. 2 Intravitreal injection of a triple phosphodegron mutant AAV2 induces humoral and cellular adaptive immune responses. Vectors were injected and blood and tissue samples were taken three weeks later for analysis. All data is presented as a bar graph showing the mean value for each group ±SEM. All statistical analyses are vs. the AAV2 group. a Injection of phosphodegron mutant AAV2 increases murine sera neutralising antibody (NAb) levels. NAb titres were assessed using a HEK-293T/1E9 VP/mL AAV2.CMV.Luciferase system. Luminescence was measured across a range of dilutions of sera, and remaining infectivity was deﬁned as the luminescent signal in each well divided by ‘Control’ values. IVT of AAV2 (TM) is associated with higher levels of retinal gliosis than wild-type AAV2 injections Having identiﬁed a possible increase in systemic immune activation after IVT of AAV2 (TM) compared to wild-type AAV2 injections, we then tested whether retina glia cell populations may also be inﬂuenced by injection of the vectors. First, cryosectioned retina tissue was stained with Iba1 antibodies to analyse expression of a microglia activation marker [16]. In mice who were injected with AAV2 (TM), an increase in Iba1 immunor- eactivity was observed vs. wild-type AAV2 injected mice (Fig. 4a, b). Next, GFAP antibodies were used to delineate Müller glia ﬁbrils in cryosectioned retinae, which display increased length and intensity of immunoreactivity when exposed to proinﬂammatory stimuli [17]. AAV2 (TM) treated mice Müller glia ﬁbrils displayed increased GFAP+ ﬁbril length and immunoreactivity compared with mice in the wild-type AAV2 group (Fig. 4–e). Finally, GFAP antibodies were used to identify astrocyte dendritic arbours in ﬂatmounted retina tissue, which may exhibit increased complexity in an inﬂammatory milieu [18]. An increase in the number of junctions and number of quadruple points in the dendritic arbours were observed in the AAV2 (TM) group compared to wild-type AAV2 control IVTs (Fig. 4f–h). In summary, this set of experiments showed that increased retinal gliosis may be associated with AAV2 (TM) vs. wild-type AAV2 IVTs, involving microglia, Müller glia and astrocyte cells. Mutation of phosphodegron residues in AAV2 capsids is associated with reduced neutralisation by soluble HS and anti- AAV2 NAbs Changes were observed in the levels of follicular helper T-cells and germinal centre B-cells, and in classical and myeloid dendritic cell MHC c. II expression. All data is displayed as column graphs, and show the mean for each group ±SEM. All statistical analyses are vs. the AAV2 group. a CD4 and CD8 T-cell gating strategy used in subsequent analyses. b Effector memory CD4 + T- cell (CD3 + CD4 + CD8- CD62Llo CD44hi) levels expressed as a percentage of total CD4 + T-cell levels, and representative ﬂow plots. c Effector memory CD8 + T-cell (CD3 + CD4- CD8+ CD62Llo CD44hi) levels expressed as a percentage of total CD8 + T-cell levels, and representative ﬂow plots. d Follicular helper CD4 + T-cell (CD3 + CD4+ CXCR5hi PD-1hi) levels expressed as a percentage of all CD4+ cells. p < 0.01, Brown- Forsythe ANOVA and Dunnett’s T3 posthoc tests (n = 4), and representative ﬂow plots. e B-cell gating strategy used for subsequent analysis. f Germinal centre B-cell (CD19+ IgM + /lo IgDlo CD95hi GL7+ ) levels as a percentage of all splenic lymphocytes. *p < 0.001, Kruskal-Wallis test and Dunn’s posthoc tests (n = 4), and representative ﬂow plots. g Levels of MHC c. II expressed on germinal centre B-cells. p < 0.01, one-way ANOVA and Dunnett’s posthoc tests (n = 4). h Percentage of class-switched B-cells (IgMlo IgDlo) in the germinal centres, as a percentage of all germinal centre B-cells. *p < 0.01, one-way ANOVA and Dunnett’s posthoc tests (n = 4), and representative ﬂow plots. i Gating strategy used to delineate DC subsets for subsequent analysis j MHC c. II expression on conventional DCs subset 1 (cDC1) (CD11chi CD8a + XCR1 + ) dendritic cells. p < 0.05, one-way ANOVA and Dunnett’s posthoc tests (n = 4). k MHC c. II expression on conventional DCs subset 2 (cDC1) (CD11chi CD11b+) dendritic cells. p < 0.05, Student’s t test (n = 4). l MHC c. II expression on plasmacytoid (CD11c+ SiglecH+) dendritic cells. AAV2 (TM) and wild-type AAV2 were compared via Iba1 immunoreactivity in this study. Microglia have a demonstrated role in initiating immune responses in the CNS [21], which may include activation of retinal endothelium, a concomitant increase in vascular permeability, and increased extravasation of various immune cell types into the retina [22]. Mutation of phosphodegron residues in AAV2 capsids is associated with reduced neutralisation by soluble HS and anti- AAV2 NAbs An alternative explanation is that attenuated HSPG binding of AAV2 (TM) in the ILM may have affected interaction with microglia cells, perhaps due to increased permeation of vector particles into the retina, and increased microglia activation was observed when Recent work has suggested that the incorporation of phosphode- gron mutations into AAV2 capsids may elicit changes in binding Gene Therapy Fig. 3 Intravitreal injection of AAV2 (TM) induces changes in splenic lymphocyte populations. Vectors were injected via IVT and spleens were harvested for analysis after three weeks. Changes were observed in the levels of follicular helper T-cells and germinal centre B-cells, and in classical and myeloid dendritic cell MHC c. II expression. All data is displayed as column graphs, and show the mean for each group ±SEM All statistical analyses are vs. the AAV2 group. a CD4 and CD8 T-cell gating strategy used in subsequent analyses. b Effector memory CD4 + T- cell (CD3 + CD4 + CD8- CD62Llo CD44hi) levels expressed as a percentage of total CD4 + T-cell levels, and representative ﬂow plots. c Effector memory CD8 + T-cell (CD3 + CD4- CD8+ CD62Llo CD44hi) levels expressed as a percentage of total CD8 + T-cell levels, and representative ﬂow plots. d Follicular helper CD4 + T-cell (CD3 + CD4+ CXCR5hi PD-1hi) levels expressed as a percentage of all CD4+ cells. p < 0.01, Brown- Forsythe ANOVA and Dunnett’s T3 posthoc tests (n = 4), and representative ﬂow plots. e B-cell gating strategy used for subsequent analysis f Germinal centre B-cell (CD19+ IgM + /lo IgDlo CD95hi GL7+ ) levels as a percentage of all splenic lymphocytes. p < 0.001, Kruskal-Wallis test and Dunn’s posthoc tests (n = 4), and representative ﬂow plots. g Levels of MHC c. II expressed on germinal centre B-cells. p < 0.01, one-way ANOVA and Dunnett’s posthoc tests (n = 4). h Percentage of class-switched B-cells (IgMlo IgDlo) in the germinal centres, as a percentage of al germinal centre B-cells. *p < 0.01, one-way ANOVA and Dunnett’s posthoc tests (n = 4), and representative ﬂow plots. i Gating strategy used f hi M. Whitehead et al. M. Whitehead et al. 8 Fig. 3 Intravitreal injection of AAV2 (TM) induces changes in splenic lymphocyte populations. Vectors were injected via IVT and spleens were harvested for analysis after three weeks. Mutation of phosphodegron residues in AAV2 capsids is associated with reduced neutralisation by soluble HS and anti- AAV2 NAbs As such, our data may be explained by a model in which AAV2 (TM) IVT increased microglia activation and retinal vascular permeability, which increased shedding of vector protein and draining of capsid antigen to the spleen. Again, this model may be consistent with the cDC1 & 2 and germinal centre reactions observed, which increased NAb and TAb titres [23]. This explanation may also explain the patterns of T-cell inﬁltration into the retina, whereby AAV2 (TM) IVTs increased CD4+ and CD8+ counts by activating microglia cells which in turn elevated vessel permeability and leucocyte extravasation. More research will be required to determine whether either of these mechanisms was responsible for the differences in immune activation observed between mutant and wild-type AAV2, or Gene Therapy M. Whitehead et al. 9 whether both pathways played a role. For instance, further work to characterise the interaction between microglia cells and blood vessel endothelium [22] after vector administration could prove a useful addition to the ﬁeld. This could be complemented by investigations into whether microglia are capable of activating inﬁltrating CD4 + T-cells, w neuroprotective or neurodeg comparable CNS immunopat prove useful in expanding presented, in which CD4+ Gene Therapy whether both pathways played a role. For instance, further work to characterise the interaction between microglia cells and blood vessel endothelium [22] after vector administration could prove a useful addition to the ﬁeld. This could be complemented by investigations into whether microglia are capable of activating inﬁltrating CD4 + T-cells, which may drive development of neuroprotective or neurodegenerative phenotype as reviewed i comparable CNS immunopathologies [24]. This information coul prove useful in expanding upon the ﬁndings of the dat presented, in which CD4+ and CD8 + T-cell inﬁltration wa whether both pathways played a role. For instance, further work to characterise the interaction between microglia cells and blood vessel endothelium [22] after vector administration could prove a useful addition to the ﬁeld. This could be complemented by investigations into whether microglia are capable of activating inﬁltrating CD4 + T-cells, which may drive development of a neuroprotective or neurodegenerative phenotype as reviewed in comparable CNS immunopathologies [24]. This information could prove useful in expanding upon the ﬁndings of the data presented, in which CD4+ and CD8 + T-cell inﬁltration was inﬁltrating CD4 + T-cells, which may drive development of a neuroprotective or neurodegenerative phenotype as reviewed in comparable CNS immunopathologies [24]. Mutation of phosphodegron residues in AAV2 capsids is associated with reduced neutralisation by soluble HS and anti- AAV2 NAbs This information could prove useful in expanding upon the ﬁndings of the data presented, in which CD4+ and CD8 + T-cell inﬁltration was Gene Therapy M. Whitehead et al. 10 Fig. 4 Intravitreal injection of a phosphodegron mutant AAV2 and high titre AAV2 leads to gliosis in the murine retina. Vectors were injected via IVT and retinas were extracted for analysis after three weeks. All data is presented as column graphs, with the mean value for each group shown ±SEM. All statistical analyses are vs. the AAV2 group. RGC, retinal ganglion cell layer; INL inner nuclear layer, ONL outer nuclear layer. a Representative 40x objective epiﬂuorescent microscopy images of retinal cryosections showing IBA1 immunoreactivity in groups receiving phosphodegron mutant AAV2 injections. b Corresponding quantiﬁcation performed in ImageJ shows the level of IBA1 immunoreactivity per ﬁeld of view (FOV). p < 0.05, *p < 0.01, Kruskal-Wallis ANOVA and Dunn’s posthoc test, n = 4–5. c Representative 40x objective epiﬂuorescent microscopy images of retinal cryosections showing GFAP immunoreactivity in groups receiving phosphodegron mutant AAV2 injections. Corresponding quantiﬁcation was performed using Simple Neurite Tracer, an ImageJ plugin (https://imagej.net/SNT). Each GFAP+ ﬁbril was identiﬁed in each FOV and the length of each ﬁbril (d) and its ﬂuorescence intensity (e) and was measured. p < 0.05, *p < 0.01, one-way ANOVA and Dunnett’s posthoc tests, n = 4. f Representative 63x images acquired on an Leiss Airyscan Confocal microscope showing increased complexity of astrocytic dendritic arbours in AAV2 (TM). Data was analysed in ImageJ using by assessing the number of junctions (g) and quadruple points (h) of skeletonized images. p < 0.05, *p < 0.01, one-way ANOVA and posthoc Dunnett’s tests, n = 5–8. number of junctions (g) and quadruple points (h) of skeletonized images. p < 0.05, *p < 0.01, one-way ANOVA and posthoc Dunnett’s tests, n = 5–8. Fig. 5 Injection of phosphodegron mutant vectors is not associated with changes in electrophysiological function in the murine retina. Vectors were injected via IVT and ERG was performed after three weeks. 250 ng of lipopolysaccharide (LPS) 24 h prior to ERG. a Average pSTRs, B-waves and A-waves from each treatment group (–4.37, –1.90 & 1.29 log cd.s/m2 light intensities, respectively). b Column charts showing peak voltages across a range of light intensities for each treatment group. Data is presented as the mean value ±SEM. Mutation of phosphodegron residues in AAV2 capsids is associated with reduced neutralisation by soluble HS and anti- AAV2 NAbs c Representative 40x confocal images of Tuj1+ cells (an RGC marker) in ﬂatmounted retina tissue. Fi 5 I j ti f h h d t t t i t i t d ith h i l t h i l i l f ti i th i ti Fig. 5 Injection of phosphodegron mutant vectors is not associated with changes in electrophysiological function in the murine retina. Vectors were injected via IVT and ERG was performed after three weeks. 250 ng of lipopolysaccharide (LPS) 24 h prior to ERG. a Average pSTRs, B-waves and A-waves from each treatment group (–4.37, –1.90 & 1.29 log cd.s/m2 light intensities, respectively). b Column charts showing peak voltages across a range of light intensities for each treatment group. Data is presented as the mean value ±SEM. c Representative 40x confocal images of Tuj1+ cells (an RGC marker) in ﬂatmounted retina tissue. Gene Therapy M. Whitehead et al. 11 Fig. 6 Mutation of AAV2 capsids at selected phosphodegron regions attenuates neutralisation by heparan sulphate (HS), and anti-AAV2 neutralising antibody-containing sera. All data is presented as bar graphs, with the mean value for each group shown ±SEM. All statistical analyses are vs. the AAV2 group. a VP3 capsid monomer showing the positioning of the three phosphodegron mutants, the ﬁve residues thought to mediate the binding afﬁnity of AAV2 capsids to HSPG (blue) and the 14 residues thought to mediate binding of AAV2 to the AAV receptor (orange) (AAVR; KIAA0319). This model was generated in PyMol using 6ih9, a 2.8 Å resolution cryoelectron microscopy-derived structure of the AAV2 VP3 monomer. b AAV2 full 60mer capsid structure with highlighted phosphodegron mutations and heparin binding domains. Red = Y444F, Green = K556E, Yellow = S662V, Blue = R484, R487, K532, R585, and R588 heparin binding domains (HBDs), Orange = R471, D528, Q589, T592, S262, Q263, G265, A266, S267, N268, H271, N382 and Q385 AAVR binding domains (AAVR BDs). c RIVEM plot showing the positioning of the three phosphodegron mutants and their proximity to HBDs and AAVR BDs (using 6IH9 AAV2 coordinates, see (b) for colouring). d Schematic representation of mutations represented in PyMol. Mutations were introduced using PyMol’s Mutagenesis Wizard, and the optimal rotamer conﬁrmation was selected in accordance with the software’s prediction. e Mutation of phosphodegron residues attenuates neutralisation by HS. AAV2 and mutant capsids were incubated with HS for 1 h, prior to addition to HEK293T cell media. Number of GFP+ cells was normalised to –HS controls for each vector group, allowing calculation of remaining infectivity (I/I0). p < 0.05, ***p < 0.0001, one-way ANOVA and a Dunnett’s posthoc tests, n = 4. f Mutation of phosphodegron residues partially rescues neutralisation by AAV2 NAbs. AAV2 and mutant capsids were incubated with sera extracted from animals that had previously been injected intravitreally with AAV2, and the samples were tested to conﬁrm the presence of NAbs. Calculation of remaining infectivity (I/I0) was performed as in (d). p < 0.05, p < 0.01, p < 0.001, *** p < 0.0001, one-way ANOVA and a Dunnett’s posthoc tests, n = 4. Fig. 6 Mutation of AAV2 capsids at selected phosphodegron regions attenuates neutralisation by heparan sulphate (HS), and anti-AAV2 neutralising antibody-containing sera. All data is presented as bar graphs, with the mean value for each group shown ±SEM. In Eye pp. 1885-9. 11. Timmers AM, Newmark JA, Turunen HT, Farivar T, Liu J, Song C, et al. Ocular inﬂammatory response to intravitreal injection of adeno-associated virus vector: relative contribution of genome and capsid. Human Gene Therapy. 2020;31:80–89. 12. Tummala G, Crain A, Rowlan J, Pepple KL. Characterization of gene therapy associated uveitis following intravitreal adeno-associated virus injection in mice. Investig Ophthalmol Visual Sci. 2021;62:41. 13. Seitz IP, Michalakis S, Wilhelm B, Reichel FF, Ochakovski GA, Zrenner E, et al. Superior retinal gene transfer and biodistribution proﬁle of subretinal versus intravitreal delivery of AAV8 in nonhuman primates. Investig Ophthalmol Visual Sci. 2017;58:5792–801. One interesting observation of the properties of AAV2 (TM) compared to wild-type AAV2 was that the vector may have been less susceptible to neutralisation by anti-AAV2 NAbs. As men- tioned, pre-existing humoral immunity to capsid protein repre- sents a key barrier to successful vector administration in ~50% of patients [30], which has led to the development of a number of strategies to circumvent the problem [31, 32]. Whilst we only utilised an in vitro neutralisation assay to demonstrate this effect, the data suggests that mutation of phosphodegron residues in AAV2 capsids may prove a useful, albeit partial, means of overcoming pre-existing NAbs in patients, highlighting a potential clinical utility for the technology in addition to improving retinal transduction [7] and increasing vector permeation to photo- receptor cells after IVT [1]. 14. Mesin L, Ersching J, Victora GD. Germinal center B cell dynamics. Immunity. 2016;45:471–82. 15. ten Broeke T, Wubbolts R, Stoorvogel W. MHC class II antigen presentation by dendritic cells regulated through endosomal sorting. Cold Spring Harbor Per- spect Biol. 2013;5:168–73. 16. Hopperton KE, Mohammad D, Trépanier MO, Giuliano V, Bazinet RP. Markers of microglia in post-mortem brain samples from patients with Alzheimer’s disease: a systematic review. Mol Psychiatry. 2018;23:177–98. 17. Shamsuddin N, Kumar A. TLR2 mediates the innate response of retinal muller glia to Staphylococcus aureus. J Immunol. 2011;186:7089–97. to Staphylococcus aureus. J Immunol. 2011;186:7089–97. 18. Giovannoni F, Quintana FJ. The role of astrocytes in CNS inﬂammation. Trends Immunol. 2020;41:805–19. 19. Pavlou M, Schön C, Occelli LM, Rossi A, Meumann N, Boyd RF, et al. Novel AAV capsids for intravitreal gene therapy of photoreceptor disorders. EMBO Mol Med. 2021;13:e13392. 20. Durai V, Murphy KM. Functions of murine dendritic cells. Immunity. 2016;45:719–36. All statistical analyses are vs. the AAV2 group. a VP3 capsid monomer showing the positioning of the three phosphodegron mutants, the ﬁve residues thought to mediate the binding afﬁnity of AAV2 capsids to HSPG (blue) and the 14 residues thought to mediate binding of AAV2 to the AAV receptor (orange) (AAVR; KIAA0319). This model was generated in PyMol using 6ih9, a 2.8 Å resolution cryoelectron microscopy-derived structure of the AAV2 VP3 monomer. b AAV2 full 60mer capsid structure with highlighted phosphodegron mutations and heparin binding domains. Red = Y444F, Green = K556E, Yellow = S662V, Blue = R484, R487, K532, R585, and R588 heparin binding domains (HBDs), Orange = R471, D528, Q589, T592, S262, Q263, G265, A266, S267, N268, H271, N382 and Q385 AAVR binding domains (AAVR BDs). c RIVEM plot showing the positioning of the three phosphodegron mutants and their proximity to HBDs and AAVR BDs (using 6IH9 AAV2 coordinates, see (b) for colouring). d Schematic representation of mutations represented in PyMol. Mutations were introduced using PyMol’s Mutagenesis Wizard, and the optimal rotamer conﬁrmation was selected in accordance with the software’s prediction. e Mutation of phosphodegron residues attenuates neutralisation by HS. AAV2 and mutant capsids were incubated with HS for 1 h, prior to addition to HEK293T cell media. Number of GFP+ cells was normalised to –HS controls for each vector group, allowing calculation of remaining infectivity (I/I0). p < 0.05, **p < 0.0001, one-way ANOVA and a Dunnett’s posthoc tests, n = 4. f Mutation of phosphodegron residues partially rescues neutralisation by AAV2 NAbs. AAV2 and mutant capsids were incubated with sera extracted from animals that had previously been injected intravitreally with AAV2, and the samples were tested to conﬁrm the presence of NAbs. Calculation of remaining infectivity (I/I0) was performed as in (d). p < 0.05, p < 0.01, p < 0.001, *** p < 0.0001, one-way ANOVA and a Dunnett’s posthoc tests, n = 4. Gene Therapy Gene Therapy M. Whitehead et al. 12 observed, yet the precise role the cells played at the site of infection was not studied. In particular, a more detailed characterisation of the CD8+ phenotype is warranted given the possibility that these cells killed AAV transduced hepatocytes in haemophilia B clinical trials, leading to diminished therapeutic beneﬁt [25]. To summarise, after IVT of AAV2 (TM), increased humoral immune activation, T-cell inﬁltration, splenic germinal centre reactions and DC activation, and retinal gliosis was observed compared to wild-type AAV2 injections. However, it is important to note that the observed immune response was not correlative with changes in electrophysiology or loss of inner retinal cells. AAV2 (TM) transduced a greater proportion of retinal cells and was more resistant to anti-AAV2 NAbs, suggesting the capsid may be useful in overcoming pre-existing humoral immunity in patients. The present study therefore highlights novel aspects of phospho- degron mutant AAV2 immunobiology relevant to preclinical and clinical applications. 21. Ousman SS, Kubes P. Immune surveillance in the central nervous system. Nat Neurosci. 2012;15:1096–101. 22. Okunuki Y, Mukai R, Nakao T, Tabor SJ, Butovsky O, Dana R, et al. Retinal microglia initiate neuroinﬂammation in ocular autoimmunity. Proc Natl Acad Sci. 2019;116:9989–98. 23. Claesson-Welsh L, Dejana E, McDonald DM. Permeability of the endothelial bar- rier: identifying and reconciling controversies. Trends Mol Med. 2021;27:314–31. 24. Schetters STT, Gomez-Nicola D, Garcia-Vallejo JJ, Van Kooyk Y. Neuroinﬂamma- tion: microglia and T cells get ready to Tango. Front Immunol. 2018;8:1905. , , j , y tion: microglia and T cells get ready to Tango. Front Immunol. 2018;8:1905. microglia and T cells get ready to Tango. Front Immunol. 2018;8:190 25. Ertl HCJ. T cell-mediated immune responses to AAV and AAV vectors. Front Immunol. 2021;12,:666666. 26. Zong H, Ward M, Madden A, Yong PH, Limb GA, Curtis TM, et al. Hyperglycaemia- induced pro-inﬂammatory responses by retinal Müller glia are regulated by the receptor for advanced glycation end-products (RAGE). Diabetologia. 2010;53:2656–66. DATA AVAILABILITY 27. Eastlake K, Banerjee PJ, Angbohang A, Charteris DG, Khaw PT, Limb GA. Müller glia as an important source of cytokines and inﬂammatory factors present in the gliotic retina during proliferative vitreoretinopathy. GLIA. 2016;64:495–506. All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. The published article includes all datasets and code generated or analysed during this study. 28. Gu R, Ding X, Tang W, Lei B, Jiang C, Xu G. A synthesized glucocorticoid- induced leucine zipper peptide inhibits retinal müller cell gliosis. Front Pharmacol. 2018;9:331. Obtaining this information may prove helpful in understanding why T-cell inﬁltration is commonly observed in ocular AAV gene therapy studies [12], but did not correlate with detectable changes in electroretinography in the present study. 4. Yu-Wai-Man P, Newman NJ, Carelli V, Moster ML, Biousse V, Sadun AA, et al. Bilateral visual improvement with unilateral gene therapy injection for Leber hereditary optic neuropathy. Sci Transl Med. 2020;12:eaaz7423. hereditary optic neuropathy. Sci Transl Med. 2020;12:eaaz7423. 5. Li C, Samulski RJ. Engineering adeno-associated virus vectors for gene therapy. Nat Rev Genet. 2020;21:255–72. 6. Büning H, Srivastava A. Capsid Modiﬁcations for Targeting and Improving the Efﬁcacy of AAV Vectors. Mol Therapy—Methods Clin Dev. 2019;12:248–65. cacy of AAV Vectors. Mol Therapy—Methods Clin Dev. 2019;12:248– 7. Petrs-Silva H, Dinculescu A, Li Q, Min SH, Chiodo V, Pang JJ, et al. High-efﬁciency 7. Petrs-Silva H, Dinculescu A, Li Q, Min SH, Chiodo V, Pang JJ, et al. High-efﬁciency transduction of the mouse retina by tyrosine-mutant AAV serotype vectors. Mol Therapy. 2009;17:463–71. transduction of the mouse retina by tyrosine-mutant AAV serotype vectors. Mol Therapy. 2009;17:463–71. In addition to microglia activation, we detected changes in GFAP+ Müller glia and astrocytes indicative of increased activation of these cell types in AAV2 (TM) vs. wild-type AAV2 injected retinae. Given the capacity of Müller cells to secrete proinﬂammatory cytokines across a range of retinopathies [26, 27] and in response to inﬂammatory stimuli [28], it is likely that the activated Müller glia identiﬁed in this study in some way participated in the anti-AAV2 innate immune response, possibly by facilitating development of an inﬂammatory microenvironment that assisted in leucocyte recruitment to the retina. The precise role that astrocyte activation played in the study is also unclear, however, future investigations into whether activation of inﬁltrat- ing CD4 + T-cells was mediated by MHC c. II-restricted AAV2 antigen expressed by these cells, which has been described in other CNS neuropathologies [29], may be beneﬁcial. 8. Ryals RC, Boye SL, Dinculescu A, Hauswirth WW, Boye SE. Quantifying transduc- tion efﬁciencies of unmodiﬁed and tyrosine capsid mutant AAV vectors in vitro using two ocular cell lines. Mol Vision. 2011;17:1090–102. using two ocular cell lines. Mol Vision. 2011;17:1090–102. 9. Martino AT, Basner-Tschakarjan E, Markusic DM, Finn JD, Hinderer C, Zhou S, et al. Engineered AAV vector minimizes in vivo targeting of transduced hepatocytes by capsid-speciﬁc CD8+ T cells. Blood. 2013;121:2224–33. p p 10. Taylor, AW (2009) Ocular immune privilege. REFERENCES 29. Rostami J, Fotaki G, Sirois J, Mzezewa R, Bergström J, Essand M, et al. Astrocytes have the capacity to act as antigen-presenting cells in the Parkinson’s disease brain. J Neuroinﬂamm. 2020;17:119. 1. Boye SL, Bennett A, Scalabrino ML, McCullough KT, Van Vliet K, Choudhury S, et al. Impact of heparan sulfate binding on transduction of retina by recombinant adeno-associated virus vectors. J Virol. 2016;90:4215–31. 1. Boye SL, Bennett A, Scalabrino ML, McCullough KT, Van Vliet K, Choudhury S, et al. Impact of heparan sulfate binding on transduction of retina by recombinant adeno-associated virus vectors. J Virol. 2016;90:4215–31. 30. Whitehead M, Osborne A, Yu-Wai-Man P, Martin K. Humoral immune responses to AAV gene therapy in the ocular compartment. Biol Rev. 2021;96:1616–44. 2. Kwon I, Schaffer DV. Designer gene delivery vectors: molecular engineering and evolution of adeno-associated viral vectors for enhanced gene transfer. Phar- maceut Res. 2008;25:489–99. 31. Leborgne C, Barbon E, Alexander JM, Hanby H, Delignat S, Cohen DM, et al. IgG- cleaving endopeptidase enables in vivo gene therapy in the presence of anti-AAV neutralizing antibodies. Nat Med. 2020;26:1096–101. 3. Russell S, Bennett J, Wellman JA, Chung DC, Yu Z-F, Tillman A, et al. Efﬁcacy and safety of voretigene neparvovec (AAV2-hRPE65v2) in patients with RPE65 -mediated inherited retinal dystrophy: a randomised, controlled, open-label, phase 3 trial. Lancet. 2017;390:849–60. 32. Mingozzi F, Anguela XM, Pavani G, Chen Y, Davidson RJ, Hui DJ, et al. Overcoming preexisting humoral immunity to AAV using capsid decoys. Sci Transl Med. 2013;5:192–4. Gene Therapy M. Whitehead et al. 13 COMPETING INTERESTS Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http:// creativecommons.org/licenses/by/4.0/. AO is an employee of Ikarovec Ltd, a company developing gene therapy technologies. PY is a consultant for Gensight Biologics. KGM is a founder of Quethera Ltd, a gene therapy company acquired by Astellas Pharma. MW and AS declare no competing interests. FUNDING Reprints and permission information is available at http://www.nature.com/ reprints Reprints and permission information is available at http://www.nature.com/ reprints This work was supported by grants from the Medical Research Council, Addenbrookes Charitable Trust, National Eye Research Council, and a core support grant from the Wellcome Trust (104001/Z/14/Z) and MRC to the Wellcome Trust–Medical Research Council Cambridge Stem Cell Institute. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. AUTHOR CONTRIBUTIONS MW, AS and AO conceived and designed the experiments. MW, AS and AO analysed the data. MW wrote the manuscript. MW, AO, PY and KGM obtained the funding. AO, PY and KGM provided feedback on the manuscript. Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41434-023-00409-x. Correspondence and requests for materials should be addressed to Michael Whitehead. Reprints and permission information is available at http://www.nature.com/ reprints ETHICAL APPROVAL All procedures performed on animals were approved by the UK Home Ofﬁce in accordance with the UK Animals (Scientiﬁc Procedures) Act, and undertaken in accordance with the Association for Research in Vision and Ophthalmology’s (ARVO) Statement for the Use of Animals in Ophthalmic and Visual Research. The sample size used in each experiment is stipulated in the ﬁgure legends. © The Author(s) 2023 Gene Therapy
https://openalex.org/W2116060098	https://europepmc.org/articles/pmc3213060?pdf=render	English	null	Sequential thrombosis and bleeding in a woman with a prolonged activated partial thromboplastin time	Thrombosis journal	2,011	cc-by	3,885	Background oral contraception for the previous three weeks, had a body mass index of 24.5 (normal 18.5-24.9) had no vari- cose veins, and had not been on any long haul trips by either plane or car since delivery. Her personal history was uneventful, this being her first pregnancy during which she had no complications. There was no family history of thrombosis or recurrent miscarriages. Her aPTTr was 3.01 (normal range 0.88 -1.16) and pro- thrombin time ratio (PTr) was normal. Her Hb was 9.1 g/dl with a slight microcytosis. Low molecular weight heparin (LMWH) at treatment dose was started embri- cated with warfarin then stopped after four days when her international normalised ratio (INR) was 2.3. A week later she re-attended casualty for spontaneous bruising on upper limbs and worsening of the pain and swelling in the right leg that was markedly ecchymotic. Blood tests on admission revealed Hb 8.1 g/dl, platelets 557 × 109/L and C-reactive protein at 247 mg/L (normal < 5 mg/L). Urine and blood cultures were negative. Since 1988 in Airedale the possibility of a bleeding dis- order was tested by comparing the clotting times of two aPTT reagents, currently Synthesil (IL) as the reagent sensitive to factor inhibitors and deficiencies and Actin FS (Dade) as the reagent insensitive to factor inhibitors and deficiencies but sensitive to factor XII deficiency [2]. Unbeknown to the laboratory personnel the same g The differential diagnosis of a prolonged aPTT (acti- vated partial thromboplastin time) is manifold and necessitates an algorithm to indicate factor deficiencies or the presence of inhibitors that could be lupus like, heparin like or specific to a clotting factor [1]. A quick and correct laboratory diagnosis is of the utmost impor- tance in the emergency setting when a clinician is faced with a critical haemorrhage in a previously healthy indi- vidual. We describe the sequential occurrence of throm- bosis and haemorrhage developing over a nine day period in a young woman with a prolonged aPTT ratio. © 2011 Spencer et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Sequential thrombosis and bleeding in a woman with a prolonged activated partial thromboplastin time Akpan Spencer1, Michael I Pearce1 and Paul RJ Ames2* Abstract Simultaneous or sequential haemorrhage and thrombosis in the presence of a prolonged activated partial thromboplastin time (aPTT) is a rare occurrence: we describe the case a 37 year old lady who developed post- delivery deep vein thrombosis treated with low molecular heparin and warfarin followed a week later by extensive bruising over legs and forearms, a significant drop in haemoglobin and a very prolonged aPTT. Further tests revealed an acquired factor VIII inhibitor at 35 Bethesda Units. We discuss the clinical and laboratory implications and provide a literature review of simultaneous thrombophilia and haemophilia in the presence of a prolonged aPTT. Keywords: thrombosis, acquired haemophilia, pregnancy, lupus anticoagulant Spencer et al. Thrombosis Journal 2011, 9:16 http://www.thrombosisjournal.com/content/9/1/16 Spencer et al. Thrombosis Journal 2011, 9:16 http://www.thrombosisjournal.com/content/9/1/16 Spencer et al. Thrombosis Journal 2011, 9:16 * Correspondence: paxmes@aol.com 2Department of Haematology, Airedale Foundation Trust, Steeton, BD20 6TD, UK Full list of author information is available at the end of the article Case presentation Description A 37 year old woman presented to casualty for pain and swelling in the right calf; her Well’s score was 5, D- dimer elevated at 422 ng/ml (cut-off limit 230 ng/ml) and a Doppler ultrasound revealed a clot in the upper portion of the right femoral vein and in the deep veins of the right calf. With regards to thrombosis risk factors, she was 5 weeks post caesarean delivery, had been on ull list of author information is available at the end of the article Spencer et al. Thrombosis Journal 2011, 9:16 http://www.thrombosisjournal.com/content/9/1/16 Page 2 of 4 was markedly abnormal at her attendance for DVT. A factor VIII level at 2% and a factor VIII inhibitor at 35 Bethesda units were unequivocal with respect to preg- nancy related acquired haemophilia: the latter is a life- threatening antibody-mediated haemorrhagic disorder that occurs prevalently in people over 50 years of age presenting with bleeding and/or with a prolonged aPTT. The FVIII inhibitor may develop during and/or after pregnancy with a prevalence of 7% to 21% according to series. It appeared in the first pregnancy in 80% (16 of 20) of women and did not seem to recur with further pregnancies [4-10] though in one survey it recurred in the three women who had successive pregnancies [11]. In general the inhibitor is identified on occasions of overt bleeding: in one series 50% of bleeding occurred in the peri-partum period and in 50% within 30 days of delivery; in another series bleeding occurred pre-partum in two women, within three days of delivery in three and within 3-12 months in nine [11,12]. Another review showed that 3% of 27 women bled during pregnancy, 3% after an abortion, 15% immediately after delivery, 30% within 4 months and another 30% between 4 to 12 months of delivery; this review also shows that post-partum factor VIII inhibitor often disappears spontaneously [12]. comparison can be used to detect a lupus anticoagulant [3]. Both assays are run on an automated coagulometer (TOP-CTS, Instrumentation Laboratories). The patient’s INR was 2.3 (due to warfarin), the Synthesil aPTTr was disproportionately prolonged at 5.03 whereas the Actin FS aPTTr was 3.21 and a thrombin time was 12.8” (nor- mal 12-16.5”) ruling out a possible residual effect of LMWH that had been stopped three days earlier. Case presentation Description A repeat Doppler US of the right lower limb did not show thrombus in the femoral vein but extensive haematoma in the calf obscuring the possibility of persistent throm- bus. The following day her Hb had dropped to 6.9 g/dl: warfarin was stopped and the patient was given a total dose of 8 mg of recombinant VIIa intravenously, repeated two hourly 12 times after transfer to the Hae- mophilia Centre at Bradford Royal Infirmary. The patient was started on oral cyclophosphamide 100 mg daily continued for six months and prednisolone 1 mg/ kg for 6 weeks, gradually tapered down over the ensuing five months: three weeks into immune suppression her factor VIII levels was 36 IU/dl and in June 2010 her fac- tor VIII was 62 IU/dl with normal aPTTs. A thrombo- philia screen done at the same time (plasma levels of protein C, protein S, antithrombin and gene testing for factor V Leiden and prothrombin mutation, anticardioli- pin antibodies and lupus anticoagulant) was negative. At 16 months follow up she is inhibitor negative with nor- mal plasma factor VIII level. However, prior to haemorrhage, our patient had suf- fered a DVT, quite a rare occurrence in the presence of a bleeding diathesis. In the absence of genetic or acquired thrombophilia, four haemophilia A patients developed spontaneous limb vein occlusions [13-16], one with a duplicated superficial femoral vein [16] whereas a haemophilia A youngster who was also factor V Leiden heterozygous suffered a spontaneous cerebral infarction [17]. Conversely two haemophilia A patients with inherited thombophilia developed a fatal pulmonary embolism and a DVT after a surgical challenge [18,19] whereas a severe haemophilia A patient also factor V Leiden het- erozygous suffered portal vein thrombosis after continu- ous infusion of F VIII [20]. DVT has been described also in a haemophilic A patient with factor VIII inhibi- tor 18 days after recombinant activated factor VII infu- sion [21]. Case presentation Description Given the prolongation of both aPTTs, the laboratory person- nel informed the consultant haematologist of a possible factor inhibitor and pursued factor assays that revealed a factor VIII at 2 IU/dl (normal range 60-180 IU/dl), factor XII at 65 IU/dl (normal range 50-180 IU/dl), fac- tor XI at 61 IU/dl (normal range 60-140 IU/dl) and fac- tor IX at 46 IU/dl (normal range 50-150 IU/dl). A factor VIII inhibitor was detected at 35 Bethesda Units. A repeat Doppler US of the right lower limb did not show thrombus in the femoral vein but extensive haematoma in the calf obscuring the possibility of persistent throm- bus. The following day her Hb had dropped to 6.9 g/dl: warfarin was stopped and the patient was given a total dose of 8 mg of recombinant VIIa intravenously, repeated two hourly 12 times after transfer to the Hae- mophilia Centre at Bradford Royal Infirmary. The patient was started on oral cyclophosphamide 100 mg daily continued for six months and prednisolone 1 mg/ kg for 6 weeks, gradually tapered down over the ensuing five months: three weeks into immune suppression her factor VIII levels was 36 IU/dl and in June 2010 her fac- tor VIII was 62 IU/dl with normal aPTTs. A thrombo- philia screen done at the same time (plasma levels of protein C, protein S, antithrombin and gene testing for factor V Leiden and prothrombin mutation, anticardioli- pin antibodies and lupus anticoagulant) was negative. At 16 months follow up she is inhibitor negative with nor- mal plasma factor VIII level. comparison can be used to detect a lupus anticoagulant [3]. Both assays are run on an automated coagulometer (TOP-CTS, Instrumentation Laboratories). The patient’s INR was 2.3 (due to warfarin), the Synthesil aPTTr was disproportionately prolonged at 5.03 whereas the Actin FS aPTTr was 3.21 and a thrombin time was 12.8” (nor- mal 12-16.5”) ruling out a possible residual effect of LMWH that had been stopped three days earlier. Given the prolongation of both aPTTs, the laboratory person- nel informed the consultant haematologist of a possible factor inhibitor and pursued factor assays that revealed a factor VIII at 2 IU/dl (normal range 60-180 IU/dl), factor XII at 65 IU/dl (normal range 50-180 IU/dl), fac- tor XI at 61 IU/dl (normal range 60-140 IU/dl) and fac- tor IX at 46 IU/dl (normal range 50-150 IU/dl). A factor VIII inhibitor was detected at 35 Bethesda Units. e ratio (PTr), activated partial thromboplastin time ratio (aPTTr) and fibrinogen (FNG) from the end of the second trimester of pregna to end of puerperium (Mar 12) Author details 1 1Department of Medicine, Airedale Foundation Trust, Steeton, BD20 6TD, UK. 2Department of Haematology, Airedale Foundation Trust, Steeton, BD20 6TD, UK. Authors’ contributions PRJA d h Within the thrombosis setting a prolonged aPTTr may indicate the presence of a lupus anticoagulant (LA). In the flurry of the Friday afternoon when the acquired fac- tor VIII inhibitor was detected, whilst the patient was administrated the first dose of recombinant factor VII and then transferred to Bradford Royal Infirmary, we overlooked that the Actin FS aPTTr was shorter than the Synthesil aPTTr (3.21 vs 5.03): a study comparing sensitive and insensitive reagents to the LA showed that the shortening of the aPTTr with Actin FS (the insensi- tive reagent) can be suggestive of LA even in patients on oral anticoagulants and/or with acquired haemophilia [3]. Hence we could not rule out that our patient also had a LA that was not detected on subsequent testing because of the immunosuppressive treatment she received. Of the four reported cases where acquired fac- tor VIII inhibitor coexisted with LA the clinical pheno- type was always bleeding [24-27]. With regards to haemophiliacs with anti-factor VIII inhibitors, one study showed a simultaneous LA detected by the dilute Rus- sell viper venom time (DRVVT) (and Staclot LA) in 12 such patients [28] whereas a positive LA was more com- mon in haemophiliacs with anti-factor VIIII inhibitors than in haemophiliacs without inhibitors either by DRVVT (IL-LAC screen and confirm) (22% vs 10%) or by Staclot LA (Stago, France) (30% vs 5%). PRJA conceived the report and coordinated the manuscript that was drafted by AS and completed by MIP. All authors read and approved the final manuscript. Discussion Our patient shifted from deep vein thrombosis (DVT) to bleeding within the span of nine days: those involved in her care had not noticed that her aPTTr had been getting higher throughout her pregnancy (Table 1) and that it Table 1 Evolution of coagulation tests since late pregnancy 2009 2010 Range Sept 30 Dec 31 Jan 2 Mar 3 Mar 10 Mar 12 Gestational age (weeks) 26 39 39 Puerperium (weeks) 5 6 6 PTr 1.00 0.90 1.03 1.26 2.05 2.32 0.9-1.12 aPTTr 1.01 1.23 1.32 3.01 9.31 5.03 0.88-1.16 FNG (mg/dl) 638 > 700 816 538 701 691 150-400 Prothrombin time ratio (PTr), activated partial thromboplastin time ratio (aPTTr) and fibrinogen (FNG) from the end of the second trimester of pregnancy (September 30) to end of puerperium (Mar 12) Table 1 Evolution of coagulation tests since late pregnancy Prothrombin time ratio (PTr), activated partial thromboplastin time ratio (aPTTr) and fibrinogen (FNG) from the end of the second trimester of pregnancy (September 30) to end of puerperium (Mar 12) Page 3 of 4 Page 3 of 4 Page 3 of 4 Spencer et al. Thrombosis Journal 2011, 9:16 http://www.thrombosisjournal.com/content/9/1/16 Spencer et al. Thrombosis Journal 2011, 9:16 http://www.thrombosisjournal.com/content/9/1/16 Spencer et al. Thrombosis Journal 2011, 9:16 http://www.thrombosisjournal.com/content/9/1/16 With regards to acquired haemophilia, a small series described a spontaneous DVT in a 60 year old woman and in an 80 year old man with splenic marginal zone lymphoma and a proximal leg vein occlusion after a femoral to popliteal by-pass in a 76 year old man [22]; pulmonary embolism developed in a 29 year old man who was on tranexamic acid to minimise bleeding epi- sodes that were managed with recombinant factor VII when occurring acutely [23]: none of these patients had inherited or acquired thrombophilia. References Scully MF, Shublaq W, Oliver GD: Acquired hemophilia A presenting as a bleeding diathesis in a postpartum patient: diagnosis and management. J Obstet Gynaecol Can 2002, 24:430-432. 8. Scully MF, Shublaq W, Oliver GD: Acquired hemophilia A presenting as a bleeding diathesis in a postpartum patient: diagnosis and management. J Obstet Gynaecol Can 2002, 24:430-432. Consent The patient gave written informed consent for publica- tion of this case report. A copy of the written consent is available for review by the Editor-in-Chief of this journal References 1. Key N, Makris M, O’Shaughnessy D, Lilicrap D: Laboratory tests of hemostasis. Practical Hemostasis and Thrombosis. Chap 2 Wiley-Blackwell; 2009, 7-16. 2. Bowyer A, Smith J, Woolley AM, Kitchen S, Hampton KK, Maclean RM, et al: The investigation of a prolonged APTT with specific clotting factor assays is unnecessary if an APTT with Actin FS is normal. Int J Lab Hematol 2011, 33:212-218. 2. Bowyer A, Smith J, Woolley AM, Kitchen S, Hampton KK, Maclean RM, et al: The investigation of a prolonged APTT with specific clotting factor assays is unnecessary if an APTT with Actin FS is normal. Int J Lab Hematol 2011, 33:212-218. 3. Brancaccio V, Ames PR, Glynn J, Iannaccone L, Mackie IJ: A rapid screen for lupus anticoagulant with good discrimination from oral anticoagulants, congenital factor deficiency and heparin, is provided by comparing a sensitive and an insensitive APTT reagent. Blood Coagul Fibrinolysis 1997, 8:155-160. 3. Brancaccio V, Ames PR, Glynn J, Iannaccone L, Mackie IJ: A rapid screen for lupus anticoagulant with good discrimination from oral anticoagulants, congenital factor deficiency and heparin, is provided by comparing a sensitive and an insensitive APTT reagent. Blood Coagul Fibrinolysis 1997, 8:155-160. 4. Santoro C, Rago A, Biondo F, De Propris MS, De Vellis A, Guarini A, et al: Efficacy of rituximab treatment in postpartum acquired haemophilia A. Haemophilia 2008, 14:147-149. 5. Mytopher K, Dudebout J, Card R, Gilliland B: Acquired hemophilia A presenting post-partum. CMAJ 2007, 177:339-340. 4. Santoro C, Rago A, Biondo F, De Propris MS, De Vellis A, Guarini A, et al: Efficacy of rituximab treatment in postpartum acquired haemophilia A. Haemophilia 2008, 14:147-149. 5. Mytopher K, Dudebout J, Card R, Gilliland B: Acquired hemophilia A presenting post-partum. CMAJ 2007, 177:339-340. 6. Jost E, Kiefer P, Neulen J, Galm O, Osieka R: Post-partum acquired haemophilia after IVF without recurrence during a second pregnancy obtained by IVF. Hum Reprod 2007, 22:2348-2349. 6. Jost E, Kiefer P, Neulen J, Galm O, Osieka R: Post-partum acquired haemophilia after IVF without recurrence during a second pregnancy obtained by IVF. Hum Reprod 2007, 22:2348-2349. 7. Koeijers JJ, Vlasveld LT: A spontaneous haemarthrosis in a post-partal woman: a case of acquired haemophilia A. Ned Tijdschr Geneeskd 2005, 149:1164-1167. 7. Koeijers JJ, Vlasveld LT: A spontaneous haemarthrosis in a post-partal woman: a case of acquired haemophilia A. Ned Tijdschr Geneeskd 2005, 149:1164-1167. 8. Competing interests h h d l h The authors declare that they have no competing interests. Received: 11 July 2011 Accepted: 27 October 2011 Published: 27 October 2011 Received: 11 July 2011 Accepted: 27 October 2011 Published: 27 October 2011 Spencer et al. Thrombosis Journal 2011, 9:16 http://www.thrombosisjournal.com/content/9/1/16 Spencer et al. Thrombosis Journal 2011, 9:16 http://www.thrombosisjournal.com/content/9/1/16 16. Stewart AJ, Manson LM, Dennis R, Allan PL, Ludlam CA: Thrombosis in a duplicated superficial femoral vein in a patient with haemophilia A. Haemophilia 2000, 6:47-49. 17. Oclay L, Gurgey A, Topaloglu H, Atay S, Parlak H, Firat M: Cerebral infarction associated with factor V Leiden mutation in a boy with haemophilia A. Am J Hematol 1997, 58:189-190. 18. Butcher JH, Pasi KJ: Fatal postoperative pulmonary embolism in mild haemophilia. Haemophilia 2006, 12:179-182. 18. Butcher JH, Pasi KJ: Fatal postoperative pulmonary embolism in mild haemophilia. Haemophilia 2006, 12:179-182. 19. Pruthi RK, Heit JA, Green MM, Emiliusen LM, Nichols WL, Wilke JL, Gastineau DA: Venous thromboembolism after hip fracture surgery in a patient with haemophilia B and factor V Arg 506 Gln (factor V Leiden) Haemophilia 2006, 6:631-634. 19. Pruthi RK, Heit JA, Green MM, Emiliusen LM, Nichols WL, Wilke JL, Gastineau DA: Venous thromboembolism after hip fracture surgery in a patient with haemophilia B and factor V Arg 506 Gln (factor V Leiden). Haemophilia 2006, 6:631-634. 20. Ettingshausen CE, Saguer IM, Kreuz W: Portal vein thrombosis in a patient with severe haemophilia A and F V G1691A mutation during continuous infusion of F VIII after intramural jejunal bleeding-successful thrombolysis under heparin therapy. Eur J Pediatr 1999, 158:180-182. 20. Ettingshausen CE, Saguer IM, Kreuz W: Portal vein thrombosis in a patient with severe haemophilia A and F V G1691A mutation during continuous infusion of F VIII after intramural jejunal bleeding-successful thrombolysis under heparin therapy. Eur J Pediatr 1999, 158:180-182. 21. Van der Planken MG, Schrovens W, Vertessen F, Michiels JJ, Berneman ZN: Distal deep venous thrombosis in a haemophilia A patient with inhibitor and severe infectious disease, 18 days after recombinant activated factor VII transfusion. Blood Coagul Fibrinolysis 2002, 13:367-370. 22. Deitcher SR, Carman TL, Kottke-Marchant K: Simultaneous deep venous thrombosis and acquired factor VIII inhibitor. Clin Appl Thromb Hemost 2002, 8:375-379. 23. Taparia M, Cordingley FT, Leahy MF: Pulmonary embolism associated with tranexamic acid in severe acquired haemophilia. Eur J Haematol 2002, 68:307-309. 24. Ballard HS, Nyamuswa G: Life-threatening haemorrhage in a patient with rheumatoid arthritis and a lupus anticoagulant coexisting with acquired autoantibodies against factor VIII. Br J Rheumatol 1993, 32:515-517. 25. doi:10.1186/1477-9560-9-16 Cite this article as: Spencer et al.: Sequential thrombosis and bleeding in a woman with a prolonged activated partial thromboplastin time. Thrombosis Journal 2011 9:16. Conclusion Thrombosis and bleeding are the yin-yang of haemosta- sis and their simultaneous occurrence must alert us to the rarity and complexity of such occurrences and induce us to have a sharp insight in these cases: acquired haemophilia A developing post-partum is not uncommon, a vascular occlusion on the background of acquired haemophilia A followed by bleeding aggravated by warfarin is unusual indeed. Given the differences in the Synthesil and Actin FS aPTT ratios, we cannot rule out that DVT was also promoted by a LA that might have disappeared after immune suppressive treatment: strict adherence to existing guidelines for LA testing [29] should have lead us to perform also a DRVVT (screen and confirm) to shed further light on the throm- botic event. 9. Santoro RC, Prejanò S: Postpartum-acquired haemophilia A: a description of three cases and literature review. Blood Coagul Fibrinolysis 2009, 20:461-465. 10. Baudo F, de Cataldo F: Italian Association of Haemophilia Centres (AICE): Register of acquired factor VIII inhibitors (RIIA). Acquired factor VIII inhibitors in pregnancy: data from the Italian Haemophilia Register relevant to clinical practice. BJOG 2003, 110:311-314. 11. Solymoss S: Postpartum acquired factor VIII inhibitors: results of a survey. Am J Hematol 1998, 59:1-4. 12. Michiels JJ, Hamulyak K, Nieuwenhuis HK, Novakova I, van Vliet HH: Acquired haemophilia A in women postpartum: management of bleeding episodes and natural history of the factor VIII inhibitor. Eur J Haematol 1997, 59:105-109. 13. Bicer M, Yanar M, Tuydes O: Spontaneous deep vein thrombosis in hemophilia A: a case report. Cases J 2009, 2:6390. 14. Dargaud Y, Meunier S, Negrier C: Haemophilia and thrombophilia: an unexpected association! Haemophilia 2004, 10:319-326. 15. Kashyap R, Sharma Lm, Gupta S, Saxena R, Srivastava DN: Deep vein thrombosis in a patient with severe haemophilia A. Haemophilia 2006, 12:87-89. Page 4 of 4 Spencer et al. Thrombosis Journal 2011, 9:16 http://www.thrombosisjournal.com/content/9/1/16 Grossmann R, Wankmüller H, Schwender S, Kirschner J, Mansouri- Taleghani B, Keller P, et al: The diagnosis and therapy of acquired factor- VIII inhibitors in combination with lupus anticoagulants. Dtsch Med Wochenschr 1996, 121:906-910. 25. Grossmann R, Wankmüller H, Schwender S, Kirschner J, Mansouri- Taleghani B, Keller P, et al: The diagnosis and therapy of acquired factor- VIII inhibitors in combination with lupus anticoagulants. Dtsch Med Wochenschr 1996, 121:906-910. 26. Biron C, Durand L, Lemkecher T, Dauverchain J, Meunier L, Meynadier J, et al: Simultaneous occurrence of lupus anticoagulant, factor VIII inhibitor and localized pemphigoid. Am J Hematol 1996, 51:250-251. 26. Biron C, Durand L, Lemkecher T, Dauverchain J, Meunier L, Meynadier J, et al: Simultaneous occurrence of lupus anticoagulant, factor VIII inhibitor and localized pemphigoid. Am J Hematol 1996, 51:250-251. 27. Taher A, Abiad R, Uthman I: Coexistence of lupus anticoagulant and acquired haemophilia in a patient with monoclonal gammopathy of unknown significance. Lupus 2003, 12:854-856. 27. Taher A, Abiad R, Uthman I: Coexistence of lupus anticoagulant and acquired haemophilia in a patient with monoclonal gammopathy of unknown significance. Lupus 2003, 12:854-856. 28. Blanco AN, Cardozo MA, Candela M, Santarelli MT, Pérez Bianco R, Lazzari MA: Anti-factor VIII inhibitors and lupus anticoagulants in haemophilia A patients. Thromb Haemost 1997, 77:656-659. 29. Pengo V, Tripodi A, Reber G, Rand JH, Ortel TL, Galli M, et al: Update of the guidelines for lupus anticoagulant detection. Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibody of the Scientific and Standardisation Committee of the International Society on Thrombosis and Haemostasis. J Thromb Haemost 2009, 7:1737-1740. doi:10.1186/1477-9560-9-16 Cite this article as: Spencer et al.: Sequential thrombosis and bleeding in a woman with a prolonged activated partial thromboplastin time. Thrombosis Journal 2011 9:16. doi:10.1186/1477-9560-9-16 Cite this article as: Spencer et al.: Sequential thrombosis and bleeding in a woman with a prolonged activated partial thromboplastin time. Thrombosis Journal 2011 9:16. 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https://openalex.org/W4379112862	https://e-journal.umaha.ac.id/index.php/deskovi/article/download/1935/1150	Indonesian	null	Bentuk Penyajian Musik Kesenian Nandong pada Upacara Khitanan di Desa Lataling Kecamatan Teupah Selatan Kabupaten Simeulue	Deskovi	2,022	cc-by	4,071	DESKOVI : Art and Design Journal Volume 5, Nomor 2, Desember 2022, 128-133 DESKOVI : Art and Design Journal Volume 5, Nomor 2, Desember 2022, 128-133 ISSN 2655 - 464X online ISSN 2654 - 5391 print ABSTRAK Kesenian Nandong adalah seni vokal yang diwariskan secara turun-tumurun pada masyarakat Simeulue. Kesenian Nandong merupakan sejenis seni tutur yang syair-syairnya berisikan karangan yang mengandung nasehat, sindiran, rintihan yang dilantunkan dengan iringan alat musik yaitu kedang/gendang dan biola. Nandong dilantunkan menggunakan bahasa Devayan khas bahasa Simeulue, namun dalam kesenian Nandong juga menggunakan bahasa Aneuk Jamee. Keunikan Nandong selain terdapat dalam bahasa Aneuk Jamee yaitu dilantunkan dengan nada yang tinggi dan melengking serta saling bersahut-sahutan oleh pria dengan suara tinggi dan melengking. Penelitian ini dilakukan dengan pendekatan kualitatif, yang berusaha mengaplikasikan teori untuk menjelaskan fenomena- fenomena yang terjadi pada objek penelitian. Metode dalam penelitian ini dilakukan secara deskriptif yang diwujudkan dalam bentuk keterangan atau gambar tentang kejadian atau kegiatan yang dilakukan menyeluruh dengan mendatangi langsung di Desa Lataling, Kecamatan Teupah Selatan, Kabupaten Simeulue. Adapun langkah tersebut dilakukan dengan tahapan yaitu: penentuan subjek penelitian; pemilihan setting dan instrumen penelitian, teknik, dan analisis data. Hasil penelitian akan menunjukkan bahwa terdapat analisis terhadap fenomena musik pada kesenian ini. Fenomena tersebut seperti pemakaian timbre low, disusul timbre high dengan berbagai intensitas kemunculan, serta diperoleh deskripsi tangga nada (scale), dan pengurutan nada-nada yang terdapat dalam Nandong tersebut dimulai dari nada terendah sampai nada yang tertinggi. K k i A li i M ik P ji M ik K i N d Kata kunci: Analisis Musik, Penyajian Musik, Kesenian Nandong. Abdul Rozak1, Haria Nanda Pratama2, Nadra Akbar Manalu3 1Seni Karawitan, Jurusan Seni Pertunjukan Institut Seni Budaya Indonesia Aceh, Kota Jantho, Aceh Besar, Aceh, Indonesia Email : abdulrozak.isbiaceh@gmail.com 1Seni Karawitan, Jurusan Seni Pertunjukan Institut Seni Budaya Indonesia Aceh, Kota Jantho, Aceh Besar, Aceh, Indonesia Email : abdulrozak.isbiaceh@gmail.com 2Seni Teater, Jurusan Seni Pertunjukan Institut Seni Budaya Indonesia Aceh, Kota Jantho, Aceh Besar, Aceh, Indonesia Email : harianandapratama@gmail.com 2Seni Teater, Jurusan Seni Pertunjukan Institut Seni Budaya Indonesia Aceh, Kota Jantho, Aceh Besar, Aceh, Indonesia Email : harianandapratama@gmail.com 3Seni Tari, Jurusan Seni Pertunjukan Institut Seni Budaya Indonesia Aceh, Kota Jantho, Aceh Besar, Aceh, Indonesia Email : nadramanalu@gmail.com Diterima : 8 Juni 2022. Disetujui : 10 Desember 2022. Dipublikasikan : 15 Desember 2022 ©2022 – DESKOVI Universitas Maarif Hasyim Latif. Ini adalah artikel dengan akses terbuka di bawah lisensi CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/) Diterima : 8 Juni 2022. Disetujui : 10 Desember 2022. Dipublikasikan : 15 Desember 2022 ©2022 – DESKOVI Universitas Maarif Hasyim Latif. Ini adalah artikel dengan akses terbuka di bawah lisensi CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/) Keyword: Music Analysis, Music Presentation, Art of Nandong. PENDAHULUAN kedang/gendang (dalam khas budaya Simeulue), biola (pada dulunya biola dimainkan sesama dengan kedang). Pada setiap lirik yang disampaikan mengandung nilai-nilai estetika antara perpaduan irama dengan makna syairnya yang mendayu-dayu. Selain itu terdapat juga makna sosial yang terkandung dalam syair-syairnya yang berupa nasehat, petuah, yang menceritakan kehidupan seseorang atau pesan dari leluhur kepada anak cucunya yang digunakan pada saat pesta perkawinan, khitanan, ataupun disaat berlangsungnya acara pesta rakyat. Perbedaan yang nyata terlihat dari kesenian ini adalah dalam hal keunikan dialeknya. Selain hal tersebut, Nandong juga memiliki fungsi dalam mengetahui gejala alam seperti gempa bumi (Lubis, 2019: 6). Kesenian merupakan salah satu isi dari kebudayaan manusia secara umum, karena kesenian adalah cerminan dari suatu bentuk peradaban yang tumbuh dan berkembang sesuai dengan cita-cita, keinginan dengan berpedoman pada nilai-nilai yang berlaku dan dilakukan dalam bentuk aktivitas berkesenian, sehingga masyarakat mengetahui bentuk keseniannya. Perkembangan kesenian Indonesia sesungguhnya banyak variasinya, namun, di antara banyaknya variasi kesenian tradisional tersebut masih banyak yang belum diketahui oleh masyarakat Indonesia secara umum. Fadinul (2016) dalam skripsinya mengakatan hal ini terjadi disebabkan oleh berbagai alasan, kesenian Nandong adalah salah satu kesenian tradisional yang masih belum diketahui oleh masyarakat luas ini disebabkan karna kurangnya minat masyarakat terhadap kesenian Nandong. Kurangnya minat masyarakat terhadap kesenian Nandong ini disebabkan pengaruh musik Barat yang terus berdatangan yang menarik sehingga perhatian masyarakat terhadap kesenian kesenian Nandong pun diabaikan dan di tinggalkan. Artinya. kurangnya kesadaran kita untuk menginventarisasi, merevitalisasi, melestarikan serta mengembangkan kesenian tersebut pada masa sekarang ini. g p ( ) Lubis dalam tesisnya (2019: 6), mengatakan bahwa kesenian Nandong termasuk ke dalam salah satu folklor lisan yakni puisi rakyat seperti pantun, gurindam dan syair. Bentuk-bentuk folklor lisan yang termasuk di dalamnya adalah: (1) bahasa rakyat seperti logat, julukan, dan titel kebangsawaan; (2) ungkapan tradisional, seperti peribahasa, pepatah dan pameo; (3) pertanyaan tradisional seperti teka-teki; (4) puisi rakyat, seperti pantun, gurindam, dan syair; (5) cerita prosa rakyat, seperti mite, legenda, dongeng, dan (6) nyayian rakyat.. James Danandjaja (1984: 2) yang menyatakan bahwa folklor adalah sebagian kebudayaan yang kolektif yang tersebar dan diwariskan turun-temurun, di antara kolektif macam apa saja, secara tradisional dalam versi yang berbeda, baik dalam bentuk lisan maupun contoh yang disertai dalam gerak isyarat atau alat pembantu pengingat. Abdul Rozak, dkk / DESKOVI, Vol. 5, No.2, Desember 2022, 128-133 Abdul Rozak, dkk / DESKOVI, Vol. 5, No.2, Desember 2022, 128-133 ABSTRACT Nandong art is a vocal art that has been passed down from generation to generation among the Simeulue people. Nandong art is a kind of speech art in which the poems contain compositions that contain advice, satire, groans sung accompanied by musical instruments, kedang/gendang and violins. Nandong is sung using the Devayan language typical of the Simeulue language, but in art, Nandong also uses the Aneuk Jamee language. The uniqueness of Nandong besides being in the Aneuk Jamee language is that it is sung in a high and shrill tone and shouted at each other by men in high and shrill voices. This research is a qualitative research, this study seeks to apply the theory to explain the phenomena that occur in the object of research. The method in this study was carried out descriptively which was manifested in the form of information or pictures about events or activities carried out thoroughly by visiting Lataling Village, Teupah Selatan District, Simeulue Regency. The steps are carried out in stages, namely: determining the research subject; selection of research settings and instruments, techniques, and data analysis. The results of the research will show that there is an analysis of the phenomenon of music in this art. Such phenomena include the use of a low timbre, followed by a high timbre with various intensities of appearance, and a description of the scale is obtained, and the order of the tones contained in Nandong starts from the lowest to the highest tone. Keyword: Music Analysis, Music Presentation, Art of Nandong. 128 PENDAHULUAN Dari pendapat James Danandjaja di atas maka peneliti menyimpulkan kesenian Nandong adalah salah satu kesenian yang termasuk dalam foklor, dilihat dari segi pertunjukan penyampaiyan dalam bentuk syairnya yang menyampaikan cerita kehidupan yang mengandung makna-makna yang tersirat dalam kesenian Nandong tersebut. Asal usul kesenian Nandong tidak diketahui dengan jelas, akan tetapi kesenian Nandong diperkirakan sudah ada pada abad ke-12 sebelum agama islam masuk di Kabupaten Simeulue. (wawancara dengan Harumsyah seniman Nandong). Nandong adalah salah satu kesenian tradisional masyarakat Kabupaten Simeulue, Provinsi Aceh. Kesenian ini diketahui memiliki nilai-nilai estetika yang tinggi serta makna yang luas bermanfaat bagi masyarakat setempat, yang disebut memiliki estetika yaitu penyampaian yang dilakukan seniman Nandong melalui syair yang dilantunkan memiliki ciri khas, seniman Nandong melantunkan syairnya menggunakan bahasa aneuk jamee dan devayan dengan bait-bait yang disajikan menggunakan nada tinggi yang mendayu- dayu diiringi tabuhan kedang di antara bait-bait syair tersebut. Kabupaten Simeulue merupakan sebuah kepulauan yang terletak di pantai bagian barat Provinsi Aceh yang berjarak 105 mil laut dari kota Meulaboh, Kabupaten Aceh Barat atau sekitar 85 mil laut dari kota Labuhan Haji Kabupaten Aceh Selatan. Kabupaten Simeulue ini terbilang jauh dari Ibu kota Provinsi sehingga membuat kesenian ini kurang dikenal oleh masyarakat luas di luar Kabupaten Simeulue (Mahyunilawati, 2016: 3). Folklor termasuk salah satu bentuk kesenian tradisional masyarakat yang sudah turun-temurun diketahui masyarakat dan merupakan wujud kebudayaan. Kaitan antara kesenian tradisional seperti Nandong ini terhadap kebudayaan adalah di dalamnya terdapat nilai-nilai budaya berupa makna tersirat yang diketahui oleh masyarakat Simeulue. Kesenian Nandong yang menceritakan tentang peruntungan atau nasib yang dialami dikenal dengan Nandong Untung dapat membuat pendengarnya menjadi sedih karena terhanyut dengan cerita yang disampaikan. Nandong yang dilantunkan menggunakan bahasa Devayan khas bahasa Simeulue, namun dalam kesenian Nandong juga menggunakan bahasa Aneuk Jamee yang juga merupakan salah satu bahasa yang digunakan di Kabupaten Simeulue, khususnya di Kota Sinabang. Keunikan Nandong selain adalah dilantunkan dengan nada yang tinggi seperti melengking dan saling bersahut-sahutan oleh pria. Keunikan Nandong yang dilantunkan oleh pria dengan suara tinggi dan melengking serta awalnya menggunakan bahasa Kesenian Nandong adalah seni vokal yang diwariskan secara turun-tumurun pada masyarakat Simeulue. Kesenian Nandong merupakan sejenis seni tutur yang dalam syair-syairnya berisikan karangan yang mengandung nasehat, sindiran, rintihan yang dilantunkan dengan diiringi alat musik yaitu 129 Abdul Rozak, dkk / DESKOVI, Vol. 5, No.2, Desember 2022, 128-133 menurut Creswell dalam Pratama (2021: 5) mengatakan minang merupakan fenomena yang menarik. A. KESENIAN NANDONG Pada penelitian ini, peneliti memfokuskan objek pada penyajian musik dari pertunjukan kesenian Nandong di Desa Lataling, Kecamatan Teupah Selatan, Kabupaten Simeulue. Maka peneliti sebelum melakukan obvervasi, wawancara serta dokumentasi terhadap kesenian Nandong, maka peneliti lebih utama menetapkan judul sebagai acuan untuk melakukan obvervasi di lapangan, maka peneliti menetapkan judul besar dalam penelitian ini yaitu “Bentuk Penyajian Musik Kesenian Nandong Pada Upacara Khitanan di Desa Lataling Kecamatan Teupah Selatan Kabupaten Simeulue”. Nandong merupakan kesenian tradisional yang tumbuh dan berkembang dalam kebudayaan masyarakat Simeulue. Kesenian Nandong memiliki arti sebagai senandung yakni nyanyian yang didendangkan pada waktu melakukan sesuatu pekerjaan yang disenangi atau untuk menghibur hati yang sedang gundah (Agur dalam Mahyunilawati 2016: 38). Nandong sebagai sebuah seni tutur yang telah menjadi kesenian daerah masyarakat Simeulue terdiri atas pantun/puisi dan mengandung nasehat, cerita dan sindiran. Kesenian Nandong bisa dimainkan dua orang atau lebih dengan diiringi pukulan gendang yang ditabuh di antara sela bait- baitnya dilantunkan. Bait-bait yang dilantunkan merupakan nasehat-nasehat, cerita, sindiran, ungkapan perasaan seseorang. Dan jenis-jenis pantun tersebut berisikan cerita, kisah kasih/ percintaan, nasib/ peruntungan dan dinamika kehidupan lainnya. Kesenian Nandong juga merupakan media dalam menyampaikan pesan-pesan moril kepada masyarakat. PENDAHULUAN Suara tinggi atau dilantunkan oleh pria dengan jenis tenor bahkan counter tenor yang memiliki makna pertunjukan yang dapat digali (Lubis, 2019: 12). bahwa dalam penelitian kualitatif peneliti adalah instrumen kunci (researcher as key instrument) yang mengumpulkan sendiri data melalui dokumentasi dan studi pustaka. Sebagai bentuk penelitian kualitatif, maka penelitian ini berusaha mengaplikasikan teori untuk menjelaskan fenomena-fenomena yang terjadi pada objek penelitian yaitu kesenian Nandong di Desa Lataling, Kecamatan Teupah Selatan, Kabupaten Simeulue. Langkah-langkah penelitian ini adalah: (a) penentuan subjek penelitian, (b) pemilihan setting, (c) Instrumen penelitian, (d) teknik pengumpulan data, (e) analisis data. Kesenian Nandong dimainkan oleh dua orang pemain atau lebih dengan menggunakan instrumen kedang (dalam khas bahasa Simeulue) atau gendang. Dimulai dengan tabuhan seuramo kedang (pembukaan) hingga syair-syair Nandong dilantunkan dan saling bersautan, dan beberapa syair rantau, kasih dan lain- lainnya dari beberapa syair pantun yang disajikan kembali berseuramo kedang yang bertanda pertunjukan kesenian Nandong segera berahir, syair tonjon yang akan menjadi penutup pertunjukan kesenian Nandong. Pertunjukan Nandong dimulai dengan melantunkan syair-syairnya yang saling bersahut-sahutan dan diakhiri dengan seuramo kedang tanda kesenian Nandong berakhir. METODE PENELITIAN Penelitian ini adalah penelitian kualitatif yaitu data yang dikumpulkan berupa kata-kata, gambar, dan bukan angka-angka. Penelitian kualitatif adalah penelitian yang bermaksud untuk memahami fenomena tentang persepsi, motivasi, tindakan dan lain-lain secara holistik, dan dengan cara deskripsi dalam bentuk kata-kata dan bahasa, pada suatu konteks khusus yang alamiah dan dengan memanfaatkan metode alamiah (Moleong, 2011: 6). Peneliti memperoleh data dari keberadaan kesenian tradisional Pengertian bentuk yang dimaksud dalam bentuk penyajian musik pada kesenian Nandong salah satunya adalah seperti diungkapkan Djelantik (1990: 14) bahwa bentuk merupakan dasar dari unsur-unsur susunan pertunjukan. Unsur-unsur penunjang yang membantu bentuk itu dalam dalam mencapai perwujudannya yang khas adalah: seniman, alat musik, kostum dan rias, lagu yang disajikan, tempat pertunjukan, waktu serta penonton. Selanjutnya, menurut Djelantik (1990: 73) penyajian yaitu bagaimana kesenian itu disuguhkan kepada penyaksinya, penonton, para pengamat, pembaca, pendengar, khalayak ramai pada umumnya. Sedangkan unsur yang berperan dalam penampilan atau penyajian adalah bakat, keterampilan, serta sarana atau media. Menurut pernyataan tersebut dapat disimpulkan bahwa penyajian merupakan penampilan yang meliputi salah satunya seperti alat musik, pemain musik, dan unsur dalam musik. Nandong di Desa Lataling, Kecamatan Teupah Selatan, Kabupaten Simeulue. Data yang dikumpulkan berupa informasi dalam bentuk deskriptif. Metode tersebut diwujudkan dalam bentuk keterangan atau gambar tentang kejadian atau kegiatan yang dilakukan menyeluruh dengan mendatangi langsung di Desa Lataling, Kecamatan Teupah Selatan, Kabupaten Simeulue. Selanjutnya, penelitian akan berisi kutipan- kutipan data untuk memberi gambaran penyajian penelitian. Data berasal dari naskah wawancara, catatan lapangan, foto, video, dokumen pribadi, catatan atau memo, dan dokumen resmi lainnya. Penelitian kualitatif lebih banyak mementingkan segi proses dari pada hasil. Hal ini disebabkan oleh hubungan bagian- bagian yang sedang diteliti akan jauh lebih jelas apabila diamati dalam proses (Moleong, 2011: 11). Selain itu 130 Abdul Rozak, dkk / DESKOVI, Vol. METODE PENELITIAN 5, No.2, Desember 2022, 128-133 Ala pui mancari untung Untung nan tidak dapek lai Sudah jerah mencari untung Untung sudah tidak dapat lagi Cubadak duo namonyo Dibali di tapi jalan Paruntungan sabui namonyo Ai mato di buang jangan Nangka dua namanya Di beli ditepi jalan Peruntungan sebut namanya Air mata di buang jangan Silaguri tumbuh dibatu Jatuh bungonyo dimuaro Sakik sanang tanggung daulu Isuk dimano kan painyo Silaguri tumbuh dibatu Jatuh bunganya di limba Sakit senang jalani dulu Besok di mana kan perginya Tinggi batang sidurian Condong rabanyo kasubarang Sakek manimbang paruntungan Kain basa karing dipinggang Tinggi batang si durian Lebih jatuhnya kesebelah Sakit menimbang peruntungan Kain basa kering di pinggang Dibaapo mambala papan Buek banang mangko basagi Di baapo manyeso badan Untung suratan pado diri Bagaimana membela kayu Buat benang baru bersegi Untuk apa menyiksa badan Untung suratan pada diri Dijamu padi ate tambak Silaguri tumbu dilamba Lawik rimbo habi ku jajak Nasib badan ku balun baruba Dijemur padi atas jalan Silaguri hidup dilimba Laut luas habis ku pergi Nasib badan ku belum berubah Makah al tersebut merupakan unsur dari bentuk penyajian musik dari kesenian Nandong. Maka peneliti menguraikan, bentuk penyajian musik pada kesenian Nandong mengandung nilai- nilai keindahan yang disampaikan melalui syair- syair yang dilantunkannya. Kesenian Nandong termasuk salah satu musik ansambel yang dimainkan secara berkelompok menggunakan alat musik kedang. Dahulunya kesenian Nandong menggunakan dua alat instrumen yaitu biola dan kedang. Dalam segi penggunaan instrumen yang berkurang tersebut disebabkan karena tidak ada lagi penerus kesenian Nandong yang bisa memainkan alat musik biola tersebut (wawancara dengan Bapak Harumsyah 2021, di Desa Lataling, pada tanggal 2 Mei 2021). ) Pertunjukan kesenian Nandong berlangsung pada malam hari, yang dilakukan setelah Shalat Isya hingga menjelang terbitnya fajar, pemain Nandong lebih mengutaman fisik karena berlangsungnya pertunjukan yang berdurasi lama. Diketahui bahwa kesenian Nandong akan berlangsung terutama para seniman Nandong menabuh Kedang tanda akan mulainya pertunjukan kesenian Nandong. Namun, kesenian Nandong yang ada di Desa Lataling tidak seperti umumnya melakukan pembukaan kesenian Nandong, dahulu pada umumnya keseniaan Nandong pada bagian awal pembukaan para seniman Nandong melakukan salam pembuka dengan Seuramo Kedang kemudian langsung disahut oleh karangan pantun Samba. Selanjutnya, seniman Nandong di Desa Lataling menabuh Kedang dengan pengulangan beberapa kali pengulangan tanda bahwa kesenian Nandong segerah akan disajikan untuk penikmat yang menonton. Abdul Rozak, dkk / DESKOVI, Vol. 5, No.2, Desember 2022, 128-133 Abdul Rozak, dkk / DESKOVI, Vol. 5, No.2, Desember 2022, 128-133 Pantun Nandong Arti dalam Bahasa Indonesia Dari mano punai malayang Dari pucuk kape panji Dari mano mulonyo sayang Dari jantung turun kahati Dari mana burung melayang Dari pucuk kapas panji Dari mana awalnya sayang Dari jantung turun ke hati Kayu di ulu tabalintang Tampek baranti burung balam Kok sunggu bakasi sayang Apo alamat kami ganggam Kayu di gunung terbelintang Tempat berhenti burung balam Kalau benar berkasih sayang Apa alamat kami pegang Padi sipului di samaikan Padi banamo pului- pului Dimului sajo mangatokan Bagai pasang mengelak surut Padi ketan di semaikan Padi bernama ketan-ketan Di mulut saja mengatakan Seperti pasang menarik surut Notasi 2. Pola Melodi Vokal pada Kesenian Nandong (Transkriptor: Zulkarnain, tahun 2021) si 2. Pola Melodi Vokal pada Kesenian Nandong (Transkriptor: Zulkarnain, tahun 2021) Notasi 2. Pola Melodi Vokal pada Kesenian Nandong (Transkriptor: Zulkarnain, tahun 2021) Jumlah ritme adalah banyaknya ritme-ritme yang dipakai secara keseluruhan dalam suatu musik baik musik instrumental atau vokal. Pada syair Nandong, penulis memperoleh 100 ritme dengan timbre low, 63 ritme dengan timbre high, dalam 1 sampiran Nandong. Ritme yang paling sering muncul pada nandong adalah timbre low, disusul timbre high. Dengan demikian, intensitas kemunculan yang paling banyak yaitu timbre low. Berdasarkan jumlah timbre yang diperoleh dalam 1 syair Nandong, maka jumlah timbre secara keseluruhan dalam 1 bait Nandong yaitu 48 nada. Pola ritme merupakan bunyi yang muncul dalam satu atau beberapa birama yang tersusun sesuai dengan pola tertentu dalam sebuah syair. Pola ritme perkusi yang terdapat dalam Nandong memiliki dua pola ritme yaitu sebagai berikut. Kayu di gunung terbelintang Tempat berhenti burung balam Kalau benar berkasih sayang Apa alamat kami pegang Padi ketan di semaikan Padi bernama ketan-ketan Di mulut saja mengatakan Seperti pasang menarik surut Notasi 3. Pola 1 Ritme Kedang Notasi 4. Pola 2 Ritme Kedang METODE PENELITIAN Salam pembuka kesenian Nandong di Desa Lataling tidak menggunkan Seuramo Kedang bukan berarti menghilangkan keasliannya melainkan disebabkan sudah terlalu lama fakum jadi banyak seniman- seniman yang lupa pola Seuramo Kedang. Itulah alasan mengapa kelompok kesenian Nandong di Desa Lataling tidak ada Seuramo Kedang di saat salam pembuka yang menandakan kesenian Nandong akan segera dimulai (wawancara dengan Bapak Harumsyah 2021, di Desa Lataling, pada tanggal 2 Mei 2021). Pada syair di atas menceritakan tentang peruntungan kehidupan yang dialami sehari-hari, para seniman mengungkapkan perasaannya dengan melantunkan syair diiringi oleh tabuhan Kedang di setiap bait-bait syairnya. Di setiap bait-bait yang dilantunkan memiliki makna tersendiri bagi seniman Nandong, Asnawi (Wawancara di Desa Lataling, pada tanggal 2 Mei 2021) juga mengungkapkan dalam syair- syair Nandong bukan menceritakan tentang peruntungan saja, melainkan juga menceritakan tentang kisah mengadu nasib di rantau orang, dalam syair Nandong itu menceritakan tentang kasih sayang dalam keluarga bahkan juga ada syair Carai atau bercerai (Wawancara dengan Sarman, di Desa Lataling, pada tanggal 2 Mei 2021). Dari penjelasan di atas maka bentuk penyajian kesenian Nandong, awal pertama dimulai menabuh Kedang beberapa kali pengulangan lalu bar kedepalan terakhir dilanjutkan dengan karangan pantun atau syair yang dilantunkan. Adapun syair yang sering dilantunkan oleh para seniman adalah sebagai berikut. Tabel 1. Syair Untung Pantun Nandong Arti dalam Bahasa Indonesia Ala pue mananam jagung Jagung nan tidak idui lai Sudah jerah menanam jagung Jagung sudah tidak hidup lagi Dialek yang digunakan dalam syair Nandong terkadang berubah, mulai dari dialek bahasa Aneuk jamee hingga menggunakan bahasa Devayan, misalnya seperti syair di bawah ini. Tabel 2. Syair dengan Dialek yang Berubah-ubah Tabel 2. Syair dengan Dialek yang Berubah-ubah 131 PENUTUP Fadinul, Andi. 2016. Sikap Masyarakat Terhadap Pelestarian Budaya Nandong di Gampong sambay Kecamatan Teluk Dalam Kabupaten Simeulue, dalam Skripsi Fakultas Ilmu Sosial dan Ilmu Politik Program Studi Ilmu Sosiologi Universitas Teuku Umar. Nandong merupakan salah satu kesenian tradisional masyarakat Kabupaten Simeulue, Provinsi Aceh. Kesenian ini diketahui memiliki nilai-nilai estetika yang tinggi (menurut ukuran seni masyarakat pendukungnya), serta makna yang luas bermanfaat bagi masyarakat Desa Lataling. Kesenian Nandong merupakan seni tutur dalam bentuk syair, yang mengandung nasehat-nasehat, petuah, nasib, paruntungan dan kasih yang diiringi oleh alat musik Kedang (gendang khas bahasa Simeulue). Harumsyah. (2021). “Penyajian Musik Pada Kesenian Nandong”. Hasil wawancara Pribadi: 2 Mei 2021, di Desa Lataling. Khatimah, Husnul. (2020). Bentuk Penyajian Meurukon dalam Maulid Nabi di Gampong Lambada Kecamatan Seulimum Kabupaten Aceh Besar. dalam Skripsi Program Studi Seni Karawitan, Jurusan Seni Pertunjukan. Institut Seni Budaya Indonesia Aceh. Lubis, Tasnim. (2019). Tradisi Lisan Nandong Simeulue: Pendekatan Antropolingustik, dalam Tesis Program Doktor S3, Fakultas Ilmu budaya Universitas Sumatera Utara. Repositori Institusi Universitas Sumatera Utara. Khatimah, Husnul. (2020). Bentuk Penyajian Meurukon dalam Maulid Nabi di Gampong Lambada Kecamatan Seulimum Kabupaten Aceh Besar. dalam Skripsi Program Studi Seni Karawitan, Jurusan Seni Pertunjukan. Institut Seni Budaya Indonesia Aceh. Ritme yang paling sering muncul pada nandong adalah timbre low, disusul timbre high. Dengan demikian, intensitas kemunculan yang paling banyak yaitu timbre low. Berdasarkan jumlah timbre yang diperoleh dalam 1 syair Nandong, maka jumlah timbre secara keseluruhan dalam 1 bait. Lubis, Tasnim. (2019). Tradisi Lisan Nandong Simeulue: Pendekatan Antropolingustik, dalam Tesis Program Doktor S3, Fakultas Ilmu budaya Universitas Sumatera Utara. Repositori Institusi Universitas Sumatera Utara. Hasil yang didapatkan dari transkripsi Nandong penulis memperoleh dalam mendeskripsikan tangga nada (scale), penulis mengurutkan nada-nada yang terdapat dalam Nandong tersebut dimulai dari nada terendah sampai nada yang tertinggi. Penulis memperoleh bahwa terdapat 1 nada dengan nada terendah adalah C dan nada tertinggi adalah G’. Mahyunilawati. (2016). Kajian Musikal dan Makna Nandong dipertunjukan Pada malam Malaulu dalam Adat Perkawinan Etnik Simeulue di Sinabang, Kecamatan Simeulue Timur, Aceh. dalam Skripsi Universitas Sumatera Utara. Repositori Institusi Universitas Sumatera Utara. Penulis menyadari banyak kekurangan dalam penulisan bentuk penyajian kesenian Nandong. Salah satunya kurangnya sumber-sumber referensi mengenai bentuk penyajian kesenian Nandong ini yang dapat mendukung dalam tulisan ini. Penulis berharap peneliti-peneliti selanjutnya bisa menyempurnakan tulisan ini. Bagi para peneliti berikutnya, penulis menyarankan beberapa hal untuk dipersiapkan dalam penyusunan tulisan ini. Pertama, kita harus mempunyai pengetahuan umum tentang kebudayaan kesenian Nandong. B. ANALISIS MUSIK NANDONG Dalam ilmu etnomusikologi, transkripsi merupakan proses penulisan bunyi-bunyian sebagai hasil dari pengamatan dan pendengaran suatu musik kedalam bentuk simbol-simbol yang disebut dengan notasi (Djamaris, 2002: 19). Untuk melakukan transkripsi pola kedang nandong, penulis memilih notasi deskriptif yang dikemukakan oleh Charles Seeger. Notasi deskriptif adalah notasi yang ditujukan untuk menyampaikan kepada pembaca tentang ciri- ciri atau detail-detail komposisi musik yang belum diketahui oleh pembaca. Dalam mendeskripsikan tangga nada (scale), penulis mengurutkan nada-nada yang terdapat dalam Nandong tersebut dimulai dari nada terendah sampai nada yang tertinggi. Penulis memperoleh bahwa terdapat 1 nada dengan nada terendah adalah C dan nada tertinggi adalah G’ pada oktaf yang berikutnya. Perantara untuk menginterprestasikan bunyi perkusi adalah dengan notasi, dalam hal ini penulis menggunakan notasi balok dengan beberapa penyesuaian yaitu menggunakan tibre low dan high. Timbre low ditetapkan dibawa dan timbre high atas, low dengan hasil bunyi bum sedangkan high dengan bunyi peng/tak perhatikan penjelasan di bawah ini. Berdasarkan struktur notasi yang digunakan, maka tangga nada Nandong dapat dikategorikan ke dalam jenis tangga nada kromatik, yaitu tangga nada dengan dua jenis interval yaitu 1/2 laras. Dalam hal ini interval tersebut adalah setengah laras. Interval adalah jarak suatu nada ke nada lain yang terdiri interval naik maupun turun. Di bawa ini tabel jumlah interval dalam satu bait syair Nandong. Tabel 3. Jumlah Interval Interval Jumlah Prime 11 Second 29 Kwart 2 Berikut adalah transkripsi pola perkusi kedang pertunjukan nandong. Notasi 1. Pola Ritme Kedang pada Kesenian Nandong (Transkriptor: Zulkarnain, tahun 2021) Notasi 1. Pola Ritme Kedang pada Kesenian Nandong (Transkriptor: Zulkarnain, tahun 2021) Notasi 1. Pola Ritme Kedang pada Kesenian Nandong (Transkriptor: Zulkarnain, tahun 2021) Melalui tabel di atas dapat diketahui bahwa interval yang paling banyak digunakan dalam penyajian Nandong adalah interval second dengan jumlah 29 kali dan interval prime dengan jumlah 11 132 Abdul Rozak, dkk / DESKOVI, Vol. 5, No.2, Desember 2022, 128-133 dapat terus mengembangkan dan melestarikan kesenian Nandong ini dapat terus mengembangkan dan melestarikan kesenian Nandong ini kali di ikuti oleh interval kuart berjumlah 2 kali. Dengan demikian dapat disimpulkan bahwa interval second dan prime memiliki peranan yang sangat penting dalam membentuk Nandong. DAFTAR PUSTAKA Berpedoman pada apa yang dikemukakan mengenai bentuk nyanyian, maka penulis menarik kesimpulan bahwa bentuk yang terdapat dalam Nandong adalah bentuk nyanyian dengan kategori strophic. Nandong terdiri dari 2 bentuk, yaitu bentuk A dan B. Namun dalam penyajiannya, bentuk A akan diulangi pada bagian akhir. Dengan demikian, Nandong memiliki bentuk A-B-A. Bentuk A merupakan bagian Nandong yang dinyanyikan pada bagian response. Sedangkan bentuk B merupakan bagian Nandong yang dinyanyikan pada bagian call. Asnawi Sarma. (2021). “Konsep Penyajian Musik Pada Kesenian Nandong”. Hasil wawancara Pribadi: 2 Mei 2021, di Desa Lataling. Danandjaja, James. (1984). Folklor Indonesia: Ilmu Gosip, Dongeng, dan lain-lain. Jakarta: Pustaka Utama Grafitri. Djamaris. 2002. Strategi Belajar. Jakarta: PT. Rineka Cipta Djelantik, A.A.M. (1990). Estetika sebuah pengantar. Bandung: Masyarakat Seni Pertunjukan Indonesia. PENUTUP Sehingga pada saat menerapkan teknik-teknik penelitian lapangan kita dapat mengetahui dan menyusun konsep pengerjaan selanjutnya secara bertahap dan sistematis, antara lain wawancara, observasi, dan dokumentasi. Penulis berharap untuk generasi-generasi selanjutnya semoga Moleong, Lexy J. (2011). Metode penelitian kualitatif (Cetakan Kedua Puluh Empat). Bandung: PT Remaja Rosdakarya Pratama, Haria Nanda, Rozak, Abdul. (2021) Karakteristik Musikal Pada Film Tenggelemnya Kapal Van Der Wijck. dalam Jurnal Gorga: Jurnal Seni Rupa, Vol. 10, No. 2, Juli-Desember 2021. Universitas Medan. DOI: https://doi.org/10.24114/gr.v10i2.29202 Sarman. (2021). “Syair Musik Pada Kesenian Nandong”. Hasil wawancara Pribadi: 2 Mei 2021, di Desa Lataling. 133
https://openalex.org/W2952288277	https://orca.cardiff.ac.uk/id/eprint/122951/9/sbz066.pdf	English	null	Oscillatory, Computational, and Behavioral Evidence for Impaired GABAergic Inhibition in Schizophrenia	Schizophrenia Bulletin	2,019	cc-by	6,085	Oscillatory, Computational, and Behavioral Evidence for Impaired GABAergic Inhibition in Schizophrenia Alexander D. Shaw1,5, Laura Knight1,2,5, Tom C. A. Freeman1, Gemma M. Williams1, Rosalyn J. Moran3, Karl J. Friston4, , James T. R. Walters2, and Krish D. Singh,1 1CUBRIC, School of Psychology, College of Biomedical and Life Sciences, Cardiff University, Cardiff, UK; 2MRC Centre for Neuropsychiatric Genetics and Genomics, Division of Psychological Medicine and Clinical Neurosciences, School of Medicine, Haydn Ellis Building, Cardiff University, Cardiff, UK; 1CUBRIC, School of Psychology, College of Biomedical and Life Sciences, Cardiff University, Cardiff, UK; 2MRC Centre for Neuropsychiatric Genetics and Genomics, Division of Psychological Medicine and Clinical Neurosciences, School of Medicine, Haydn Ellis Building, Cardiff University, Cardiff, UK; 3Centre for Neuroimaging Science, Denmark Hill, Kings College London, London, UK; 4Wellcome Trust Centre for Neuroimaging, University College London, London, UK 5Joint first authors. To whom correspondence should be addressed; CUBRIC, School of Psychology, College of Biomedical and Life Sciences, Cardiff University, Maindy Road, Cardiff CF24 4HQ, UK; tel: +44-(0)-2920-874690, fax: +44 (0)29 2087 4679, e-mail: singhkd@cardiff.ac.uk predict behavioral performance or oscillatory measures. These findings endorse the importance, and behavioral rel- evance, of GABAergic synaptic disconnection in schizo- phrenia that underwrites excitation–inhibition balance. AbstractThe dysconnection hypothesis of schizophrenia (SZ) proposes that psychosis is best understood in terms of aberrant connectivity. Specifically, it suggests that dysconnectivity arises through aberrant synaptic modu- lation associated with deficits in GABAergic inhibition, excitation–inhibition balance and disturbances of high- frequency oscillations. Using a computational model combined with a graded-difficulty visual orientation dis- crimination paradigm, we demonstrate that, in SZ, per- ceptual performance is determined by the balance of excitation–inhibition in superficial cortical layers. Twenty- eight individuals with a DSM-IV diagnosis of SZ, and 30 age- and gender-matched healthy controls participated in a psychophysics orientation discrimination task, a visual grating magnetoencephalography (MEG) recording, and a magnetic resonance spectroscopy (MRS) scan for GABA. Using a neurophysiologically informed model, we quantified group differences in GABA, gamma measures, and the predictive validity of model parameters for orien- tation discrimination in the SZ group. MEG visual gamma frequency was reduced in SZ, with lower peak frequency as- sociated with more severe negative symptoms. Orientation discrimination performance was impaired in SZ. Dynamic causal modeling of the MEG data showed that local syn- aptic connections were reduced in SZ and local inhibi- tion correlated negatively with the severity of negative symptoms. Oscillatory, Computational, and Behavioral Evidence for Impaired GABAergic Inhibition in Schizophrenia The effective connectivity between inhibitory interneurons and superficial pyramidal cells predicted ori- entation discrimination performance within the SZ group; consistent with graded, behaviorally relevant, disease- related changes in local GABAergic connections. Occipital GABA levels were significantly reduced in SZ but did not Key words: schizophrenia/GABA/inhibition/dysconnect ion/oscillatory/behavioral Schizophrenia Bulletin vol. 46 no. 2 pp. 345–353, 2020 doi:10.1093/schbul/sbz066 Advance Access publication 20 June 2019 Schizophrenia Bulletin vol. 46 no. 2 pp. 345–353, 2020 doi:10.1093/schbul/sbz066 Advance Access publication 20 June 2019 © The Author(s) 2019. Published by Oxford University Press on behalf of the Maryland Psychiatric Research Center. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Introduction The neurobiological underpinnings of schizophrenia (SZ) are currently poorly understood, with sev- eral competing synaptic hypotheses; including dys- function of GABAergic neuromodulation,1 NMDA receptor hypofunction (review2) and aberrant dopa- mine regulation.3 Reconciling these various models, the dysconnection hypothesis4 proposes that SZ results from synaptic dysconnectivity—both between brain regions and between the layers of cortical columns—caused by aberrant synaptic modulation. Physiologically, synaptic modulation refers to the balance of excitation and inhi- bition, whereby GABAergic neurons exert control (and synchrony) over the firing of principal cells in cortex. In rodent, NMDA receptor hypofunction reduces excita- tion of GABAergic cells, which results in a disinhibited, hyperdopaminergic state.5 As such, cortical deficits in NMDA, GABA, or both, may underlie the pathophysi- ology of schizophrenia. GABAergic theories of SZ have recently gained sup- port from genetic studies, including evidence for a pri- mary GABAergic hypothesis, rather than secondary, by implication in large scale studies of both rare6 and © The Author(s) 2019. Published by Oxford University Press on behalf of the Maryland Psychiatric Research Center. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 345 A. D. Shaw et al A. D. Shaw et al Psychiatric Genomics Consortium17), with full details of the case sample recruitment and procedures are pro- vided in Lynham et al.18 Controls were recruited prima- rily through an advert placed on the Cardiff University Noticeboard system and opportunistically from CUBRIC, Cardiff University. This study was approved by the NHS ethics Board and also the Cardiff University School of Psychology Ethics Board. Full details of the inclusion and exclusion criteria can be found in the sup- plementary materials. common7 genetic variation. In vivo studies of altered bulk GABA concentrations in schizophrenia are, how- ever, equivocal, with a recent meta-analysis suggesting no detectable differences in GABA levels across prefrontal, parietal/occipital cortex, and striatum using proton mag- netic resonance spectroscopy (1H-MRS).8 p py ( ) Performance on visual orientation discrimination paradigms is known to correlate with occipital GABA concentrations, making these paradigms useful probes of the GABAergic system9 without putative confounding effects from dopaminergic influences. Behavioral Visual Orientation Discrimination Task Orientation discrimination thresholds were measured using a 2-alternative forced choice procedure as shown in figure 1A. Stimuli were programmed using DELPHI and consisted of 2 sequentially presented circular grating patches (width 4°, contrast 80%, 3 cycles per degree, refresh rate 80 Hz, mean luminance 44.5 cd/m2). The task used 2 stimulus types, an easier vertical condition in which the ori- entation difference between the 2 patches varied around a constant mean of 0° and a more difficult oblique condition where the orientation between the 2 patches varied around a constant mean of 45°. Further details on this paradigm can be found in the supplementary materials. Introduction Performance tends to correlate with peak oscillation frequency in the gamma range (30+ Hz) in the visual cortex.9 Because gamma oscillations are also dependent on excitation– inhibition balance—under the pyramidal-interneuron gamma (PING) model of oscillatory activity10,11— gamma oscillations have been proposed as a consequence of the synaptic modulatory mechanisms thought to be disrupted in SZ.12 Successful Data Collection One male control participant was unable to undertake ei- ther the MEG experiment reported here or a GABA MR Spectroscopy assessment, but did complete the behav- ioral orientation discrimination experiment. Similarly, 2 SZ participants (1 male, 1 female) did not complete the MEG data collection, but MRS data and behav- ioral data were successfully acquired. This means for the multimodal comparisons reported here, the total cohort sizes were 29 controls and 27 SZ participants. The dem- ographics of these participants are outlined in table 1. Details of the acquisition parameters of the GABA MRS scan are found in supplementary materials. Neurophysiologically, the PING model proposes that gamma frequency oscillations are generated in superficial layers (2/3) of cortex and mediated by fast GABA-A and AMPA receptors as well as slower NMDA receptors.13 Deficits in GABA may therefore explain reports of reduced gamma oscillatory power in SZ.11,14–16 Here, we address the relationship between gamma oscillations and orientation discrimination perfor- mance in SZ, using an integrated framework. Using neurophysiologically informed modeling based on dy- namic causal modeling (DCM), we demonstrate how GABA-mediated local dysconnectivity leads to altered gamma oscillatory activity that predicts behavioral per- formance in a graded, disease-specific manner. Participant Sample and Recruitment Participants consisted of 28 schizophrenia participants (8 female, mean age males = 44.2 [SD = 8.6] years, females = 45.9 [SD = 8] years) and 30 control participants (11 female, mean age males = 42.4 [SD = 10] years, females = 40.6 [SD = 10.2] years). The age range was 22–58 for the SZ group and 24–58 for the control group. There was no difference in age between the groups (P > .1). Participants were recruited from the Cognition in Psychosis study (Schizophrenia Working Group of the Neurophysiologically Informed Modeling Using a convolution-based canonical microcircuit model (CMC),22 optimized to reflect the known properties of pri- mary visual cortex (V1) and implemented within the dynamic causal modeling (DCM) framework of SPM8 (https://www. fil.ion.ucl.ac.uk/spm/software/spm8/), we modeled the spec- tral density recorded by the MEG virtual sensors, to better characterize the neuronal population-level interactions under- lying the oscillatory responses. A brief overview of the mod- eling framework is included in the supplementary materials. MEG data were collected using a 275-channel CTF MEG system sampled at 1200 Hz. Datasets were epoched into trials of 4 s centered on stimulus onset. Trials were visually inspected for gross recording artifact on a trial- by-trial basis. Synthetic aperture magnetometry (SAM) beamformer20 analysis was used to compare activity for 2 s of baseline (−2 to 0 s) and 2 s of visual stimula- tion (0 to 2 s). Pseudo t-statistics were used to compute differences between baseline and stimulation activity. Volumetric SAM images were computed in a 30–80 Hz frequency band. The peak location of gamma ac- tivity in each participant’s visual cortex, defined by the peak occipital t-statistic, was located and virtual sensors constructed for these locations using covariance matrices low-pass filtered at 100 Hz. Time-frequency analysis of these virtual sensors used Hilbert envelopes of bandpass filtered data21 between 1 and 100 Hz in 0.5 Hz steps to produce spectra in which the responses at each time/fre- quency point were expressed as mean percentage change from baseline. The peak frequency and amplitude spec- tral features used for statistical analysis were derived by identifying the peak after averaging the Hilbert envelope at the time window of interest (0–300 ms for transient responses, 300–800 ms for sustained/induced responses). g pp y Within the CMC, responses were driven by endogenous afferent input to spiny stellate cells, formed of a mixture of scale free fluctuations. The CMC featured 4 inter- connected cell populations (layer 2/3 and 5/6 pyramidal cells, layer 4 spiny-stellate cells and multi-layer inhibitory interneurons; figure 5A), each modeled by parameterized first-order differential equations describing the evolu- tion of states (membrane potentials and postsynaptic currents). Model parameters control the strength of connections between the cell populations, the synaptic time constants of these interactions and parameters encoding the form of endogenous input. Gamma Acquisition and Analysis Gamma Acquisition and Analysis Gamma Acquisition and Analysis The MEG paradigm was based on the paradigm of Hoogenboom,19 where stimuli consisted of a centrally presented, circular sine wave grating (diameter 5°, spatial fre- quency 2 cpd and maximum contrast). The circular grating Table 1. Sample Ages by Group and Gender Group Number Mean Age (y) SD Age (y) Independent T-Test SZ Vs Controls (P value) All controls 29 41.1 10.1 All SZ 27 44.7 8.5 0.16 Female controls 11 40.2 12.1 Female SZ 8 45.9 8.0 0.3 Male controls 18 41.6 9.4 Male SZ 19 44.2 8.8 0.4 Table 1. Sample Ages by Group and Gender 346 Modeling GABAergic Inhibition in Schizophrenia Fig. 1. Group averaged, percentage-change time–frequency spectrograms, with visual grating pattern onset at 0 s. Both the transient and sustained gamma responses are evident, with marginal reductions in the SZ group, compared with the control group. Fig. 1. Group averaged, percentage-change time–frequency spectrograms, with visual grating pattern onset at 0 s. Both the transient and sustained gamma responses are evident, with marginal reductions in the SZ group, compared with the control group. Accordingly, peak frequency measures are in units of hertz (Hz), while amplitude measures are in units of percentage-change from baseline (%). drifted inwards toward the fixation point and the speed of this contraction increased (velocity step at 2.2 deg/s) at a randomized time of between 50 and 3000 ms after stimulus onset. The participant was instructed to press a button with their right index finger as soon as they noticed a speed change. Stimulus offset was followed by a resting period of 1000 ms, in which subjects were given visual feedback of either “OK,” “early,” or “late” depending on their response. For further details on this paradigm, see supplementary materials. Orientation Discrimination Performance Is Impaired in Schizophrenia Orientation Discrimination Performance Is Impaired in Schizophrenia Group SAM beamformer reconstructions—with weights optimized for the gamma range—demonstrated bilateral gamma increases in or close to the calcarine sulcus, con- sistent with a primary visual cortex generator (figure 2B). A 2-by-2 ANOVA was used to examine between group differences in orientation discrimination performance. This revealed a group-by-condition interaction effect [F(1, 28) = 8, P = .007]. A significant effect of condition (vertical vs oblique) demonstrated “the oblique-effect,” in Two-way repeated measures ANOVAs were used to deter- mine between-group differences in peak oscillatory features, Fig. 2. (A) Schematic of the stimuli and trial timings. (B) Group SAM beamformer reconstructions of the stimulus-induced gamma changes (30–80 Hz) during the sustained stimulus presentation time (50–3000 ms). For both participant cohorts, these group SAM maps are shown on an MNI template brain, for each of the three experimental runs. Bilateral gamma increases were seen in or close to the calcarine sulcus. (C) Quantification of SZ-related differences in gamma amplitude and frequency. These were assessed separately for the transient broadband gamma “spike” (0–300 ms) and the initial sustained gamma period (300–800 ms). Fi 2 (A) S h ti f th ti li d t i l ti i (B) G SAM b f t ti f th ti l i d d Fig. 2. (A) Schematic of the stimuli and trial timings. (B) Group SAM beamformer reconstructions of the stimulus-induced gamma changes (30–80 Hz) during the sustained stimulus presentation time (50–3000 ms). For both participant cohorts, these group SAM maps are shown on an MNI template brain, for each of the three experimental runs. Bilateral gamma increases were seen in or close to the calcarine sulcus. (C) Quantification of SZ-related differences in gamma amplitude and frequency. These were assessed separately for the transient broadband gamma “spike” (0–300 ms) and the initial sustained gamma period (300–800 ms). Fig. 2. (A) Schematic of the stimuli and trial timings. (B) Group SAM beamformer reconstructions of the stimulus-induced gamma changes (30–80 Hz) during the sustained stimulus presentation time (50–3000 ms). For both participant cohorts, these group SAM maps are shown on an MNI template brain, for each of the three experimental runs. Bilateral gamma increases were seen in or close to the calcarine sulcus. (C) Quantification of SZ-related differences in gamma amplitude and frequency. Neurophysiologically Informed Modeling This model has previously been used to recover perturbations of neu- ronal population-level dynamics induced by GABAergic manipulation following administration of tiagabine,22 making it a sensitive and specific model for assaying cor- tical GABAergic dynamics. Coupled with a weighted- contribution observation model,22 the model was inverted 347 A. D. Shaw et al A. D. Shaw et al to fit the spectral density of the virtual sensor over the 4–90 Hz range using DCM for cross spectral responses.23 to fit the spectral density of the virtual sensor over the 4–90 Hz range using DCM for cross spectral responses.23 calculated as percentage-change. A main effect of group was observed for sustained (induced) frequency [F(1, 29) = 4.15, P = .047] with the SZ group showing an estimated mean reduc- tion of 3 Hz (Controls mean = 58, SE = 0.92, SZ mean = 55, SE = 0.95). Trends in the same direction were observed for both transient amplitude [F(1, 29) = 3.6, P = .065] and sus- tained amplitude [F(1, 29) = 3.5, P = .068]. This is most ev- ident in the group-mean time-frequency representations of figure 3. No differences in prestimulus gamma features were observed between groups (supplementary materials). Statistical Analyses Details of the statistical analyses performed are described in supplementary materials. All results are Bonferroni corrected for multiple comparisons. Impairments of Superficial Pyramidal-Interneuron Connectivity Predict Negative Symptom Severity The strength of the G4 parameter showed a signifi- cant negative correlation with negative symptom score, as measured with the SANS (r = −.7, P = 5.7e-04, Bonferroni corrected 0.0046) (figure 4C). Group differences in synaptic connectivity parameters were assessed using repeated-measures ANOVA (left columns of table 2). There was no effect of session (radial 1–3) [group-by-session-by-parameter F(4.429) = 0.54, P = .73, parameter-by-session F(4.429) = 2.18, P = .06) but a parameter-by-group effect (F = 4.893, P < .001). Orientation Discrimination Performance Is Impaired in Schizophrenia These were assessed separately for the transient broadband gamma “spike” (0–300 ms) and the initial sustained gamma period (300–800 ms). 348 Modeling GABAergic Inhibition in Schizophrenia which performance is much poorer for oblique targets [F(1, 28) = 16.34, P < .001]. A significant effect of group revealed that the SZ group were significantly impaired (higher thresholds) across conditions [F(1, 28) = 20.9, P < .001]. We used JASP to compute Bayes factors (BF) for each parameter (right side of table 2, unpaired t-test, averaging over sessions as no session effect). This revealed that the G4 parameter had the highest BF (8.3, “moderate”), followed by G7 (BF = 2.7, “weak”). The relative evidence for G4 having a stronger effect than G7 (and hence any other pa- rameter) had a Bayes Factor of 3.0, ie, “moderate.” Impairments of Superficial Pyramidal-Interneuron Connectivity Predict Negative Symptom Severity P = survives Bonferroni correction. P = survives Bonferroni correction. Behaviorally Relevant, Disease-Related Dysconnectivity of Local GABA Function Main effects of group on parameter were observed for G4 (P = .005), G6 (P = .029), G7 (P = .021) and G12 (P = .05), table 2, indicating reductions of connec- tivity strength in the SZ group. Only G4 was significant after Bonferroni correction for the number of coupling parameters (8 parameters, G4 P = .04). The inhibitory connection between the inhibitory interneurons and the superficial pyramidal cell popu- lation (G11, shown in figure 5B) consistently predicted behavioral performance, in the SZ group, across the 3 MEG sessions. Bayes factors for each parameter Fig. 3. (A) Participants viewed a sequential presentation of 2 circular grating patches, each presented for 250 ms at a frame rate of 80 Hz, with a grating contrast of 80%, a spatial frequency of 3 cycles/degree, a mean luminance of 44.5 cd/m2 and subtending 4° of visual angle. Participants were instructed to respond which of the 2 sequentially presented gratings was oriented more to the right in a 2-alternative forced choice design. (B) Results of the task for SZ and controls in both vertical and oblique conditions. Fig. 3. (A) Participants viewed a sequential presentation of 2 circular grating patches, each presented for 250 ms at a frame rate of 80 Hz, with a grating contrast of 80%, a spatial frequency of 3 cycles/degree, a mean luminance of 44.5 cd/m2 and subtending 4° of visual angle. Participants were instructed to respond which of the 2 sequentially presented gratings was oriented more to the right in a 2-alternative forced choice design. (B) Results of the task for SZ and controls in both vertical and oblique conditions. Table 2. Group Effects on Parameters Table 2. Group Effects on Parameters Table 2. Group Effects on Parameters Parameter F (ANOVA) P (ANOVA) BF (T-Test) BF (Best)/BF G4 8.583 0.005* 8.3 1.0 G7 5.683 0.021 2.7 3.0 G6 5.0773 0.029 2.1 3.9 G12 4.039 0.05 1.4 5.8 G8 0.686 0.411 0.4 22.7 G9 0.561 0.457 0.3 23.9 G11 0.449 0.506 0.3 25.0 G5 0.048 0.828 0.3 29.5 349 A. D. Shaw et al Fig. 4. (A) DCM of the MEG data was performed using a CMC comprising 4 neural populations, with plausible neurophysiological connections and intrinsic time-constants. (B) Demonstrating the difference in synaptic connection strengths between SZ and controls. Gamma and Task Performance Deficits Peak gamma frequency was reduced in SZ for sustained- induced (nonstimulus locked) responses, and a trend was observed in the same direction for amplitude, however this did not survive Bonferroni correction (corrected P = .065). Both the frequency and amplitude of gamma oscillations in visual cortex are thought to reflect the encoding of stimulus properties27 and feature integration,28 while peak frequency is known to depend on both the size of primary visual cortex (V1)29 and the density of GABA receptors in V1.30 As such, gamma frequency is interesting in the con- text of perceptual diseases such as SZ (reviewed in ref.31), because it represents an intermediate phenotype linking cortical physiology and cognition. Under the disconnection hypothesis of SZ, hypofunction of NMDA receptors leads to reduced acti- vation of GABAergic interneurons, which in turn results in disinhibition of pyramidal cells.5,24 This aberrant state means impaired synchrony, which in turn suggests im- paired oscillatory activity. We demonstrate significant reductions in the peak frequency of induced gamma oscillations in SZ (corrected P = .047), along with trends toward reduced amplitude (corrected P = .065), in ac- cordance with previous reports.25 Moreover, as discussed in the supplementary materials, we demonstrated a re- duction in the total concentration of GABA in the same region; however, this did not correlate with reductions of gamma frequency (r = −.1) or amplitude (r = −.13). Reduced peak gamma frequency in SZ has been re- ported during perception of Gestalt visual stimuli, using EEG,32 leading to the proposition that reduced oscillatory frequency is a specific index of impaired feature-binding processes.32 Our results extend these neural correlates of perception; demonstrating reductions in primary visual cortex gamma frequency in SZ are also induced by inward-drifting annular grating stimuli. Employing a neurobiologically grounded neuronal model to examine population interactions underlying visual cortical responses, we find reductions in key syn- aptic parameters in SZ (in silico), which include inhibi- tory modulation of inhibitory interneuron and superficial pyramidal populations as well as excitatory projections from deep pyramidal to interneuron populations, al- though only the former survive correction for multiple comparisons. Notably, the synaptic parameters related to granular and deep cells (layers 4, 5/6) did not show this association with gamma. The amplitude of the initial evoked gamma “spike” demonstrated a trend to toward reduction in the SZ group, however this did not survive correction for multiple comparisons (corrected P = .065). Behaviorally Relevant, Disease-Related Dysconnectivity of Local GABA Function (C) The strength of inhibitory self-modulation of inhibitory interneuron populations predicts negative symptom score (SANS) in SZ (r = −.7, P = 5.7e-4, Bonferroni corrected P = .0046). MEG data was performed using a CMC comprising 4 neural populations, with plausible neurophysiological time-constants. (B) Demonstrating the difference in synaptic connection strengths between SZ and controls. tory self-modulation of inhibitory interneuron populations predicts negative symptom score (SANS) in SZ erroni corrected P = .0046). Downloaded from https://academic.oup.com/schizophreniabulletin/article-abstract/46/2/345/5521107 by Acquisitions user on 02 Marc Fig. 4. (A) DCM of the MEG data was performed using a CMC comprising 4 neural populations, with plausible neurophysiological connections and intrinsic time-constants. (B) Demonstrating the difference in synaptic connection strengths between SZ and controls. (C) The strength of inhibitory self-modulation of inhibitory interneuron populations predicts negative symptom score (SANS) in SZ (r = −.7, P = 5.7e-4, Bonferroni corrected P = .0046). Fig. 5. Demonstrating the negative correlation between synaptic coupling strength of inhibitory interneurons and superficial pyramidal cells (G11) and performance on each of the trials, for both vertical and oblique conditions, for the SZ group. etin/article-abstract/46/2/345/5521107 by Acquisitions user on 02 March 2020 Fig. 5. Demonstrating the negative correlation between synaptic coupling strength of inhibitory interneurons and superficial pyramidal cells (G11) and performance on each of the trials, for both vertical and oblique conditions, for the SZ group. Fig. 5. Demonstrating the negative correlation between synaptic coupling strength of inhibitory interneurons and superficial pyramidal cells (G11) and performance on each of the trials, for both vertical and oblique conditions, for the SZ group. G11 over all other parameters is strong in all oblique sessions (BFG11/BF >12). fi h l i b demonstrated strong (oblique session 1 and 2), mod- erate (oblique session 3) and anecdotal evidence (ver- tical sessions 1–3) for G11, but no evidence for any other parameters (table 3), with anecdotal evidence in some for the null. Note that the relative evidence for In figures 5C–E, the correlation between G11 parameter and behavioral performance is shown for the oblique ver- sion of the task. Cases with stronger inhibitory connections 350 Modeling GABAergic Inhibition in Schizophrenia Table 3. Discussion Using MEG imaging and DCM to “computationally assay” cortical GABAergic inhibition in the visual cortex in SZ, we have demonstrated that synaptic disinhibi- tion of superficial layer pyramidal populations (in silico) predicts performance during an orientation discrimina- tion task, in a graded and disease-specific manner. Behaviorally Relevant, Disease-Related Dysconnectivity of Local GABA Function Bayes Factors for Predicting Performance in the SZ Group Parameter G4 G5 G6 G7 G8 G9 G11 G12 Oblique Session 1 0.264 0.329 0.259 0.288 0.31 0.392 11.354a 0.297 Session 2 0.366 0.267 0.319 0.281 0.302 0.696 12.029a 0.262 Session 3 0.521 0.28 0.32 0.415 0.495 0.702 8.482b 0.275 Vertical Session 1 0.259 0.364 0.314 0.28 0.289 0.321 1.239 0.285 Session 2 0.288 0.3 0.259 0.329 0.275 0.421 1.499 0.372 Session 3 0.343 0.259 0.89 0.498 0.349 0.967 2.961 0.77 aBF > 10 = “strong” evidence. bBF3-10 = “moderate.” Table 3. Bayes Factors for Predicting Performance in the SZ Group g bBF3-10 = “moderate.” find that the synaptic strength of inhibitory connections from inhibitory interneurons to superficial layer pyram- idal cells (II → SP, G11, figure 5) predicts performance across 3 runs of the task. Strikingly, this parameter corresponds to the aforementioned mechanism of disin- hibition and has been reported as a key determinant of gamma frequency.22,26 As such, our findings formalize a framework whereby disturbances of gamma, and impairments in orientation discrimination performance may be understood as consequences of GABAergically mediated dysconnectivity in SZ. (II → SP) were better at the task, with this model param- eter explaining 44%, 40%, and 32% of the variance in the behavioral performance when derived from MEG runs 1, 2, and 3, respectively. By using the 3 runs separately, we demonstrate repeatability of this result within the sample. These model results demonstrate the key importance of local, GABA-mediated, connectivity in determining low-level visual performance in this SZ cohort. Supplementary Material Supplementary data are available at Schizophrenia Bulletin online. Gamma and Task Performance Deficits Given reduced ampli- tude reflects reduced synchrony, this trend aligns with the aforementioned NMDAR mediated disinhibition hy- pothesis, whereby NMDAR hypofunction leads to disin- hibition of pyramidal cells5 by GABAergic interneurons, which leads to aberrant activity, and therefore reduced Assessing the relationship between synaptic parameters and performance during orientation discrimination, we 351 A. D. Shaw et al A. D. Shaw et al synchrony. This finding is also in accordance with pre- vious reports of reduced gamma amplitude in SZ during face processing,33 suggesting this result is generalizable to all visual processing and not stimulus-specific. The age range (22–58) could be seen as introducing un- necessary variance to the neuroimaging signals (eg, ref.37). However, a broad age range means our sample is heter- ogenous and inclusive of the individual differences found within the normal population. In this respect, our results should have increased generalizability to the population. It is plausible that the SZ group differed from the controls in their attentiveness to the MEG visual par- adigm, despite the button press required on each trial. However, attention has been shown not to modulate sus- tained low-frequency gamma oscillatory responses in pri- mary (V1) visual cortex.34 The relative simplicity of the CMC model used in this study was guided by existing literature.22,38,39 While com- plex enough to enable identification of the key synaptic determinants of oscillatory features, it lacks the complexity of multicompartment and detailed spatial models such as the Human Neocortical Neurosolver (https://hnn.brown.edu/). Acknowledgments We thank Professor Peter Uhlhaas and Dr Heng-Ru May Tan for providing the moving visual stimulus. Drs Knight, Shaw, Freeman, Williams, Friston, Moran, Walters, and Singh all reported no biomedical financial interests or po- tential conflicts of interest. Funding This work was supported by CUBRIC and the Schools of Psychology and Medicine at Cardiff University, the MRC/EPSRC funded UK MEG Partnership Grant (MR/K005464/1). A.D.S. and G.M.W. are supported by a Wellcome Trust Strategic Award (104943/Z/14/Z). K.J.F. is funded by a Wellcome Trust Principal Research Fellowship (088130/Z/09/Z). ( ) In the SZ group, the GABAergic synaptic connection from interneurons to superficial pyramidal cells (G11) was found to predict orientation discrimination performance across 3 repeated runs, although parameter Bayes factors indicated strong and moderate evidence only for the oblique (hard) condition. This is important, because this synaptic parameter is instrumental in the generation of gamma oscillations,10,26 mediated by GABAergic inhibition and forms a key component of the disinhibition/dysconnectivity hypothesis. Thus, its ability to predict perceptual perfor- mance links noninvasive evidence of the dysconnectivity hy- pothesis to behavior in vivo, for the first time. Conclusion Neurophysiologically informed modeling suggested local dysconnectivity in SZ, mediated by reduced inhibitory gain control of inhibitory interneuron populations (G4, corrected P = .04, BF = 8.3), and trends toward reduced inhibitory gain control of superficial pyramidal cells (G7, P = .021, corrected P = ns) and the excitatory projection from deep layer pyram- idal cells to inhibitory interneurons (G6, P = .029, corrected P = ns). Both G4 and G7 are inhibitory connections located in superficial cortical layers and are major determinants of gamma amplitude and frequency, respectively.22 In conclusion, these results establish—for the first time—a 4-way construct validity between (1) a visual psychophysical test dependent on local gain control, (2) a noninvasive syn- aptic assay (DCM), (3) a neurotransmitter deficit (GABA MRS), and (4) the functional anatomy of SZ (in terms of dysconnectivity that speaks to cortical gain control and fast synchronous activity). Crucially, all of these measures offer noninvasive endophenotypes or biomarkers—that may find a powerful application in schizophrenia research. Interactions of these populations underlie the “disinhi- bition hypothesis,”5,35 whereby hypofunction of NMDA receptors on interneurons results in pyramidal cells reaching a disinhibited, aberrant state. Thus, within the context of our model, we find evidence linking the disin- hibition hypothesis of SZ with altered gamma oscillations. An analysis of parameter Bayes factors, to assess each parameter in explaining group, revealed that the G4-only model had the most evidence (BF = 8.3), with moderate evidence that this was better than the next best parameter, G7 (BFG4/BFG7 = 3.1). Modeling GABAergic Inhibition in Schizophrenia 22. Shaw AD, Moran RJ, Muthukumaraswamy SD, et al. Neurophysiologically-informed markers of individual vari- ability and pharmacological manipulation of human cortical gamma. Neuroimage. 2017;161:19–31. 4. Stephan KE, Friston KJ, Frith CD. Dysconnection in schizo- phrenia: from abnormal synaptic plasticity to failures of self- monitoring. Schizophr Bull. 2009;35(3):509–527. 5. Homayoun H, Moghaddam B. NMDA receptor hypofunction produces opposite effects on prefrontal cortex interneurons and pyramidal neurons. J Neurosci. 2007;27(43):11496–11500. 23. 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https://openalex.org/W4248405143	http://vital.lib.tsu.ru/vital/access/services/Download/vtls:000644507/SOURCE1	English	null	THE INFLUENCE OF PYROLYSIS CONDITIONS ON HYDROCARBONS COMPOSITION OF THE SHALE OIL (ALEKSINAC OIL SHALE, SERBIA)	Perspektivnye materialy s ierarkhicheskoy strukturoy dlya novykh tekhnologiy i nadezhnykh konstruktsiy» i «Khimiya nefti i gaza» v ramkakh Mezhdunarodnogo simpoziuma «Ierarkhicheskie materialy: razrabotka i prilozheniya dlya novykh tekhnologiy i nadezhnykh konstruktsiy	2,018	cc-by	867	DOI: 10.17223/9785946217408/556 THE INFLUENCE OF PYROLYSIS CONDITIONS ON HYDROCARBONS COMPOSITION OF THE SHALE OIL (ALEKSINAC OIL SHALE, SERBIA) 1Gajica G., 1Šajnović A., 2Stojanović K., 2Jovančićević B. 1University of Belgrade, Institute of Chemistry, Technology and Metallurgy, Center of Chemistry, Belgrade, Serbia 2University of Belgrade, Faculty of Chemistry, Belgrade, Serbia ggajica@chem.bg.ac.rs, sajnovica@chem.bg.ac.rs, ksenija@chem.bg.ac.rs, bjovanci@chem.bg.ac.rs 1Gajica G., 1Šajnović A., 2Stojanović K., 2Jovančićević B. 1University of Belgrade, Institute of Chemistry, Technology and Metallurgy, Center of Chemistry, Belgrade, Serbia 2University of Belgrade, Faculty of Chemistry, Belgrade, Serbia ggajica@chem.bg.ac.rs, sajnovica@chem.bg.ac.rs, ksenija@chem.bg.ac.rs, bjovanci@chem.bg.ac.rs The amount and the composition of liquid hydrocarbons (HCs) obtained by pyrolysis of oil shale depend on kerogen type, as well as pyrolytic system and conditions [1]. The aim of this study was to compare HCs composition of bitumen isolated from raw oil shale samples (osh) and shale oils obtained by pyrolysis of oil shales in an open system (os) and close system (cs) (Table 1). Investigation has been made on immature outcrop oil shale samples (vitrinite reflectance of 0.41 % Rr) from the Aleksinac deposit (Serbia). Pyrolysis experiments were performed on the two selected samples, which have shown the highest quantity of total organic carbon (TOC > 13 %) and high HCs generation potential (Hydrogen Index, HI > 615 mg HCs/g TOC) in the studied sample set [2]. The HCs composition of the shale oils obtained by open system pyrolysis indicates low maturity. They are similar to distributions of HCs in bitumens isolated from raw (initial) oil shales (Table 1). Therefore open system pyrolysis can be useful for assessment of source and depositional environment of organic matter. On the other head, shale oils obtained by pyrolysis in the close system have distributions of HCs which correspond to higher maturity and they are similar to composition of HCs in crude oil, generated in early stage of “oil window” (Table 1). Therefore, for artificial generation of shale oil (from immature oil shale), having composition comparable to crude oil, the close system pyrolysis is required. DOI: 10.17223/9785946217408/556 THE INFLUENCE OF PYROLYSIS CONDITIONS ON HYDROCARBONS COMPOSITION OF THE SHALE OIL (ALEKSINAC OIL SHALE, SERBIA) 1Gajica G., 1Šajnović A., 2Stojanović K., 2Jovančićević B. 1University of Belgrade, Institute of Chemistry, Technology and Metallurgy, Center of Chemistry, Belgrade, Serbia 2University of Belgrade, Faculty of Chemistry, Belgrade, Serbia ggajica@chem.bg.ac.rs, sajnovica@chem.bg.ac.rs, ksenija@chem.bg.ac.rs, bjovanci@chem.bg.ac.rs Table 1 – Source and maturity parameters of bitumen isolated from raw oil shale (osh) and shale o obtained by open (os) and close system (cs) pyrolysis Source parameters Maturity parameters Sample CPI (C15-C35) %C27 ααα(R)- sterane %C28 ααα(R)- sterane %C29 ααα(R)- sterane Steranes/ Hopanes C29 ααα 20S/ (20S+20R)- steranes C29αββ/(αββ+ ααα)- steranes Rc (%) C31 αβ 22S/(22S+22R) -hopanes C30 βα/C30αβ- hopanes D13 osh 1.87 20.07 42.29 37.65 5.29 0.14 / 0.41 0.11 1.93 D16 osh 1.37 32.74 20.80 46.47 0.50 0.07 / 0.36 0.12 0.43 D13 os 1.13 25.96 45.60 28.44 0.33 0.18 / 0.44 0.30 4.25 D16 os 1.08 37.26 25.10 37.65 0.07 0.12 / 0.39 0.23 2.27 D13 cs 1.03 30.20 33.62 36.18 0.31 0.47 0.50 0.76 0.55 0.74 D16 cs 1.06 34.60 30.22 35.18 0.26 0.55 0.57 0.92 0.54 0.27 urce and maturity parameters of bitumen isolated from raw oil shale (osh) and shale oil obtained by open (os) and close system (cs) pyrolysis CPI – Carbon Preference Index, calculated from distributions of n-alkanes; Rc – calculated vitrinite reflectance = 0.49 x C29ααα20S/20R + 0.33; / – Not determined due to the absence of C29 αββ 20R- sterane in these samples. R f CPI – Carbon Preference Index, calculated from distributions of n-alkanes; Rc – calculated vitrinite reflectance = 0.49 x C29ααα20S/20R + 0.33; / – Not determined due to the absence of C29 αββ 20R- sterane in these samples. ТЕЗИСЫ ДОКЛАДОВ Секция С. Рациональное использование углеводородного сырья DOI: 10.17223/9785946217408/556 THE INFLUENCE OF PYROLYSIS CONDITIONS ON HYDROCARBONS COMPOSITION OF THE SHALE OIL (ALEKSINAC OIL SHALE, SERBIA) 1Gajica G., 1Šajnović A., 2Stojanović K., 2Jovančićević B. 1University of Belgrade, Institute of Chemistry, Technology and Metallurgy, Center of Chemistry, Belgrade, Serbia 2University of Belgrade, Faculty of Chemistry, Belgrade, Serbia ggajica@chem.bg.ac.rs, sajnovica@chem.bg.ac.rs, ksenija@chem.bg.ac.rs, bjovanci@chem.bg.ac.rs 2. Gajica G., Šajnović A., Stojanović K., Kostić A., Slipper I., Antonijević M., Nytoft H.P., Jovančićević, B. Organic geochemical study of the upper layer of Aleksinac oil shale in the Dubrava block, Serbia // Oil Shale. – 2017. – No 3. Pp. 197–218. 1. Sari A., Aliyev S.A. Organic geochemical characteristics of the Paleocene–Eocene oil shales in the Nallıhan Region, Ankara, Turkey // Journal of Petroleum Science and Engineering. – 2006. – No. 1-2. Pp. 123–134. CPI – Carbon Preference Index, calculated from distributions of n-alkanes; Rc – calculated vitrinite reflectance = 0.49 x C29ααα20S/20R + 0.33; / – Not determined due to the absence of C29 αββ 20R- sterane in these samples. References 1. Sari A., Aliyev S.A. Organic geochemical characteristics of the Paleocene–Eocene oil shales in the Nallıhan Region, Ankara, Turkey // Journal of Petroleum Science and Engineering. – 2006. – No. 1-2. Pp. 123–134. Š 2. Gajica G., Šajnović A., Stojanović K., Kostić A., Slipper I., Antonijević M., Nytoft H.P., Jovančićević, B. Organic geochemical study of the upper layer of Aleksinac oil shale in the Dubrava block, Serbia // Oil Shale. – 2017. – No 3. Pp. 197–218. 809
https://openalex.org/W4315640719	https://www.frontiersin.org/articles/10.3389/fpls.2022.1072232/pdf	English	null	Transposable elements are associated with genome-specific gene expression in bread wheat	Frontiers in plant science	2,023	cc-by	15,383	OPEN ACCESS OPEN ACCESS EDITED BY Gennady I. Karlov, All-Russia Research Institute of Agricultural Biotechnology, Russia REVIEWED BY Zujun Yang, University of Electronic Science and Technology of China, China Nikolai Borisjuk, Huaiyin Normal University, China CORRESPONDENCE Khalil Kashkush kashkush@bgu.ac.il SPECIALTY SECTION This article was submitted to Plant Systems and Synthetic Biology, a section of the journal Frontiers in Plant Science RECEIVED 17 October 2022 ACCEPTED 20 December 2022 PUBLISHED 12 January 2023 EDITED BY Gennady I. Karlov, All-Russia Research Institute of Agricultural Biotechnology, Russia Inbar Bariah, Liel Gribun and Khalil Kashkush Inbar Bariah, Liel Gribun and Khalil Kashkush* Department of Life Sciences, Ben-Gurion University, Beer-Sheva, Israel Introduction: Recent studies in wheat emphasized the importance of TEs, which occupy ~85% of the wheat genome, as a major source of intraspeciﬁc genetic variation due to their recent activity and involvement in genomic rearrangements. The contribution of TEs to structural and functional variations in bread wheat genes is not fully understood. Methods: Here, publicly available RNA-Seq databases of bread wheat were integrated to identify TE insertions within gene bodies (exons\ introns) and assess the impact of TE insertions on gene expression variations of homoeologs gene groups. Overall, 70,818 homoeologs genes were analyzed: 55,170 genes appeared in each one of the three subgenomes (termed ABD), named triads; 12,640 genes appeared in two of the three subgenomes (in A and B only, termed AB; or in A and D only, termed AD; or in B and D only, termed BD);, named dyads; and 3,008 genes underwent duplication in one of the three subgenomes (two copies in: subgenome A, termed AABD; subgenome B, termed ABBD; or subgenome D, termed ABDD), named tetrads. Bariah I, Gribun L and Kashkush K (2023) Transposable elements are associated with genome-speciﬁc gene expression in bread wheat. Front. Plant Sci. 13:1072232. doi: 10.3389/fpls.2022.1072232 © 2023 Bariah, Gribun and Kashkush. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Results: To this end, we found that ~36% of the 70,818 genes contained at least one TE insertion within the gene body, mostly in triads. TYPE Original Research PUBLISHED 12 January 2023 DOI 10.3389/fpls.2022.1072232 TYPE Original Research PUBLISHED 12 January 2023 DOI 10.3389/fpls.2022.1072232 TYPE Original Research PUBLISHED 12 January 2023 DOI 10.3389/fpls.2022.1072232 transposable elements, wheat, genome evolution, allopolyploidy, genome-speciﬁc, Triticum aestivum, gene expression, copy number variation 1 Introduction allopolyploidization in wheat (ampliﬁcation bursts vs. slow accumulation), there is no doubt that rapid TE turnover occurred during the wheat group evolution (Wicker et al., 2018; Bariah et al., 2020). The great contribution of TEs to genome plasticity might affect the ability of the new polyploid species to survive and rapidly adapt to various biological, environmental, and even cultivation environment stress (Van De Peer et al., 2017; Levy and Feldman, 2022). Transposable elements (TEs) are a major component of plant genomes (Mhiri et al., 2022), e.g., they account for ~85% of the bread wheat genome (Appels et al., 2018; Wicker et al., 2018). Once thought of as “junk DNA” and “parasites”, today, a growing body of evidence reveals that TEs have a prominent role in genome evolution (Avni et al., 2017; Bourque et al., 2018; Dubin et al., 2018). TEs are capable of moving and increasing their copy number within the host genome mainly through copy and paste (Class I, retrotransposons) or cut and paste (Class II) mechanisms (Wicker et al., 2007). The highly repetitive nature, high abundance, and activity of TEs might trigger massive structural genomic rearrangements (Gray, 2000; Bourque et al., 2018; Krasileva, 2019). They are considered a great source for genetic variation, mainly creating new alleles by transposing within gene bodies (Bourque et al., 2018; Dubin et al., 2018). y The huge number of TE insertions adjacent to wheat genes led researchers to investigate the possible role of TEs in gene regulation (Wicker et al., 2018; Keidar-Friedman et al., 2020). Wicker et al. (2018) found no strong associations between speciﬁc TE families found near promoters and various expression modules. Additionally, a study by Ramı́rez- González et al., 2018 focused on the effect of TE insertions within triads (homeologs with a 1:1:1 correspondence across the three bread wheat sub-genomes- ABD) genes promoters and found no correlation between the presence of TEs in gene promoters and altered expression patterns between the three homeolog genes. However, Ramı́rez-González et al., 2018 did observe that higher TE abundance in the vicinity of the translation start site correlated with triads that showed more dynamic expression patterns across different tissues. This observation led Ramı́rez-González et al., 2018 to suggest a possible role for TEs in gene regulation as cis-regulatory elements or through other epigenetic mechanisms in a tissue- speciﬁc manner. 1 Introduction Moreover, recent studies showed that TEs, speciﬁcally MITEs (Miniature Inverted repeat TEs), which are prevalent in the vicinity of wheat genes, might act as miRNAs precursors in wheat and thus can potentially shape regulatory gene networks (Poretti et al., 2019; Crescente et al., 2022). While the effect of TE insertions into promoter regions in wheat has been well-investigated, very little is known about the possible effect of TE insertions within gene bodies (Li et al., 2014; Xi et al., 2016; Keidar et al., 2018; Keidar-Friedman et al., 2018; Domb et al., 2019; Jiang et al., 2019). Here, the analysis of a large amount of publicly available databases in bread wheat facilitated the assessment of the functional impact of TE insertions within gene bodies in a genome-speciﬁc manner. The high abundance of TEs near and within plant genes might impact the function of those genes by inﬂuencing both transcriptional and post-transcriptional levels and lead to the creation of novel transcripts (Schrader and Schmitz, 2019; Bariah et al., 2020; Zhang et al., 2021). The mere presence of TE, adjacent or within the transcribed region, might result in gene downregulation or silencing due to epigenetic modiﬁcations or interfering with enhancers or regulatory promoter elements (Dubin et al., 2018). Furthermore, TEs contain regulatory sequences such as promoters, transcription factors binding sits, and target sites for post-transcriptional degradation, which might affect adjacent gene expression or even modulate gene expression through complex transcriptional regulatory networks (Bourque et al., 2018; Dubin et al., 2018; Qiu and Köhler, 2020; Zhang et al., 2021). Additionally, the insertion of TE into a gene body might result in the creation of new isoforms through exonization, truncation, alternative splicing, or even by the domestication of TE-derived coding sequences into host genes, potentially altering the gene function (Keidar et al., 2018; Poretti et al., 2019; Crescente et al., 2022). Wheat (Triticum- Aegilops group) is among the world’s most widely grown crops, providing a signiﬁcant portion of daily human caloric intake (Shewry and Hey, 2015; Levy and Feldman, 2022). The most widely grown bread wheat, Triticum aestivum, is a relatively new polyploid species that has been generated by two subsequent allopolyploidization events between members of two closely related genera, Triticum and Aegilops (Avni et al., 2017; Appels et al., 2018; Levy and Feldman, 2022). Allopolyploidization is the only mechanism that enables the formation of a new species in one step (Feldman and Levy, 2005). OPEN ACCESS Analysis of 14,258 triads revealed that the presence of TE insertion in at least one of the triad genes (7,439 triads) was associated with balanced expression (similar expression levels) between the homoeolog genes. TE insertions within the exon or in the untranslated regions (UTRs) of one or more of the homoeologs in a triad were signiﬁcantly associated with homoeolog expression bias. Furthermore, we found a statistically signiﬁcant correlation between the presence\absence of TEs insertions belonging to six TE superfamilies and 17 TE subfamilies and the suppression of a single homoeolog gene. A signiﬁcant association was observed between the presence of TE insertions from speciﬁc superfamilies and the expression of genes that are associated with biotic and abiotic stress responses. Conclusion: Our data strongly indicate that TEs might play a prominent role in controlling gene expression in a genome-speciﬁc manner in bread wheat. Frontiers in Plant Science 01 frontiersin.org Bariah et al. 10.3389/fpls.2022.1072232 1 Introduction The rapid genomic structural and functional alterations accompanied with an allopolyploidization process have been intensively studied in recent years (Ramı́rez-González et al., 2018; Salina and Adonina, 2018; Fox et al., 2020; Juery et al., 2020). While currently there is still a debate regarding the extent of TE activity following 2.3 Correlation between polymorphic TE insertions within gene bodies and homoeolog expression bias Following the identiﬁcation of TE insertions within gene bodies and the characterization of TE insertions distribution, we wanted to assess the polymorphism(s) generated by TE insertions between the homoeologous copies in dyads, triads, and tetrads. For this, we used the pandas Python package (RRID : SCR_018214) (Reback et al., 2021) to organize the genes (see Supplementary Table S1) as homoeologous groups (Supplementary Table S2) according to the homoeologous group IDs and to sum the number of genes which contained one or more TE insertion within the gene body in each expressed homoeologous group (a group that includes one or more expressed gene, not expressed groups were removed from the analysis). For each of the homoeologous groups, we determined whether it was a polymorphic or monomorphic group. If all the homoeologs in a speciﬁc homoeologous group contained TE insertion (not depending on TE type or insertion location within the gene), the homoeologous group was considered as monomorphic. However, if one or more, but not all, of the homoeologs in the group contained TE insertion, the homoeologous group was considered polymorphic. To assess the possible impact of TE insertional polymorphism within gene bodies on the relative gene expression in the homoeologous groups, we used summarized data “relative contribution category in brief” retrieved from Juery et al. (2020), on the assignment of each of the homoeologous groups to relative contribution categories. Based on this analysis, if all the homoeologs in a speciﬁc homoeologous group had similar relative abundance, the group was assigned to the balanced category, while groups in which different relative abundance was observed between the homoeologs were assigned to one of the non-balanced categories (Juery et al., 2020). Speciﬁcally, triads were assigned to the balanced category, homoeolog-suppressed category, or homoeolog-dominant category, dyads were assigned to a balanced category or homoeolog-suppressed category, and tetrads were assigned to either one of the following categories: balanced category, tetrads with one suppressed copy, tetrads with two suppressed copies, and tetrads with one dominant copy (Juery et al., 2020). Additionally, some of the homoeologous groups were referred to as not expressed and thus were excluded from further analysis. The assignment of the homoeologous groups to relative contribution categories was performed according to the calculation method described by Ramı́rez- González et al. (2018) and based on the same RNA-seq data used by Ramı́rez-González et al. 2.1 Identiﬁcation of TE insertions within gene bodies To identify TE insertions within gene bodies (exons\ introns) in the Chinese Spring bread wheat cultivar (CS42), we integrated data from two publicly available databases (Appels et al., 2018; Juery et al., 2020). The name, homoeologous group IDs, assignment to one of the ﬁve chromosomal regions (R1, R2a, C, R2b, and R3), and the start and stop positions of 70,818 wheat genes belonging to 6,320 dyads (12,640 genes belongs to Frontiers in Plant Science 02 frontiersin.org frontiersin.org 10.3389/fpls.2022.1072232 Bariah et al. homoeologous groups ﬁt the expected numbers calculated based on the proportions of gene bodies that contain TE insertions. The expected number of monomorphic and polymorphic homoeologous groups was calculated assuming a binomial distribution of the presence \ absence of TE insertions within a gene body. First, the probability of a single gene including TE insertion was calculated based on the number of genes containing TE insertions and belonging to TE containing homoeologous group and the total number of genes belonging to TE containing homoeologous group. Then, the expected number of monomorphic and polymorphic homoeologous groups was calculated according to binomial distribution using the probability of a single gene including TE insertion and then divided by the probability of a homoeologous group to have at least one TE insertion (conditional probability) and multiplied by the number of groups containing one or more TE insertion. The observed numbers of polymorphic and monomorphic homoeologous groups were compared with the calculated expected numbers using the chisquare function from Python SciPy (RRID : SCR_008058). Stats module (Virtanen et al., 2020). homoeologous groups that underwent elimination of a single gene), 18,390 triads (55,170 genes), and 752 tetrads (3,008 genes, belongs to homoeologous groups that underwent duplication of a single gene) were retrieved from Juery et al. (2020) and integrated with the IWGSC RefSeq v1.0 assembly coordinates for TEs (Appels et al., 2018) using python 3.7 (Guttag, 2021) scripts. Prior to the data integration, the IWGSC RefSeq v1.0 assembly annotations for TEs were organized using pandas, a Python package used for data analysis (Reback et al., 2021), and ﬁltered to include only repeats deﬁned as “repeat region” (nested repeats and repeat fragments were removed). Next, overlaps between repeat regions and each of the 70,818 genes were detected based on the genes and TEs coordinates and summarized in Supplementary Table S1. 2.1 Identiﬁcation of TE insertions within gene bodies To compare the proportions of TE-containing genes between dyads, triads, and tetrads, the Chi-square test of independence of variables was performed using the chi2_contingency function from Python SciPy (RRID : SCR_008058). Stats module (Virtanen et al., 2020). Frontiers in Plant Science 2.4 Correlation between TE insertion within gene bodies and homoeolog expression bias in triads Here we used data on the relative expression abundance of the homoeologs in each of the triads (Ramı́rez-González et al., 2018) and the IWGSC RefSeq v1.0 assembly annotations for genes and TEs (Appels et al., 2018) to assess the possible impact of TE insertions on gene expression variations of homoeologous groups. We used 55,422 genes that had a 1:1:1 correspondence across the three homoeologous subgenomes (A, B, and D) of bread wheat (18,474 homoeolog triads) from Ramı́rez-González et al. (2018) and identiﬁed TE insertion within each of the genes bodies as described in Supplementary Table S1 (see Supplementary Table S3). For each of the triads, the TE classiﬁcation (superfamily and subfamily) was determined for elements that were found to be inserted within the gene bodies of the genes in the triad (Supplementary Tables S4, S5). TE subfamily names were according to the ClariTeRep naming system (Wicker et al., 2018), in which the three ﬁrst letters of the subfamily name represent the TE superfamily, and the number at the end of the name represents the family and in some cases is followed by a dot and a number, which represents speciﬁc subfamily within the TE family. For the following analysis, the three homoeolog genes in each one of the 14,258 triads were combined, meaning that a triad was referred to as a triad that included TE insertions if one or more TE insertions were found within the sequence of at least one of the triad genes. The dependency between the presence\ absence of TE insertions within the gene bodies, exons, or UTRs and different contribution categories was analyzed using the Chi-square test of independence of variables with the chi2_contingency function from Python SciPy. Stats module (Virtanen et al., 2020). The dependency between the TE superfamilies\ subfamilies from which insertions were present\ absent in at least one of the triad genes and the different contribution categories was analyzed using the Chi-square test of independence of variables with the chi2_contingency function from Python SciPy. Stats module and corrected for multiple testing using the multipletests function from the Python statsmodels module (RRID : SCR_016074) with the Benjamini/ Hochberg Procedure (non-negative) (Virtanen et al., 2020). 2.3 Correlation between polymorphic TE insertions within gene bodies and homoeolog expression bias (2018) for 123 samples of bread wheat (Chinese Spring) taken from 15 different tissues under To test whether TE insertions were randomly distributed between the genes or rather tend to be more\ less polymorphic than expected, we focused only on homoeologous groups that included TE insertions in one or more of the homoeologs gene bodies (referred to as homoeologous groups that include TE insertions) and were determined to be expressed (include one or more expressed gene). Then, we performed the Chi-square Goodness of Fit Test separately for dyads, triads, and tetrads, to test whether the numbers of monomorphic and polymorphic Frontiers in Plant Science 03 frontiersin.org Bariah et al. 10.3389/fpls.2022.1072232 10.3389/fpls.2022.1072232 above categories based on ternary diagrams representing the relative expression of each homoeolog and by comparison to the ideal normalized expression bias for the seven categories as described by Ramı́rez-González et al., (Ramı́rez-González et al., 2018). The analysis was performed for RNA-seq data from several different studies (Ramı́rez-González et al., 2018) (a total of 850 wheat RNA-sequencing samples), which were organized into partly overlapping datasets. Here we focused on data generated from 123 RNA-Seq samples of bread wheat (Chinese Spring) (Ramı́rez-González et al., 2018). The 123 samples were derived from 15 different tissues under non- stress conditions. For our analysis, we focused on 14,258 triads which were found to be syntenic and expressed in at least 6 out of 15 tissues tested for this dataset (see Supplementary Table S3) (Ramı́rez-González et al., 2018). non-stress conditions (Juery et al., 2020). The dependency between polymorphism and balanced\ non-balanced expression of the homoeologs was tested using the Chi-square test of independence of variables with the chi2_contingency function from Python SciPy. Stats module (Virtanen et al., 2020). 2.4 Correlation between TE insertion within gene bodies and homoeolog expression bias in triads In addition to the identiﬁcation of TE insertions within gene bodies, we identiﬁed TE insertions found speciﬁcally within exons and within the UTRs using a similar approach, combining the exons, 5’ UTRs, and 3’ UTRs coordinates for each gene according to IWGSC RefSeq v1.0 HC genes annotations with TEs coordinates (Appels et al., 2018). Then, we integrated data from ﬁles dividing the homoeolog triads into seven relative contribution categories (Ramı́rez-González et al., 2018), to create Supplementary Table S3. 2.5 Gene ontology enrichment analysis Gene ontology (GO) provides structured, computable knowledge regarding the functions of genes and gene products in three non‐overlapping domains of molecular biology (Carbon et al., 2019). The three domains are Biological Process (BP), which refers to a biological objective to which the gene or gene product contributes, Molecular Function (MF), deﬁned as the biochemical activity of a gene product and Cellular Component (CC), which refers to the location in the cell where a gene product is active (Ashburner et al., 2000). GO enrichment analysis is used to ﬁnd over-represented GO terms in a gene set compared to a reference set. The seven ﬁles divided the triads into contribution categories as follows: triads for which a similar abundance of transcripts was observed from each of the three homoeologs were assigned to a balanced category, while triads that showed a higher or lower abundance of transcripts from a single homoeolog relative to the other two, were assigned to one of six non-balanced categories. The non-balanced categories include three homoeolog-dominant categories (A dominant, B dominant, and D dominant) and three homoeolog-suppressed categories (A suppressed, B suppressed, and D suppressed) (Ramı́rez- González et al., 2018). Each triad was attributed to one of the Here, we performed GO enrichment analysis for triads, including TE insertions from each of the 14 TE superfamilies (see Table 1). Additionally, we selected triads that belonged to Frontiers in Plant Science 04 frontiersin.org frontiersin.org Bariah et al. 10.3389/fpls.2022.1072232 TABLE 1 Analysis of the 7,439 expressed and syntenic triads which contained TE insertions belonging to 14 different TE superfamilies. 3 Results speciﬁc relative contribution categories and included TE insertions from superfamilies that showed a correlation to the mentioned category (see Table 2). The reference set for all the GO enrichment analyses performed in this study was the whole set of 14,258 expressed and syntenic triads. GO Singular Enrichment Analysis (SEA) was performed using the AgriGO toolkit (RRID : SCR_006989) (Tian et al., 2017) with Fisher’s exact test to identify enriched GO terms. 2.5 Gene ontology enrichment analysis BLE 1 Analysis of the 7,439 expressed and syntenic triads which contained TE insertions belonging to 14 different TE superfamil GO classes3 Code1 Class Order Superfamily Triads2 BP CC MF RLG Class I (retrotransposons) LTR Gypsy 1,134 438 70 148 RLC Copia 1,359 371 67 157 RLX Unclassiﬁed LTR-retrotransposons 474 132 15 84 RIX non-LTR (LINE) Long interspersed nuclear elements 1,458 416 97 209 SIX non-LTR (SINE) Short interspersed nuclear elements 20 19 11 23 DTC Class II (DNA transposons) TIR CACTA 2,960 734 140 327 DTM Mutator 428 185 11 80 DTX unknown 2,099 606 89 248 DTH Harbinger 456 142 21 94 DTT Mariner 4,576 956 156 400 DTA hAT 9 7 1 4 DXX unknown unknown 105 68 15 22 DHH Helitron Helitron 2 – – – XXX unknown unknown unknown 1,347 266 16 225 1 The three letters code represents the class (ﬁrst letter), order (second letter) and superfamily (third letter) of the TE (Wicker et al., 2007). 2 Number of triads in which at least one of the genes includes TE insertion from the speciﬁc superfamily. Note that the sum of the triad column is larger than 7,439. This is since some triads include insertions from more than one subfamily. 3 Number of signiﬁcantly enriched GO terms found in GO SEA preformed for triads which include TE insertions from mentioned superfamily in each of the three biological objective to which the gene or gene product contributes: BP, Biological Process, CC, Cellular Component and MF, Molecular Function.”-” notes missing values due to short query list which did not met the criteria for enrichment analysis. 1 The three letters code represents the class (ﬁrst letter), order (second letter) and superfamily (third letter) of the TE (Wicker et al., 2007). 2 Number of triads in which at least one of the genes includes TE insertion from the speciﬁc superfamily. Note that the sum of the triad column is larger than 7,439. This is since some triads include insertions from more than one subfamily. 3 Number of signiﬁcantly enriched GO terms found in GO SEA preformed for triads which include TE insertions from mentioned superfamily in each of the three biological objective to which the gene or gene product contributes: BP, Biological Process, CC, Cellular Component and MF, Molecular Function.”-” notes missing values due to short query list which did not met the criteria for enrichment analysis. frontiersin.org 3.1 Different TE insertion patterns within gene bodies in dyads, triads, and tetrads TABLE 2 TE superfamilies for which the presence\absence of TE insertion in at least one of the triad genes correlated with speciﬁc triad expression patterns TABLE 2 TE superfamilies for which the presence\absence of TE insertion in at least one of the triad genes correlated with spe expression patterns. TE insertion TE superfamily1 corrected p-values2 Yes No Yes No Balanced triads3 Non-balanced triads4 RLC 0.008837323 1132 5252 227 828 DTT 6.26E-06 4001 2383 575 480 DTM 0.000236883 338 6046 90 965 DTX 0.01607886 1837 4547 262 793 SIX 0.008282664 12 6372 8 1047 RLX 0.00382206 382 6002 92 963 XXX 0.01015199 1123 5261 224 831 Suppressed triads5 Not suppressed triads6 RLC 0.014455 192 690 1167 5390 DTT 0.000675 488 394 4088 2469 DTM 0.023678 68 814 360 6197 DTX 0.047574 220 662 1879 4678 RLX 0.000675 83 799 391 6166 XXX 0.006588 194 688 1153 5404 Dominant triads7 Not dominant triads8 DTT 0.013897 87 86 4489 2777 DTM 0.001364 22 151 406 6860 1 The three letters code represents the class (ﬁrst letter), order (secuned letter) and superfamily (third letter) of the TE (Wicker et al., 2007). 2 c2 corrected p-values for multiple tests using Benjamini/Hochberg Procedure (non-negative). 3,5,7 Number of triads belonging to the mentioned category (balanced, suppressed, or dominant), in which at least one homoeolog contains TE insertion from the mention superfamily (Yes) or none of the homoeologs contain TE insertion from the mention superfamily (No). 4,6,8 Number of triads that does not belong to the mentioned category, in which at least one homoeolog contains TE insertion from the mention superfamily (Yes) or none of the homoeologs contain TE insertion from the mention superfamily (No). For the balanced category it will refer to the number of triads from the homoeolog-dominant or homoeolog- suppressed categories, for the suppressed categories it will refer to the number of triads from balanced or to one of the homoeolog-dominant categories and for the dominant categories it will include triads belong to either the balanced or to one of the homoeolog- suppressed categories. the whole genome. Out of the 41,503 genes (4,781 belonging to dyads, 35,447 to triads, and 1,275 to tetrads) found in the proximal region, 40.29% included TE insertion within the gene body, with signiﬁcant differences in proportion (c2 = 135.78, p < 0.001) between dyads (1,665 genes, 34.83%), triads (14,675 genes, 41.40%), and tetrads (380 genes, 29.80%) genes. 3.1 Different TE insertion patterns within gene bodies in dyads, triads, and tetrads 3.1 Different TE insertion patterns within gene bodies in dyads, triads, and tetrads In order to perform a genome-wide analysis of TE insertions within wheat gene bodies, 70,818 bread wheat genes belonging to 6,320 dyads, 18,390 triads, and 752 tetrads were analyzed. TE insertions within gene bodies were identiﬁed based on the IWGSC RefSeq v1.0 assembly coordinates for HC genes and TEs (Appels et al., 2018). We found that ~36% of the 70,818 genes (25,811 genes) contain at least one TE insertion within the gene body, with higher proportions of TE containing genes observed for triads (20,975 genes, 38.02%) relative to dyads (3,972 genes, 31.42%) and tetrads (864 genes, 28.72%) (Figure 1A; Supplementary Figure S1). The difference in the proportions of TE containing genes between the dyads, triads, and tetrads genes was statistically signiﬁcant (c2 = 273.99, p < 0.001). TE insertions were found either in all the homoeologous copies in the group (i.e., for triads: monomorphic insertion in the three sub-genomes) or only in some of the homoeolog genes (i.e., for triads: polymorphic insertion in the three sub-genomes). Following the GO SEA, enriched GO terms for each GO category (Biological function, Cellular component, and Molecular function) were visualized as a scatter plot generated by REVIGO (RRID : SCR_005825) (Supek et al., 2011). REVIGO summarizes the GO terms lists generated from the GO SEA by reducing functional redundancies based on the value provided and visualizes the remaining GO terms as a scatterplot, where more semantically similar GO terms are found closer to each other in the plot. For each GO SEA, we provided REVIGO, a list of GO terms that were found to be signiﬁcantly enriched with false discovery rate (FDR) less or equal to 0.05 and their FDR value which is an adjusted p-value that enables us to have less false positive results then if the p-value was used. The scatterplots generated by REVIGO were imported into R, where wanted labels were added, and others were moved manually to slightly different coordinates to better visualize all the labels. The differences in TE abundance between the dyads, triads, and tetrads categories might be the result of the different Frontiers in Plant Science 05 frontiersin.org 10.3389/fpls.2022.1072232 Bariah et al. 3.1 Different TE insertion patterns within gene bodies in dyads, triads, and tetrads The boxplots depict the ﬁrst quartile (Q1) and the third quartile (Q3) of the data with the median between them. The whiskers extend from the box to 1.5x the interquartile range (IQR). Rhombuses represent values found past the end of the whiskers. boxplots were drawn using the boxplot function from the seaborn python package (Waskom, 2021). A B D E C FIGURE 1 The distribution of genes belonging to the dyads, triads, or tetrads categories in the ﬁve chromosomal regions (R1, R2a, C, R2b, and R3). Gene distribution was calculated individually for genes that belong to the same category and found in the same and chromosomal region in each one of 18 bread wheat chromosomes. Genes found in chromosomes 1A, 1B, and 1D were eliminated from the analysis due to the lack of tetrads i h f h 1A d 1B (A) P f TE i i f l (B) P f A B C D E D FIGURE 1 The distribution of genes belonging to the dyads, triads, or tetrads categories in the ﬁve chromosomal regions (R1, R2a, C, R2b, and R3). Gene distribution was calculated individually for genes that belong to the same category and found in the same and chromosomal region in each one of 18 bread wheat chromosomes. Genes found in chromosomes 1A, 1B, and 1D were eliminated from the analysis due to the lack of tetrads genes in the centromeres of chromosomes 1A and 1B. (A) Percentage of TE containing genes out of total genes. (B) Percentage of genes belonging to polymorphic group out of genes that found in TE containing group. (C) Percentage of genes belonging to balanced group out of total genes. (D) Percentage of genes belonging to balanced group out of genes that found in TE containing group. (E) Percentage of genes belonging to balanced and polymorphic group out of genes that found in TE containing group. The boxplots depict the ﬁrst quartile (Q1) and the third quartile (Q3) of the data with the median between them. The whiskers extend from the box to 1.5x the interquartile range (IQR). Rhombuses represent values found past the end of the whiskers. boxplots were drawn using the boxplot function from the seaborn python package (Waskom, 2021). 3.1 Different TE insertion patterns within gene bodies in dyads, triads, and tetrads A lower proportion of TE containing genes was observed in the distal regions, where only 31.01% of the 29,315 genes (7,859 belonging to dyads, 19,723 to triads, and 1,733 to tetrads) included TE insertion. However, the signiﬁcant differences in proportion (c2 = 25.77, p < 0.001) of TE containing genes were still observed in the distal region, with a higher proportion of TE insertion in triad genes (6,300 genes, 31.94%) relative to dyads (2,307 genes, 29.35%) and tetrads (484 genes, 27.93%). chromosomal distribution patterns of the genes between the categories. While triads are more abundant in the proximal region (R2a, C and R2b), which contains a higher proportion of TEs, dyads, and tetrads are most abundant in the distal region (R1 and R3), which was found to have lower TE density (Wicker et al., 2018; Juery et al., 2020). The proportions of TEs containing genes belonging to each of the three categories in each of the ﬁve chromosomal regions are shown in Figure 1A. To test whether the difference in TEs abundant between the dyads, triads, and tetrads is mainly due to the chromosomal distribution of the genes, we performed the analysis separately for the proximal and distal regions. Signiﬁcant differences in TEs abundant within gene bodies from dyads, triads, and tetrads were observed for each region separately, displaying the same pattern observed for Frontiers in Plant Science 06 frontiersin.org Bariah et al. 10.3389/fpls.2022.1072232 A B D E C FIGURE 1 The distribution of genes belonging to the dyads, triads, or tetrads categories in the ﬁve chromosomal regions (R1, R2a, C, R2b, and R3). Gene distribution was calculated individually for genes that belong to the same category and found in the same and chromosomal region in each one of 18 bread wheat chromosomes. Genes found in chromosomes 1A, 1B, and 1D were eliminated from the analysis due to the lack of tetrads genes in the centromeres of chromosomes 1A and 1B. (A) Percentage of TE containing genes out of total genes. (B) Percentage of genes belonging to polymorphic group out of genes that found in TE containing group. (C) Percentage of genes belonging to balanced group out of total genes. (D) Percentage of genes belonging to balanced group out of genes that found in TE containing group. (E) Percentage of genes belonging to balanced and polymorphic group out of genes that found in TE containing group. Frontiers in Plant Science 3.2 Polymorphic TE insertions within gene bodies and homoeologous group expression patterns Focusing only on the 2,386 dyads that include TE insertions, we found that 54.99% (1,312 dyads) were polymorphic and included TE insertions in only one of the homoeologs gene bodies, a result that did not ﬁt our module (c2 = 136.78, p < 0.001) which predicted that only 43.13% (~1,029 dyads) would be polymorphic. While the percentage of polymorphic dyads was higher than our module anticipated, the opposite trend was observed for triads and tetrads. Out of 17,676 expressed triads, 59.24% (10,471 triads) included TE insertions, with 64.08% of the triads identiﬁed as polymorphic (6,710 triads), a distribution that did not match our module (c2 = 212.36, p < 0.001), which predict that 70.57% of the triads (~7390 triads) will be polymorphic. For tetrads, we found that out of the 667 expressed tetrads, 58.17% (388 tetrads) included TE insertions, and 82.73% (321 tetrads) of the TE insertions containing tetrads were found to be polymorphic. This result also did not ﬁt our module (c2 = 43.47, p < 0.001), which predicts that 91.88% of the tetrads (~356 tetrads) will be polymorphic. Next, we aimed to assess whether polymorphic TE insertions affect the relative expression within the homoeologous group. The data on the relative expression within each homoeologous group was retrieved from Juery et al. (2020), which performed the analysis for 123 RNA-Seq samples of bread wheat (Chinese Spring) taken from 15 different tissues under non-stress conditions, and was integrated into Supplementary Table S2. Expressed homoeologous groups were assigned as balanced if all the homoeologs showed similar transcript abundance or as homoeolog-dominant or homoeolog-suppressed (non- balanced), based on the relative higher\ lower transcript abundance of each homoeolog (Figure 1C) (Juery et al., 2020). To learn about the possible effect of TE insertions within gene bodies on the relative expression, we focused only on TE containing homoeologous groups and tested the correlation between polymorphism and expression patterns separately for dyads, triads, and tetrads (Figures 1B, D, E). Our analysis revealed that a higher percentage of the polymorphic homoeologous groups belonged to one of the non-balanced expression categories relative to monomorphic groups, with a signiﬁcant difference in proportions for dyads and triads (dyads: c2 = 11.34, p < 0.001, triads: c2 = 73.45, p < 0.001), while for tetrads the differences in proportions were not statistically signiﬁcant (c2 = 0.32, p = 0.57). 3.2 Polymorphic TE insertions within gene bodies and homoeologous group expression patterns homoeologs copies from each homoeologous group and determined for each expressed group (a group that includes one or more expressed gene) whether it is monomorphic or polymorphic. Homoeologous group was considered as monomorphic if all the homoeologs copies in the group included at least one TE insertion or polymorphic if at least To assess the associations between TE insertion patterns and gene expression in dyads, triads, and tetrads, we ﬁrst grouped the Frontiers in Plant Science 07 frontiersin.org 10.3389/fpls.2022.1072232 Bariah et al. number of polymorphic triads was lower than the number predicted by our module (proximal region: c2 = 154.72, p < 0.001, distal region: c2 = 28.96, p < 0.001), with 59.78% polymorphic triads (3,978 triads) at the proximal region and 72.84% polymorphic triads (2,113 triads) in the distal region, versus 66.96% in the proximal region (~4455 triads) and 77.04% in the distal region (~2,235 triads) predicted by our module. The distribution of polymorphic tetrads also did not ﬁt the numbers predicted by our module (proximal region: c2 = 25.53, p < 0.001, distal region: c2 = 16.65, p < 0.001). Out of the 388 expressed tetrads which include TE insertions, 132 tetrads included only genes located in the proximal region, and 79.55% (105 tetrads) of them were found to be polymorphic, while our module predicted that 91.69% of the tetrads (~121 tetrads) in the proximal region would be polymorphic. Moreover, out of the 181 TE containing expressed tetrads that included only genes located in the distal region, only 83.43% were found to be polymorphic, while our module predicted that 91.76% of the tetrads in the distal region (~166 tetrads) would be polymorphic. one but not all the homoeologs copies in the group included TE insertion within the gene body (Figure 1B, Supplementary Table S2). Then, the number of monomorphic and polymorphic homoeologous groups was compared with the numbers predicted by our module, which was based on the assumption that the presence\ absence of TE insertions within a gene is random. Out of 5,059 expressed dyads, 47.16% (2,386 dyads) included TE insertions, meaning at least one TE insertion was present within one or more of the homoeologs gene bodies. 3.2 Polymorphic TE insertions within gene bodies and homoeologous group expression patterns However, different results were obtained when the analysis was performed separately for the proximal and the distal chromosomal regions. For dyads, 37.88% (497 dyads) of the 1,312 polymorphic groups showed non-balanced expression, while only 31.19% (335 dyads) of the 1,074 monomorphic groups showed non-balanced expression. A statistically signiﬁcant correlation (c2 = 9.96, p = 0.002 < 0.05) was also identiﬁed between polymorphism and relative expression pattern for dyads that contained only genes found in the proximal region (872 dyads), with 32.55% of the To reduce the effect of the different chromosomal distribution patterns of dyads, triads, and tetrads on our analysis, we reperformed the analysis focusing on homoeologous groups in which all the homoeologs copies were found in the same chromosomal region (proximal\ distal). Thus, the numbers of monomorphic and polymorphic homoeologous groups and the predicted distribution were counted and calculated separately for dyads, triads, and tetrads in each chromosomal region. Similar patterns to those observed for the whole chromosome were observed separately for the proximal and distal regions. Out of the 2,386 expressed dyads that include TE insertions, 872 included only genes found in the proximal region, and 1,288 included only genes found in the distal region. The numbers of polymorphic dyads in both the proximal and distal regions were higher than expected by our module. They did not match our predictions (proximal region: c2 = 33.11, p < 0.001, distal region: c2 = 95.11, p < 0.001), with 48.62% polymorphic dyads (424 dyads) at the proximal region and 59.24% polymorphic dyads (763) at the distal region. In contrast, our module predicted that 39.19% of the dyads found in the proximal region (~341 dyads) and 45.70% of the dyads in the distal region would be polymorphic. For triads, we found that out of the 10,471 expressed TE containing groups, 6,654 triads included only genes located in the proximal region and 2,901 triads included only genes located in the distal region. The Frontiers in Plant Science 08 frontiersin.org 10.3389/fpls.2022.1072232 Bariah et al. polymorphic dyads (138 dyads) and 22.77% of the monomorphic dyads (102 dyads) found in one of the non- balanced categories. 3.2 Polymorphic TE insertions within gene bodies and homoeologous group expression patterns However, no signiﬁcant correlation was found between polymorphism and relative expression patterns for dyads which include only genes located in the distal region (c2 = 0.05, p = 0.82 > 0.05), although higher proportions of polymorphic dyads were found in non-balanced categories (37.75%, 288 dyads) relative to monomorphic dyads (36.95%, 194 dyads). Similar to dyads, for triads, 18.21% (1,222 triads) of the 6,710 polymorphic groups were classiﬁed as non-balanced, compared to 11.81% (444 triads) of the 3,761 monomorphic triads that were classiﬁed as non-balanced. The correlation between non-balanced expression and polymorphism in triads was also observed separately for triads that include only genes located in the proximal region (c2 = 52.98, p < 0.001) and for triads that include only genes located at the distal region (c2 = 5.54, p = 0.02 < 0.05). At the proximal region, 16.37% of the polymorphic triads (651 triads) and 10.05% of the monomorphic triads (269 triads) were classiﬁed as non- balanced, and at the distal region, 21.15% of the polymorphic triads (447 triads) and 17.13% of the monomorphic triads (135 triads) were classiﬁed as non-balanced. Finally, for tetrads, 77.26% (248 tetrads) of the 321 polymorphic groups and 73.13% (49 tetrads) of the 67 monomorphic groups were assigned to one of the non-balanced categories. No statistically signiﬁcant dependency between polymorphism and homoeologous group expression pattern was identiﬁed upon performing the analysis separately for tetrads which include genes located only at the proximal (c2 = 0.43, p = 0.51 > 0.05) or only at the distal (c 2 = 0.02, p = 0.89 > 0.05) chromosome regions. bodies of triads genes were identiﬁed based on the IWGSC RefSeq v1.0 assembly coordinates for high conﬁdence (HC) genes and TEs (Appels et al., 2018). The analysis of the 14,258 expressed and syntenic triads revealed that the presence of TE insertions in at least one of the triad genes (7,439 triads) correlated to balanced expression between the homoeolog genes. Out of the 14,258 expressed and syntenic homoeolog triads, 52.17% (7,439 triads) contain one or more TE insertions (based on repeat regions coordinates) within the gene body sequence of at least one of the genes in the triad (triads that include TE insertions). 3.2 Polymorphic TE insertions within gene bodies and homoeologous group expression patterns A higher proportion of triads that include TE insertions are found in the balanced expression category (6,384, 85.82%) relative to triads that don’t include TE insertions (5,450, 79.92%) with a statistically signiﬁcant difference in proportions (c2 = 87.18, p < 0.001). signiﬁcant difference in proportions (c 87.18, p < 0.001). The TEs that were found to be within gene bodies represented all the 14 TE superfamilies identiﬁed in the wheat genome (see Table 1) and belonged to 455 subfamilies out of the 570 subfamilies annotated by the IWGSC as “repeat region”, as was counted from the annotation ﬁle (Appels et al., 2018). To learn about the possible association between TE type and the relative expression contribution of each of the homoeologs in the triad, we tested separately for each TE superfamily and subfamily whether the presence\ absence of TE insertions from said type within gene bodies correlated with balanced, suppressed, or dominant relative expression of the homoeologs. Here, we focused only on TE groups (superfamily or subfamily) that had sufﬁcient sample size, mining 5 or more cases were observed for all the combinations of the tested conditions for the group with the examined relative expression category. For instance, the number of triads in which TE insertions from speciﬁc TE superfamily were presence\ absent must be ﬁve or higher both in balanced and non-balanced categories for the superfamily to be included in the analysis against the balanced relative expression category. Out of the 14 TEs superfamilies, 12 were found adequate for analysis against the balanced expression category (DTA and DHH were removed from the analysis), and 7 TEs superfamilies showed a statistically signiﬁcant correlation (Chi-square corrected p-value ≤0.05, Table 2) with balanced\ non-balanced expression categories. The correlation between superfamily and balanced expression was negative for 5 (SIX, DTM, RLX, RLC, and XXX, Table 2) of the 7 superfamilies and positive for the remaining 2 superfamilies (DTT and DTX). The same 12 superfamilies that were found adequate for analysis against the balanced expression category were also found adequate for comparison against homoeolog-suppressed\ non- suppressed expression categories, with the remaining 2 superfamilies (DTA and DHH, Table 2) excluded from the analysis due to a low number of cases. Speciﬁc superfamilies also showed a statistically signiﬁcant correlation with homoeolog-suppressed\ non-suppressed expression categories. In total, 6 superfamilies showed statistically signiﬁcant correlation with homoeolog-suppressed\ non-suppressed 3.4 Triads which include TE insertions belonging to speciﬁc TE superfamilies were associated with various GO terms To assess the association between the presence of TE insertions from speciﬁc types within gene bodies and gene function, we tested whether triads that include TE insertions from each of the 14 TEs superfamilies were associated with speciﬁc cellular functions. GO SEA conducted by AgriGO toolkit (Tian et al., 2017) against the database of the 14,258 expressed and syntenic triads revealed that triads which include TEs from each of 13 speciﬁc superfamilies were enriched for numerous GO terms from the BP, MF, and CC domains (Table 5; Figure 2 and Supplementary Figures S2-S13, Supplementary Tables S6- S25). The DTA superfamily was excluded from the analysis due to the small sample size. In the BP domain, a signiﬁcantly correlation was found between the presence of TEs from speciﬁc superfamilies within the triad and basic cell processes like gene silencing by RNA, cell cycle, organelle organization, recombinational repair, DNA recombination, telomere organization, DNA-templated DNA replication, and DNA methylation. Additionality, a signiﬁcant correlation was found between the presence of TE insertions from speciﬁc superfamilies and response to biotic and abiotic stress, such as response to virus, response to nematode, vernalization response, and response to symbiotic fungus. Interestingly, triads that include TEs from speciﬁc superfamilies were also found to be associated with GO terms from the BP domine associated with the transposition mechanisms of the two TE classes, including transposition, RNA-mediated, and DNA-mediated (Table 5; Figure 2 and Supplementary Figures S2-S13, Supplementary Tables S6-S25). For the MF domain association was observed between the presence of TE insertions from speciﬁc superfamilies within the triad and enzymes activities and that carry out basic cell processes, including ligase activity, helicase activity, and DNA- directed DNA polymerase activity and with DNA repair, including DNA insertion or deletion binding (tale 5). Similarly, for the CC domain, an association was observed with the RNA polymerase I complex, responsible for basic cell activity, and with the DNA repair complex (Table 5; Supplementary Figures S2- S13, Supplementary Tables S6-S25). In addition, our analysis revealed enrichment in terms associated with the regulation of gene expression, such as the RISC complex (CC) and RNAi effector complex (CC), and transposase activity (MF) (Table 5; Supplementary Figures S2-S13, Supplementary Tables S6-S25). Next, we performed a similar analysis for TE subfamilies. 3.3 TE content within gene bodies and triad expression patterns Here, we aimed to study the possible effect of TE insertions on gene expression in wheat. We analyzed 14,258 expressed and syntenic triads that were assigned to 7 relative contribution categories according to the calculation method described by Ramı́rez-González et al. (2018). Most of the 14,258 triads (11,834 triads, 83%) showed balanced expression, meaning a similar relative abundance of transcripts was observed for the three homoeologs. The remaining 2,424 triads were divided between 6 non-balanced categories, with 13.99% of the triads (1,995 triads) assigned to one of the homoeolog-suppressed relative contribution categories (5.16% of the triads belonged to the A suppressed category, 5.31% to the B suppressed category and 3.52% to the D suppressed category) and 3.01% of the triads (429 triads) assigned to one of the homoeolog-dominant relative contribution categories (0.90% of the triads belonged to the A dominant category, 1.05% to the B dominant category and 1.07% to the D dominant category). TE insertions within the gene Frontiers in Plant Science 09 frontiersin.org Bariah et al. 10.3389/fpls.2022.1072232 10.3389/fpls.2022.1072232 expression categories (Chi-square corrected p-value ≤0.05, Table 2), all of them also showed correlation with balanced\ non-balanced expression categories. TEs superfamilies that showed a positive correlation with balanced expression showed a negative correlation with suppressed expression (DTT and DTX, Table 2), while TEs superfamilies that showed a negative correlation with balanced expression showed a positive correlation with suppressed expression (DTM, RLX, RLC and XXX, Table 2). Finally, only 10 of the 14 superfamilies were found ﬁtted for analysis against the homoeolog-dominant\ non- dominant expression categories (SIX, DTA, DXX, and DHH were removed from the analysis), with only 2 TE superfamilies (DTM and DTT) showing statistically signiﬁcant correlation with homoeolog-dominant\ non-dominant expression categories (Chi-square corrected p-value ≤0.05, Table 2), both also found to correlate with balanced expression signiﬁcantly. The DTM superfamily showed a positive correlation with dominant expression and a negative correlation with balanced expression, while the DTT superfamily showed a negative correlation with dominant expression and a positive correlation with balanced expression. correlation with balanced expression, and only two subfamilies, RIX_famc8 and DTT_famn14, showed a positive correlation with homoeolog-balanced expression. Frontiers in Plant Science 3.4 Triads which include TE insertions belonging to speciﬁc TE superfamilies were associated with various GO terms The majority of the 455 TE subfamilies found within gene bodies were excluded from the analysis due to the small sample size: out of the 455 TE subfamilies, 303 subfamilies were eliminated from the analysis for the balanced expression category, 323 subfamilies were excluded from the analysis for the suppressed expression categories, and 433 subfamilies were excluded from the analysis for the dominant expression categories. Out of the TEs subfamilies which were found adequate for analysis, 19 subfamilies showed a statistically signiﬁcant correlation (Chi- square corrected p-value ≤0.05, Table 3) with balanced\ non- balanced expression categories, 17 subfamilies showed a statistically signiﬁcant correlation (Chi-square corrected p- value ≤0.05, Table 4) with homoeolog-suppressed\ non- suppressed expression categories and none of the TE subfamilies showed statistically signiﬁcant correlation with homoeolog-dominant\ non-dominant expression categories. Fourteen of the subfamilies that showed a signiﬁcant correlation between presence\absence of TE insertions and suppression of a single homoeolog gene also showed a correlation with balanced relative expression of the homoeologs, while the other subfamilies were found in correlation only to suppressed (3 subfamilies) or balanced (5 subfamilies) relative expression. Of the 17 subfamilies that showed statistically signiﬁcant correlation with homoeolog- suppressed expression categories, only the DTT_famn14 subfamily showed a negative correlation with homoeolog- suppressed expression, while insertions of the remaining 16 subfamilies appeared in higher proportions than expected in the homoeolog-suppressed categories. Similarly, 17 of the 19 subfamilies that showed a statistically signiﬁcant correlation with homoeolog-balanced expression showed a negative While some of the TE superfamilies were found to be signiﬁcantly enriched for most of the mentioned GO terms, Frontiers in Plant Science 10 frontiersin.org Bariah et al. 10.3389/fpls.2022.1072232 bfamilies for which the presence/absence of TE insertion in at least one of the triad genes correlated with balanced relative e three homoeologs. TABLE 3 TE subfamilies for which the presence/absence of TE insertion in at least one of the triad genes correlated with balanced relative expression of the three homoeologs. 3.4 Triads which include TE insertions belonging to speciﬁc TE superfamilies were associated with various GO terms CLARITE name1 corrected p-values2 TE insertion Yes No Yes No Balanced triads3 Non-balanced triads4 DTC_famc11.1 0.046018 23 6361 11 1044 RLC_famc6 0.000736 18 6366 13 1042 RLC_famc1.6 0.00292 6 6378 7 1048 RLC_famc20 9.35E-11 22 6362 24 1031 RLC_famc7.1 0.034407 7 6377 6 1049 DTC_famc4.3 0.00766 5 6379 6 1049 DTM_famc9 0.04758 20 6364 10 1045 RLG_famc1.1 0.002862 13 6371 10 1045 RIX_famc1 0.004706 212 6172 59 996 RLC_famc8 0.001236 14 6370 11 1044 RIX_famc8 0.029024 693 5691 82 973 DTM_famc8 0.04758 20 6364 10 1045 DTT_famn14 0.000511 721 5663 72 983 XXX_famc13 0.000302 99 6285 38 1017 SIX_famc1 0.026102 9 6375 7 1048 XXX_famc16 3.10E-05 119 6265 46 1009 RLX_famc22 0.001572 29 6355 16 1039 XXX_famc112 0.001236 14 6370 11 1044 RIX_famc15 3.23E-10 24 6360 24 1031 1 According to Wicker et al. (Wicker et al., 2018). TE names were selected based on the ClariTeRep naming system, which assigns simple numbers to individual families and subfamilies. 2 c2 corrected p-values for multiple tests using Benjamini/Hochberg Procedure (non-negative). 3 Number of triads belonging to the homoeolog-balanced category, in which at least one homoeolog contains TE insertion from the mention subfamily (Yes) or none of the homoeologs contain TE insertion from the mention subfamily (No). 4 Number of triads belonging to one of the non-balanced categories, meaning to one of the homoeolog-dominant or homoeolog-suppressed categories, in which at least one homoeolog contains TE insertion from the mention subfamily (Yes) or none of the homoeologs contain TE insertion from the mention subfamily (No). relative to all the triads which include TE insertions from the same superfamily. For this purpose, we focused on triads that include TE insertions from superfamilies that we found that their presence within a triad correlated with speciﬁc relative expression patterns and are found in the relevant expression category (shown in Table 2). For example, triads that include DTT insertions were signiﬁcantly more likely to be found in the balanced relative expression category compering to triads that included TE insertions but did not include insertions of DTT TEs, and thus, the analysis was performed for triads that included insertions belonging to the DTT superfamily and showed balanced expression of the homoeologs (Figure 2A; Supplementary Figures S2-S4, S11-S13, Supplementary Tables S12 and S20). Frontiers in Plant Science 1 According to Wicker et al. (Wicker et al., 2018). TE names were selected based on the ClariTeRep naming system, which assigns simple numbers to individual families and subfamilies. 2 c2 corrected p-values for multiple tests using Benjamini/Hochberg Procedure (non-negative). 3 Number of triads belonging to the homoeolog-balanced category, in which at least one homoeolog contains TE insertion from the mention subfamily (Yes) or none of the homoeologs contain TE insertion from the mention subfamily (No). 4 Number of triads belonging to one of the non-balanced categories, meaning to one of the homoeolog-dominant or homoeolog-suppressed categories, in which at least one homoeolog contains TE insertion from the mention subfamily (Yes) or none of the homoeologs contain TE insertion from the mention subfamily (No). 3.4 Triads which include TE insertions belonging to speciﬁc TE superfamilies were associated with various GO terms However, for triads that include insertions belonging to the DTM superfamily, the analysis was others showed enrichment for only a few of the GO terms we decided to focus on or even only for one of the mentioned terms (Table 5). For instance, triads that included TE insertions belonging to the DTT superfamily (Figure 2A) were found to be enriched for all the GO terms mentioned in Table 5 except for transposition, RNA-mediated (BP), and chromosome (CC), while triads that included TE insertions belonging to the DTH superfamily showed association with only 2 of the GO terms from Table 5, gene silencing by RNA (BP) and DNA methylation (BP). Following the GO SEA performed for triads that included TE insertions from speciﬁc superfamilies within the gene bodies, we further examined whether triads that showed a speciﬁc relative expression pattern and included TE insertions from speciﬁc superfamilies would associate with different GO terms Frontiers in Plant Science 11 frontiersin.org Bariah et al. 10.3389/fpls.2022.1072232 TABLE 4 TE subfamilies for which the presence/absence of TE insertion in at least one of the triad genes correlated with the suppression of a single homoeolog gene TABLE 4 TE subfamilies for which the presence/absence of TE insertion in at least one of the triad genes correlated with the s single homoeolog gene. CLARITE name1 corrected p-values2 TE insertion Yes No Yes No Suppressed triads3 Not suppressed triads4 DTC_famc11.1 0.028255 10 872 24 6533 RLC_famc6 0.013022 10 872 21 6536 RLC_famc1.6 0.000371 7 875 6 6551 RLC_famc20 3.85E-10 21 861 25 6532 XXX_famc33 0.013022 5 877 5 6552 RLC_famc7.1 0.008919 6 876 7 6550 RLG_famc1.1 0.018009 8 874 15 6542 RIX_famc1 0.017541 49 833 222 6335 RLC_famc8 0.008831 9 873 16 6541 DTT_famn14 0.000371 57 825 736 5821 RLG_famc15 0.026756 8 874 16 6541 XXX_famc13 3.68E-05 35 847 102 6455 XXX_famc16 1.02E-06 43 839 122 6435 XXX_famc140 0.018009 8 874 15 6542 RLX_famc22 0.000371 15 867 30 6527 XXX_famc112 7.95E-05 11 871 14 6543 RIX_famc15 1.88E-10 22 860 26 6531 1 According to Wicker et al. (Wicker et al., 2018). TE names were selected based on the ClariTeRep naming system, which assigns simple numbers to individual families and subfamilies. 2 c2 corrected p-values for multiple tests using Benjamini/Hochberg Procedure (non-negative). 3.4 Triads which include TE insertions belonging to speciﬁc TE superfamilies were associated with various GO terms 3 Number of triads belonging to one of the homoeolog-suppressed categories, in which at least one homoeolog contains TE insertion from the mention subfamily (Yes) or none of the homoeologs contain TE insertion from the mention subfamily (No). 4 Number of triads belonging to the balanced category or to one of the homoeolog-dominant categories, in which at least one homoeolog contains TE insertion from the mention subfamily (Yes) or none of the homoeologs contain TE insertion from the mention subfamily (No). GO terms production of siRNA involved in RNA interference (GO:0030422), regulation of DNA methylation (GO:0044030), and posttranscriptional gene silencing by RNA (GO:0035194) were found to be signiﬁcantly enriched when the analysis was performed for all the DTM insertions containing triads they were missing from the results of the analysis for only triads from the dominant relative expression categories, and from the results of the analysis for only triads from the suppressed relative expression categories (Figure 2B; Supplementary Figures S2- S10, Supplementary Tables S6, S21, S22). However, signiﬁcant association with GO terms that were not found to be enriched for all the DTM continuing triads was identiﬁed for the triads that include DTM insertions and belonging to one of the homoeolog-dominant expression categories, mainly associated with response to biotic and abiotic factors and aging (aging (GO:0007568), leaf senescence (GO:0010150), organ senescence (GO: 0010260), response to metal ion (GO:0010038), response to oxidative stress (GO:0006979), response to biotic stimulus performed separately for triads that belonged to the suppressed and the dominant relative expression categories since triads that include DTM insertions were signiﬁcantly more likely to be found in suppressed or dominant relative expression category in comparison to triads that included TE insertions but did not include insertions of DTM TEs (Figure 2B; Supplementary Figures S2-S10, Supplementary Tables S6, S21, S22). Generally, similar GO terms were found to be signiﬁcantly enriched for the same TE superfamily when all the TE containing triads were tested and upon focusing on triads from a speciﬁc relative expression contribution category (Supplementary Figures S2-S13; Supplementary Tables S6- S25). However, we noticed that in some cases, speciﬁc terms were found to be enriched by the analysis performed for all the triads with TE insertions from speciﬁc TE superfamily and were missing from the results when the analysis was performed only for triads from speciﬁc relative expression category, or the other way around. Frontiers in Plant Science 1 According to Wicker et al. (Wicker et al., 2018). TE names were selected based on the ClariTeRep naming system, which assigns simple numbers to individual families and subfamilies. 2 c2 corrected p-values for multiple tests using Benjamini/Hochberg Procedure (non-negative). 3 Number of triads belonging to one of the homoeolog-suppressed categories, in which at least one homoeolog contains TE insertion from the mention subfamily (Yes) or none of the homoeologs contain TE insertion from the mention subfamily (No). 4 Number of triads belonging to the balanced category or to one of the homoeolog-dominant categories, in which at least one homoeolog contains TE insertion from the mention subfamily (Yes) or none of the homoeologs contain TE insertion from the mention subfamily (No). + + + + + + – – + – – + + – – + + – – + + + + – r (second let be enriched 3.4 Triads which include TE insertions belonging to speciﬁc TE superfamilies were associated with various GO terms 10.3389/fpls.2022.1072232 A B A B C FIGURE 2 Signiﬁcantly enriched GO BP (Biological prosses) terms found in GO SEA preformed for triads which include TE insertions from the DTT (A), DTM (B) and RLC (C) superfamilies (TE codes are based on Wicker et al., 2007). GO SEAs were preformed using AgriGO toolkit with Fisher’s exact test (FDR ≤0.05). Following the GO SEA, the enriched GO terms for BP for each superfamily were visualized as a scatter plot generated by REVIGO. Closer GO terms in the plot are showing higher semantically similarity to each other. The bubble color indicates the FDR value and the size indicates the frequency of the GO term in the GOA database, bubbles of more general terms are larger. A B C C C FIGURE 2 Signiﬁcantly enriched GO BP (Biological prosses) terms found in GO SEA preformed for triads which include TE insertions from the DTT (A), DTM (B) and RLC (C) superfamilies (TE codes are based on Wicker et al., 2007). GO SEAs were preformed using AgriGO toolkit with Fisher’s exact test (FDR ≤0.05). Following the GO SEA, the enriched GO terms for BP for each superfamily were visualized as a scatter plot generated by REVIGO. Closer GO terms in the plot are showing higher semantically similarity to each other. The bubble color indicates the FDR value and the size indicates the frequency of the GO term in the GOA database, bubbles of more general terms are larger. FIGURE 2 Signiﬁcantly enriched GO BP (Biological prosses) terms found in GO SEA preformed for triads which include TE insertions from the DTT (A), DTM (B) and RLC (C) superfamilies (TE codes are based on Wicker et al., 2007). GO SEAs were preformed using AgriGO toolkit with Fisher’s exact test (FDR ≤0.05). Following the GO SEA, the enriched GO terms for BP for each superfamily were visualized as a scatter plot generated by REVIGO. Closer GO terms in the plot are showing higher semantically similarity to each other. The bubble color indicates the FDR value and the size indicates the frequency of the GO term in the GOA database, bubbles of more general terms are larger. (GO:0009607), innate immune response (GO:0045087)) (Figure 2B; Supplementary Figures S2-S10, Supplementary Tables S6, S21). 3.4 Triads which include TE insertions belonging to speciﬁc TE superfamilies were associated with various GO terms For example, for the DTM superfamily, while the Frontiers in Plant Science 12 frontiersin.org RLC1* RLX1* RIX1* SIX1* DTC1* DTM1* DTX1* DTH1* DTT1* DTA1* DXX1* XXX1* + – + – + – + + + – – + + – + – + – + – + – – + + + + – + – + – + – – – + – + – + – + – + – – + + – + – + – + – + – + + + – + – + – + – + – – – – – – – + – + – + – – – + – – – + – – – – – – + – + – + – + + + – – + – – – – – – + – + – – – + – + – + – + – + – – + – – + – + – – + – – + + + – – + – – – + – – – – – – – – – + – + – + – – – – – – – + – + – – – + – + – + – + – + – – – + + + – + – + – + – – + – – + – – – + – + – – – – – – – + – + – + – – – + – + – + – + – + – – – + – – – + – + – + – – – + – – – + – + – + – – – + – + – + – + – – – – – + – – – – + – + – – – ond letter) and superfamily (third letter) (Wicker et al., 2007). “+” notes that the respective GO term was found to be enriched in triads that include TE insertion nriched in triads which include TE from the speciﬁc superfamily. GO SEA was preformed using AgriGO toolkit (Tian et al., 2017) with Fisher’s exact test (FDR ≤ G1* R + + + + + + – – + – – + + – – + + – – + + + + – (second let be enriched Bariah et al. Frontiers in Plant Science 3.4 Triads which include TE insertions belonging to speciﬁc TE superfamilies were associated with various GO terms Go terms directly related to the regulating gene expression, DNA modiﬁcations, and response to biotic and abiotic stress (cellular response to glucose stimulus (GO:0071333)gene silencing by RNA (GO:0031047), chromatin silencing (GO:0006342), DNA methylation (GO:0006306), gene silencing(GO:0016458), RNA interference (GO:0016246), response to dsRNA(GO:0043331), response to virus (GO:0009615), response to nematode (GO:0009624)) were found to be signiﬁcantly enriched in the analysis performed for all the XXX insertions containing triads and missing from the results obtained from the GO SEA for triads belonging to an homoeolog-suppressed expression category that include XXX insertions (Supplementary Figures S2-S4, S8-S10, Supplementary Tables S18, S25). Meanwhile, GO terms related to aging and some abiotic stress (aging (GO:0007568), negative regulation of growth (GO:0045926), leaf senescence (GO:0010150) response to salt stress (GO:0009651), cellular response to alcohol (GO:0097306)) were found to be enriched in the list of triads belonging to one of the homoeolog-suppressed expression categories and containing XXX insertions, and missing from the GO SEA results obtained for all the XXX insertions containing triads (Supplementary Figures S2-S4, S8-S10, Supplementary Tables S18, S25). for homoeolog-suppressed vs. non-suppressed (balanced and dominant relative contribution) categories (c2 = 30.65, p < 0.001) and for homoeolog-dominant vs. non-dominant (balanced and suppressed relative contribution) categories (c2 = 18.64, p < 0.001). Out of the triads that contained TE insertions but did not contain insertions within exons, only 11.14% (741) were found in one of the homoeolog-suppressed categories, and 2.06% (137) were found in one of the homoeolog-dominant categories, while triads that include TE insertion within an exon were more likely to be found in both the homoeolog-suppressed (17.96%, 141 triads) and the homoeolog- dominant (4.59%, 36 triads) categories. More speciﬁcally, of the 608 triads that include TE insertions within an exon of at least one of the genes in the triad, 10.53% (64) include TE insertions within the 5’ UTR and 61.02% (371) include TE insertions within the 3’ UTR (Table S3). A lower proportion of the triads that include TE insertions within the 5’ UTR (46, 71.88%) and 3’ UTR (299, 80.59%) was found in the balanced expression category compared to triads that contain TE insertions but did not contain insertions within the 5’ UTRs (6,338, 85.94%) or within the 3’ UTR (6085, 86.09%), respectively. 3.4 Triads which include TE insertions belonging to speciﬁc TE superfamilies were associated with various GO terms Similarly, signiﬁcant association with GO terms that were not found to be enriched for all the DTM continuing triads was identiﬁed for the triads that include DTM insertions and belonging to one of the homoeolog-suppressed expression categories, including GO terms related to pollen formation and circadian rhythm regulation (negative regulation of circadian rhythm (GO:0042754), pollen exine formation (GO:0010584), pollen wall assembly (GO:0010208), Frontiers in Plant Science 14 frontiersin.org 10.3389/fpls.2022.1072232 Bariah et al. pollen development (GO:0009555)) (Figure 2B, Supplementary Figures S2-S10; Supplementary Tables S6, S22). Another example is the differences observed in the GO SEA results performed for all triads that include TE insertions from unknown class (XXX- unclassiﬁed repeats) versus the results obtained only for triads which include TE insertions from unknown class (XXX) and assigned to one of the homoeolog- suppressed expression categories (Supplementary Figures S2-S4 , S8-S10, Supplementary Tables S18, S25). Go terms directly related to the regulating gene expression, DNA modiﬁcations, and response to biotic and abiotic stress (cellular response to glucose stimulus (GO:0071333)gene silencing by RNA (GO:0031047), chromatin silencing (GO:0006342), DNA methylation (GO:0006306), gene silencing(GO:0016458), RNA interference (GO:0016246), response to dsRNA(GO:0043331), response to virus (GO:0009615), response to nematode (GO:0009624)) were found to be signiﬁcantly enriched in the analysis performed for all the XXX insertions containing triads and missing from the results obtained from the GO SEA for triads belonging to an homoeolog-suppressed expression category that include XXX insertions (Supplementary Figures S2-S4, S8-S10, Supplementary Tables S18, S25). Meanwhile, GO terms related to aging and some abiotic stress (aging (GO:0007568), negative regulation of growth (GO:0045926), leaf senescence (GO:0010150) response to salt stress (GO:0009651), cellular response to alcohol (GO:0097306)) were found to be enriched in the list of triads belonging to one of the homoeolog-suppressed expression categories and containing XXX insertions, and missing from the GO SEA results obtained for all the XXX insertions containing triads (Supplementary Figures S2-S4, S8-S10, Supplementary Tables S18, S25). pollen development (GO:0009555)) (Figure 2B, Supplementary Figures S2-S10; Supplementary Tables S6, S22). Another example is the differences observed in the GO SEA results performed for all triads that include TE insertions from unknown class (XXX- unclassiﬁed repeats) versus the results obtained only for triads which include TE insertions from unknown class (XXX) and assigned to one of the homoeolog- suppressed expression categories (Supplementary Figures S2-S4 , S8-S10, Supplementary Tables S18, S25). 3.4 Triads which include TE insertions belonging to speciﬁc TE superfamilies were associated with various GO terms The difference in proportions of the triads that include TE insertions within the 5’ UTR (c2 = 9.19, p = 0.0024 < 0.05) and within the 3’ UTR (c2 = 8.31, p = 0.0039 < 0.05) in the balanced expression category relative to the proportions of triads with no TE insertions in mentioned regions, were statistically signiﬁcant. We observed that triads that include TE insertions within the 5’ UTR were more likely to be found both in the homoeolog-suppressed (15.63%, 10 triads) and the homoeolog- dominant (12.50%, 8 triads) categories, relative to triads that contain TE insertions but did not contain insertions the 5’ UTRs, with only 11.82% of the triads (872) assigned to one of the homoeolog-suppressed categories and 2.24% of the triads (165) assigned to one of the homoeolog-dominant categories. Similar results were observed for triads which include TE insertions within the 3’ UTR, which were also found in higher proportions in the homoeolog-suppressed (15.09%, 56 triads) and the homoeolog-dominant (4.31%, 16 triads) categories, in comparison to triads that contain TE insertions but did not contain insertions the 3’ UTRs, for which only 11.69% of the triads (826) were assigned to one of the homoeolog-suppressed categories and only 2.22% of the triads (157) were assigned to one of the homoeolog-dominant categories. While a signiﬁcant difference was observed for the proportions of triads which include TE insertions within the UTRs and triads that contain TE insertions but did not contain insertions in the UTRs within homoeolog-dominant categories (for 5’ UTR: c2 = 25.08, p < 0.001 and for 3’ UTR: c2 = 5.90, p = 0.0152 < 0.05), the difference in proportion in the homoeolog-suppressed categories were not statistically signiﬁcant (for 5’ UTR: c2 = 0.55, p = 0.46 > 0.05 and for 3’ UTR: c2 = 3.60, p = 0.058 > 0.05). 4.2 Polymorphic TE insertions within gene bodies associated with non- balanced expression within the homoeologous group Since the three diploid genome donors of bread wheat originated from a common ancestor, it is not surprising that a high percentage of wheat HC genes are conserved and syntenic between the three subgenomes (Appels et al., 2018). Similarly, the abundances of 76% of TEs families were found to be similar between the A, B, and D sub-genomes of bread wheat and TE families distribution in promoter regions was found to be highly conserved between subgenomes (Wicker et al., 2018). However, almost no conserved TE insertions were observed between the three subgenomes, and more speciﬁcally, no conservation of TE insertions was observed between homeologous promoters (Wicker et al., 2018). To address the possible effect of TE insertions within gene bodies on gene expression, we ﬁrst identiﬁed TE insertions within dyads, triads, and tetrads genes. We found that a high percentage of the examined genes contained TE insertions within exons and introns, with the highest proportions of TE insertions found in triads genes. Additionally, genes found in the proximal region were more likely to include TE insertions within the gene body, suggesting that TE distribution within the gene body is in accordance with TE distribution across the chromosome, with lower density in the distal regions. However, the higher percentage of TE insertions in triads genes relative to dyads and tetrads genes persist throughout the different chromosomal regions. Therefore, the difference in the abundance of TE insertions within gene bodies between triads and dyads and tetrads genes is not only due to the higher abundance of triads genes in the TE rich proximal regions. Here, we assigned homoeologous groups as monomorphic or polymorphic based only on the presence of TE insertions within all or only some of the gene bodies of the homoeologs in the group. While the TE insertions found within the homoeologs in a monomorphic group were not necessarily inherited from the common ancestor, did not necessarily belong to the same TE family, and might have been inserted in different locations in the sequence, in this part of our analysis we focused on the effect of the mere presence of TE insertion on the relative expression within the homoeologous group. 4.1 TE insertions are highly abundant within gene bodies Together, dyads (11.7%), triads (51.1%), and tetrads (2.8%) are accounted for 65.6% of all bread wheat HC genes, while the rest of the genes deviate from these rations (Juery et al., 2020). While triads genes were kept in a 1:1:1 ratio between the three bread wheat subgenomes, dyads and tetrads homoeologous groups underwent copy number variations during the wheat group evolution (Juery et al., 2020). A study by Juery et al. (2020) showed that triads are diverse from dyads and tetrads in various characteristics, including conservation, chromosomal distribution, epigenetic modiﬁcation, gene ontology, and expression patterns. Their ﬁndings led Juery et al. (2020) to suggest that the highly conserved triads belong to the bread wheat core genome, while dyads and tetrads are parts of the dispensable genome. 4 Discussion instance, triads genes were found to be enriched in the H3K9ac active euchromatin mark and expressed at a higher level and higher breadth relative to dyads and tetrads genes, which were more associated with repressive H3K27me3 modiﬁcation (Juery et al., 2020). There is evidence that some TEs are preferentially inserted into transcriptionally active regions near active histone marks (Bennetzen, 2000; Hirsch and Springer, 2017; Sultana et al., 2017), conditions that ﬁt better to triads genes. Speciﬁcally, TEs belonging to the Mariner superfamily (DTT), the most abundant superfamily in triad genes, are known to be enriched in genes with high expression (Sultana et al., 2017). Moreover, the higher conservation of triads genes might contribute to the persistence of the TE insertion, provided that the insertion did not result in loss of ﬁtness. Alternatively, the presence of TE insertion within the gene body might impact various characteristics of the gene and maybe ultimately on gene conservation. Further study is necessary for a better understanding of the processes leading to the unique TE distribution pattern observed in this study. As an allohexaploid species, the bread wheat contains three subgenomes, A, B, and D, which originated from three diploid genome donors that diverged from a common progenitor ~7 MYA (Million Years Ago) (Levy and Feldman, 2022). While high conservation in gene content and order was observed between the three subgenomes, almost no sequence conservation was found in the intergenic regions, containing mostly TEs (Appels et al., 2018; Wicker et al., 2018). The contribution of TEs to the differentiation between the three subgenomes of the young allohexaploid bread wheat might facilitate genetic and cytological diploidization, which is essential for the survival of the new species. 3.5 TE insertions site within the gene body and triad expression patterns Next, we tested for a possible association between TE insertion context within the gene body (exon, 5’ UTR, or 3’ UTR) of one or more of the homoeologs in a triad and homoeolog expression bias. Of the 7,439 triads that include TE insertions, 10.55% (785) include TE insertion within an exon of at least one of the genes (Supplementary Table S3). A lower proportion of the triads that include TE insertion within an exon was found in the balanced expression category (608, 77.45%) compared to triads that contain TE insertions but did not contain TE insertions within exons (5,776, 86.80%) with a signiﬁcant difference in proportions (c2 = 49.70, p < 0.001). The signiﬁcant difference in proportions of the triads that include TE within an exon in balanced vs. non-balanced relative contribution categories was also observed separately Frontiers in Plant Science 15 frontiersin.org 10.3389/fpls.2022.1072232 Bariah et al. Frontiers in Plant Science 4.2 Polymorphic TE insertions within gene bodies associated with non- balanced expression within the homoeologous group Triads that contained TE insertions from each one of the 13 different TE superfamilies showed enrichment for a unique set of GO terms. Triads which include insertions of DTT and DTX, superfamilies for which a positive correlation was identiﬁed between TE presence within gene bodies and balanced expression of the triad, are found in association with numerous GO terms related to basic cell processes (Figure 2A). Additionally, triads which include insertions belonging to TE superfamilies that showed a positive correlation between TE presence within gene bodies and suppressed or dominant relative expression of the homoeologs (DTM, RLX, RLC, and XXX) were enriched for multiple GO terms. Speciﬁcally, triads that contained TE insertions from each of those 4 superfamilies (DTM, RLX, RLC, and XXX) were enriched for GO terms related to response to biotic and \ or abiotic stimulus (Figures 2B, C). characteristics of the homoeologs affected the probability of TEs to insert into each one of the genes in the homoeologous group. The percentage of polymorphic groups was lower than expected for triads and tetrads and higher than expected for dyads, which were found to be the least conserved relative to triads and tetrads (Juery et al., 2020). Additionally, our analysis revealed a strong signiﬁcant correlation between polymorphic TE insertions and non-balanced expression patterns of triads. We suggest that the mentioned correlation might be a result of either the effect of TE insertions on gene expression and \ or TE target site preference inﬂuenced by gene expression patterns and expression breadth. 4.3 Strong association between TE insertions within gene bodies and homoeolog expression bias Since TE insertions were found to be abundant in triads, and a clear correlation was observed between TE insertion pattern and relative expression of the homoeolog within the triad, we focused on triads to further learn about the possible impact of TE insertions on gene expression, using existing data regarding the relative contribution of each homoeolog to the overall triad expression. Our analysis revealed that a great variety of TEs inserted within wheat gene bodies, both into introns and exons. Here, we observed a strong correlation between the presence of TE insertions in gene bodies and the balanced expression of the three homoeologs in the triad. Similar to the differences in TE abundant in triads vs. dyads and tetrads genes, the unique characteristics of each relative expression category might explain the difference in TE content. Syntenic triads that were classiﬁed as balanced showed higher expression levels and had higher levels of active histone markers than syntenic triads in the homoeolog-dominant and homoeolog-suppressed categories (Ramı́rez-González et al., 2018). As we suggested for triads vs. dyads and tetrads, those characteristics, together with the balanced triads over representation in low recombination regions (Ramı́rez- González et al., 2018), might lid to higher insertion rate and higher persistent of TE insertions in the balanced triads genes relative to triads from the non-balanced categories. This claim is supported by the very high abundant of insertions from the Mariner superfamily (DTT), which was found to be enriched in genes with high expression (Sultana et al., 2017), in balanced triads and by the strong correlation observed speciﬁcally between the presence of TE insertions from the DTT superfamily and balanced homoeologs expression. Additionally, we suggest that the presence of TE insertions within gene bodies might result in a change in gene expression, resulting in balanced expression of the homoeologs. 5 Conclusions In this study, the integration and analysis of data from several publicly available databases revealed signiﬁcant correlations between the presence of TE insertions within gene bodies, gene expression and gene function in a genome-speciﬁc manner in wheat. We found that TE insertion site within the gene (exon\ intron) and TE type (superfamily\ subfamily) correlate strongly with homoeolog expression bias. Additionally, presence of TE insertion from all tested TE superfamilies were found to associate with numerous gene functions. Future studies are needed to decipher the causes for such correlations. In addition, comparative analysis between bread wheat accessions might shed light on the evolutionary time frame for TE insertions into gene bodies and on the involved mechanisms connecting TE presence within the gene body, gene expression, and gene function. 4.2 Polymorphic TE insertions within gene bodies associated with non- balanced expression within the homoeologous group However, the fact that the proportion of polymorphic groups did not match a module describing the random distribution of presence\ absence of TE insertions leads us to suggest that some of the TE insertions are indeed having a common origin, or alternatively, that common The higher abundance of TE insertions within triads genes relative to dyads and tetrads genes might be attributed to some of the distinguish characteristics of each of the categories. For Frontiers in Plant Science 16 frontiersin.org 10.3389/fpls.2022.1072232 Bariah et al. expression. However, while considering the insertion site and TE type, a more complex relationship between the presence of TE insertion and homoeolog expression bias is revealed. We found that speciﬁc TE superfamilies and families were enriched in triads which showed speciﬁc relative expression patterns. Furthermore, the presence of TE insertions from 13 out of the 14 TE superfamilies within a triad associated with multiple GO terms enriched both in basic cellular functions and in response to environmental factors. Triads that contained TE insertions from each one of the 13 different TE superfamilies showed enrichment for a unique set of GO terms. Triads which include insertions of DTT and DTX, superfamilies for which a positive correlation was identiﬁed between TE presence within gene bodies and balanced expression of the triad, are found in association with numerous GO terms related to basic cell processes (Figure 2A). Additionally, triads which include insertions belonging to TE superfamilies that showed a positive correlation between TE presence within gene bodies and suppressed or dominant relative expression of the homoeologs (DTM, RLX, RLC, and XXX) were enriched for multiple GO terms. Speciﬁcally, triads that contained TE insertions from each of those 4 superfamilies (DTM, RLX, RLC, and XXX) were enriched for GO terms related to response to biotic and \ or abiotic stimulus (Figures 2B, C). expression. However, while considering the insertion site and TE type, a more complex relationship between the presence of TE insertion and homoeolog expression bias is revealed. We found that speciﬁc TE superfamilies and families were enriched in triads which showed speciﬁc relative expression patterns. 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https://openalex.org/W4308446467	https://www.mdpi.com/1424-8220/22/21/8516/pdf?version=1667740365	English	null	An Effective Self-Configurable Ransomware Prevention Technique for IoMT	Sensors	2,022	cc-by	9,719	Article An Effective Self-Conﬁgurable Ransomware Prevention Technique for IoMT Usman Tariq 1 , Imdad Ullah 2 , Mohammed Yousuf Uddin 2 and Se Jin Kwon 3,* 1 Department of Management Information Systems, CoBA, Prince Sattam bin Abdulaziz University, Al-Khraj 16278, Saudi Arabia 2 College of Computer Engineering and Sciences, Prince Sattam bin Abdulaziz University, Al-Khraj 16278, Saudi Arabia 3 Department of AI Software, Kangwon National University, Samcheok 25913, Korea * Correspondence: sjkwon@kangwon.ac.kr 1 Department of Management Information Systems, CoBA, Prince Sattam bin Abdulaziz Universi Al-Khraj 16278, Saudi Arabia 3 Department of AI Software, Kangwon National University, Samcheok 25913, Korea * Correspondence: sjkwon@kangwon.ac.kr * Correspondence: sjkwon@kangwon.ac.kr Abstract: Remote healthcare systems and applications are being enabled via the Internet of Medical Things (IoMT), which is an automated system that facilitates the critical and emergency healthcare services in urban areas, in addition to, bridges the isolated rural communities for various healthcare services. Researchers and developers are, to date, considering the majority of the technological aspects and critical issues around the IoMT, e.g., security vulnerabilities and other cybercrimes. One of such major challenges IoMT has to face is widespread ransomware attacks; a malicious malware that encrypts the patients’ critical data, restricts access to IoMT devices or entirely disable IoMT devices, or uses several combinations to compromise the overall system functionality, mainly for ransom. These ransomware attacks would have several devastating consequences, such as loss of life-threatening data and system functionality, ceasing emergency and life-saving services, wastage of several vital resources etc. This paper presents a ransomware analysis and identiﬁcation architecture with the objective to detect and validate the ransomware attacks and to evaluate its accuracy using a comprehensive veriﬁcation process. We ﬁrst develop a comprehensive experimental environment, to simulate a real-time IoMT network, for experimenting various types of ransomware attacks. Following, we construct a comprehensive set of ransomware attacks and analyze their effects over an IoMT network devices. Furthermore, we develop an effective detection ﬁlter for detecting various ransomware attacks (e.g., static and dynamic attacks) and evaluate the degree of damages caused to the IoMT network devices. In addition, we develop a defense system to block the ransomware attacks and notify the backend control system. To evaluate the effectiveness of the proposed framework, we experimented our architecture with 194 various samples of malware and 46 variants, with a duration of sixty minutes for each sample, and thoroughly examined the network trafﬁc data for malicious behaviors. Citation: Tariq, U.; Ullah, I.; Yousuf Uddin, M.; Kwon, S.J. An Effective Self-Conﬁgurable Ransomware Prevention Technique for IoMT. Sensors 2022, 22, 8516. https:// doi.org/10.3390/s22218516 Keywords: Internet of Medical Things (IoMT); ransomware; Cyber-Security; Tizen OS; Cuckoo Sandbox Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. sensors sensors sensors sensors Article An Effective Self-Conﬁgurable Ransomware Prevention Technique for IoMT The evaluation results show more than 95% of accuracy of detecting various ransomware attacks. Citation: Tariq, U.; Ullah, I.; Yousuf Uddin, M.; Kwon, S.J. An Effective Self-Conﬁgurable Ransomware Prevention Technique for IoMT. Sensors 2022, 22, 8516. https:// doi.org/10.3390/s22218516 1. Introduction Unlike conventional malware attacks, which often target a single layer of an IoT network, ransomware may spread across the network, wreaking havoc on several levels before ultimately compromising the network’s security in the process e.g., integrity, conﬁdentiality etc. This has a disastrous effect on essential real-time systems such as IoMT since it leads to money losses and vital information leaks. A ransomware attack not only takes control of the network infrastructure of IoT network, it can also take control of the user data, resulting in limited or no access to the service data and operations. If the victim refuses to pay, the ransomware attack may result in extended period of attack, increased demand, or the data may be deleted by the attacker [11]. p y y The ransomware attacks can be divided into three categories: Crypto ransomware [12] where the attackers use the encryption and decryption algorithm (i.e., public-private key) to encrypt the data and ﬁles, hold the decryption key, and demand for ransomware. The decryption key is handed over once the victims pay the ransom amount. In case of the IoT networks (e.g., IoMT), the crypto ransomware is launched over the backend application servers instead of IoT devices since the sensor devices do not contain large amounts of data. Locker ransomware blocks user access to IoT devices, disables IoT sensor devices, and controls system performance. In addition, this attack can be combined with the DDoS attacks to disable user interface with the IoT devices or inactivate the sensor nodes, thus denying legitimate user access. The Hybrid ransomware encrypts the important devices’ data (i.e., IoMT devices) and uses locking mechanism, which is severely devastating since both the data and overall system functionality is compromised. y y p In this paper, we develop a ransomware analysis and identiﬁcation architecture that detects ransomware attacks and ensures their validity and accuracy using a comprehensive veriﬁcation process, by experimenting with various types of malwares. We took ten different malwares, e.g., GPcode, Filecoder, etc., and experimented on their various samples (respectively with an Avg. and St. Dev. of 21 and 18 samples) and variants (respectively with an Avg. and St. Dev. of ﬁve and two malware variants). Hence, we evaluated our ransomware analysis and identiﬁcation architecture with a total of 194 various samples of malware and a total of 46 variants. 1. Introduction The Internet of Things (IoT) has successfully managed the growing needs of societies by improving quality of life and has efﬁciently deployed a range of services, such as smart cities, agriculture, healthcare and emergency health services etc. Among the various applications of IoT, the implementation of the e-health Internet of Medical Things (IoMT) is playing a vital role in the healthcare industry with the aim to gain efﬁcient health access, improve the hospital healthcare quality, and improve the productivity of medical equipment [1,2]. To enable IoMT, a diverse range of sensor devices are connected together with IoT-enabled technologies in order to deliver reliable healthcare services e.g., distantly monitoring patients [3,4] and effectively communicating and advising [5]. Researchers have Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/sensors Sensors 2022, 22, 8516. https://doi.org/10.3390/s22218516 Sensors 2022, 22, 8516 2 of 18 been working to create healthcare frameworks that link medical equipment to a variety of healthcare services in a way that is accurate, reliable, and most importantly secure, all in an effort to enhance the healthcare industry as a whole via IoMT [6]. Various types of security attacks over IoT [7], especially controlling the medical devices in an IoMT e.g., for ransomware, have been affecting the communication of IoMT devices that could threaten the patients’ lives and disable the entire healthcare system. been working to create healthcare frameworks that link medical equipment to a variety of healthcare services in a way that is accurate, reliable, and most importantly secure, all in an effort to enhance the healthcare industry as a whole via IoMT [6]. Various types of security attacks over IoT [7], especially controlling the medical devices in an IoMT e.g., for ransomware, have been affecting the communication of IoMT devices that could threaten the patients’ lives and disable the entire healthcare system. The ransomware attacks are rapidly growing and are threats to individuals, businesses, and government and private sectors, where the attackers infect their deployed infrastructure and demand large amounts of ransom [8–10]. Due to an excess of such attacks, newer versions of the ransomware attacks appear frequently and have been responsible for millions of dollars of losses annually [11]. 1. Introduction For each of the selected malwares, we considered various types of ransomware attacks e.g., ‘Encrypting Files’, ‘Deleting Files’, ‘Stealing Files’. We conducted our experiments using a Cuckoo Sandbox [13] that helps analyze the network trafﬁc data, speciﬁcally encrypted with Secure Sockets Layer (SSL) and Transport Layer Security (TLS) protocols, for malicious behavior of network peers. In addition, for each of the 194 samples, we ran our experiment for a total for sixty minutes and thoroughly examined the network trafﬁc data for malicious behaviors. The primary goal of this malware analysis is multifold: How a malware attack interacts with ﬁle system associated with IoMT devices, the nature of attack e.g., changing metadata of a ﬁle system, to evaluate the robustness of a secure software system e.g., OpenSSL, against eavesdropping, to differentiate between static and dynamic ransomware attacks, and to verify the integrity via registry of a ﬁle system. In addition, we followed various guidelines [14,15] in order to ensure the validity and accuracy of ransomware detection analysis i.e., ensuring that the testing dataset comprises of diverse range of malwares, the malware attack is robust against different system conﬁgurations to experiment various settings and security measures, the target is within the vicinity of connected IoMT devices, Sensors 2022, 22, 8516 3 of 18 3 of 18 in addition to guaranteeing that the detection ﬁlter has access to optimize various signatures of source ﬁles. During each trial session, the system log was copied to a parallel storage location, where the responses were subsequently aggregated, evaluated, and audited. This was done in order to improve anomaly detection and make our detection process as effective as possible. Throughout this process of detection and optimization process, we took several measures and steps: to see if the ﬁles were setup in diverse locations, separate ﬁle setup was implemented to launch a different attack, search for corrupted ﬁles, and the ﬁles are concurrently generated. After every iteration of our experimental session, we record a detection rate which has a precision that is more than 85% of the detection accuracy. Similarly, for each of the iterations, we note an average latency of malware detection was recorded nearly 0.03 and 0.025 ms, respectively for continuous and triggered scanning of malware. Furthermore, for various timestamps of the experiments, we found an increase of malware detection of our proposed detection model. 1. Introduction We argue that this performance is satisfactory: it demonstrates the system’s consistency once an anomaly is initiated by the ransomware. Our main contributions are: Our main contributions are: • Developed a ransomware analysis and identiﬁcation architecture that detects ran- somware attacks within an IoMT network • Developed a comprehensive experimental environment to simulate a real-time IoMT network • Constructed a comprehensive set of ransomware attacks and analyze their effects over an IoMT network devices • Developed an effective Detection Filter for successfully detecting various ransomware attacks and evaluating the degree of damages caused to the IoMT network devices • Developed a defense system to block the ransomware attacks and notify. We organize the paper in the following sections: Section 2 presents the related work. The proposed methodology along with the experimental setup is presented in Section 3. Section 4 presents the performance analysis of our proposed ransomware analysis and identiﬁcation architecture. Section 5 presents experimental results; we conclude in Section 6. 2. Related Work Results for new ransomware detection rate are 99% of CryptoWall, 75% of TorrentLocker and 92% of Sage. This study focused on fog layer nodes, which holds sensitive data [19]. Azmoodeh et al. proposed a method in which energy consumption patterns are monitored to detect the ransomware. Energy consumption rate of different process is used to classify ransomware from non-malicious applications on Android devices. PowerTutor is used monitor power consumption of all running processes in 500 ms intervals. Ransomware samples from VirusTotal, Intelligence API are applied with active Command and Control (C2). This study demonstrated proposed approach outperforms K-Nearest Neighbors, Neural Networks, Support Vector Machine and Random Forest, in detection rate, recall rate, precision rate and F-measure [20]. Hatzivasilis et al. studied the security and privacy requirements and best practices, which can be adopted to safeguard the users and stakeholders along with IoMT system. Authors consider this study could be used as practical guide for developing IoMT appli- cation. Security areas considered by most of the popular vendors are devices, security at connectivity and cloud levels and security aspects studied here are conﬁdentiality, in- tegrity, and availability. Different protection mechanisms discussed in this study. Privacy of personal data stored in IoMT devices is equally a high priority requirement. There are established standards for privacy protection of Personal Identiﬁable Information (PII), similar to the ISO/IEC standards 27018 and 29100 and regulation efforts such as the General Data Protection Regulation of European Union Regulation (EC) 2016/679 was established. Data is categorized into personal sensitive, sensitive and statistical. Highest privacy pro- tection is given to personal sensitive data followed by the two others. Three aspects of privacy studied in this paper are data-collection, data-access, and data-usage. Protection mechanisms for these aspects are discussed and described [21]. Tervoort et al. reviewed options for mitigating cybersecurity risks from legacy medical device software. This study found eighteen solutions for intrusion detection and prevention, communication tunneling insecure wireless communications. This scoping review focus on vulnerabilities in medical devices due to lack of security features, legacy operating systems, unsupported software and inability to apply patches. To deal with the security issues of medical devices that run legacy software a scoping review using a bidirectional citation searching method. Search started with three relevant studies and discovered 121 articles cited by these three studies and 725 articles cite these studies. 2. Related Work Urooj et al. studied the dynamic analysis for ransomware detection across multiple platforms, dataset collection and application of machine learning, deep learning and hybrid approaches in dynamic analysis of ransomware detection. This study also proposes future research directions. This study listed twenty-three windows platform datasets, two Android, four cloud and IoT datasets and two network based datasets used in dynamic analysis. Dynamic analysis executes the malicious code in a controlled environment to capture the ransomware behavior. Pre-encryption detection studies are very limited according to this study. This study addressed the open issues in ransomware detection for future research: real-time detection, time-complexity, implementation on low speciﬁcation hardware, evasion and obfuscation-tolerant systems. This article provides comprehensive study on dynamic analysis for ransomware detection [16]. Humayun et al. provided a wide-ranging analysis on evolution, prevention and mitigation of ransomware in IoT. This study reported the current research directions for ransomware during the period of 2014 to 2018, the penetration in different countries and statistical report on ransomware attacks. Further ransomware propagation through various sources and most affected sectors. Healthcare sector is top most priority for the attackers including government and education sectors. This survey revels more priority should be given in studying prevention and mitigation of ransomware attacks on IoT enabled environments [17]. Alrawashdeh et al. offered a rapid method of ransomware detection using Memory- Assisted-Stochastic-Dynamic-Fixed-Point arithmetic using a four-layer Deep Belief Net- work (DBN) structure. In this approach, efﬁcient cross-correlation for the stochastic com- Sensors 2022, 22, 8516 4 of 18 putation in FPGA is produced by storing random bit-streams in memory. The memory technique for the Deep Belief Network (DBN) is trained with stochastic computation with dynamic ﬁxed-point arithmetic. Precision rate 91% and detection speed of 0.006 ms. is reported. This approach improves ransomware detection of ransomware in Internet of Things (IoTs) [18]. Homayoun et al. presented ’Deep Ransomware Threat Hunting and Intelligence System (DRTHIS)’ to detect ransomware and its family using Long Short-Term Memory (LSTM) and Convolutional Neural Network (CNN) deep learning techniques, for classiﬁca- tion using the softmax algorithm. Training dataset includes 220 Locky, 220 Cerber and 220 TeslaCrypt ransomware samples, and 219 benign samples. Test results achieve F-measure of 99.6% with a true positive rate of 97.2%. This model is even tested with previously untrained variants of ransomware families CryptoWall, TorrentLocker and Sage. 2. Related Work Results classiﬁed based on four criteria: (a) Application area (b) Risk type (c) Solution type and (d) Method of analysis. The authors found 18 studies that address the risk caused by legacy software in medical devices [22]. Fernandez et al. [23] proposed an intelligent and automatic solution to detection, classiﬁcation and mitigation of ransomware attacks in integrated clinical environments (ICE++). ICE++ combines the mobile edge computing (MEC), software deﬁned networking (SDN), and Network Function Virtualization (NFV) provides adjustable, cost-effective, and self-regulating administration of security system to mitigating the ransomware attacks in ICE. The proposed system consists of four modules: the monitoring module collects the network trafﬁc from medical devices and generates feature vectors, the ofﬂine model gener- Sensors 2022, 22, 8516 5 of 18 5 of 18 ation module receives feature vectors generated training dataset and selects a classiﬁcation algorithm to train a model and the analyzer module accepts the qualiﬁed ML models from and, in real time, the current feature vectors thereafter detects the anomalies and labels the trafﬁc as ransomware or benign. Finally, the decision and reaction module assesses the risk of having a functioning ransomware attack. This system uses One Class SVM for anomaly detection and Naive Bayes for probability valuation of the association of the new models to the class with the best analogous trafﬁc pattern. Performance of the proposed model is signiﬁcant: it achieved 92.32% of accuracy and 99.97% of precision in abnormality recognition; however Naive Bayes achieved a 99.99% of classiﬁcation precision. g y p Baek et al. [24] suggested a two-step hybrid malware detection system (2-MaD) to guard IoT nodes against obscured malware in a smart city environment. The 2-MaD program was split into two steps. First, in which the detection of malicious software is carried out using static analysis, and second, where the detection of malware is carried out using a dynamic scan. The correctness of the false negative rate (FNR) was used as a performance indicator. The performance assessment for 2-MaD exhibited that the malware discovery precision was 94.46%, which exceeded detection based on static analysis. The shortcoming of the projected scheme is that it failed to emphasized on involved application interface calls, instruction trace logs and track the registry changes. pp g g y g Damien et al. 2. Related Work [25] offered a feature section architecture (FeSA) that aims to discover a set of ransomware features that helped to develop the sustainability of the applied machine learning classiﬁer. The functional technique was equated to other systems such as evolutionary search, harmony search, etc. in order to measure the ransomware exposure degree, recall, false negative and accuracy. The projected mechanism failed to highlight the exploitation impact of victim node’s boot record. Moreover, the ransomware identiﬁcation and prevention paradigm was not thoroughly investigated at gateway that is prime spot for effective anomaly detection and response. Farnoush et al. [26] identiﬁed ransomware only consuming the headers of the exe- cutable ﬁle by creating and mapping feature vector graph using ‘Power Iteration’ technique. For evaluation purpose, three datasets were compiled. The ﬁrst data set contained 12,000 portable executables whereas the second dataset was based on 2000 executables. The third data set was assembled with malwares such as Wannacry, Cryptowall, etc. with ﬁle sizes ranging from 1kb to 26mb. The core beneﬁts of the offered technique are satisfactory computational convolution and satisfactory ransomware discovery rates. Zahoora et al. [27] proposed a novel Deep Contractive Autoencoder based Attribute Learning (DCAE-ZSL) system. The projected method was able to efﬁciently discover code insertion that can study the semantic depiction of zero-day attacks in an unsubstan- tiated style. To examine the method effectiveness the applied dataset comprises of 582 ransomware and 942 normal software samples based on ofﬁce productivity apps, mobile gaming APK and Microsoft Windows OS compatible widgets. Scheme accomplished a sub- stantial conciliation between false positive and false negative as associated to the shallow baseline prototypes. DCAE-ZSL is only effective on executable ﬁles, and does not show promising outcome for diverse family of ransomware anomalies. Manabu et al. [28] presented an open data set about hypervisor-based ransomware storage intake behaviors. The dataset contains entree conﬁgurations of ransomware options that considers variable OS versions and encryption applied methodologies as a benchmark- ing criterion for sample segmentation. Dataset validity was examined by applying feature engineering, and confusion matrices, which allowed to gain ﬁve-dimensional data vectors. The main limitation of focused research is that the dataset was compiled and assembled in consideration with an obsolete operating system (i.e., Windows 7) and will not be helpful to train system against emerging ransomware tools. 2. Related Work y g g g The Industrial Control System (ICS) links the virtual and natural worlds with various physical components, such as sensors and controllers. The present installation of the ICS is mostly housed in what is known as the “plant”, which is a technical name for the facility’s physical infrastructure. Inputs, outputs, and logic are the three components that Sensors 2022, 22, 8516 6 of 18 make up control systems in IoT enabled facilities. Control inputs transmit plant status. Zhang et al. [29] claims control logic is PLC software that can repeatedly receive, calculate, and deliver control signals. This article introduces ICS-ARC, an innovative ransomware attack mechanism that can automatically construct network packets unique to speciﬁc control logic. The approach is presented in this article. A cyberattack using ransomware is carried out by ICS-ARC in a four-step process. Both an Arduino with pre-installed OpenPLC and a separate tap water treatment system were constructed by the authors so that they could test the systems’ ability to exploit ICS-ARC vulnerabilities. The ﬁndings of the demonstrated operational anomaly reveals that ICS-ARC substantially increases the fault tolerance of the attempt while simultaneously decreasing the cost of the attack. The extensive implementation of various information technology platforms across a wide range of enterprise sectors is what is meant to be referred to as “digitization”. The process of transformation often makes use of several sorts of systems, such as software platforms, computer networks, and other types of network infrastructure. To identify phish- ing simulation research methods, technologies and research gaps in practically evaluated literature, a comprehensive review was carried out by Yeng et al. [30]. Phishing serves as the most prevalent ransomware assault because it exploits the weakest link. An SMS-based spooﬁng scenario research was conducted on Ghanaian medical practitioners using state- of-the-art and quantitative techniques. For evaluation, the following roadmap was adopted: (a) create content that is intentionally misleading, distribute a leaked document to the authorities, and get approval in advance; (b) examine the safety of the individuals involved as well as the security of their private details; and (c) claim about doing a debrieﬁng, getting post-consent feedback, and protecting your data. Research concludes that 61 percent of recruited healthcare professionals were vulnerable. Alqahtani et al. 2. Related Work [12], offered a survey that is dedicated to studying and assessing the state-of-the-art in ransomware detection and prevention in the interest of aiding the scientists that attempts to disrupt this extremely signiﬁcant and rising malware issue. The emphasis is on cryptographic ransomware since it is the kind that is the most common, damaging, and difﬁcult to deal with. This article reviews the methods used for ransomware identiﬁcation modeling to provide suggestions for the focus and orientation of research directions. Furthermore, concerns about ransomware early detection were addressed in this article. Approaches pertinent to the various detection method stages have been investigated. Authors have extensively commented on the current efforts that aim to enhance feature extraction, selection, and behavioral modeling. Article suggested that innovative techniques and solutions are still required, particularly those that examine combined data from various sources and systems to strengthen and enhance malevolent software’s behavioral traces. Table 1 summarizes the purpose of various research works, characterized over various features of ransomware, e.g., objective, contagion, malevolent events and extortion, along with various tested environments and platforms i.e., PCs, mobile device, and IoT. Sensors 2022, 22, 8516 7 of 18 Table 1. Assessment of similarities and differences of Related Work. Research Narrative Progression Covered Features of Ransomware Covered Platforms Objectives Contagion Malevolent Events Extortion PCs Mobile Devices IoT Alrawashdeh et al. [18] To effectively detect malicious activity, used deep learning to extract the latent representation of high-dimensional data. N I I I N I I F Homayoun et al. [19] Reveals IoT Ransomware development. I I I F I N I F Azmoodeh et al. [20] Demonstrated an Android power consumption-based machine learning method to identify ransomware assaults. I F I I N N F F Fernandez et al. [24] Examined how genetic and nature-inspired attribute selection methods work in systems with unpredictable forecasting modiﬁcations. I I I I N N N I Damien et al. [27] Examined how evolutionary and nature-inspired attribute selection techniques work in settings where the forecasting model evolves unexpectedly. I I I I N I I I Umme et al. [27] Used Zero-shot Learning (ZSL) to identify ransomware. I I I I I I I I Manabu et al. [28] Presented a novel dataset of ransomware conﬁgurations on several operating systems and storage system with complete ciphering. 3. Proposed Method Today, more than twelve billion devices are interlinked with each other to sense, aggregate, process, analyze and exchange valuable streams of data [31]. Each device has a unique identiﬁer which enables the functional user to seamlessly operate and perform rightful action. Considering the actionable gains, researchers are experimenting different use cases that will enhance the productivity, efﬁciency, and effectiveness of healthcare infrastructure. Mobility-aware digital healthcare appliances (i.e., Internet of Medical Things (IoMT)) are helpful in ‘remote patient monitoring (RPM)’ by leveraging the connected devices equipped with IoT sensors to monitor the ﬁtness of a person and feed the sensed data directly to ‘electronic health record (EHR)’ system. EHR is a vital decision-making tool for physicians. p y IoMT sensors help caregivers to capture reliable and contextual data feeds, such as a patient’s status of his/her physiological, heart rate, glucose level in blood, oxygen saturation, etc. Automation in hospitals reduces the cost and enhances the quality of care. For hospital staff, their prime focus is to keep the patients happy and healthy. Unfortunately, security and privacy are an exploitable consideration. Connected devices, such as ﬁtness monitors, sensors, afﬁliate data sources can be maliciously abused and disrupt the ﬂow of information to/from the needed platform. One of the emerging risks is associated with ‘ransomware’ that locks the victim machine to support adversary’s illicit intentions. Malwares (such as constructor, behavior analyzers, backdoors, SQL slammers, and Crypto- Lockers) are able to deploy evasion methods, infection broadcasting, and distributed Denial of Service (DDoS) attacks. Unlike traditional malwares, ransomware target ‘Command and Control (C&C)’ servers and health monitoring interconnected devices to exhaust and lock the daily operations of an organization. Proposed research focuses on devices mounted with Tizen [32] operating system. Tizen was programmed in consideration with rapidly changing mobility aware sensing devices and for afﬁliate data-driven systems. Tizen module supports Linux-based essential libraries that are helpful for EHR databases, data parsing, connectivity-related functionali- ties, personal information management (PIM), etc. It sustains an applied method and its properties using cgroup (i.e., control group). The signiﬁcance of the process is stimulated to lessen the likelihood that the OS event progression is killed in a low memory condition. Moreover, sensor frameworks furnish instrument proceedings to applications and platform modules. A sensor operation can be evaluated by engaging hardware or simulated sensors. Table 2 describes the application & system states as illustrated in Figure 1. Table 2. 2. Related Work F I I F I I I I N = No details are furnished, I = Incomplete data furnished, F = Full details are furnished. Table 1. Assessment of similarities and differences of Related Work. Sensors 2022, 22, 8516 8 of 18 3. Proposed Method System Service States. State Description Auto-Restart On-Boot On-Reboot Post Package Installation Pre Package Updates Ready Application has begun (i.e., setting up the dbus connection) No No Not auto launched when reboot Not auto-launched callback (i.e., service_app) when reboot Not launched inevitably Created Application initiates the core loop. No Yes Launched automatically Initiated callback (i.e., service_app) Launched inevitably Running Application is functional. Yes No Not Launched Not launched the callback (i.e., service_app) Launched inevitably Terminated Application’s functional operation has ended Yes Yes Not Launched Launched callback (i.e., service_app) Launched inevitably Table 2. System Service States. 9 of 18 Sensors 2022, 22, 8516 Figure 1. Module based application statuses. Figure 1. Module based application statuses. By extracting application statues, information such as Resource ID, Resource Type, URI (i.e., provides data related to Authority, application Path, system Query), Multipur- pose Internet Mail Extensions (MIME) type, application & message keys (for example: app_control_data, etc.), and launch mode settings (i.e., privileges) can be revealed. In pro- jected technique, for messaging ‘remote & local’ ports were accessible. Tizen platform secures accessible data and mounted applications by storing them in a secure repository and crypto modules. h l O SS d l l d k Figure 1. Module based application statuses. By extracting application statues, information such as Resource ID, Resource Type, URI (i.e., provides data related to Authority, application Path, system Query), Multipur- pose Internet Mail Extensions (MIME) type, application & message keys (for example: app_control_data, etc.), and launch mode settings (i.e., privileges) can be revealed. In pro- jected technique, for messaging ‘remote & local’ ports were accessible. Tizen platform secures accessible data and mounted applications by storing them in a secure repository and crypto modules. yp Using the popular OpenSSL command line program, we implemented key manage- ment, data integrity, ciphering, and decoding. The following cryptographic techniques were utilized: (a) Data Encryption Standard (DES), (b) RSA (Rivest–Shamir–Adleman), and (c) a digital signature algorithm (DSA). Tizen has a built-in Content Screening and Reputation (CSR) framework that grants privilege to installed application to ﬁlter, audit and block malicious event calls. Each application is eligible to code its malware scanning criteria by (a) forming content screening context, (b) applying content scanning (i.e., mem- ory, ﬁle, & directory), (c) detect a malware by signature veriﬁcation (i.e., size, exploit path, event list & size, and device privilege policy). Detection Filter We assume that if malware is present in the system, it inﬂuences more than one application-driven data set. Whether the adversary attempt was successful or failed, the corresponding data were logged. If a violation (i.e., malicious event or behavior) is observed, the defense system will block the event and notify the base station. ∑ Executions kO × 1 mK × 1 pos = Mo with ∑ Executions kO × 1 mK × 1 pos = Mo with (1) (1) kO = 1 if the execution comprehending ’H’ failed, (1) kO = 1 if the execution comprehending ’H’ failed, (1) kO = 1 if the execution comprehending ’H’ failed, (1) therwise Here, ‘kO’ is the entropy (i.e., state of disorder), where ‘O’ is the unique group of samples, ‘H’ is the associated malicious event linked with ransomware, and ‘mK’ is the analyzed log ﬁle. As per Equation (1), the number of unsuccessful executions in which ‘H’ is available (Mo) over several executions that exist in (M). ‘pos’ is represented as a Boolean function that signiﬁes the ‘open or false’ state of policy logic. k = K(H, i.e., is the malicious activity linked to ransomware ) = Mo + ∑execution kO × (1/mK)α × (1/pos)β 2M (2) (2) According to the Equation (2), there is a strong chance that the fault/Trojan horse is embedded in the system routine if there are abuses signaling a similar procedure. Thus, the use of probabilistic score estimation is beneﬁcial in the process of assigning relative ratings to all interconnected devices. For a given IoMT ad-hoc network ‘N = (H, P)’ with \|L\| devices, let ‘T = (tyx)’ be the parsimony model; if vertex ‘y’ is linked to ‘x’, so ‘tyx = 1’, and ‘tyx = 0’ otherwise. Here ‘N’ is the entropy rate of the ad-hoc network, and ‘P’ is the state of malware transmission matrix. dy = 1 ∃∑ xϵU(y) dx = 1 ∃∑ xϵN tyxdx (3) (3) where ∃refers to existential quantiﬁcation that was used to identify predicate variable and U(y) is a regular dataset of related event log associated to constants ‘y and ∃’. Application call-related event data require a ranking matrix to identify the importance of generated data logs. 3. Proposed Method To verify the integrity of registry keys, registry process, Dynamic-link library (DLL), UNICODE strings, information about TCP connections, TCP dumps, data related to ‘ﬁle_change_monitor’, TCP port listener, and unpacked applications. 5. To verify the integrity of registry keys, registry process, Dynamic-link library (DLL), UNICODE strings, information about TCP connections, TCP dumps, data related to ‘ﬁle_change_monitor’, TCP port listener, and unpacked applications. 3. Proposed Method Contrasting most of current malware attacks, ransomware threats are not typically considered to be furtive after the septicity level, as the entire idea of the attack is to report targets that their computing infrastructure is maliciously compromised with the aim to encrypt or destroy the data. To understand the ransomware triggering behavior of malicious software we have analyzed the following (Table 3) original and upgraded malware: Table 3. Analyzed malware that was used in experiments. Table 3. Analyzed malware that was used in experiments. Attack Types Malware Samples Variant Encrypting Files Deleting Files Stealing Files GPcode 55 9 Yes No No Filecoder 46 5 Yes Yes No Cryptolocker 30 5 Yes No Yes Tobfy (screen locker) 15 4 Yes No Yes Cryptowall 11 3 Yes No No AutoKMS 9 6 No Yes Yes Wacatac.B!ml 11 4 Yes Yes Yes Obfuscator 7 5 Yes No No Daemon_Tools_Lite_BundleInstaller 10 5 Yes Yes Yes Table 3. Analyzed malware that was used in experiments. Experiments were conducted using modular Cuckoo Sandbox [33] that permits anal- ysis of memory, behavior of malicious ﬁles and network trafﬁc especially when the data stream is cyphered with Secure Sockets Layer (SSL)/Transport Layer Security (TLS). Each sample was rigorously examined for sixty minutes. After each session, the system was Sensors 2022, 22, 8516 10 of 18 10 of 18 refreshed and rolled back to normal state to diminish or avoid any intrusion by secondary evaluations. The prime goal of malware analysis was: refreshed and rolled back to normal state to diminish or avoid any intrusion by secondary evaluations. The prime goal of malware analysis was: 1. To deﬁne by what means a malevolent procedure interacts with the ﬁle system after a victim IoMT device is experiencing a ransomware attack. 1. To deﬁne by what means a malevolent procedure interacts with the ﬁle system after a victim IoMT device is experiencing a ransomware attack. p g 2. To evaluate I/O callback routines that handles the ‘feature change call’ to the ﬁle system. p g 2. To evaluate I/O callback routines that handles the ‘feature change call’ to the ﬁle system. y 3. To analyze the reliability of customized cryptosystems (i.e., OpenSSL and YACA). 4. To understand the static (code) and dynamic (behavior) of ransomware. 5. Detection Filter KBy = 1 −A M + a ∑ xϵH(y) KB(x) outQ(x) (4) (4) where ‘a’ is the datasets, ‘H(y)’ is a set of relationship among data connections to ‘y’, ‘OutQ(x)’ is the outer links to external data sources available of node ‘x’, and ‘M’ is the total number of associated interlinked devices. Sensors 2022, 22, 8516 11 of 18 11 of 18 During experiments, we observe that during malware transaction, the short-time power consumption and frequency-domain function outcome data are drastically variable in comparison from the routine behavior. The Laplace transform function was applied to projected method to analyze continuously generated variable case data to resolve the initial value problem. D(w) = M−1 ∑ w=0 d(w)p−x2πxw/m, (0 ≤w ≤M −1) (5) (5) Equation (5) indicates the possibility to observe and evaluation of linear continuum ‘D(w)’ which indicates exposure of observed system to the malevolent entity. To match the signature, a requirement-focused characteristic database is required and was maintained. For this reason, Ay( f y) = probabilitydistribution( f y\|dy; ∃) (6) (6) where ‘y’ represents the chosen preparation data section, and ‘f’ is the classiﬁcation in which this dataset can ﬁt in to, and Ay refers to a deﬁnite likelihood distribution of ‘f’. where ‘y’ represents the chosen preparation data section, and ‘f’ is the classiﬁcation in which this dataset can ﬁt in to, and Ay refers to a deﬁnite likelihood distribution of ‘f’. measured variable (θ) = maxθ ∑ y ∑ f y Ay( f y) log probability(dy, f y; ∃) Ay( f y) (7) (7) For analysis, we collected, processed, and analyzed data captured in context of CPU energy consumption when speciﬁc health monitoring applications were active. In health- care facility, EHR was enriched by real-time sensor feeds (e.g., biosensor data ﬂow, images, accelerometer, temperature, and pressure). Sampling frequency for data aggregation of required data to identify ransomware was 200 ms per sample. Nominated ﬂags to identify the ransomware existence in the host device are identiﬁed in Table 4. Table 4. Observed attributes for malicious software and/or process call. Table 4. Observed attributes for malicious software and/or process call. Detection Filter Operation Purpose DLL removable execution Hijack Execution Flow Unwanted Executable Image Exploit targets Outdated Application Version Inject executable malicious code Size of Executable Code Identify and/or payload exploitation Size of Reserved Stack Off-by-one overﬂow indicator Size of Heap Stack Off-by-one overﬂow indicator Size of Reserved Commit Log4Shell exploitation Size of Heap Commit Heap-based buffer overﬂow Address of Entry Point Understanding Privilege Escalation Data Source Understanding Privilege Escalation, Command Injection Attacks Application Loader Flags Identiﬁcation of Code Flaws, and Insecure Code Data Relocation Calls Identify attack vectors Pointer to Raw Identify attack vectors Data Applied Primary & Sub Languages by Application Identify ‘System call parameters and application ‘Conﬁguration settings’ Timestamp of Executable Code Generation Exploit DB to launch Denial-of-Service The ransomware executable packages itself with numerous executables that are prereq- uisites for it to achieve its operational requirements. During the experiment, the projected ransomware used SHA-256 to partially encrypt each ﬁle separately, change and rename the ﬁle type extension identiﬁer, change the wallpaper on the device, programmatically avoid the caching suspension, and skew the examination of the encrypted binary ﬁle. Sensors 2022, 22, 8516 12 of 18 12 of 18 Here, we assumed that skewness is a degree of regularity in a dissemination. Momen- tal skewness (E) can be evaluated as: αu = 1/2E1=µ3/3σ3 (8) (8) where σ is the regular eccentricity, µ is the mean and ‘E’ is referred as skewness. Since the suggested experimental system could provide a mean and a standard deviation, the skew- ness of the data set at the time of evaluation could be calculated. In consideration of Equation (8), where σ is the regular eccentricity, µ is the mean and ‘E’ is referred as skewness. Since the suggested experimental system could provide a mean and a standard deviation, the skew- ness of the data set at the time of evaluation could be calculated. In consideration of Equation (8), E3 = ∑M y (Dy −D)3 (M −1) × σ3 (9) (9) where ‘D’ is random variable, and ‘D’ is the mean of distribution. where ‘D’ is random variable, and ‘D’ is the mean of distribution. In general, proposed technique considers an unfamiliar executable (i.e., mentioned in Tables 3 and 4) as apprehensive, for the reason that these kinds of process complication practices are commonly applied by malwares. 2. Malware is effective in different system conﬁgurations, 1. Veriﬁcation of testing dataset by ensuring if it contains dissimilar malwares, 4. Performance Analysis In consideration with Tizen ransomware attack phases, the malware identiﬁcation, tolerance, and countermeasure mechanisms were coded for characterization and evolution purpose. Various conﬁguration settings for the experimental evaluations are given in Table 5. Dataset (i.e., Table 3) analysis was conducted for the following system conﬁgura- tions: Table 5. Conﬁguration for experimental evaluation. Table 5. Conﬁguration for experimental evaluation. Operating System Tizen 6.0 M2 OS Kernel Linux LTS with multiuser support File format Tizen Package Kit (TPK) De-compilation Tool dnSPY CPU AMD A10-4600M, AMD A6-3400M Data Width 64 bits L1 Data 4 * 64 Kbytes (2-way) CPU Cores & Threads 4 Frequency 1400 to 2300 MHz Bus Speed 99.82 MHz Thermal Power 35 Watt Power state levels Screen (OFF, DIM, NORMAL, AWAKE) RAM 8 GB Devices X86 supportive architecture (wearables, mobile, etc.) Transistor Count 1303 million Total Data/Code Samples 194 Analyzed Ciphered File Extensions .FOX, .KOKO8, FASTB, .ANN Targeted Extensions .txt, .doc, .docx, .xls, .xlsx, .jpg, jpeg, .mdb, .zip, .rar Data Mining Tool (Testing and Validation) KNIME (Konstanz Information Miner) Before performing feature assortment, we have prepared our data set by eradicating duplicate features. For applied architecture mentioned in Figure 2, dataset weight was approximately 149 GB, which contains source ﬁles, data ﬁles, database backups, xml supported multimedia ﬁles, TPK, etc. To ensure validity and accuracy of ransomware detection analysis, we followed pre-declared guidelines, such as: 13 of 18 Sensors 2022, 22, 8516 13 of 18 3. Intended target is linked with Prince Sattam University’s internal or external network devices, and 3. Intended target is linked with Prince Sattam University’s internal or external network devices, and , 4. Detection ﬁlter is capable of editing system ‘access control policy’ source ﬁles in order to tune and optimize certain signature or behavior. 4. Detection ﬁlter is capable of editing system ‘access control policy’ source ﬁles in order to tune and optimize certain signature or behavior. Figure 2. Ransomware analysis and identiﬁcation architecture (i.e., before Denial-o-Service). Figure 2. Ransomware analysis and identiﬁcation architecture (i.e., before Denial-o-Service). The following factors are maintained constant during the entire experiment. The following factors are maintained constant during the entire experiment. 1. The experimental conﬁguration on which the test is performed. 2. The ransomware agent used to attack the target system. 3. The mechanism that detects ransomware. 4. The timing mechanism for system shutdowns. 4. The timing mechanism for system shutdowns. 4. Performance Analysis To apply weights to each evaluated feature, we used Logistic Regression (LR) model. LR provides an effective understanding of relationship among dissimilar dataset variables that helped project system to decide whether certain system call (e.g., ‘feature change call’) is malicious or legitimate. ρ(d) = 1 1 + p−d , d = β0 + β1d1 + . . . + βmdn (10) (10) where βm the erudite ratio for feature dn. This ratio is erudite through repetitions in order to diminish the inaccuracy between the anticipated values and the deﬁnite values. 5. Experimental Results Malware implies the use of common techniques, as illustrated in Table 3. To determine how the malware binary is generated and responded, three experimental trials were carried out. For each session, 60% of the sample set was used for training and 40% for testing the intended method. The results of each experimental session are set out in Figure 3. The primary aims to conduct detection trails were (a) to scan folders using signature-based detection to identify ﬁles and patterns related with ransomware, and (b) by using rule- based analytic, ﬁnd ransomware strains that have not been seen before. After each session, the system response log was aggregated, analyzed, and audited to enhance the anomaly detection system. Analyzed ransomware accesses a variety of directories to establish an appropriate environment for ciphering. Sensors 2022, 22, 8516 14 of 18 14 of 18 (a) (b) (c) Figure 3. Ransomware detection rate (a) Trial 1, (b) Trial 2, (c) Trial 3. (b) (a) (a) (b) ( ) ( (c) (c) Figure 3. Ransomware detection rate (a) Trial 1, (b) Trial 2, (c) Trial 3. Figure 4 (i.e., extension of Figure 2) focuses on the feature extraction that is required to identify and optimize vulnerability detection process. The following characteristics were exclusively (but not limited to) evaluated for each iteration: 1. If directories/ﬁles are set up in various locations? 2. If the created folders begin with a distinctive character? 3. If a separate ﬁling setup is implemented? 4. Do ﬁles that are created have understandable names? 5. Are various ﬁle formats taken into consideration? 6. Whether the ﬁles are unusable (corrupted) ﬁles? 7. If more than 50 ﬁles are generated concurrently? 8. If an alert is triggered when detection log ﬁles are viewed? Figure 4. Ransomware’s Feature detection and Optimization Process. Figure 4. Ransomware’s Feature detection and Optimization Process. Figure 5 indicates the mean latency, which is logged and compared after the detection technique has been carried out to determine whether the performance of other transactions is stimulated by the applied detection approach. In terms of performance measurement after registering the aforementioned activities, devices were periodically exposed to predeﬁned vulnerabilities. Once the infection scenario begins, the start and end times of the events are recorded to calculate the average overhead. 15 of 18 Sensors 2022, 22, 8516 Figure 5. Average Latency impact on Tizen mounted devices. 5. Experimental Results Figure 6 shows how the model performs on different timestamps. The output is not constant every second, but it is usually high because it is equal to or over 90 percent. For quick malware recognition, this performance is satisfactory because it demonstrates consistency once anomaly is initiated by the ransomware. Figure 5. Average Latency impact on Tizen mounted devices. Figure 5. Average Latency impact on Tizen mounted devices. Figure 5. Average Latency impact on Tizen mounted devices. Figure 6 shows how the model performs on different timestamps. The output is not constant every second, but it is usually high because it is equal to or over 90 percent. For quick malware recognition, this performance is satisfactory because it demonstrates consistency once anomaly is initiated by the ransomware. Figure 6. Early Detection Accuracy. Figure 6. Early Detection Accuracy. Figure 6. Early Detection Accuracy. Figure 6. Early Detection Accuracy. Figure 7 exhibits a comparison of the suggested technique with that of previously published studies. It is apparent that there are currently no solutions that can suit all the requirements that have been described. Nevertheless, the proposed technique has the potential to dramatically increase the IoMT system’s security and dependability while simultaneously minimizing threat likelihood. Figure 6. Early Detection Accuracy. Figure 6. Early Detection Accuracy. Figure 7 exhibits a comparison of the suggested technique with that of previously published studies. It is apparent that there are currently no solutions that can suit all the requirements that have been described. Nevertheless, the proposed technique has the potential to dramatically increase the IoMT system’s security and dependability while simultaneously minimizing threat likelihood. Figure 7 exhibits a comparison of the suggested technique with that of previously published studies. It is apparent that there are currently no solutions that can suit all the requirements that have been described. Nevertheless, the proposed technique has the potential to dramatically increase the IoMT system’s security and dependability while simultaneously minimizing threat likelihood. Figure 7. Comparative Analysis [18–20,24,25,27,28]. Figure 7. Comparative Analysis [18–20,24,25,27,28]. Sensors 2022, 22, 8516 16 of 18 16 of 18 To further support our evaluations, we carry out a t-test (note that other hypothesis testing can be carried out) to test the null hypothesis whether the proposed framework and literature works would provide the same level of protection to the IoMT systems. 5. Experimental Results Various statistics are given in Table 6, such as t Stat, t Critical two-tail, and the Mean Differences. It can be observed that t Stat values compared to t Critical two-tail, for the proposed framework, are consistently higher than the previously published works. As an example, examin- ing the ﬁrst column i.e., comparing proposed framework with Alrawashdeh, et al. [18], the tStat > t Critical two −tail are evaluated to 3.55 > 2.57, hence we reject the null hy- pothesis, whereas carrying a huge mean difference of 0.15. Similarly, for some other proposals e.g., [19,20,24], there is a huge difference among various statistics, which is convincing enough to claim that the security provision with the proposed approach is signiﬁcantly different from these proposals. Similar trends can also be observed with other proposals i.e., [25,27,28]. Table 6. Statistical hypothesis testing for proposed framework and previously published works. Statistical Symbols Proposed Research t Stat 3.55 11.49 12.82 15.64 8.06 9.74 6.67 t Critical two-tail 2.57 2.31 2.31 2.31 2.36 2.36 2.45 Mean Differences 0.15 0.26 0.25 0.36 0.19 0.19 0.20 [18] [19] [20] [24] [25] [27] [28] Table 6. Statistical hypothesis testing for proposed framework and previously published works. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Deanship of Scientiﬁc Research, Prince Sattam Bin Abdulaziz University, Saudi Arabia. 6. Conclusions Large or small, regardless of size, each institute is vulnerable to devastating effects of ransomware attack. Cyber criminals have been developing vulnerabilities at a more rapid pace than system developers have programmed protections in several recent incidents. The risk associated with ransomware begins with a preliminary infection and compromise, but it does not end there. In order to avoid vulnerability, a defense system should be autonomic and autonomous. The proposed technique leveraged multiple capabilities, such as monitoring, identiﬁcation and alerting of abnormal sourcing patterns for incident response. Vulnerability analysis enabled the ransomware prevention system to respond in an active manner. During the experimental assessment, the proposed methodology was reviewed and trained to achieve resiliency with integrated recovery skills. The malware prevention shield limits the ability of the adversary to create highly targeted attacks. To eliminate blind spots, in future we aim to focus on: 1. Explore opportunities to bridge security monitoring gaps. 2. Examine the impact of packet trafﬁc monitoring to purpose secure hybrid environment (i.e., on-premises and in the cloud) that can operate without friction, 2. Examine the impact of packet trafﬁc monitoring to purpose secure hybrid environment (i.e., on-premises and in the cloud) that can operate without friction, 3. Indicate appropriate methodology to gain scalable system audit log in order to gain valuable insights. g 4. Detect next-generation and emerging ransomware threats in real-time with higher efﬁciency and diminished false positives. Author Contributions: Conceptualization, U.T.; Data curation, M.Y.U.; Formal analysis, U.T., I.U. and S.J.K.; Funding acquisition, U.T., M.Y.U. and S.J.K.; Investigation, M.Y.U.; Methodology, U.T., I.U. and S.J.K.; Project administration, S.J.K.; Resources, S.J.K.; Software, I.U. and M.Y.U.; Supervision, S.J.K.; Validation, M.Y.U.; Visualization, M.Y.U.; Writing—original draft, U.T. and S.J.K.; Writing— review & editing, I.U. and S.J.K. All authors have read and agreed to the published version of the manuscript. Author Contributions: Conceptualization, U.T.; Data curation, M.Y.U.; Formal analysis, U.T., I.U. and S.J.K.; Funding acquisition, U.T., M.Y.U. and S.J.K.; Investigation, M.Y.U.; Methodology, U.T., I.U. and S.J.K.; Project administration, S.J.K.; Resources, S.J.K.; Software, I.U. and M.Y.U.; Supervision, S.J.K.; Validation, M.Y.U.; Visualization, M.Y.U.; Writing—original draft, U.T. and S.J.K.; Writing— review & editing, I.U. and S.J.K. All authors have read and agreed to the published version of the manuscript. Funding: The funding for this research is from the Deanship of Scientiﬁc Research at Prince Sattam Bin Abdulaziz University under the research project ‘2021/01/17623’. Funding: The funding for this research is from the Deanship of Scientiﬁc Research at Prince Sattam Bin Abdulaziz University under the research project ‘2021/01/17623’. References 1. Alattas, K.; Wu, Q. A framework to evaluate the barriers for adopting the internet of medical things using the extended generalized TODIM method under the hesitant fuzzy environment. Appl. Intell. 2022, 52, 13345–13363. [CrossRef] 2. Zikria, Y.B.; Afzal, M.K.; Kim, S.W. Internet of multimedia things (IoMT): Opportunities, challenges and solutions. Sensors 2020, 20, 2334. [CrossRef] [PubMed] 3. Manickam, P.; Mariappan, S.A.; Murugesan, S.M.; Hansda, S.; Kaushik, A.; Shinde, R.; Thipperudraswamy, S. 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https://openalex.org/W4377038204	https://zenodo.org/records/7947908/files/2022.bucc-1.5.pdf	English	null	Multilingual Comparative Analysis of Deep-Learning Dependency Parsing Results Using Parallel Corpora	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	7,816	1. Introduction Therefore, our aim in this paper is to propose a multi- lingual analysis of dependency parsing results consid- ering the syntactic structure of languages (using head directionality parameter). By using parallel annotated corpora, our idea is to scrutinize parsing results ob- tained with a deep learning model to check how dif- ferent language structures influence the performance of the chosen tool. Also, our aim is to correlate it with the syntactical characterization of languages concern- ing the specific syntactic feature of head and dependent position. As presented by Jurafsky and Martin (Juraf- sky and Martin, 2021) this is one of the features that plays a role in the performance of graph-based parsers. The idea is to check the degree of influence in depen- dency parsing of this specific language characteristic. The paper is composed as follows: Section 2 presents an overview of the related work to this topic. Section 3 describes the campaign design: language and data-sets selection, dependency parsing annotation, and syntac- tic typological characterization; Section 4 present the obtained results which are discussed in Section 5. In Section 6 we provide conclusions and possible future directions for research. Therefore, our aim in this paper is to propose a multi- lingual analysis of dependency parsing results consid- ering the syntactic structure of languages (using head directionality parameter). By using parallel annotated corpora, our idea is to scrutinize parsing results ob- tained with a deep learning model to check how dif- ferent language structures influence the performance of the chosen tool. Also, our aim is to correlate it with the syntactical characterization of languages concern- ing the specific syntactic feature of head and dependent position. As presented by Jurafsky and Martin (Juraf- sky and Martin, 2021) this is one of the features that plays a role in the performance of graph-based parsers. The idea is to check the degree of influence in depen- dency parsing of this specific language characteristic. The paper is composed as follows: Section 2 presents an overview of the related work to this topic. Section 3 describes the campaign design: language and data-sets selection, dependency parsing annotation, and syntac- tic typological characterization; Section 4 present the obtained results which are discussed in Section 5. In Section 6 we provide conclusions and possible future directions for research. Proceedings of the 15th Workshop on Building and Using Comparable Corpora (BUCC 2022) @LREC2022 , pages 33–42 Marseille, June 25, 2022 © European Language Resources Association (ELRA), licensed under CC-BY-NC Proceedings of the 15th Workshop on Building and Using Comparable Corpora (BUCC 2022) @LREC2022 , pages 33–42 Marseille, June 25, 2022 © European Language Resources Association (ELRA), licensed under CC-BY-NC 1. Introduction Dependency parsing is an important part of Natural Language Processing (NLP) chains which consist of annotating raw texts from tokenization to dependency relations. This specific task concerns the process to an- alyze the grammatical structure in a sentence and iden- tify syntactic heads as well as the type of the relation- ship between them (syntactical analysis) (Jurafsky and Martin, 2009). Since the 1980s, the NLP field has increasingly relied on statistics, probability, and machine learning meth- ods which require a large amount of linguistic data. Unlike other annotation tasks such as POS tagging, de- pendency parsing annotation is much more complex and expensive. Furthermore, from 2015 onward, the usage of deep learning techniques has been dominant in this field which has provided a great improvement in overall results even for under-resourced languages (Otter et al., 2018). The paper is composed as follows: Section 2 presents an overview of the related work to this topic. Section 3 describes the campaign design: language and data-sets selection, dependency parsing annotation, and syntac- tic typological characterization; Section 4 present the obtained results which are discussed in Section 5. In Section 6 we provide conclusions and possible future directions for research. The focus of the majority of studies regarding depen- dency parsing is on new methods to improve overall results using existing data. Methods and algorithms are compared in terms of results, however, usually, there is no comparison or analysis of the obtained results con- sidering the syntactic complexity of languages. This is due to the fact that, in general, systems are trained using different data-sets (in terms of size and content) for different languages. The lack of data for under- resourced languages is the usual explanation for worse results with respect to dependency parsing metrics. It is undeniable that the amount of training data plays a cru- cial role in the performance of deep learning models, however, it is not clear how models deal with different structures of languages when the same type and amount of linguistic data is provided for different languages. Abstract This article presents a comparative analysis of dependency parsing results for a set of 16 languages, coming from a large variety of linguistic families and genera, whose parallel corpora were used to train a deep-learning tool. Results are analyzed in comparison to an innovative way of classifying languages concerning the head directionality parameter used to perform a quantitative syntactic typological classification of languages. It has been shown that, despite using parallel corpora, there is a large discrepancy in terms of LAS results. The obtained results show that this heterogeneity is mainly due to differences in the syntactic structure of the selected languages, where Indo-European ones, especially Romance languages, have the best scores. It has been observed that the differences in the size of the representation of each language in the language model used by the deep-learning tool also play a major role in the dependency parsing efficacy. Other factors, such as the number of dependency parsing labels may also have an influence on results with more complex labeling systems such as the Polish language. Keywords: dependency parsing, typology, multilingualism Keywords: dependency parsing, typology, multilingualism 2. Related Work One example is the method proposed by Agi´c (Agi´c, 2017) where he combines three language comparison techniques to determine the best single source for an unannotated language: part-of-speech trigrams, a lan- guage identification software (lang.py tool, developed by (Litschko et al., 2020)), and WALS features. It con- siders the whole corpus of the unannotated language to determine the best (most similar in terms of the de- scribed comparison features) training corpus. Later, it has been showed by (Litschko et al., 2020) that bet- ter results are obtained when typologically analysing each sentence of the unannotated language in compar- ison with the available annotated corpora, defining, for each instance the best model (and not the same one for the whole text). In both studies, only qualitative typo- logical features and surface level word order (part-of- speech trigrams) are considered. In the article ”Evaluating Language Tools for Fifteen EU-official Under-resourced Languages” (Alves et al., 2020), the authors have compared tools to check the reproducibility of presented results in the official re- spective articles. The authors, however, used the same heterogeneous corpora as the developers of the tools to train the models, the focus was on the analysis of the discrepancy between obtained results and claimed ones by the tool creators. While the studies mentioned in the previous paragraph focus on part-of-speech trigrams to compare languages, (Wang and Eisner, 2018) proposed a method to com- pare word order (in terms of part-of-speech possi- ble combinations) by using a deep-learning algorithm (multilayer perceptron architecture) that classify lan- guages in an unsupervised way with the information extracted from delexicalized corpora. This model is, then, used to the identification of the best language to serve as the source of the best training corpus for the target one. Their major aim was to prove that part-of- speech (POS) sequences carry useful information about the syntax of a language. Parallel corpora are most often used in machine trans- lation (MT) tasks. Therefore, many studies considering the quality, availability, and performance of tools using this type of data-set do not consider dependency pars- ing. It is the case of the studies presented by Heiki-Jaan Kaalep and Kaarel Veskis (Kaalep and Veskis, 2007) and Wolfgang Teubert (Teubert, 1996). When paral- lel corpora are considered for parsing, the analysis is, most generally, focused on the improvement of over- all results, not on language comparison, as in (Kuhn, 2005). 2. Related Work The Universal Dependencies (UD) framework (Nivre et al., 2016) proposes a robust set of rules for annotat- ing parts of speech, morphological features, and syn- tactic dependencies across different human languages allowing multi-lingual data to be annotated with the same set of tags. If the framework can be used to anno- tate, in a homogeneous way, different languages, there is a lack of annotated parallel corpora that can be used for more precise multilingual comparison studies. As mentioned in the previous section, many studies 33 automatic annotation, especially regarding unannotated languages (which do not have any corpora annotated in terms of syntactic relations) and low-resource ones. One example is the method proposed by Agi´c (Agi´c, 2017) where he combines three language comparison techniques to determine the best single source for an unannotated language: part-of-speech trigrams, a lan- guage identification software (lang.py tool, developed by (Litschko et al., 2020)), and WALS features. It con- siders the whole corpus of the unannotated language to determine the best (most similar in terms of the de- scribed comparison features) training corpus. Later, it has been showed by (Litschko et al., 2020) that bet- ter results are obtained when typologically analysing each sentence of the unannotated language in compar- ison with the available annotated corpora, defining, for each instance the best model (and not the same one for the whole text). In both studies, only qualitative typo- logical features and surface level word order (part-of- speech trigrams) are considered. concerning dependency parsing metrics present multi- lingual perspectives but results cannot be compared in terms of language structure as training sets come from different sources and present different sizes and gen- res. An example of it is the article presenting UDify tool (Kondratyuk and Straka, 2019) which is a soft- ware conceived for PoS-MSD and dependency parsing tagging integrating Multilingual BERT (mBERT) lan- guage model (104 languages) (Pires et al., 2019). It is also the case for mainstream NLP tools such as Stan- ford Core NLP (Manning et al., 2014), UDPipe (Straka and Strakov´a, 2017), sPacy (Honnibal and Montani, 2017) and NLPCube (Boros, et al., 2018). automatic annotation, especially regarding unannotated languages (which do not have any corpora annotated in terms of syntactic relations) and low-resource ones. 2. Related Work A different approach, using only typological informa- tion from URIEL database (lang2vec tool, (Littell et al., 2017)), was presented by (Glavaˇs and Vuli´c, 2021). Their method consists of comparing the vector com- posed by the values of the linguistic features of the target language with vectors from other well-resourced languages. The idea is not to select the best corpus but to combine the most similar ones from different lan- guages as long as the similarity respects a determined threshold. Liu and Xu proposed a quantitative syntactic typolog- ical analysis of Romance languages using information from corpora annotated for dependency syntactic rela- tions (Liu and Xu, 2012). They have analyzed the over- all distribution of dependency directions which enabled them to correlate with the degree of inflectional varia- tion of a language and to classify them diachronically (compared to Latin) and synchronically. Moreover, in a different article (Alzetta et al., 2020), the authors pre- sented a study whose main objective was to identify cross-linguistic quantitative trends in the distribution of syntactic relations in annotated corpora from dis- tinct languages (4 Indo-European ones) by using an al- gorithm (LISCA - LInguiStically– driven Selection of Correct Arcs) (Dell’Orletta et al., 2013) capable of de- tecting patterns of syntactic constructions in large data- sets. However, results were not correlated to scores of dependency parsing tools and corpora used were not parallel, thus the content part of texts was not a con- trolled variable. These studies have in common the objective of choos- ing the best combination of languages to improve de- pendency parsing results, there is no specific analysis concerning the influence of the chosen features used to compare languages on the final results. In a different perspective, (de Lhoneux et al., 2018) compared how typological features are related to the dependency parsing results when twenty-seven di- verse deep-learning parameters are used for cross- lingual parameters sharing. They were divided in three sets: character based one-layer (bidirectional LSTM), word based two-layer (bidirectional LSTM), and multi- layered perceptron (MLP) with a single layer. The authors have shown that the linguistic intuition that character- and word-level LSTMs are highly sensitive Typological information has been used in different ways in many studies intending to improve depen- dency parsing results. 1https://github.com/UniversalDependencies/UD English- PUD 2https://wals.info/ 3. Experimental Design In this section, we describe the corpora that have been used in this study, the dependency parsing task evalua- tion using UDify software, and the typological classifi- cation method that has been employed. 2. Related Work It has been proved that typolog- ical comparison of languages is a powerful way of in- crease overall metrics concerning dependency parsing 34 Language Code Family Genus Arabic arb Afro- Asiatic Semitic Chinese cmn Sino- Tibetan Chinese Czech ces Indo- European Slavic English eng Indo- European Germanic Finnish fin Uralic Finnic French fra Indo- European Romance German deu Indo- European Germanic Hindi hin Indo- European Indic Icelandic isl Indo- European Germanic Indonesian ind Austro- nesian Malayo- Sumbawn Italian ita Indo- European Romance Japanese jpn Japanese Japanese Korean kor Korean Korean Polish pol Indo- European Slavic Portuguese por Indo- European Romance Russian rus Indo- European Slavic Spanish spa Indo- European Romance Swedish swe Indo- European Germanic Thai tha Tai-Kadai Kam-Tai Turkish tur Altaic Turkic Table 1: List of languages, the respective ISO-639-3 code and the genealogical information to phonological and morphosyntactic differences (such as word order), whereas the MLP learns to predict less idiosyncratic, hierarchical relations from relatively ab- stract representations of parser configurations. Lan- guages were compared in terms of their genealogical family and subject, verb and object order (qualitative classification). 3.1. Languages and Data-set Selection The data-sets used for all experiments are part of the Parallel Universal Dependencies (PUD) tree-banks cre- ated for the CoNLL 2017 shared task on Multilin- gual Parsing from Raw Text to Universal Dependen- cies. They are composed of 1000 sentences for each language, always in the same order, coming from the news domain and Wikipedia (Zeman et al., 2017). The first 750 sentences are originally in English and the rest are in German, French, Italian or Spanish. Sen- tences were mostly translated by professional transla- tors via the English text. The data has been annotated morphologically and syntactically by Google accord- ing to Google universal annotation guidelines, after- wards, labels were converted to Universal Dependen- cies v2 guidelines1. The corpora were composed to serve as test sets to the mentioned shared task. Due to their relatively small size, the creators have suggested that a ten-fold cross- validation should be employed should these sets be used as training ones (as it is the case in this article). As our aim was to focus on one specific syntactic feature, the idea was to use only parallel corpora so that there would be no bias concerning the size or the domain of corpora. No data augmentation technique was used as there is no other parallel annotated corpora covering all PUD languages. Table 1: List of languages, the respective ISO-639-3 code and the genealogical information The list of PUD languages, their ISO-639-3 code and their genealogical information according to WALS2 (Dryer and Haspelmath, 2013) is presented in the table 1. Although WALS database provides valuable infor- mation of word order patterns, there is no information regarding the relative position of the head and depen- dent in a broader way. Their focus is on word order position of subject, object and verb (and other type of syntactic functions), not exactly specifying the behav- ior of the ensemble of heads and dependents. on the language, their distribution is presented in the tables 2 and 3. Languages Number of UPOS labels kor 13 cmn, tur 15 arb, ces, fin, hin, jpn, spa, swe 16 eng, fra, deu, ita, pol, por, rus, tha 17 isl, ind 18 Table 2: Distribution of the number of UPOS labels (core part-of-speech) for each language in PUD data- set The CoNLL-U format also presents a column for on the language, their distribution is presented in the tables 2 and 3. 3.1. Languages and Data-set Selection Language Size Range (GB) eng [11.314, 22.627] deu, fra, spa, rus [2.828, 5.657] cmn, ita, jpn, pol, por [1.414, 2.828] arb, ces, swe [0.707, 1.414] fin, ind, kor, tur [0.354, 0.707] tha [0.177, 0.354] hin [0.088, 0.177] isl [0.022, 0.044] Table 4: List of languages we consider in mBERT and its pre-training corpus size (Wu and Dredze, 2020) Language Size Range (GB) eng [11.314, 22.627] deu, fra, spa, rus [2.828, 5.657] cmn, ita, jpn, pol, por [1.414, 2.828] arb, ces, swe [0.707, 1.414] fin, ind, kor, tur [0.354, 0.707] tha [0.177, 0.354] hin [0.088, 0.177] isl [0.022, 0.044] Table 4: List of languages we consider in mBERT and its pre-training corpus size (Wu and Dredze, 2020) Table 4: List of languages we consider in mBERT and its pre-training corpus size (Wu and Dredze, 2020) Table 4: List of languages we consider in mBERT and its pre-training corpus size (Wu and Dredze, 2020) Table 3: Distribution of the number of deprel labels for each language in PUD data-set It is possible to notice that there is a huge discrepancy regarding the amount of data from different languages used to generate multilingual BERT language model. language-specific part-of-speech tag (XPOS). For this feature, not all languages follow the same labeling sys- tem. Arabic, Chinese, English, French, German, Hindi, Italian, Korean, Portuguese, Spanish, Turkish and Thai use the same tags to characterize language specific POS, the other languages in PUD either present dif- ferent sets of tags, or, as it is the case of Finnish and Indonesian, no information at all is provided concern- ing this feature. As expected, English is the language which has the largest pre-training corpus size, followed by German, French, Spanish and Russian. It is possible to observe that the largest mBERT pre-training corpora come from Indo-European languages, only Chinese and Japanese languages are also quite well represented. Icelandic is the one with the smaller pre-training corpus, therefore, not as well represented in this language model as the other languages from PUD corpora. 3.2. Dependency Parsing Annotations Thus, even though we use parallel data to understand the influence of the position of head and dependent fea- ture, by using a system based on mBERT introduce a bias regarding the discrepancy of the training data used in this language model. The importance of this bias will be analysed further in this article. We could have cho- sen a tool which does not depend on language models to conduct our experiments, however, as these models are part of the state-of-the-art concerning dependency parsing, we decided to keep our initial choice to verify how the chosen syntactic feature influences the results of parsing, if it plays an important role or if it is com- pletely minimized. UDify tool (Kondratyuk and Straka, 2019) proposes an architecture aimed for PoS-MSD and dependency pars- ing tagging integrating Multilingual BERT language model (104 languages). It can be fine-tuned using spe- cific corpora (mono or multilingual) to enhance overall results. We have selected this tool as it presents the state-of- the-art algorithms concerning the specific task of de- pendency parsing Annotation. Training parameters were defined as: • Number of epochs: 80 • Warmup: 500 • Warmup: 500 3.1. Languages and Data-set Selection on the language, their distribution is presented in the tables 2 and 3. Languages Number of UPOS labels kor 13 cmn, tur 15 arb, ces, fin, hin, jpn, spa, swe 16 eng, fra, deu, ita, pol, por, rus, tha 17 isl, ind 18 All corpora have been tagged in terms of core part- of-speech categories (UPOS) and dependency relation (deprel) using Universal Dependencies labels. The number of UPOS and deprel labels varies depending Table 2: Distribution of the number of UPOS labels (core part-of-speech) for each language in PUD data- set The CoNLL-U format also presents a column for The CoNLL-U format also presents a column for 35 Languages Nb. of deprel types Nb. of deprel sub-types arb 34 8 cmn 32 12 ces 31 12 eng 36 12 fin 30 14 fra 31 14 deu 33 12 hin 28 10 isl 31 5 ind 33 14 ita 33 7 jpn 25 0 kor 26 8 pol 28 31 por 33 9 rus 31 8 spa 32 9 swe 33 9 tha 33 10 tur 34 7 Table 3: Distribution of the number of deprel labels for each language in PUD data-set Languages Nb. of deprel types Nb. of deprel sub-types arb 34 8 cmn 32 12 ces 31 12 eng 36 12 fin 30 14 fra 31 14 deu 33 12 hin 28 10 isl 31 5 ind 33 14 ita 33 7 jpn 25 0 kor 26 8 pol 28 31 por 33 9 rus 31 8 spa 32 9 swe 33 9 tha 33 10 tur 34 7 Table 3: Distribution of the number of deprel labels for each language in PUD data-set iment, 600 sentences were used for training, 200 for validation and 200 for testing. We have considered the LAS (labelled attachment score) value, which is the percentage of words that are assigned both the correct syntactic head and the cor- rect dependency label, as the main dependency parsing metric metric. Since UDify uses Multilingual BERT, and knowing that languages are not equally represented in this model, it is important to present the data distribution of the selected languages inside it (table 4), as it may have an impact on the final dependency parsing results. 3.3. Syntactic Typological Characterisation • ADV aux precedes ADJ - head-final or left- branching - It means that the dependent, which is an adverb (ADV) precedes the head which is an adjective (ADJ) and has the syntactic function of an auxiliary (aux). The dependent can be in any position of the sentence previous to the head, not necessarily right before. Table 5: LAS and standard deviation results obtained for each language of PUD data-set using UDify and 10- fold cross-validation. Results are presented from low- est to highest LAS score. Table 5: LAS and standard deviation results obtained for each language of PUD data-set using UDify and 10- fold cross-validation. Results are presented from low- est to highest LAS score. • CCONJ cc follows NOUN - head-initial or right- branching - In this case, the dependent, a coor- dinating conjunction (CCONJ), comes after the head, which is a noun (NOUN), and has the func- tion of coordination (cc). The dependent can be in any position after the head, not necessarily being right next to it. Even though parallel corpora were used to train UD- ify tool, LAS results vary considerably among PUD languages. The lowest LAS score was obtained for Chinese language (72.98) and the highest for French language (90.82), difference of 15.38 points which is much higher than the calculated standard deviation val- ues. Even though parallel corpora were used to train UD- ify tool, LAS results vary considerably among PUD languages. The lowest LAS score was obtained for Chinese language (72.98) and the highest for French language (90.82), difference of 15.38 points which is much higher than the calculated standard deviation val- ues. LAS results are higher than 85 for 11 out of the 16 PUD languages considered in this part of the study, which can be considered as relatively satisfying scores con- sidering the small size of the training corpora. Therefore, for each language, we have obtained a vec- tor containing all the existing patterns and their fre- quency. The distances between languages were calcu- lated using R dist() function (Euclidean) and from the obtained distance matrix, we generated a plot with lan- guage clusters using R hclust() function, which uses the complete linkage method for hierarchical clustering by default. This particular clustering method defines the cluster distance between two clusters to be the maxi- mum distance between their individual components. 3.3. Syntactic Typological Characterisation Analysing Indo-European languages, Romance lan- guages tend to have better LAS scores (higher than 90 for French and Spanish), followed by Germanic and Slavic languages, the exception being Icelandic which has the second lowest LAS value (78.12) among the considered languages. Indonesian and Korean have scores comparable to other Indo-European languages such as Swedish, Pol- ish and Czech (around 85). Finnish and Arabic have lower scores than Indo-European languages but higher than 80 and, therefore, better than Icelandic and Turk- ish languages. 4. Results In this section, first, we present the LAS results ob- tained using UDify trained with the different parallel corpora from PUD data-set. Then, we display the re- sults of the typological analysis (clusters in the format of a dendogram). When we analyse these LAS results together with the training size of mBERT (mean value of the size range), it is possible to calculate the following correlation co- efficients: 3.3. Syntactic Typological Characterisation 3.3. Syntactic Typological Characterisation To analyse the dependency parsing results obtained from different languages using parallel corpora, we propose a quantitative typological approach concerning syntax, more specifically the head directionality param- eter, whether the head precedes the dependent (right- Other parameters remained the same as proposed by the authors in their configuration file. As previously mentioned, the size of the PUD corpora is relatively small (1.000 sentences), therefore, a 10- fold-cross-validation was employed. For each exper- 36 Language LAS Std. Dev. cmn 72.98 2.08 tur 75.34 2.11 hin 76,12 1.12 isl 77.80 2.56 fin 81.15 1.88 arb 82.37 0.70 kor 84.55 1.33 swe 85.13 1.53 ind 85.51 1.26 pol 86.08 1.59 ces 86.34 1.00 eng 87.39 1.28 deu 88.22 0.85 rus 88.22 0.97 por 88.88 0.85 ita 89.74 0.86 spa 90.23 1.20 jpn 90.75 2.11 fra 90.84 1.36 branching) or is after it (left-branching) in the sentence (F´abregas et al., 2015). The extraction of parameters reflect the directionality observed at the surface level (position of head and dependent observed at the sen- tence level). The corpora being parallel, therefore containing the same semantic information, allows us to focus on the syntax differences among the selected languages. Using a python script, we have extracted for each lan- guage the existing patterns concerning the relative po- sition in the sentence of the heads and the dependents, Using a python script, we have extracted for each lan- guage the existing patterns concerning the relative po- sition in the sentence of the heads and the dependents, as well as the frequency of occurrence of each pattern. All observed patterns concerning the relative position of head and in PUD corpora have been considered. All observed patterns concerning the relative position of head and in PUD corpora have been considered. All observed patterns extracted from the PUD corpora (2,890 in total) have been included in the language vec- tors. Our aim is to verify the relevance of this quantita- tive method to predict LAS results. p An example of extracted pattern is the following: • ADV aux precedes ADJ - head-final or left- branching - It means that the dependent, which is an adverb (ADV) precedes the head which is an adjective (ADJ) and has the syntactic function of an auxiliary (aux). The dependent can be in any position of the sentence previous to the head, not necessarily right before. • Spearman’s correlation: 0.73 5. Discussion Comparing the results obtained in our campaign to the scores presented by the developers of UDify (Kon- dratyuk and Straka, 2019), it is possible to notice that, in general, our LAS values for PUD corpora are higher. It may be due to the fact of using different strategies for using PUD as training set. Also, as expected, LAS scores using PUD are not as high as compared to results from other models trained with larger corpora. Beside the classification presented in the figure 1, with the syntactic information extracted for each language, it is also possible to analyse the overall tendency of left- branching or right-branching. The table 6 presents the percentage of cases inside each language corpus where the dependent comes before the head in the sentence (left-branching). We observed that, as expected, the size of the corpus used to train mBERT has a strong positive correlation with the LAS scores, however, it does not explain the ensemble of the results as English has the biggest train- ing corpus but do not provide the best score concerning UDify. With the results presented in the table 6 and in the Fig- ure 2, it is possible to check whether PUD languages are head-initial or head-final. Arabic, Thai and Indone- sian are head-initial languages, Japanese also tends to- wards being head-initial. Oppositely, Turkish and Ko- rean are distinctly head-final languages. Chinese, Ro- mance and Germanic languages, except Icelandic, have a tendency of being head-final (percentage superior to 55 in the table 6). Slavic languages present different patterns, Polish does not present any tendency, Russian and Czech tend to be head-final because of more re- laxed word order in Slavic languages. Analysing Indo-European languages results, it is pos- sible to see that, overall, Romance languages are the ones with the highest LAS values. In terms of multilin- gual BERT, all of them have large pre-training mBERT corpora. French and Spanish have larger pre-training corpora when compared to Portuguese and Italian and UDify performs better for these two languages. Ro- mance languages are grouped in the figure 1, showing similar distribution of patterns concerning head and de- pendents position when compared to other PUD lan- guages. The correlation coefficients (Spearman’s and Pear- son’s) were calculated using the percentage of heads preceeding dependents and the delta concerning a bal- anced distribution of directionality (50/50). 4.1. Dependency Parsing Results using UDify Using UDify with 10-fold cross-validation, we were able to obtain LAS results for all PUD languages. LAS scores and the respective standard deviation values are presented in the table 5. • Pearson’s correlation = 0.37 • Spearman’s correlation: 0.73 37 Language % arb 36.33 tha 39.05 ind 41.91 jpn 45.85 pol 49.21 isl 51.08 rus 54.50 fin 55.85 hin 56.10 ita 57.09 ces 57.13 spa 57.79 por 57.94 fra 58.28 swe 58.75 cmn 60.06 eng 63.77 deu 66.81 tur 69.96 kor 79.86 Thus, it seems that these two variables are strongly cor- related following a non-linear monotonic function (as it is attested by the value obtained for Spearman’s coeffi- cient). 4.2. Quantitative Syntactic Language Classification As explained previously in this article, languages were compared and classified considering quantitative infor- mation of the patterns of the position of heads and de- pendents. The figure 1 presents the clusters of lan- guages generated using R’ hclust function. It is possible to observe in this dendogram the main central cluster corresponding to most of Indo-European languages (except for Hindi). Romance languages are grouped in a sub-cluster of the Indo-European one. We can also notice the proximity of English and Swedish (both Germanic) and Russian and Czech (both Slavic). Icelandic, although being a Germanic language, is closer to Polish language when this specific syntactic feature is analysed. Icelandic is presented in the den- dogram grouped with the other Slavic languages. Ger- man, also a Germanic language, is grouped closer to the Romance group (specially with Italian and French) and not with the other Germanic languages from PUD corpora. Table 6: Total percentage of occurrences where the de- pendent precedes the head (left-branching / head-final) in each selected language corpus Table 6: Total percentage of occurrences where the de- pendent precedes the head (left-branching / head-final) in each selected language corpus Close to the Germanic/Slavic cluster, it is possible to notice the group containing Thai, Arabic and Indone- sian which have no genealogical relation. The two ex- tremes groups are composed, on the left, by Hindi and Japanese, and, on the left by two sub-clusters: Finnish and Turkish (which is expected as similarities between these languages have been previously observed) and Chinese and Korean. 5. Discussion The ob- tained results are lower than 0.1, thus, no correlation English language, which is the one with the largest pre- training mBERT corpus, does not have the highest LAS 38 Figure 1: PUD language clusters generated using quantitative analysis of head and dependent position features Figure 1: PUD language clusters generated using quantitative analysis of head and dependent position features Figure 1: PUD language clusters generated using quantitative analysis of head and dependent position features Figure 1: PUD language clusters generated using quantitative analysis of head and dependent position features corpus which has probably strongly contributed to the low LAS value obtained using UDify. score. Its result can be compared to other languages with much smaller mBERT corpus such as Russian, German, Czech and Polish. Thus, it seems that size of the representation of a language in mBERT may play a role only to a certain point when it is used for fine- tuning in parsing tools. On the left of the main cluster of Indo-European lan- guages in the Figure 1, we have the sub-cluster formed by Arabic and Indonesian. Both languages have lower LAS scores when compared to Indo-European ones, In- donesian having a better performance even though its mBERT representation is smaller and its number of de- prel is higher (47 for Indonesian and 42 for Arabic). When we observe, more precisely, Germanic and Slavic languages, it is possible to notice that although English and Swedish languages form a sub-cluster, their LAS scores are slightly different. In this case, it may be caused by the discrepancy of the representa- tion of the languages in mBERT. It is the same when we consider Russian and Czech languages. Considering the cluster on the right side of the figure 1, formed by Finnish, Turkish, Chinese and Korean, these languages tend to present lower LAS scores. Finnish, Turkish and Korean have similar size of mBERT repre- sentations, but smaller than Indo-European languages. However, their size is comparable to Indonesian which presents better LAS value and, in the figure 1, this lan- guage is clustered closer to the Indo-European group. As seen in the table 4, Turkish is a head-final language, it may influence the results. However, Korean language is even more head-final when compared to Turkish and has a better LAS score, however, Korean presents only 34 deprel, while Turkish has 41. 5. Discussion It is also interesting to observe the sub-cluster formed by Polish and Icelandic inside the group of Indo- European languages. Polish language has a mBERT representation size comparable to Portuguese and Ital- ian, however, its LAS value is much lower. It may be due to its specific syntactic structures as well as to elevated number of deprel labels (59) which is much higher than all the other PUD languages. Icelandic has the second lowest LAS score among PUD languages. Although being a Germanic language, it is not similar in its syntactic structure of heads and dependents when compared to English, Swedish nor German. In addi- tion, Icelandic has the smallest mBERT pre-training In light of these results, it is possible to notice that dif- ferences in the syntactic structures concerning head and dependents may play a role in dependency parsing tools overall results. The size of the language representation 39 Figure 2: Distribution of the directionality of head and dependent in PUD language corpora Figure 2: Distribution of the directionality of head and dependent in PUD language corpora in the language model, however, plays a major role as it helps improving final scores (as it is the case for French and Spanish). Nevertheless, it may not be sufficient to guarantee satisfying LAS result (as it is the case for Turkish). We have observed that, even though parallel corpora were used, different languages present considerably different LAS results. Indo-European languages tend to present better LAS scores, inside this group, Romance languages are the ones that performed the best. UDify tool uses multilingual mBERT and as the sizes of each language inside this language model are not homogeneous, it was possible to notice that this dis- crepancy plays a major role in the LAS scores. As ex- pected, languages with larger mBERT representation tend to perform better. Also, the parameter of head and dependent position is not the only syntactic feature playing an important role in the observed LAS results, the complexity of the language, represented by the number of deprel labels should be considered as it may have caused the lower LAS value for Polish language (which has a high num- ber of deprel subtypes). However, the size of the language in mBERT is not the only parameter playing a role in the overall results. 5. Discussion En- glish has, by far, the largest size in mBERT and still has a lower LAS score when compared to Romance lan- guages which were all classified the same cluster in our typological study. It is also the case for Russian, which has a mBERT size comparable to French and Spanish for which LAS values are comparable to Portuguese and Italian with smaller mBERT size. Furthermore, it is important to mention that morpho- logical aspects (whether the language has synthetic or analytical morphology), can also influence the efficacy of the parser. As it can be observed for Finnish and Turkish (both synthetic languages), LAS results are low. However, it is also the case for Chinese, which is an analytical language. In addition to that, Arabic language has a mBERT rep- resentation comparable to Czech and Swedish but its LAS results are not as good as these two languages. Arabic language forms a sub-cluster with Indonesian, not as close to other languages with better performance as it is the case for Czech and Swedish. Also, it is possi- ble to conclude that the size of the language in mBERT and the head and dependent position are not the only aspects influencing the results. Polish language is an example of that, and the reason for the lower LAS value obtained for this language may be the higher number of dependency parsing labels specific of this language. 8. Bibliographical References Agi´c, ˇZ. (2017). Cross-lingual parser selection for low-resource languages. In Proceedings of the NoDaLiDa 2017 Workshop on Universal Dependen- cies (UDW 2017), pages 1–10, Gothenburg, Sweden, May. Association for Computational Linguistics. Jurafsky, D. and Martin, J. H. (2021). Speech and Lan- guage Processing: An Introduction to Natural Lan- guage Processing, Computational Linguistics, and Speech Recognition. 3rd. (draft) edition. Kaalep, H.-J. and Veskis, K. (2007). Comparing par- allel corpora and evaluating their quality. In Pro- ceedings of Machine Translation Summit XI: Papers, Copenhagen, Denmark, September 10-14. Alves, D., Thakkar, G., and Tadi´c, M. (2020). Eval- uating language tools for fifteen EU-official under- resourced languages. In Proceedings of the 12th Language Resources and Evaluation Conference, pages 1866–1873, Marseille, France, May. European Language Resources Association. Kondratyuk, D. and Straka, M. (2019). 75 languages, 1 model: Parsing universal dependencies universally. Kuhn, J. (2005). Parsing word-aligned parallel corpora in a grammar induction context. In Philipp Koehn, et al., editors, Proceedings of the Workshop on Build- ing and Using Parallel Texts@ACL 2005, Ann Arbor, Michigan, USA, June 29-30, 2005, pages 17–24. As- sociation for Computational Linguistics. Alzetta, C., Dell’Orletta, F., Montemagni, S., Osenova, P., Simov, K., and Venturi, G. (2020). Quantitative linguistic investigations across universal dependen- cies treebanks. In Johanna Monti, et al., editors, Pro- ceedings of the Seventh Italian Conference on Com- putational Linguistics, CLiC-it 2020, Bologna, Italy, March 1-3, 2021, volume 2769 of CEUR Workshop Proceedings. CEUR-WS.org. Litschko, R., Vuli´c, I., Agi´c, ˇZ., and Glavaˇs, G. (2020). Towards instance-level parser selection for cross- lingual transfer of dependency parsers. In Proceed- ings of the 28th International Conference on Com- putational Linguistics, pages 3886–3898, Barcelona, Spain (Online), December. International Committee on Computational Linguistics. Boros,, T., Dumitrescu, S. D., and Burtica, R. (2018). NLP-cube: End-to-end raw text processing with neural networks. In Proceedings of the CoNLL 2018 Shared Task: Multilingual Parsing from Raw Text to Universal Dependencies, pages 171–179, Brus- sels, Belgium, October. Association for Computa- tional Linguistics. Littell, P., Mortensen, D. R., Lin, K., Kairis, K., Turner, C., and Levin, L. (2017). URIEL and lang2vec: Representing languages as typological, geographi- cal, and phylogenetic vectors. In Proceedings of the 15th Conference of the European Chapter of the As- sociation for Computational Linguistics: Volume 2, Short Papers, pages 8–14, Valencia, Spain, April. Association for Computational Linguistics. de Lhoneux, M., Bjerva, J., Augenstein, I., and Søgaard, A. (2018). Parameter sharing between de- pendency parsers for related languages. 7. Acknowledgements The work presented in this paper has received fund- ing from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska- Curie grant agreement no. 812997 and under the name CLEOPATRA (Cross-lingual Event-centric Open Ana- lytics Research Academy). Honnibal, M. and Montani, I. (2017). spaCy 2: Natu- ral language understanding with Bloom embeddings, convolutional neural networks and incremental pars- ing. To appear. Jurafsky, D. and Martin, J. H. (2009). Speech and language processing : an introduction to natural language processing, computational linguistics, and speech recognition. Pearson Prentice Hall, Upper Saddle River, N.J. 6. Conclusions and Perspectives In this article we have presented a detailed analysis of dependency parsing results obtained for 16 languages using parallel corpora to understand the differences in the obtained scores considering the specific syntactic feature of head directionality parameter with which we have conducted a quantitative syntactic typological classification. Thus, we have conducted a series of experiments us- ing UDify tool, using a 10-fold cross-validation to ob- tain LAS metric for the selected languages. In parallel, we have extracted patterns concerning the position of head and dependents (left or right branching) to gen- erate vectors which were used to compare and classify languages into clusters. For future research, it would be interesting to observe how these languages perform in systems which either use more homogeneous language models in terms of language representation or that do not depend on lan- 40 guage models at all. Furthermore, specific analysis could be done considering only subject-verb or object- verb directionality. Glavaˇs, G. and Vuli´c, I. (2021). Climbing the tower of treebanks: Improving low-resource dependency parsing via hierarchical source selection. In Find- ings of the Association for Computational Linguis- tics: ACL-IJCNLP 2021, pages 4878–4888, Online, August. Association for Computational Linguistics. 8. Bibliographical References In Proceed- ings of the 2018 Conference on Empirical Methods in Natural Language Processing, pages 4992–4997, Brussels, Belgium, October-November. Association for Computational Linguistics. Liu, H. and Xu, C. (2012). Quantitative typological analysis of romance languages. Pozna´n Studies in Contemporary Linguistics, 48(4):597–625. Dell’Orletta, F., Venturi, G., and Montemagni, S. (2013). Linguistically–driven selection of correct arcs for dependency parsing. Computaci´on y Sis- temas, 17. Manning, C., Surdeanu, M., Bauer, J., Finkel, J., Bethard, S., and McClosky, D. (2014). The Stanford CoreNLP natural language processing toolkit. In Proceedings of 52nd Annual Meeting of the Associa- tion for Computational Linguistics: System Demon- strations, pages 55–60, Baltimore, Maryland, June. Association for Computational Linguistics. Matthew S. Dryer et al., editors. (2013). WALS On- line. Max Planck Institute for Evolutionary Anthro- pology, Leipzig. F´abregas, A., Mateu, J., and Putnam, M. T. (2015). Contemporary Linguistic Parameters: Contempo- rary Studies in Linguistics. Bloomsbury Academic, London. Nivre, J., de Marneffe, M.-C., Ginter, F., Goldberg, Y., Hajiˇc, J., Manning, C. D., McDonald, R., Petrov, S., 41 Pyysalo, S., Silveira, N., Tsarfaty, R., and Zeman, D. (2016). Universal Dependencies v1: A multilingual treebank collection. In Proceedings of the Tenth In- ternational Conference on Language Resources and Evaluation (LREC’16), pages 1659–1666, Portoroˇz, Slovenia, May. European Language Resources As- sociation (ELRA). Otter, D. W., Medina, J. R., and Kalita, J. K. (2018). A survey of the usages of deep learning in natural language processing. CoRR, abs/1807.10854. Pires, T., Schlinger, E., and Garrette, D. (2019). How multilingual is multilingual BERT? In Proceedings of the 57th Annual Meeting of the Association for Computational Linguistics, pages 4996–5001, Flo- rence, Italy, July. Association for Computational Linguistics. Straka, M. and Strakov´a, J. (2017). Tokenizing, pos tagging, lemmatizing and parsing ud 2.0 with ud- pipe. In Proceedings of the CoNLL 2017 Shared Task: Multilingual Parsing from Raw Text to Univer- sal Dependencies, pages 88–99, Vancouver, Canada, August. Association for Computational Linguistics. Teubert, W. (1996). Comparable or Parallel Corpora? International Journal of Lexicography, 9(3):238– 264, 09. Wang, D. and Eisner, J. (2018). Surface statistics of an unknown language indicate how to parse it. Trans- actions of the Association for Computational Lin- guistics, 6:667–685. Wu, S. and Dredze, M. (2020). Are all languages cre- ated equal in multilingual bert? 8. Bibliographical References q g Zeman, D., Popel, M., Straka, M., Hajiˇc, J., Nivre, J., Ginter, F., Luotolahti, J., Pyysalo, S., Petrov, S., Potthast, M., Tyers, F., Badmaeva, E., Gokir- mak, M., Nedoluzhko, A., Cinkov´a, S., Hajiˇc jr., J., Hlav´aˇcov´a, J., Kettnerov´a, V., Ureˇsov´a, Z., Kan- erva, J., Ojala, S., Missil¨a, A., Manning, C. D., Schuster, S., Reddy, S., Taji, D., Habash, N., Le- ung, H., de Marneffe, M.-C., Sanguinetti, M., Simi, M., Kanayama, H., de Paiva, V., Droganova, K., Mart´ınez Alonso, H., C¸ ¨oltekin, C¸ ., Sulubacak, U., Uszkoreit, H., Macketanz, V., Burchardt, A., Har- ris, K., Marheinecke, K., Rehm, G., Kayadelen, T., Attia, M., Elkahky, A., Yu, Z., Pitler, E., Lertpra- dit, S., Mandl, M., Kirchner, J., Alcalde, H. F., Str- nadov´a, J., Banerjee, E., Manurung, R., Stella, A., Shimada, A., Kwak, S., Mendonc¸a, G., Lando, T., Nitisaroj, R., and Li, J. (2017). CoNLL 2017 shared task: Multilingual parsing from raw text to Universal Dependencies. In Proceedings of the CoNLL 2017 Shared Task: Multilingual Parsing from Raw Text to Universal Dependencies, pages 1–19, Vancouver, Canada, August. Association for Computational Lin- guistics. Zeman, D., Popel, M., Straka, M., Hajiˇc, J., Nivre, J., Ginter, F., Luotolahti, J., Pyysalo, S., Petrov, S., Potthast, M., Tyers, F., Badmaeva, E., Gokir- mak, M., Nedoluzhko, A., Cinkov´a, S., Hajiˇc jr., J., Hlav´aˇcov´a, J., Kettnerov´a, V., Ureˇsov´a, Z., Kan- erva, J., Ojala, S., Missil¨a, A., Manning, C. D., Schuster, S., Reddy, S., Taji, D., Habash, N., Le- ung, H., de Marneffe, M.-C., Sanguinetti, M., Simi, M., Kanayama, H., de Paiva, V., Droganova, K., Mart´ınez Alonso, H., C¸ ¨oltekin, C¸ ., Sulubacak, U., Uszkoreit, H., Macketanz, V., Burchardt, A., Har- ris, K., Marheinecke, K., Rehm, G., Kayadelen, T., Attia, M., Elkahky, A., Yu, Z., Pitler, E., Lertpra- dit, S., Mandl, M., Kirchner, J., Alcalde, H. F., Str- nadov´a, J., Banerjee, E., Manurung, R., Stella, A., Shimada, A., Kwak, S., Mendonc¸a, G., Lando, T., Nitisaroj, R., and Li, J. (2017). CoNLL 2017 shared task: Multilingual parsing from raw text to Universal Dependencies. 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https://openalex.org/W2524350035	https://europepmc.org/articles/pmc6319689?pdf=render	English	null	Case Report of Recurrent Metastatic Pancreatic Neuroendocrine Tumor with Gastric Invasion: Consequences of Potential Needle-Tract Seeding from Fine-Needle Aspiration	Case reports in pancreatic cancer	2,016	cc-by	2,438	Case Reports in Pancreatic Cancer Case Reports in Pancreatic Cancer Case Reports in Pancreatic Cancer Volume 2.1, 2016 DOI: 10.1089/crpc.2016.0012 CASE REPORT Open Access ª Richard Zheng et al. 2016; Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms of the Creative Commons License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. Abstract Background: Pancreatic neuroendocrine tumors (PNETs) are relatively rare, and data guiding management of metastatic lesions are scarce. Hepatic metastases are most common; here we describe a case of metastatic PNET implanted into the posterior gastric cardia. Case Presentation: This case study describes the progression of a 44-year-old man with a history of pancreatic neuroendocrine tumor (PNET) resected through distal pancreatectomy and splenectomy who developed recurrent disease in his stomach with extension into the left adrenal fossa 17 months after initial resection. He subsequently underwent a total gastrectomy and left adrenalectomy with en bloc resection of this recurrence without compli- cation. Final pathology revealed a morphologically similar PNET with positivity for CAM5.2, chromogranin A, and synaptophysin. Conclusion: The unusual location of his recurrence could suggest that his preoperative endoscopic ultrasound and ﬁne-needle aspiration may have had a role in seeding the posterior gastric wall, highlighting the risk of per- forming this diagnostic procedure in the setting of suspected pancreatic malignancy. Keywords: metastatic pancreatic neuroendocrine tumor; PNET and ﬁne-needle aspiration (FNA) may be implicated in the local implantation of this unusual lesion. Introduction Pancreatic neuroendocrine tumors (PNETs) are fairly rare, accounting for less than 1–2% of all pancreatic masses. Although they tend to have a more favorable prognosis than pancreatic ductal adenocarcinoma, he- patic metastases are observed in more than 50% of pa- tients with PNETs and are associated with a 5-year survivalofonly30–40%whenuntreated.1 Data surround- ing treatment of other sites of metastatic spread are lim- ited. Here, we report a case of metastatic PNET in the gastric cardia with extension into the left adrenal fossa, 17 months after distal pancreatectomy and splenec- tomy. Pre-operative endoscopic ultrasound (EUS) 1Department of Surgery, Thomas Jefferson University Hospital, Philadelphia, Pennsylvania. 2Pancreas, Biliary and Related Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania. *Address correspondence to: Richard Zheng, MD, Department of Surgery, Thomas Jefferson University Hospital, 111 S. 11th Street, Philadelphia, PA 19147, E-mail: richard.zheng@jefferson.edu Richard Zheng, MD, Department of Surgery, Thomas Jefferson University Hospital, 111 S. 11th Street, Philadelphia, PA 19147, E-mail Case Report A 48-year-old man initially presented with lightheaded- ness, palpitations, and tarry stools. He was found to be anemic with a hemoglobin of 5.5 g/dL. Upper endoscopy revealed nonbleeding gastric varices. Further workup through abdominal CT and MRI demonstrated a large arterial-enhancing pancreatic mass, thought to be a PNET. No metastases were seen. The mass appeared to abut the splenic vein and portosplenic conﬂuence with evidence of thrombus within the lumen of the portal 58 59 Zheng et al.; Case Reports in Pancreatic Cancer 2016, 2.1 http://online.liebertpub.com/doi/10.1089/crpc.2016.0012 FIG. 1. Hypervascular pancreatic body mass with intraluminal enhancement within portal vein consistent with tumor thrombus (arrow). FIG. 2. CgA and PP levels before, during, and after treatment course. CgA, chromogranin A; PP, pancreatic polypeptide. FIG. 2. CgA and PP levels before, during, and after treatment course. CgA, chromogranin A; PP, pancreatic polypeptide. FIG. 2. CgA and PP levels before, during, and after treatment course. CgA, chromogranin A; PP, pancreatic polypeptide. FIG. 2. CgA and PP levels before, during, and after treatment course. CgA, chromogranin A; PP, pancreatic polypeptide. tic endoscopy to localize the source of bleeding revealed a 6 cm malignant-appearing friable mass with central ulceration in the gastric cardia. CT scan ﬁndings con- ﬁrmed the presence of this gastric soft tissue mass and also revealed a posterior nodular extension toward the upper pole of the left kidney (Fig. 3). This lesion demon- strated uptake on octreotide scan, suggesting a PNET origin; a focus of uptake was also noted in the periportal region, suggesting the possibility of a metastatic lymph node. Upon histopathological review of the material re- trieved at the endoscopy, the gastric lesion was identiﬁed to be morphologically similar to his previous PNET, with positivity for CAM5.2, chromogranin A, and synaptophysin. FIG. 1. Hypervascular pancreatic body mass with intraluminal enhancement within portal vein consistent with tumor thrombus (arrow). vein (Fig. 1). Endoscopic ultrasound was performed with FNA of the pancreatic mass. Histopathological review of the FNA sample revealed neoplastic cells, positive for Anti-Pan Cytokeratin Antibody, synapto- physin, and CD56, suggesting PNET. An octreotide scan showed focally increased signal at the site of the pancreatic mass, without dissemination. After extensive discussion with the patient regarding his treatment options, he opted for reresection through an open total gastrectomy. After exploratory laparotomy and extensive lysis of adhesions, the tumor was noted to invade into the capsule of the left adrenal gland. Case Report Both the stomach and the left adrenal gland were removed en bloc. No hepatic lesions were noted. Alimentary tract recon- struction was performed through a Roux-En-Y esopha- gojejunostomy. The periportal region was skeletonized withremovalofallnodaltissue.Thepatient’spostoperative course was uncomplicated. He was ultimately discharged to home on postoperative day 6 on a low-volume clear liquid diet and total parenteral nutrition supplementation. Surgical pathology revealed a pancreatic neuroendo- crine carcinoma in the gastric wall and adrenal gland with negative resection margins. The sampled peripor- tal lymph nodes were free of disease. The lesion was again conﬁrmed to be morphologically similar to the patient’s previous PNET (Fig. 4). Shortly thereafter, pancreatic polypeptide and chromogranin A normal- ized to 96 pg/mL and 5 ng/mL, respectively. The patient underwent resection of the tumor through distal pancreatectomy and splenectomy withen bloc por- tal vein resection and reconstruction as the tumor was di- rectly invading the splenic vein with thrombus extending into the portal vein. The patient tolerated this procedure well and his postoperative course was uncomplicated. Pathology from this initial procedure revealed a grade II PNET with 2/15 adjacent lymph nodes positive for metastatic carcinoma and negative margins with a Ki- 67 proliferative index of 15% (grade II). Seventeen months after resection, the patient began to have complaints of fatigue, exercise intolerance, and pal- pitation. He was again found to be anemic with a hemo- globin of 6.6 g/dL. Before the aforementioned initial resection, serum pancreatic polypeptide and chromogra- nin A levels had been elevated to 547 pg/mL and 15.8ng/ mL, respectively; with the onset of these new symptoms, the pancreatic polypeptide level was found to be only 226 pg/mL, but the chromogranin A level was elevated to 33ng/mL (Fig. 2). CA 19–9 levels remained normal throughout the patient’s full treatment course. Diagnos- Zheng et al.; Case Reports in Pancreatic Cancer 2016, 2.1 Zheng et al.; Case Reports in Pancreatic Cancer 2016, 2.1 http://online.liebertpub.com/doi/10.1089/crpc.2016.0012 60 FIG. 3. (A) Initial abdominal CT scan demonstrating pancreatic body lesion (yellow circle). (B) Abdominal CT scan 1 year after distal pancreatectomy and splenectomy with gastric mass visible (yellow circle). (C) Octreotide scan with enhancement of the posterior gastric wall (yellow circle). (D) Sagittal cut of abdominal CT scan showing gastric mass extending posteriorly (yellow arrows) toward the left adrenal gland. (E) Postoperative gastrographin swallow showing anatomical reconstruction through esophagojejunostomy. FIG. 3. (A) Initial abdominal CT scan demonstrating pancreatic body lesion (yellow circle). Case Report (B) Abdominal CT scan 1 year after distal pancreatectomy and splenectomy with gastric mass visible (yellow circle). (C) Octreotide scan with enhancement of the posterior gastric wall (yellow circle). (D) Sagittal cut of abdominal CT scan showing gastric mass extending posteriorly (yellow arrows) toward the left adrenal gland. (E) Postoperative gastrographin swallow showing anatomical reconstruction through esophagojejunostomy. cal management of hepatic metastases through partial hepatectomy has been shown to control symptoms of hormonal hypersecretion in patients with functional tumors and improve overall survival. Hepatic-directed therapy such as transarterial chemoembolization has also been utilized for control of liver metastases. Discussion PNETs are rare, and metastatic spread linked to FNA is not well documented in the literature. Surgical resec- tion remains the gold standard of treatment for func- tioning and nonfunctioning tumors. Management of these metastases has not yet been standardized. Surgi- FIG. 4. (A) Original pancreatic mass with synaptophysin staining, 20· magniﬁcation. (B) Original pancreatic mass with HE staining, 40· magniﬁcation. (C) Recurrent gastric mass with HE staining, 40· magniﬁcation. HE, hematoxylin and eosin. FIG. 4. (A) Original pancreatic mass with synaptophysin staining, 20· magniﬁcation. (B) Original pancreatic mass with HE staining, 40· magniﬁcation. (C) Recurrent gastric mass with HE staining, 40· magniﬁcation. HE, hematoxylin and eosin. FIG. 4. (A) Original pancreatic mass with synaptophysin staining, 20· magniﬁcation. (B) Original pancreatic mass with HE staining, 40· magniﬁcation. (C) Recurrent gastric mass with HE staining, 40· magniﬁcation. HE, hematoxylin and eosin. Zheng et al.; Case Reports in Pancreatic Cancer 2016, 2.1 http://online.liebertpub.com/doi/10.1089/crpc.2016.0012 61 A case similar to ours was described in 2014. In that re- port,2 a 78-year-old man was found to have a T3N0M0 moderate-to-well differentiated pancreatic adenocarci- noma sampled through EUS/FNA biopsy through the stomach wall. Nine months after resection of the lesion, the patient was found to have a submucosal gastric re- currence upon follow-up endoscopy. This was man- aged through a subtotal gastrectomy, whereupon ﬁnal pathology demonstrated a morphologically identical le- sion to his initial cancer. In comparison, our lesion had expanded aggressively into the retroperitoneum and appeared to involve the adrenal gland, so a total gas- trectomy and en bloc adrenalectomy were required for full resection. Furthermore, as in the previously mentioned case report, FNA played an important role in the preliminary diagnosis of PNET. Given the atyp- ical foci of recurrence in the gastric cardia, this scenario brings to mind the rare phenomenon of needle-tract seeding that has been documented in sarcoma, prostate cancer, hepatocellular carcinoma, and other lesions.3 ing may be, it is up to clinicians to be selective in obtaining biopsies of lesions where a tissue diagnosis may change management and not subject a patient to undue risk. In this particular case, one could ques- tion the utility of two interventions before the initial operation—the distal pancreatectomy and splenecto- my: the FNA done as part of the EUS, and the octreo- scan. Discussion The patient had a classic appearance of a PNET on CT and MRI, and he was symptomatic from anemia related to splenic vein occlusion. Did the FNA results alter management? Did the octreoscan provide essen- tial information? In this era of cost consciousness in surgical care, we need to be asking these questions. References 1. Ikezawa K, Uehara H, Sakai A, et al. Risk of peritoneal carcinomatosis by endoscopic ultrasound-guided ﬁne needle aspiration for pancreatic can- cer. J Gastroenterol. 2013;48:966–972. 1. Ikezawa K, Uehara H, Sakai A, et al. Risk of peritoneal carcinomatosis by endoscopic ultrasound-guided ﬁne needle aspiration for pancreatic can- cer. J Gastroenterol. 2013;48:966–972. p Despite the theoretical fears of needle-tract seeding, however, data validating this phenomenon with regard to some forms of pancreatic cancer are generally limited tocase reports.4,5 Furthermore, this is the ﬁrst report of a pancreatic neuroendocrine neoplasm recurrence poten- tially from needle-tract seeding. A retrospective study of 256 patients who underwent surgical resection of ma- lignant pancreatic neoplasms concluded that undergoing EUS/FNA of these lesions was not associated with a sta- tistically signiﬁcant increase in the rate of either perito- neal or gastric metastases.6 Similarly, use of EUS/FNA for diagnosis of pancreatic cancer was not associated with any difference in survival.6 Multiple studies have also corroborated that the use of EUS/FNA for both pan- creatic cancer and intraductal papillary mucinous neo- plasm sampling was not associated with a statistically signiﬁcant increase in peritoneal seeding when com- pared with patients who were not sampled.7,8 Alternative approaches for tissue sampling of pancreatic masses have not proven to be of beneﬁt; one study demon- strated an increase in the rate of peritoneal carcinomato- sis in patients who had undergone a percutaneous FNA when compared with those who had endoscopic tissue sampling.9 Although large-scale data on pancreatic nee- dle biopsy seeding is yet unavailable, it is likely a rare occurrence as needle biopsy of pancreatic lesions is com- monplace and gastric metastases are seldom seen. In most cases, the beneﬁts of direct tissue diagnosis out- weigh the theoretical risk of needle-tract seeding. However, no matter how remote the chance for seed- 2. Yoon WJ, Daglilar ES, Fernandez-del Castillo C, et al. Peritoneal seeding in intraductal papillary mucinous neoplasm of the pancreas patients who underwent endoscopic ultrasound-guided ﬁne-needle aspiration: The PIPE study. Endoscopy. 2014;46:382–387. 2. Yoon WJ, Daglilar ES, Fernandez-del Castillo C, et al. Peritoneal seeding in intraductal papillary mucinous neoplasm of the pancreas patients who underwent endoscopic ultrasound-guided ﬁne-needle aspiration: The PIPE study. Endoscopy. 2014;46:382–387. 3. Chong A, Venugopal K, Segarajasingam D, et al. Tumor seeding after EUS- guided FNA of pancreatic tail neoplasia. Gastrointest Endosc. 2011;74:933– 935. 3. Chong A, Venugopal K, Segarajasingam D, et al. Author Disclosure Statement No competing ﬁnancial interests exist. References Tumor seeding after EUS- guided FNA of pancreatic tail neoplasia. Gastrointest Endosc. 2011;74:933– 935. 4. Dickson PV, Behrman SW. Management of pancreatic neuroendocrine tumors. Surg Clin North Am. 2013;93:675–691. 4. Dickson PV, Behrman SW. Management of pancreatic neuroendocrine tumors. Surg Clin North Am. 2013;93:675–691. 5. Volanis D, Neal DE, Warren AY, et al. Incidence of needle-tract seeding following prostate biopsy for suspected cancer: a review of the literature. BJU Int. 2015;115:698–704. 6. Tomonari A, Katanuma A, Matsumori T, et al. Resected tumor seeding in stomach wall due to endoscopic ultrasonography-guided ﬁne needle as- piration of pancreatic adenocarcinoma. World J Gastroenterol. 2015;21:8458–8461. 6. Tomonari A, Katanuma A, Matsumori T, et al. Resected tumor seeding in stomach wall due to endoscopic ultrasonography-guided ﬁne needle as- piration of pancreatic adenocarcinoma. World J Gastroenterol. 2015;21:8458–8461. 7. Kudo T, Kawakami H, Kuwatani M, et al. Inﬂuence of the safety and diagnostic accuracy of preoperative endoscopic ultrasound-guided ﬁne-needle aspiration for resectable pancreatic cancer on clinical performance. World J Gastroenterol. 2014;20:3620–3627. 8. Paquin SC, Gariepy G, Lepanto L, et al. A ﬁrst report of tumor seeding because of EUS-guided FNA of a pancreatic adenocarcinoma. Gastrointest Endosc. 2005;61:610–611. 9. Ngamruengphong S, Xu C, Woodward TA, et al. Risk of gastric or peritoneal recurrence, and long-term outcomes, following pancreatic cancer resec- tion with preoperative endosonographically guided ﬁne needle aspiration. Endoscopy. 2013;45:619–626. 9. Ngamruengphong S, Xu C, Woodward TA, et al. Risk of gastric or peritoneal recurrence, and long-term outcomes, following pancreatic cancer resec- tion with preoperative endosonographically guided ﬁne needle aspiration. Endoscopy. 2013;45:619–626. Cite this article as: Zheng R, Tannouri S, Lavu H (2016) Case report of recurrent metastatic pancreatic neuroendocrine tumor with gastric invasion: consequences of potential needle-tract seeding from ﬁne- needle aspiration, Case Reports in Pancreatic Cancer 2:1, 58–61, DOI: 10.1089/crpc.2016.0012.
https://openalex.org/W4220712107	https://zenodo.org/records/6390603/files/MMMS4013.pdf	Russian	null	ИҚЛИМ ЎЗГАРИШЛАРИ ШАРОИТИДА ЭКОЛОГИК ХАВФСИЗЛИКНИ ҲУҚУҚИЙ ТАЪМИНЛАШ МАСАЛАЛАРИ	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	2,814	MODELS AND METHODS IN MODERN SCIENCE International scientific-online conference ИҚЛИМ ЎЗГАРИШЛАРИ ШАРОИТИДА ЭКОЛОГИК ХАВФСИЗЛИКНИ ҲУҚУҚИЙ ТАЪМИНЛАШ МАСАЛАЛАРИ Қаюмов Шуҳрат Бахром ўғли1, Жуманазар Холмўминов2 1Термиз давлат университети Юридик факультетининг 1-босқич талабаси 2Илмий раҳбар: ю.ф.д.,проф. https://doi.org/10.5281/zenodo.6390603 Бугунги кунда биз яшаш турмуш шароитимизда “иқлим ўзгаришлари”, “глобал исиш”, “экологик танглик”, “экологик таҳдид”, “экологик офат”, “экологик инқироз” сингари тушунчаларни кўп эшитамиз ва амалиётда уни гувоҳи бўлиб турибмиз. Хў д й ў ? MODELS AND METHODS IN MODERN SCIENCE International scientific-online conference Бугунги кунда биз яшаш турмуш шароитимизда “иқлим ўзгаришлари”, “глобал исиш”, “экологик танглик”, “экологик таҳдид”, “экологик офат”, “экологик инқироз” сингари тушунчаларни кўп эшитамиз ва амалиётда уни гувоҳи бўлиб турибмиз. Хўш шундай экан иқлим ўзгариши нима? 60 1Ўзбекистон Президенти Шавкат Мирзиѐев БМТ Бош Ассамблеясининг 72-сессиясида сўзлаган нутқи. https://uza.uz/uz/posts/zbekiston-prezidenti-shavkat-mirziyeev-bmt-bosh-assambleyasi-20-09-2017 2“2019-2030 йиллар даврида Ўзбекистон Республикасининг “яшил” иқтисодиѐтга ўтиш стратегиясини тасдиқлаш тўғрисида”ги Ўзбекистон Республикаси Президентининг қарори.// Қонун ҳужжатлари маълумотлари миллий базаси, 05.10.2019 й., 07/19/4477/3867-сон 3Ўзбекистон Республикаси Президентининг “2022-2026 йилларга мўлжалланган Янги Ўзбекистоннинг тараққиѐт Стратегияси тўғрисида”ги фармони.// Қонунчилик маълумотлари миллий базаси, 29.01.2022 й., 06/22/60/0082-сон Хўш шундай экан иқлим ўзгариши нима? Иқлим муайян жойдаги кўплаб йиллар давом этган об ҳавонинг ўртача кўрсаткичидир. Иқлим ўзгариши эса ана шу ўртачалардаги ўзгаришдир. Бугун иқлим ўзгариши инсоният олдида турган энг жиддий муаммо экани жаҳон ҳамжамияти томонидан тан олинган. Иқлим ўзгариши инсон ҳаётининг барча соҳаларига таъсир кўрсатмоқда ва иқлим ўзгаришининг салбий оқибатларини олдини олиш ҳамда янги ҳаёт шароитларига мослашиш бўйича кечиктирилмайдиган чоралар кўришни талаб этмоқда. 59 59 MODELS AND METHODS IN MODERN SCIENCE International scientific-online conference Дунё эколог олимлари ва замонавий илм-фан инсоннинг авваламбор қазиб олинадиган ёқилғини ёндириш натижасида иссиқхона газларининг чиқарилиши билан боғлиқ хўжалик фаолияти иқлимга сезиларли таъсир кўрсатаётганини тасдиқловчи жиддий асослар тақдим қилмоқда. Ўзбекистон Республикаси Президенти Шавкат Мирзиёев томонидан БМТ Бош Ассамблеясининг 72-сессиясида сўзлаган нутқи1да жаҳон ҳамжамиятини, шу жумладан минтақа мамлакатларини экологик масалаларда, жумладан Орол денгизнинг қуриши билан боғлиқ оқибатларни бартараф этишда халқаро миқёсдаги саъй-ҳаракатларни фаол бирлаштириш зарурлигини таъкидлаб ўтилган эди. Бугунги кунда мазкур масалаларда амалий ишларни барчамиз гувоҳи бўлиб турибмиз. Биргина мисол сифатида БМТ шафелиги остида Инсон хавфсизлиги бўйича кўпшерикли Траст фондининг ташкил этилиши ёки мустақилликнинг 27 йили давомида Оролнинг қуриган тубида 400 минг гектарда ўрмонзор ташкил этилган бўлса, сўнгги икки йилда қарийб 1200 минг гектарга саксовул ва бош чўл ўсимликлари уруғлари сепилганлиги ва кўчатлари экилганлигини келтириш мумкин. Шунингдек, бу соҳада Юртбошимизнинг “2019-2030 йиллар даврида Ўзбекистон Республикасининг “яшил” иқтисодиётга ўтиш стратегиясини тасдиқлаш тўғрисида”ги қарори2 ва Янги Ўзбекистоннинг тараққиёт Стратегияси3нинг бир қатор бандларида атроф-муҳитни муҳофаза қилиш ва экологик ҳолатни яхшилашга эътиборни кучайтириш масалалари белгиланганлиги иқлим ўзгаришларининг олдини олиш ва унга мослашиш учун бевосита ҳуқуқий асос бўлиб хизмат қилади. Мутахассисларнинг таҳлили ва фикрига кўра 2030-2050 йилларда минтақада ҳаво ҳарорати яна 1,5-3°С га кўтарилиши мумкин. Ҳаво ҳароратининг айниқса Оролбўйида кўтарилиши кутилмоқда, бу эса маҳаллий иқлим ўзгаришлари билан боғлиқ муаммоларни келтириб чиқаради. Ҳукуматлараро экспертлар гуруҳи Ер илгари ҳисоб-китоб қилинганидан кўра тезроқ исиб бораётганини маълум қилишмоқда. Дунё бўйича ўртача ҳарорат 1,1 даражага кўтарилган. Бу эса 2040 йилга бориб MODELS AND METHODS IN MODERN SCIENCE International scientific-online conference ўртача ҳарорат 1,5 даражага ошишини билдиради. Иссиқ тўлқинлар, кучли шамоллар, қурғоқчилик, сув тошқинлари ва ёнғинлар янада кўпроқ содир бўла бошлади, музликлар эриши янада кучайди. Айниқса, жорий йилда бу жараён жудаям тезлашганини кузатишимиз мумкин. Экспертларнинг баҳоларига кўра атмосферада иссиқхона газлари концентрациясининг бундан кейин ҳам ортиши, қурғоқчилик натижасида сув ва озиқ-овқат танқислиги хавфини кўпайишига, иссиқ мавсум давомийлиги ва қизғинлигининг ошиши туфайли аҳоли сонининг ортишига, шунингдек селлар, тошқинлар ва бошқа хавфли ҳодисаларнинг такрорланишига олиб келади. Хўш шундай экан иқлим ўзгариши нима? Ундан ташқари, бундай илиш экотизимлар ҳолатига салбий таъсир кўрсатади, ва Оролбўйи, Қорақалпоғистон, Сурхондарё, Бухоро ва Хоразм вилоятлари каби минтақаларда экологик вазиятнинг кескинлашувига олиб келиши мумкин. Ўзгидромет маълумотларига кўра, глобал иқлим ўзгариши ва мамлакат табиий-ресурс комплексининг ана шу ўзгаришларга таъсирчанлиги изчил иқлимий сиёсатни шакллантириш заруриятини белгилайди. Иқлим ўзгаришининг асосий сабаби нимада деб ўйласиз? Иқлим ўзгаришининг асосий сабаби нимада деб ўйласиз? Мутахассислар иқлим ўзгаришининг асосий омили иссиқхона эффекти эканлигини таъкидлашади. Қуёшдан келган иссиқликнинг Ер Мутахассислар иқлим ўзгаришининг асосий омили иссиқхона эффекти эканлигини таъкидлашади. Қуёшдан келган иссиқликнинг Ер 61 MODELS AND METHODS IN MODERN SCIENCE International scientific-online conference сатҳида жамланиб, димланиб қолиши иссиқхона эффекти дейилади. Яъни қуёшдан келган нурни Ер ҳам ўз навбатида атмосфера орқали коинотга қайтаради. Ушбу нурларнинг бир қисми коинотга чиқиб кетиш ўрнига одамлардан чиқарилган турли газларга ютилади. Унинг коинотга қайтиб чиқиб кетмаслиги оқибатида Ер юзи меъёридан ортиқ қизийди ва иқлимга таъсир кўрсатадиган иссиқхона қатлами ҳосил бўлади. Натижада сутка мобайнида энг юқори ва энг паст ҳарорат орасида кам фарқ бўлади. Яъни одамлар ва табиат тунда ҳам кундузги каби иссиқ ва дим ҳаво таъсирида қолади. Бундай суткалик иссиқлик эса кескин исиш ҳодисасини келтириб чиқаради. Иссиқхона эффектини ҳосил қилувчи асосий газ бу-карбонат ангидрид. У атмосферага ҳам табиий, ҳам сунъий йўл билан қўшилади. Метан, азот оксиди каби бошқа ифлос газлар ҳавога инсон омили туфайли чиқарилиб, булар бир бутун иссиқхона эффекти даражасини белгилайди. Иссиқхона эффектини ҳосил қилувчи газ концентрацияларининг ортиши, сайёрада табиий иссиқлик балансини бузади ва антропоген иссиқхона эффектини келтириб чиқаради. Ҳисоб-китобларга кўра, 2100 йилга бориб иссиқхона эффекти оқибатида глобал ялпи ички маҳсулот 20 фоиздан ортиққа пасайиши мумкин. Шунингдек, ҳозирги кунда асосий муаммолар сифатида антропоген омиллар таъсири ва қурғоқчилик натижасида углерод газини ютувчи ўрмон майдонлари кескин қисқариб кетиши, озон қатламининг емирилиши, ёввойи табиат майдонларининг қисқариб бораётганини ҳам келтириб ўтиш мумкин. Дунёда олиб борилаётган тадбирлар ва ўтказилаётган Саммитлар ва қабул қилинаётган халқаро ҳужжатлар иш бераяптими? Ўтган асрнинг охирларида глобал исишнинг олдини олиш дунё ҳамжамиятини эътиборини тортди. 1992 йил Бразилиянинг Рио-де- Жанейро шаҳрида БМТнинг иқлим ўзгаришлари бўйича доиравий конвенцияси қабул қилинди. 1997 йили Японияда иқлим ўзгаришларига олиб келувчи “парник газ” ларини атмосферага чиқаришни қисқартириш юзасидан Киото протоколи имзоланди. 2005 йил 16 февралдан расмий кучга кирган протоколни дунёнинг 156 давлати ратификация қилган. Киото протоколига мувофиқ саноат ривожланган давлатлар 2008-2012 йилларгача ўрта ҳисобда атмосферага “парник газ”лари чиқаришни 1990 62 р ўр ф р р р р MODELS AND METHODS IN MODERN SCIENCE International scientific-online conference йилдаги кўрсаткичдан 5 фоизга қисқартириши лозим эди. Бунинг учун ҳар бир давлатга аниқ квоталар белгиланди. Аммо, бу келишув амалда кутилган натижаларни бермади. йилдаги кўрсаткичдан 5 фоизга қисқартириши лозим эди. Бунинг учун ҳар бир давлатга аниқ квоталар белгиланди. Аммо, бу келишув амалда кутилган натижаларни бермади. 2015 йилнинг 30 ноябрь-12 декабрь кунлари ушбу протоколнинг давоми ўлароқ, Парижда ўтказилган саммитда 80 мамлакат вакиллари қатнашди. Битимдан кўзланган мақсад ҳароратнинг глобал ўсишини Цельсий шкаласи бўйича ўртача 2 даражадан паст ҳолатда ушлаб туриш ва ҳарорат ўсиши 1,5 даражада чекланиши учун саъй-ҳаракатларни ишга солишдан иборат. Таъкидлаш лозимки,Табиатда “парник газ”лари камайишини тезлаштириш учун конференцияда иштирок этган давлатлар ўз зиммаларига янги мажбуриятларни олган. Масалан, Россия 2030 йилга бориб атмосферага СО2 (карбонат ангидрид) гази чиқаришни 1990 йилдаги кўрсаткичдан 30 фоиз камайтириши жоиз. Атмосферага углерод газлари чиқариш бўйича дунёда Хитойдан кейин иккинчи ўринда турувчи АҚШ эса 2025 йилга бориб, 2005 йилдаги кўрсаткични 26-28 фоиз қисқартириши шарт. Албатта, тараққиётни тўхтатиб бўлмайди. Аҳоли сонининг ўсиб бориши ишлаб чиқариш ва саноатни ҳам ривожлантиришини талаб этмоқда. Бироқ, техноген жараёнлар шу тахлит ривожланаверса сайёрамизни муқаррар таҳликали экологик хавф- хатарлар кутиш эҳтимоли мавжуд4. Шу боис,ҳам углеводород унинг ўрнини босувчи, нисбатан арзон, атроф-муҳит учун хавфсиз ресурслар, жумладан, қуёш, шамол, сув, атом ва бошқа энергия манбаридан фойдаланиш ҳақидаги таклифлар илгари сурилмоқда. Мутахассисларнинг фикрига кўра, иқлим ўзгариши туфайли қандай салбий оқибатлар мавжуд бўлиши мумкин? Дунёда олиб борилаётган тадбирлар ва ўтказилаётган Саммитлар ва қабул қилинаётган халқаро ҳужжатлар иш бераяптими? 63 4 Холмўминов Ж.Т.Экологик таҳдидларни олдини олиш ва уларни бартараф этиш ҳуқуқий муаммоларининг илмий-назарий таҳлили.Монография.Тошкент, ТДЮУ,2016.-344-б MODELS AND METHODS IN MODERN SCIENCE International scientific-online conference -ҳозирги юз йилликнинг ўзида океанлар сатҳи 1 м кўтарилиши; -СО2 концентрацияси атмосфера ҳавосида ортиши (1870 йилдан ери уни миқдори 30 фоиз ортди); -Ернинг устки қобиғи ҳарорати +0,7°С га ўзгарган(сўнгги юз йил ) MODELS AND METHODS IN MODERN SCIENCE International scientific-online conference -ҳозирги юз йилликнинг ўзида океанлар сатҳи 1 м кўтарилиши; -СО2 концентрацияси атмосфера ҳавосида ортиши (1870 йилдан бери уни миқдори 30 фоиз ортди); -ҳозирги юз йилликнинг ўзида океанлар сатҳи 1 м кўтарилиши; -СО2 концентрацияси атмосфера ҳавосида ортиши (1870 йилдан бери уни миқдори 30 фоиз ортди); -Ернинг устки қобиғи ҳарорати +0,7°С га ўзгарган(сўнгги юз йил ичида). -сув ресурслари тақчиллигининг кучайиши, шунингдек ҳудудларда ичимлик сувининг етишмаслиги ҳамда музлик ва қор қоплами кунларининг 7-10 кунга камайиб бориши, қурғоқчилик такрорланишининг кўпайиши (ҳозирги даврда ҳар 10 йилнинг 3 йилида). -табиий офатларнинг ортиши-селлар, тошқинларнинг тез-тез такрорланиши (сўнгги 40 йил ичида табиий офатдан бўлган зарар миқдори 10 маротабага ошган); -ўртача ҳароратнинг ошиши, йиллик иссиқ даврларнинг 10-15 кунга ўсиб бориши; -ўртача ҳароратнинг ошиши, йиллик иссиқ даврларнинг 10-15 кунга ўсиб бориши; -ёғингарчиликнинг йиллик ўртача даражасини мамлакатни бутун ҳудуди бўйлаб пасайиши ва уларнинг ҳудудлар, вақт ҳамда миқдори бўйича нотекис тарқалиши, ҳаддан ташқари кучли ёғингарчиликли ва умуман ёғингарчиликсиз кунлар сонининг ортиши; -қишлоқ хўжалигида етиштириш учун мақбул экинлар таркиби бутунлай ўзгариши; -қишлоқ хўжалигида етиштириш учун мақбул экинлар таркиби бутунлай ўзгариши; -юқори даражадаги жазирамалар такрорланишининг ошиши, ҳарорат режими кучайиши натижасида аҳоли саломатлиги билан боғлиқ муаммолар кўпайиши; 64 у р ў ; 64 у р ў MODELS AND METHODS IN MODERN SCIENCE International scientific-online conference MODELS AND METHODS IN MODERN SCIENCE International scientific-online conference -ўсимликлар ва ҳайвонларнинг аксарият турлари яшайдиган ареалларнинг қайта тақсимланиши, демак, экологик жараёнлар, бериладиган маҳсулотлар ва бажариладиган функцияларнинг тубдан ўзгариши; -саҳроланиш жараёнларининг кучайиши, яъни, яшаш ва хўжалик юритиш мумкин бўлган ерларнинг камайиши; -саҳроланиш жараёнларининг кучайиши, яъни, яшаш ва хўжалик юритиш мумкин бўлган ерларнинг камайиши; -иқтисодиёт секторлари таъсирининг тўлиқ қайта тақсимланиши ва олдиндан аниқ айтиш қийин бўлган бошқа кўпгина оқибатлар ва ҳоказо. Иқлим ўзгаришларининг олдини олиш ва унга мослашиш учун қандай чора-тадбирларни амалга ошириш талаб этилиши мумкин? Биринчидан, иссиқхона газларини камайтириш. Бунинг учун биринчи навбатда ишлаб чиқариш корхоналари, ИЭС ва бошқа атмосферага ифлослантирувчи моддалар чиқарувчи ташкилотларда экологик тоза технологияларни жорий этиш орқали атмосферага ташланаётган иссиқхона газлари миқдорини камайтириш зарур. Иккинчидан, ёқилғилар сифатини янада яхшилаш ҳамда экологик тоза транспорт воситаларини кўпайтириш, шу жумладан кенг жамоатчилик учун қулай бўлган велойўлакчалар ташкил этиш ўз навбатида иссиқхона газлари миқдорини камайтиради. Учинчидан, кўкаламзорлаштириш ишларини амалга ошириш. Табиий дренаж ҳисобланган кўп йиллик дарахтларнинг экилиши 65 Учинчидан, кўкаламзорлаштириш ишларини амалга ошириш. Табиий дренаж ҳисобланган кўп йиллик дарахтларнинг экилиши MODELS AND METHODS IN MODERN SCIENCE International scientific-online conference MODELS AND METHODS IN MODERN SCIENCE International scientific-online conference MODELS AND METHODS IN MODERN SCIENCE International scientific-online conference Табиийки иқлим ўзгаришлари кейинги ярим аср ичида бизнинг минтақага ҳам сезиларли таъсир ўтказди. Бир пайтлар мавжланиб ётган Орол денгизи майдони ўнлаб бараварга қисқариб, унинг ўзани қақроқ саҳрога айланганлигини гувоҳи бўлиб турибмиз. Сўнгги йилларда ҳаво ҳароратининг кўтарилиши натижасида Сирдарё ва Амударёни сув билан таъминлайдиган Помир ва Тяньшань музликлари шиддат билан эриб бормоқда. Тожикистон ҳамда Қирғизистон вакиллари баъзи музликлар 20-30% эриганини айтишмоқда. Бир неча ўн йилликлар ичида эса бу музликларнинг ярмидан кўпи эриб битиши мумкин. Минтақамизда ичимлик сувининг 80 фоизи ана шу музликлардан келишини ҳисобга олсак, уларнинг эриши яқин келажакда сув танқислигини келтириб чиқариши мумкин. “Ўзгидромет” маълумотлари Ўзбекистонда ҳам иқлим ўзгаришлари ўз таъсирини кўрсатаётганини исботламоқда, Мамлакатимизда об-ҳавони кузатиш 1872 йилдан бошланган. Биргина Тошкент шаҳрида ўртача йиллик ёғин миқдори 423,4 миллиметрни ташкил этган бўлса, ўтган бир аср давомида бу кўрсаткич 60 мм.га ошган. Сўнгги юз йиллик кузатишлар шуни кўрсатмоқдаки, биргина Тошкент шаҳрида ўртача йиллик ҳарорат 1.7 даражага ошган. Айни пайтда сайёрамиз миқёсида бу кўрсаткич 0.7-0.8 даражани ташкил қилади. Ўтган ёзда ҳам ҳаво ҳароратининиг кескин кўтарилганига гувоҳ бўлдик. Жойларда 45 даражадан юқори ҳарорат кузатилмоқда. Кўпчиликни узоқ келажак эмас, яқин кунларда кутилаётган ўзгаришлар қизиқтириши табиий. Кейинги йилларда октябрь ойи ҳам анча илиқ келган. 2002-2003 йилларда ушбу ойда ҳарорат +34-35 даражага кўтарилган. Ойи охирида ҳарорат +31-32 даражани ташкил этган. Октябрь ойи учун энг паст ҳарорат сўнгги даврларда 2016 йилда кузатилган бўлиб, ҳаво ҳарорати -2-4 даража совуққача тушиб кетган. Ноябрь ойи 2010 йил жуда қуруқ келган бўлса, 2011 йилда серёғин келганлиги ҳам ижобий ҳол эмас. 67 Иқлим ўзгаришлари оқибатида вужудга келган салбий аҳволни бартараф этиш учун нима қилмоқ керак? MODELS AND METHODS IN MODERN SCIENCE International scientific-online conference MODELS AND METHODS IN MODERN SCIENCE International scientific-online conference ҳудуддаги микроиқлимни юмшатишга сабаб бўлади. Шунингдек, кўкаламзорлаштирилган ҳудудларни, айниқса шаҳар ва автомобил йўллари четида яшил ҳудудларни кенгайтириш зарарли моддалар миқдорини камайишига хизмат қилади. Тўртинчидан, сувдан оқилона фойдаланиш. Томчилаб суғоришни ташкил этиш, сув ресурсларидан оқилона фойдаланиш ҳам ўз навбатида иқлим ўзгаришларига мослашиш учун хизмат қилади. Бешинчидан, яшил иқтисодиётни ривожлантириш, яъни чиқиндисиз технологиялар. энергиятежамкор, ресурстежамкор, чиқиндисиз, кам чиқиндили технологиялардан фойдаланишни жорий этиш ёки бошқачароқ қилиб айтганда мамлакатда яшил иқтисодиётни жорий этиш зарур. Олтинчидан, қайта тикланувчи энергия манбаларидан фойдаланиш. Қуёш, шамол энергияларидан фойдаланиш билан биргаликда атом энергетикасини ривожлантириш орқали атмосферага иссиқхона газлари чиқарилишини олдини олиш орқали иқлим ўзгаришлари оқибатларини маълум бир даражада камайтириш мумкин ва бошқалар5. 66 Иқлим ўзгаришлари оқибатида бизнинг минтақада аҳвол қандай? 5Холмўминов Ж.Т.Энергетика соҳасидаги муносабатларни ҳуқуқий тартибга солиш. Монография. Тошкент, ТДЮУ,2019.-215-б Иқлим ўзгаришлари оқибатида бизнинг минтақада аҳвол қандай? 66 5Холмўминов Ж.Т.Энергетика соҳасидаги муносабатларни ҳуқуқий тартибга солиш. Монография. Тошкент, ТДЮУ,2019.-215-б 66 5Холмўминов Ж.Т.Энергетика соҳасидаги муносабатларни ҳуқуқий тартибга солиш. Монография. Тошкент, ТДЮУ,2019.-215-б MODELS AND METHODS IN MODERN SCIENCE International scientific-online conference Ҳудудларда, айниқса шаҳар жойларида дарахтзорларни янада кўпайтириш, дарахтларнинг асоссиз кесилишига йўл қўймаслик керак. Зотан, табиий бойликларимизга эҳтиёткорона муносабатда бўлсак, сув, газ, электр энергиясининг ортиқча сарфига йўл қўймасак, ўзимиз яшаётган ҳудуд, ҳовли ёки кўчанинг тоза ва озодалигига эътибор қаратсак, нур устига нур бўлур эди. Иқлим ўзгариши муаммоси Ўзбекистонга ҳам ўз таъсирини кўрсатиши мумкинми? Содир бўлиши мумкин бўлган оқибатлар қаторида қуйидагиларни кўрсатиш мумкин: cув ресурслари тақчиллигининг кучайиши; ўртача ҳароратнинг ошиши; ёғингарчиликларнинг нотекис тақсимланиши- ёғингарчиликларсиз қурғоқчилик узоқ давом этиши ва бир мартада кўп миқдорда ёғингарчиликлар бўлиши; қишлоқ хўжалигида етиштириш учун мақбул экинлар таркиби бутунлай ўзгариши; ҳарорат режими кучайиши натижасида аҳоли саломатлиги билан боғлиқ муаммолар кўпайиши; ўсимликлар ва ҳайвонларнинг аксарият турлари яшайдиган ареаллар қайта тақсимланиши, демак, экологик жараёнлар, бериладиган маҳсулотлар ва бажариладиган функциялар тубдан ўзгариши; саҳролашиш жараёнларининг кучайиши, демак, яшаш ва хўжалик юритиш мумкин бўлган ерлар камайиши; иқтисодиёт секторлари таъсирининг тўлиқ қайта тақсимланиши ва бошқалар. Иқлим ўзгаришлари оқибатида вужудга келган салбий аҳволни бартараф этиш учун нима қилмоқ керак? 67 MODELS AND METHODS IN MODERN SCIENCE International scientific-online conference MODELS AND METHODS IN MODERN SCIENCE International scientific-online conference International scientific-online conference International scientific online conference Иқлим ўзгаришларининг нохуш оқибатларига қарши курашиш мақсадида Ўзбекистон ҳукумати томонидан халқаро ҳамжамият билан ҳамкорлик фаол йўлга қўйилган. Ўзбекистон юқорида тилга олинган халқаро шартнома ва битимларга қўшилган. Жумладан, 2016 йил 19 апрель куни Ўзбекистон Париж битимига қўшилди. Жойларда кейинги йилларда минтақа мамлакатлари ўртасида ўзаро ҳамкорлик муҳитининиг ривожланиши, муносабатларнинг илиқлашиши иқлим ўзгаришларини мувофиқлаштиришга ҳам ижобий таъсир қилиши мумкин. Хусусан август ойида Туркманистонда Орол муаммосига бағишлаб ўтказилган саммитда ҳам минтақадаги давлат раҳбарлари ана шу масалаларни кенг муҳокама қилиб, зарур чора-тадбирларни белгилаб олишди. Бироқ, бу масала фақат давлат идоралари, соҳа мутахассислари зиммасидаги вазифа эмас. Ҳар бир инсон глобал исиш, иқлим ўзгаришларининг олдини олишга қўлидан келганча ҳисса қўшиши шарт. Ишлаб чиқариш корхоналарида асосан қайта тикланувчи энергия манбаларидан фойдаланиш, имкон қадар экологик тоза транспорт воситалари (велосипед, электромобил) хизматидан фойдаланиш, йирик шаҳаларимизда автомобилсиз кунларни жорий этиш, кўп миқдорда ёқилғи сарфлайдиган автомашиналар учун тўловларни ошириш сингари чоралар ҳам маълум маънода иқлим ўзгаришларини чеклашга ёрдам бериши мумкин. 68 р у Иқлим ўзгаришининг олдини олиш учун қандай тавсиялар бериш мумкин? Қазиб олинадиган ёқилғидан фойдаланишни камайтириш ва қайта тикланадиган энергия манбаларига ўтиш; энергия самарадорлигини ошириш ва соҳаларни энергия тежовчи технологиялар билан модернизация қилиш; табиатда яшилликни кўпайтириш, ўрмон ёнғинларининг олдини олиш, дарахтзорларни кўпайтириш; экологик тоза қишлоқ хўжалигига ўтиш; тупроқ таркибидаги органик моддаларни сақлаб қолиш (чунки уларнинг йўқолиши тўғридан тўғри иссиқхона эффектига таъсир қилади); экологик тежамкор транспорт турларига ўтиш. 69 Иқлим ўзгаришининг олдини олиш учун инсонлар нима қилиши мумкин? 69 Иқлим ўзгаришининг олдини олиш учун инсонлар нима қилиши мумкин? Иқлим ўзгаришининг олдини олиш учун инсонлар мумкин? MODELS AND METHODS IN MODERN SCIENCE International scientific-online conference MODELS AND METHODS IN MODERN SCIENCE International scientific-online conference Олимларнинг иқлим ўзгариши борасидаги билими ўсиб бормоқда. Масалан, ҳозир улар иқлим ўзгариши ва кучли ёқингарчилик ёки аномал иссиқ каби алоҳида ҳолатлар ўртасидаги алоқани аниқлай олади. Келажакда улар бундай кескин ўзгаришларни олдиндан яхшироқ башорат қила олишига умид қилинмоқда. Олимларнинг тахминига қараганда одамлар: Жамоат транспорти ёки велосипеддан кўпроқ фойдаланиб, шахсий автомашинага қарамликни камайтириш; Уйларининг иссиқлик сақлашини яхшилаш; Камроқ парвоз қилиш; Гўшт ва сут маҳсулотлари истеъмолини камайтириши мумкин. Иқлим ўзгариши: Жаҳонда +50 градусли кунлар сони икки баравар кўпайган. р р ў Хулоса Хулоса Иқлим кўрсаткичларининг йил сайин тобора ёмонлашиб бораётганини инсоният Ердан шафқатсизларча фойдаланишни давом эттираётгани билан боғлаш мумкин. Кўпчилик давлатлар томонидан бу жараён хавфсизликка қарши энг катта таҳдид сифатида кўрилмоқда. Дунё бўйлаб содир бўлган аянчли ҳодисалар экологик хавфсизликни таъминлашнинг қўшимча механизмларини ишлаб чиқишга етарли даражада кучли туртки бериши керак. Йўқса кейинчалик жуда кеч бўлиши мумкин. Иқлим ўзгаришлари шароитида экологик хавфсизликни ҳуқуқий таъминлашга оид қабул қилинган экологик қонунлар талабларига қатиий риоя қилиш ва бажариш лозим бўлади. 70 риоя қилиш ва бажариш лозим бўлади. MODELS AND METHODS IN MODERN SCIENCE International scientific-online conference Хулоса қилиб айтганда, глобал тусдаги экологик муаммони бир ёки икки давлатнинг саъй-ҳаракати билан бартараф этиб бўлмайди. Бунга барча давлатлар ҳамжиҳатликда экологик муаммони бартараф этиш учун курашиш талаб этилади. Келгуси авлодлар учун мусаффо осмон, гўзал табиат ва унинг бебаҳо неъматларини бекаму кўст ҳолда етказиш барчамизнинг умуминсоний бурчимиз эканлигини ҳеч қачон унутмаслигимиз лозим6. References: References: 1.Ўзбекистон Президенти Шавкат Мирзиёев БМТ Бош Ассамблеясининг 72-сессиясида сўзлаган нутқи. https://uza.uz/uz/posts/zbekiston-prezidenti- shavkat-mirziyeev-bmt-bosh-assambleyasi-20-09-2017 2.“2019-2030 йиллар даврида Ўзбекистон Республикасининг “яшил” иқтисодиётга ўтиш стратегиясини тасдиқлаш тўғрисида”ги Ўзбекистон Республикаси Президентининг қарори.// Қонун ҳужжатлари маълумотлари миллий базаси, 05.10.2019 й., 07/19/4477/3867-сон 3.Ўзбекистон Республикаси Президентининг “2022-2026 йилларга мўлжалланган Янги Ўзбекистоннинг тараққиёт Стратегияси тўғрисида”ги фармони.// Қонунчилик маълумотлари миллий базаси, 29.01.2022 й., 06/22/60/0082-сон 5.Холмўминов Ж.Т.Энергетика соҳасидаги муносабатларни ҳуқуқий тартибга солиш. Монография. Тошкент, ТДЮУ,2019.-215-б 6.Холмўминов Ж.Т.Қишлоқ хўжалигини барқарорлаштириш ва иқлим ўзгаришлари оқибатларини юмшатиш. Рисола. Тошкент, ТДЮУ,2018.-98-б 71 6 Холмўминов Ж.Т.Қишлоқ хўжалигини барқарорлаштириш ва иқлим ўзгаришлари оқибатларини юмшатиш. Рисола. Тошкент, ТДЮУ,2018.-98-б
https://openalex.org/W4362461665	https://figshare.com/articles/journal_contribution/Supplementary_Table_2_from_Notch3_Pathway_Alterations_in_Ovarian_Cancer/22399149/1/files/39844878.pdf	English	null	Supplementary Table 1 from Notch3 Pathway Alterations in Ovarian Cancer	null	2,023	cc-by	414	Supplementary Table 2. The siRNA and Primer Sequences siRNA Vendor Sequence (5”-3”) Control sense TTCTCCGAACGTGTCACGT [dT][dT] Control antisense ACGTGACACGTTCGGAGAA [dT][dT] Notch3 #1sense GGUAGUAAUGCUGGAGAUU[dT][dT] Notch3 #1antisense AAUCUCCAGAUUACUACC [dT][dT] Notch3 #2 sense CUGUCUUGCUGCUGGUCAU[dT][dT] Notch3 #2 antisense AUGACCAGCAGCAAGACAG [dT][dT] Notch3 #3 sense CCGUUAUGCCCGUGCUGAU[dT][dT] Notch3 #3 antisense AUCAGCACGGGCAUAACGG[dT][dT] Jagged-1 #1sense GAAUGUGAGGCCAAACCUU[dT][dT] Jagged-1 #1antisense AAGGUUUGGCCUCACAUUC[dT] [dT] Jagged-1 #2 sense CCUGUAACAUAGCCCGAAA[dT] [dT] Jagged-1 #2 antisense UUUCGGGCUAUGUUACAGG[dT][dT] Jagged-1 #3 sense CAUCGAUUAUUGUGAGCCU[dT] [dT] Jagged-1 #3 antisense AGGCUCACAAUAAUCGAUG[dT] [dT] RPS6KB1 #1sense GGAAUAUUUAUGGAAGACA[dT] [dT] RPS6KB1 #1antisense UGUCUUCCAUAAAUAUUCC[dT] [dT] RPS6KB1 #2 sense GAUAGUAAGAAAUGCUAAA [dT] [dT] RPS6KB1 #2 antisense UUUAGCAUUUCUUACUAUC[dT] [dT] Dynamin1 #1sense CAGCUAUGCUAUCAAGAAU[dT] [dT] Dynamin1 #1antisense AUUCUUGAUAGCAUAGCUG[dT] [dT] Dynamin1 #2 sense GAGAUUCUGGUCAUCCGCA[dT][dT] Dynamin1 #2 antisense UGCGGAUGACCAGAAUCUC[dT] [dT] Dynamin1 #3 sense CUUUCGAGCUGGUCAAGAU[dT][dT] Dynamin1 #3 antisense AUCUUGACCAGCUCGAAAG[dT] [dT] Dynamin2 #1sense CCCUUGACACCAUCCUGAA[dT][dT] Dynamin2 #1antisense UUCAGGAUGGUGUCAAGGG[dT][dT] Dynamin2 #2 sense CCCUCAAGGAGGCGCUCAA[dT] [dT] Dynamin2 #2 antisense UUGAGCGCCUCCUUGAGGG[dT][dT] Dynamin2 #3 sense CCAACAUGGACCUGGCCAA[dT] [dT] Dynamin2 #3 antisense UUGGCCAGGUCCAUGUUGG[dT][dT] Dynamin3 #1sense CCCAUUAAUUUACGAGUCU[dT] [dT] Dynamin3 #1antisense AGACUCGUAAAUUAAUGGG[dT] [dT] Dynamin3 #2 sense CCUUAUGAUCAAUAACGUU[dT] [dT] Dynamin3 #2 antisense AACGUUAUUGAUCAUAAGG[dT] [dT] Dynamin3 #3 sense GAUCUACCUGGAAUAACUA[dT] [dT] Dynamin3 #3 antisense UAGUUAUUCCAGGUAGAUC[dT] [dT] Supplementary Table 2. The siRNA and Primer Sequences siRNA Vendor Sequence (5”-3”) Control sense TTCTCCGAACGTGTCACGT [dT][dT] Control antisense ACGTGACACGTTCGGAGAA [dT][dT] Notch3 #1sense GGUAGUAAUGCUGGAGAUU[dT][dT] Notch3 #1antisense AAUCUCCAGAUUACUACC [dT][dT] Notch3 #2 sense CUGUCUUGCUGCUGGUCAU[dT][dT] Notch3 #2 antisense AUGACCAGCAGCAAGACAG [dT][dT] Notch3 #3 sense CCGUUAUGCCCGUGCUGAU[dT][dT] Notch3 #3 antisense AUCAGCACGGGCAUAACGG[dT][dT] Jagged-1 #1sense GAAUGUGAGGCCAAACCUU[dT][dT] Jagged-1 #1antisense AAGGUUUGGCCUCACAUUC[dT] [dT] Jagged-1 #2 sense CCUGUAACAUAGCCCGAAA[dT] [dT] Jagged-1 #2 antisense UUUCGGGCUAUGUUACAGG[dT][dT] Jagged-1 #3 sense CAUCGAUUAUUGUGAGCCU[dT] [dT] Jagged-1 #3 antisense AGGCUCACAAUAAUCGAUG[dT] [dT] RPS6KB1 #1sense GGAAUAUUUAUGGAAGACA[dT] [dT] RPS6KB1 #1antisense UGUCUUCCAUAAAUAUUCC[dT] [dT] RPS6KB1 #2 sense GAUAGUAAGAAAUGCUAAA [dT] [dT] RPS6KB1 #2 antisense UUUAGCAUUUCUUACUAUC[dT] [dT] Dynamin1 #1sense CAGCUAUGCUAUCAAGAAU[dT] [dT] Dynamin1 #1antisense AUUCUUGAUAGCAUAGCUG[dT] [dT] Dynamin1 #2 sense GAGAUUCUGGUCAUCCGCA[dT][dT] Dynamin1 #2 antisense UGCGGAUGACCAGAAUCUC[dT] [dT] Dynamin1 #3 sense CUUUCGAGCUGGUCAAGAU[dT][dT] Dynamin1 #3 antisense AUCUUGACCAGCUCGAAAG[dT] [dT] Dynamin2 #1sense CCCUUGACACCAUCCUGAA[dT][dT] Dynamin2 #1antisense UUCAGGAUGGUGUCAAGGG[dT][dT] Dynamin2 #2 sense CCCUCAAGGAGGCGCUCAA[dT] [dT] Dynamin2 #2 antisense UUGAGCGCCUCCUUGAGGG[dT][dT] Dynamin2 #3 sense CCAACAUGGACCUGGCCAA[dT] [dT] Dynamin2 #3 antisense UUGGCCAGGUCCAUGUUGG[dT][dT] Dynamin3 #1sense CCCAUUAAUUUACGAGUCU[dT] [dT] Dynamin3 #1antisense AGACUCGUAAAUUAAUGGG[dT] [dT] Dynamin3 #2 sense CCUUAUGAUCAAUAACGUU[dT] [dT] Dynamin3 #2 antisense AACGUUAUUGAUCAUAAGG[dT] [dT] Dynamin3 #3 sense GAUCUACCUGGAAUAACUA[dT] [dT] Dynamin3 #3 antisense UAGUUAUUCCAGGUAGAUC[dT] [dT] Supplementary Table 2. The siRNA and Primer Sequences Primers Primer Sequence 5”-3” PSEN1 for TGCTCCTGCCGTTCTCTATT PSEN1 rev AGTTACCTGCACCGTTGTCC NCSTN for ACGTGGATAACCCCTGTGTC NCSTN rev ACGTGGATAACCCCTGTGTC APH1A for GGACCAAGATGAACCAGACC APH1A rev CTTGATCACTGTGGCTGGG APH1B for ATGCCCCAGAATACATGCAG APH1B rev AGTGGGCATTCATGGAGATT PSENEN for TGACCAACCAGAGAAAAGGC PSENEN rev ATGAACCTGGAGCGAGTGTC Dynamin1 for CTCGGCATATTCTGTGGTTG Dynamin1 rev GCTCGAGAATTTCGTAGGCA Dynamin2 for TCGGCATGTTCTGTTTTTGA Dynamin2 rev GGTGCTGGAGAACTTCGTG Dynamin3 for TTCAGGTTGTCCAAGGGAAG Dynamin3 rev CTCGAAGGGATACTGGTTCG Jagged 1 for ACTGTCAGGTTGAACGGTGTC Jagged 1 rev ATCGTGCTGCCTTTCAGTTT RPS6KB1 for TTCTCACAATGTTCCATGCC RPS6KB1 rev CTCTGAGGATGAGCTGGAGG 18S for CGCCGCTAGAGGTGAAATTC 18S rev TTGGCAAATGCTTTCGCTC
https://openalex.org/W2971736753	https://repository.ubn.ru.nl/bitstream/handle/2066/212259/1/212259.pdf	English	null	Attentional bias for food cues in advertising among overweight and hungry children: An explorative experimental study	Food quality and preference	2,020	cc-by	8,375	Attentional bias for food cues in advertising among overweight and hungry children: An explorative experimental study Folkvord, F.; Anschutz, D.J.; Buijzen, M.A. 2020, Article / Letter to editor (Food Quality and Preference, 79, (2020), article 103792) Doi link to publisher: https://doi.org/10.1016/j.foodqual.2019.103792 Version of the following full text: Publisher’s version Downloaded from: http://hdl.handle.net/2066/212259 Download date: 2024-10-24 Frans Folkvorda,b,⁎, Doeschka J. Anschützc, Moniek Buijzenc Frans Folkvorda,b,⁎, Doeschka J. Anschützc, Moniek Buijzenc a Tilburg School of Humanities and Digital Sciences, Department Communication and Cognition, Tilburg University, Warrandelaan 2, 5037 AB Tilburg, the Netherlands b Open Evidence Research, Ramble Poblenou 22a, 08018 Barcelona, Spain c The Behavioural Science Institute, Radboud University, Montessorilaan 3, 6525 HR Nijmegen, the Netherlands Humanities and Digital Sciences, Department Communication and Cognition, Tilburg University, Warrandelaan 2, 5037 AB Tilburg, the Netherlands earch, Ramble Poblenou 22a, 08018 Barcelona, Spain a Tilburg School of Humanities and Digital Sciences, Department Communication and Cognition, Tilburg University, Warrandelaan 2, 5037 AB b Open Evidence Research, Ramble Poblenou 22a, 08018 Barcelona, Spain c The Behavioural Science Institute, Radboud University, Montessorilaan 3, 6525 HR Nijmegen, the Netherlands A R T I C L E I N F O Keywords: Food cues Energy-dense snacks Childhood obesity Attentional bias Advertising Keywords: Food cues Energy-dense snacks Childhood obesity Attentional bias Advertising People with an increased motivation for rewarding substances show increased automatic selective attention towards cues that are related to that speciﬁc substance. The aim of this study was to explore if overweight and hungry children have an attentional bias for food cues in food advertising. A randomized between-subject design was used with 95 children who played an advergame that promoted either energy-dense snacks or non-food products. Overweight children had a higher gaze duration for the food cues compared to normal weight children. No eﬀects were found of overweight on the attentional bias measurements for the non-food cues. Furthermore, hungrier children had a higher gaze duration, a higher number of ﬁxations, and a faster latency of initial ﬁxation on the food cues than less hungry children, while we found the opposite results for the non-food cues. The ﬁndings largely conﬁrm our expectations, adding important knowledge about individual susceptibility to food advertisements. Overweight and hungrier children seem to be stronger aﬀected by food advertising than normal weight and less hungry children. This study is the ﬁrst that examined attentional bias in a food advertisement that is highly comparable to advertisements that are used by food companies, thereby increasing the external validity of the ﬁndings. The second strength is that the development of an attentional bias for food cues is developing at a young age and it was examined in a real-life situation. https://doi.org/10.1016/j.foodqual.2019.103792 Received 23 June 2019; Received in revised form 6 September 2019; Accepted 6 September 2019 ⁎ Corresponding author at: Tilburg School of Humanities and Digital Sciences, Department Communication and Cognition, Tilburg University, Warrandelaan 2, 5037 AB Tilburg, the Netherlands E-mail address: fransfolkvord@gmail.com (F. Folkvord). Available online 07 Septem ber 2019 © h h bl hdb l d h l d h l Note: To cite this publication please use the final published version (if applicable). Food Quality and Preference 79 (2020) 103792 Contents lists available at ScienceDirect ⁎ Corresponding author at: Tilburg School of Humanities and Digital Sciences, Department Communication and Cognition, Tilburg University, Warrandelaan 2, 5037 AB Tilburg, the Netherlands E-mail address: fransfolkvord@gmail.com (F. Folkvord). https://doi.org/10.1016/j.foodqual.2019.103792 Received 23 June 2019; Received in revised form 6 September 2019; Accepted 6 September 2019 Available online 07 Septem ber 2019 0950-3293/ © 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecom m ons.org/licenses/BY/4.0/). https://doi.org/10.1016/j.foodqual.2019.103792 Received 23 June 2019; Received in revised form 6 September 2019; Accepted 6 September 2019 Available online 07 Septem ber 2019 0950-3293/ © 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecom m ons.org/licenses/BY/4.0/). 2. Theory Attentional bias theory suggests that when people have an increased motivation to receive or avoid a rewarding substance, like palatable food, they show increased attention towards cues that are related to that substance (Field & Cox, 2008; Werthmann et al., 2011). Further- more, attention biases and craving for the rewarding substance are assumed to be reciprocally related. Attention biases for food may elicit craving, and craving can in turn trigger attentional biases for food. This self-stimulating circle may cause a preoccupation with palatable food (Nijs, Franken, & Muris, 2010). Subsequently, the child had a small break for 5 min. After the break, the experimenter and the child ﬁlled out the second part of the online questionnaire. The second part of the questionnaire consisted of ques- tions that assessed brand recognition and attitude to the brand shown in the advergame. The experimenter read the questions and answers aloud, and the children gave their answers to the experimenter who ﬁlled the answers in the computer. When the questionnaire was ﬁn- ished, the experimenter measured height and weight of the children. The children were then accompanied back to their (class)rooms, and the experimenter invited the next child to participate. The experimenter requested that all children refrained from discussing the experiment with their classmates. The incentive-sensitization model of obesity suggests that repeated pairings of reward from food intake and cues that predict future caloric intake result in increased attention to these food cues (Berridge, 2009). In line with this model, numerous studies have found that overweight individuals have more attention for food cues than normal weight in- dividuals (Castellanos et al., 2009; Nijs, Muris, et al., 2010; Nijs, Muris, Euser & Franken, 2010; Werthmann et al., 2011). For example, over- weight individuals directed their ﬁrst gaze more often toward food pictures than healthy-weight individuals (Castellanos et al., 2009). Furthermore, obese individuals retained incentive salience for food cues despite feeding and decreased self-reported hunger levels, while normal weight individuals showed no attentional bias for food cues after eating and decreased hunger (Castellanos et al., 2009). In addition to over- weight people, hungrier people also seem to have an automatic selec- tive preference for food cues compared to less hungry people (Nijs, Franken, et al., 2010). This can be explained from an evolutionary perspective, because a selective detection of food seems to be one of the most adaptive characteristic of humans and animals2. 1. Introduction behaviour of children and found that food advertising techniques are eﬀective (Boyland et al., 2016; Folkvord, Anschütz, Boyland, Kelly, & Buijzen, 2016, Folkvord & van‘t Riet, 2018). Considering that most food advertising promotes unhealthy, palatable, and rewarding food pro- ducts, it is considered to be an important factor in the current obesity epidemic (Boyland et al., 2016; Folkvord, 2019; Folkvord et al., 2016; Harris, Bargh, & Brownell, 2009). Food commercials inﬂuence chil- dren’s preferences (Borzekowski & Robinson, 2001), requests for (Buijzen & Valkenburg, 2002), and consumption of advertised products (Folkvord, Anschütz, Buijzen, & Valkenburg, 2013, Folkvord, Anschütz, Nederkoorn, Westerik, & Buijzen, 2014, 2015, 2016, 2017). Several studies have shown that children with overweight or obesity may be particularly vulnerable to these eﬀects (Folkvord et al., 2015, 2016; Halford et al., 2004, 2007). Childhood obesity remains a major public health priority worldwide (Silventoinen et al., 2004). A central factor in understanding the impact of the environment on the obesity epidemic will be to explore the in- ﬂuence of industry-developed food cues on the brain and behaviour (Brownell & Gold, 2014; Folkvord, 2019). In an “obesogenic” society that is characterized by an abundance of highly palatable food items and the presence of food-related cues, people are frequently exposed to attractive food and food-related cues (Wardle, Carnell, Haworth, & Plomin, 2008). Food advertisements are omnipresent and contrived to be attention-grabbing, activating people’s consumer behaviours. These food advertisements are mostly promoting energy-dense snacks, high in salt, sugar and fat, and have low nutritional value (WHO, 2009). Remarkably, there is very little research about attentional bias for food cues among children, in particular in real-life contexts, such as food advertising. A recent systematic review assessing the eﬀect of food marketing on children’s attitudes, preferences and consumption showed Motivational stimuli, like food advertisements, capture attention and trigger neurological reward systems, promoting craving and moti- vating behaviour toward the foods (Castellanos et al., 2009). Multiple studies have examined the eﬀects of food marketing on eating Food Quality and Preference 79 (2020) 103792 F. Folkvord, et al. hungry children (H3), while no diﬀerences are expected in attentional bias for the non-food cues (H4). hungry children (H3), while no diﬀerences are expected in attentional bias for the non-food cues (H4). that there is a lack of use of physiological methodologies in researching food marketing eﬀects (Smith, Kelly, Yeatman, & Boyland, 2019). 1. Introduction The development of attentional biases is a process of classical conditioning (Field & Cox, 2008), which starts at a very young age. For example, children who show elevated reward circuitry responsitivity during food cue exposure also show increased attention for food cues and have a greater risk for future weight gain (Yokum, Ng, & Stice, 2012a; Yokum, Ng, & Stice, 2012b). Next, there are no studies reported that examined attentional bias for food cues in food advertisements, although food advertisements are speciﬁcally designed to capture attention and overweight and hungry children are more susceptible to food adver- tisements (Bruce et al., 2013; Forman, Halford, Summe, Macdougall, & Keller, 2009; Folkvord et al., 2015, 2016, 2019; Halford, Gillespie, Brown, Pontin, & Dovey, 2004). 3.1. Procedure The committee for ethical concerns in the Behavioral Science Institute at the Radboud University approved the current study. After obtaining consent from the schools to participate, parents were sent a letter with detailed information regarding the study. Around 90% of the children participated. It was emphasized to the parents and the children on beforehand that the data would remain conﬁdential and that chil- dren could cease participation at any moment. In this study eye movements during exposure to an advergame promoting energy-dense snacks or non-food products were measured, to assess duration of ﬁxation, number of ﬁxations, and the latency of initial ﬁxations on food or non-food cues (for more information see Field & Cox, 2008; Mogg, Bradley, Field, & de Houwer, 2003). Adver- games are online electronic games that are used to advertise a product or a brand, frequently used by food companies. Experimental studies have found that advergames that promote food increase subsequent food intake among children (Folkvord et al., 2013; Folkvord et al., 2014; Folkvord, Anschütz, Wiers, & Buijzen, 2015). Until now, no study has examined an attentional bias for food cues during exposure to food advertisements among children. Children were individually tested at their schools during regular school hours. The teacher assigned the children (in alphabetical order) to the experimenter and the experimenter brought each child to a se- parate (class)room containing a computer. The children started with an online questionnaire to assess sex, age, group and pre-experimental hunger. Subsequently, the child was placed behind a separate computer to play one version of the advergames and the child’s eyes were cali- brated and validated. After calibration, the experimenter instructed the child to play the advergame. The experimenter read the instructions from the screen. The child would be playing a memory game for 5 min and should at- tempt to ﬁnish as many games as possible, which were unlimited. The experimenter left the room until the child ﬁnished playtime. When the playtime was ﬁnished, the child signaled the experimenter. Children had a 5 min break after playing the advergame, before we continued the rest of the experiment, where they could snack ad libitum from two bowls containing energy-dense snacks for 5 min. The children were not told about the break before the advergame. The eﬀect of the adver- games on the snack intake is described elsewhere (Folkvord et al., 2015). 2. Theory Diﬀerent studies have found that the motivational state of hunger is associated with an attentional bias toward food-related cues (Castellanos et al., 2009; Mogg, Bradley, Hyare, & Lee, 1998; Nijs, Muris, et al., 2010; Veenstra & de Jong, 2010). 3.3. Measures Attentional bias. To measure the participants’ attention to food-re- lated or non-food cues, three dependent variables were used: (1) the duration of ﬁxations (i.e. gaze duration), (2) the number of ﬁxations on the cues, and (3) the latency of initial ﬁxations on the cues. A partici- pant’s gaze duration was deﬁned as the overall amount of time that the participant’s gaze was directed to the food or non-food cues. Number of ﬁxations was determined by counting the total number of times the participant ﬁxated on the food or non-food cues. Initial ﬁxation was deﬁned as the time of the ﬁrst ﬁxation of the cue after its appearance. The food or non-food cues remained visible at the screen at the same place throughout the game. To compute groups of high versus low at- tention to the food or non-food cues M split was conducted. BMI. BMI was measured as weight (kg)/height2 (cm). Weight was measured to the nearest 0.1 kg while the children were wearing clothing but no shoes. Height was also measured according to standard procedures to the nearest 0.5 cm. Additionally, BMI was divided in two diﬀerent groups by calculating whether the children were underweight or normal weight (n = 79), and overweight or obese (n = 16), using international cut-oﬀscores (Cole, Bellizzi, Flegal, & Dietz, 2000). In this study 6 children were underweight, 73 children were normal weight, 15 children were overweight, and 1 child was obese. Hunger. Hunger was measured by presenting the children with a visual analogue scale (VAS; 14 cm) to measure the extent to which they felt hungry before the experiment began. VAS are widely used and are reliable and valid rating scales for measuring subjective experiences related to food intake (King & Hill, 2004; Van Laerhoven, Van der Zaag- Loonen, & Derkx, 2004). The anchors were “not hungry at all” and “very hungry”. M split-method was used to divide the children in high (n = 61) vs. low (n = 34) hunger levels. The cut-oﬀscore was a hunger level of 3.2 cm ( ± 3.7 cm). The advergame was developed by a professional game designer. The two versions of the game were identical, except for the advertised brands and products that were shown on the side of the screen. The game involved a memory game with 16 cards with unbranded toys on the backside of the cards. 3.3. Measures The front sides of the cards were plain white. The brands (popular candy brand, Fig. 1; or popular toys brand, Fig. 2) appeared on the left and on the right side of the screen, at random on the upper or lower part of the screen, and the individual products (candy or toys) appeared on the opposite side (respectively lower or upper part) of the screen. The cues of the food and non-food brands and products were displayed continuously, for the total remaining of the advergame. Similar to advergames from food companies, two speciﬁc features to immerse the children into the game were integrated. First, a digital timer appeared on the top left of the screen, and a time bar appeared in the top center of the screen to exert time pressure on the children. Second, the game played an unpleasant sound when a child selected a false pair and a pleasant sound when a child selected a correct pair. 3.2. Experimental design and stimulus materials This study was part of a larger study (Folkvord et al., 2015), which focused on the role of attentional bias in the eﬀect of food advergames on snack intake among children. In the present study two factorial between-subject designs were used to test our hypotheses: a 2(type of advergame: energy-dense snacks vs. non-food products) × 2(weight status: overweight vs. normal weight) design and a 2(type of adver- game: energy-dense snacks vs. non-food products) × 2(hunger status: hungry vs. satiated) design. Children were randomly assigned to one of two conditions, which involved playing (1) the energy-dense snacks advergame (i.e., promoting a popular candy brand and diﬀerent gummy and jelly sweets from this popular candy brand); (2) the non- food advergame (i.e., promoting a popular toy brand and individual toys from this brand). Counterbalancing was conducted to start with a diﬀerent condition every day. Furthermore, the order of conditions was also counterbalanced to avoid any order eﬀects. The dependent vari- ables were the measurements of attentional bias. While playing one of the advergames, children’s eye movements were recorded with a Based on the attentional bias theory and the incentive-sensitization model, it is hypothesized that overweight children have a greater at- tentional bias for the food cues compared to normal weight children (H1), while no diﬀerences are expected in attentional bias for the non- food cues (H2). Furthermore, it is hypothesized that hungry children have a greater attentional bias for the food cues compared to less 2 F. Folkvord, et al. Food Quality and Preference 79 (2020) 103792 Food Quality and Preference 79 (2020) 103792 corneal reﬂection eyetracker (Tobii T120 Eyetracker, Tobii Technology, Danderyd, Sweden). The Tobii eye-tracking system with a 17″ TFT ﬂat screen monitor on which the stimuli were presented was used. The eye- tracking system recorded gaze data of both eyes at 60 Hz with an average accuracy of 0.5° visual angle. We have used 60 Hz because this increases the track box, allowing minor movements by the children. We preferred not to use chin support (which is needed with 120 Hz) to make the experiment more realistic. Tobii studio was used as software. Before beginning the experiment, children’s gazes were calibrated with a nine-point calibration procedure, in which an expanding–contracting circle appeared in every position of a screen-wide 3 × 3 grid of cali- bration points on a white background. Children were asked to accu- rately ﬁxate the circle. 3.2. Experimental design and stimulus materials When seven or fewer points were calibrated successfully, the calibration was repeated for the missing calibration points; otherwise the experiment commenced. Areas that were speciﬁed as food cues or non-food cues during playtime were measured if both of the participant’s eyes overlapped with the display of the food or non- food cues. The memory game that the children played required their attention continuously, so every time the children directed their eyes towards one of the nongame related cues, it aﬀected their game results in a negative way. Children were motivated to ﬁnish as many games as possible, thereby trying to inﬂuence their concentration to play the game. During playtime, every 16.667 ms the eyetracker measured where the eyes of the children were located. 3.4. Analyses strategy Randomization checks using a one-way analysis of variance was conducted for sex, age, hunger, brand recognition, candy brand atti- tude, gaze duration, number of ﬁxations, latency, and BMI. In Table 1, means and standard deviations are presented for all variables separately for each condition. Data was tested for normality and homogeneity of variance (Levene’s test). Distribution of the data was normal and the Levene’s test showed no signiﬁcant diﬀerences in homogeneity of variance. To test our hypotheses, three separate multivariate analyses of covariance were conducted, with age and sex as covariates. The ﬁrst multivariate analysis was conducted with BMI, hunger, and type of advergame as independent variables, and the attentional bias measurements (gaze duration, number of ﬁxations, and latency of initial ﬁxation) as de- pendent variables, see Table 2. The second multivariate analysis was conducted with an interaction between BMI and type of advergame, and the attentional bias measurements as dependent variable, see Table 3. The third multivariate analysis was conducted with an inter- action between hunger levels and type of advergame, and the atten- tional bias measurements as dependent variables, see Table 4. Bonfer- roni corrections were used to correct for multiple comparisons. The hypotheses were tested with one-tailed analyses, the adjusted p-value that was considered signiﬁcant was 0.05. We calculated eﬀect sizes for Cohen’s d and Cohen’s f2. Fig. 1. A visualization of the advergame promoting energy-dense snacks. 4.2. Main analyses Table 2 Results from the ﬁrst multivariate analysis of covariance, with attentional bias measures as dependent variables.1 Gaze duration (ms) Number of ﬁxations Latency (ms) Sex (boy = 1, girl = 0) F (1, 89) = 0.002 F (1, 89) = 0.083 F (1, 89) = 0.499 Age (y) F (1, 89) = 0.156 F (1, 89) = 1.879 F (1, 89) = 0.073 Hunger on VAS3 (cm) F (1, 89) = 0.026 F (1, 89) = 1.717 F (1, 89) = 0.004 BMI F (1, 89) = 8.3522 F (1, 89) = 2.795 F (1, 89) = 0.083 Advergames F (1, 89) = 0.402 F (1, 89) = 0.316 F (1, 89) = 0.042 Explained variance (%) 8.9 7.6 9 1 n = 95. 2 p < 0.01. 3 VAS, visual analog scale. Table 2 Results from the ﬁrst multivariate analysis of covariance, with attentional bias measures as dependent variables.1 Results from the ﬁrst multivariate analysis of covariance, with attentional bias measures as dependent variables.1 The results (see Table 2) from the ﬁrst multivariate analysis of covariance yielded a signiﬁcant eﬀect for BMI on gaze duration (P < 0.001, Cohen’s d = 0.70). Overweight children had a higher gaze duration for the commercial cues (food as well as non-food) in the advergames (M = 8807.3 ms, SD = 5087.5 ms) than normal weight children (M = 5689.0 ms, SD = 3642.6 ms). In agreement with H1, the post hoc analysis showed that overweight children who played the en- ergy-dense advergame (M = 9652.1 ms, SD = 6602.2 ms) had a higher gaze duration for food cues (P < 0.05, Cohen’s d = 0.72) than normal weight children who played the energy-dense advergame (M = 5802.3 ms, SD = 3642.1 ms), see also Fig. 3. Overweight children who played the non-food advergame did not have a higher gaze dura- tion to the non-food cues (P > 0.05) than normal weight children, thereby supporting H2. Furthermore, no main eﬀect of BMI was found on number of ﬁxations and latency of initial ﬁxation (P > 0.05), which contradicts H1 and supports H2. No eﬀect for hunger was found. Fi- nally, no eﬀect of sex and age on the attentional bias measurements (P > 0.05) was found. 4.1. Descriptives The total sample consisted of 116 children (grades 2, 3, and 4) from 3 primary schools in the Netherlands, 44% of the participants were boys. 15 children were excluded from the analyses because calibration Fig. 1. A visualization of the advergame promoting energy-dense snacks. 3 F. Folkvord, et al. Food Quality and Preference 79 (2020) 103792 Fig. 2. A visualization of the advergame promoting non-food. Fig. 2. A visualization of the advergame promoting non-food. The percentage of overweight children in the current study was higher than the percentage of overweight children in the Netherlands (13.3%). It is assumed that schools with a higher percentage of overweight children were more likely to participate in our study because the board was more interested in possible factors that could explain obesity among children. No diﬀerences were found between the experimental conditions for sex, age, hunger, candy brand attitude, gaze duration, number of ﬁxations, latency, and BMI. More children (P < 0.05) re- cognized the advertised candy brand in the advergame compared to the toys brand. Correlations were calculated between BMI and hunger and no signiﬁcant association between BMI and hunger (r = −0.093) was found. Sex and age were included in the models as covariates because other studies have shown that these variables are associated with at- tentional bias for food cues5,7. Table 1 Variables measured, by condition.1 Table 1 Variables measured, by condition.1 Energy-dense snack (n = 52) Nonfood (n = 43) Sex (boy = 1, girl = 0) 44% 47% Age (y) 8.5 ± 1.1 8.4 ± 1.1 Hunger on VAS2 (cm) 2.9 ± 3.5 3.4 ± 4.5 Brand recognition 77% 46% Candy brand attitude 11.0 ± 1.9 10.5 ± 1.7 Gaze duration (ms) 6394.6 ± 4374.3 5996.1 ± 3693.5 Number ﬁxations 57 ± 37.6 64.3 ± 36.1 Latency (ms) 11734.3 ± 24183.9 11016.7 ± 18111.0 BMI 17.1 ± 2.5 16.9 ± 2.1 1 n = 95. 2 VAS, visual analog scale. Variables measured, by condition.1 4.2. Main analyses was not successfully conducted, 4 children because they had not ﬁn- ished the session completely and 2 children because they had outlying scores on gaze duration (Ms + 2 × SD). The ﬁnal sample consisted of 95 children. The mean ( ± SD) age of the children in grade 2 (n = 45) was 7.6 ± 0.54 years, in grade 3 (n = 23) was 8.6 ± 0.73 years, and in grade 4 (n = 27) was 9.6 ± 0.56 years. In the current sample, 81.4% of the children were normal weight, 18.6% were overweight or obese. In the second multivariate analysis of covariance (see Table 3) no signiﬁcant interaction between BMI and type of advergame on gaze duration (P > 0.05), between BMI and number of ﬁxations (P > 0.05), and between BMI and latency of initial ﬁxation (P > 0.05) was found. The diﬀerence in attentional bias between overweight 4 Food Quality and Preference 79 (2020) 103792 F. Folkvord, et al. Table 3 Results from the second multivariate analysis of covariance, with attentional bias measures as dependent variables.1 Gaze duration (ms) Number of ﬁxations Latency (ms) Sex (boy = 1, girl = 0) F (1, 88) = 0.011 F (1, 88) = 0.138 F (1, 88) = 0.511 Age (y) F (1, 88) = 0.132 F (1, 88) = 1.749 F (1, 88) = 0.068 Hunger on VAS4 (cm) F (1, 88) = 0.005 F (1, 88) = 1.338 F (1, 88) = 0.008 BMI F (1, 88) = 8.1852 F (1, 88) = 2.8903 F (1, 88) = 0.079 Advergames F (1, 88) = 0.801 F (1, 88) = 1.174 F (1, 88) = 0.031 BMIAdvergames F (1, 88) = 0.405 F (1, 88) = 1.004 F (1, 88) = 0.031 Explained variance (%) 8.7 9.3 1.0 1 n = 95. 2 p < 0.01. 3 p < 0.05. 4 VAS, visual analog scale. Table 3 Results from the second multivariate analysis of covariance, with attentional bias measures as dependent variables.1 Fig. 3. Gaze duration between conditions for weight status. children and normal weight children in the advergame promoting en- ergy-dense snacks was not signiﬁcantly diﬀerent than the diﬀerence in attentional bias between overweight children and normal weight chil- dren in the advergame promoting non-food products. 4.2. Main analyses Results show that hungry children who played the energy-dense advergame (M = 7826.5 ms, SD = 5314.3 ms) had a higher gaze duration to the food cues than less hungry children (M = 5699.1 ms, SD = 3726.7 ms), while hungry children who played the non-food advergame (M = 4743.2 ms, SD = 2843.4 ms) had a lower gaze duration to the non-food cues than less hungry children (M = 6815.4 ms, SD = 3997.3 ms), see also Fig. 4. Furthermore, hungry children who played the energy-dense adver- game had a higher number of ﬁxations (M = 60.3, SD = 28.9) to the food cues then less hungry children (M = 55.4, SD = 41.5), while hungry children who played the non-food game had a lower number of ﬁxations (M = 46.2, SD = 24.5) to the non-food cues than less hungry children (M = 69.8, SD = 33.2), see Fig. 5. Finally, hungry children who played the energy-dense advergame (M = 6326.5 ms, SD = 21773.9 ms) had a faster initial ﬁxation to the food cues than less hungry children (M = 14360.9 ms, SD = 25148.7 ms), while hungry children (M = 15317.8 ms, SD = 24185.2 ms) who played the non-food advergame had a slower initial ﬁxation to the non-food cues than less hungry children (M = 8204.5 ms, SD = 12485.9 ms). No eﬀects were found for sex and age (P > 0.05) Fig. 6.. Fig. 3. Gaze duration between conditions for weight status. incentive-sensitization model (Berridge, 2009), it was expected to ﬁnd that overweight and hungry children had a greater attentional bias for the food cues compared to normal weight and less hungry children, while it was expected no diﬀerences in attentional bias for the non-food cues. The results showed that overweight children had a higher gaze duration for the food cues than normal weight children. No eﬀect was found of overweight for number of ﬁxations or latency of initial ﬁxa- tions for the food cues. These results partly support H1, because no eﬀect was found of BMI on all attentional bias measures. No eﬀect was found of overweight for gaze duration, number of ﬁxations or latency of initial ﬁxation for the non-food cues, thereby supporting H2. The in- teraction between BMI and type of advergame was not signiﬁcant. Furthermore, no main eﬀect of hunger was found on the attentional bias measurements. 4.2. Main analyses However, signiﬁcant interactions between hunger 4.2. Main analyses In the third multivariate analysis of covariance (see Table 4) a sig- niﬁcant interaction between hunger and type of advergame on gaze duration (P < 0.05, Cohen’s f2 = 0.18), number of ﬁxations (P < 0.05, Cohen’s f2 = 0.19) and latency of initial ﬁxation (P < 0.05, Cohen’s f2 = 0.15) was found. Although the post hoc analyses showed no signiﬁcant diﬀerences between the individual cells (P < 0.05), it is important to show the results of these analyses, see also Fig. 4. Results show that hungry children who played the energy-dense advergame (M = 7826.5 ms, SD = 5314.3 ms) had a higher gaze duration to the food cues than less hungry children (M = 5699.1 ms, SD = 3726.7 ms), while hungry children who played the non-food advergame (M = 4743.2 ms, SD = 2843.4 ms) had a lower gaze duration to the non-food cues than less hungry children (M = 6815.4 ms, SD = 3997.3 ms), see also Fig. 4. Furthermore, hungry children who played the energy-dense adver- game had a higher number of ﬁxations (M = 60.3, SD = 28.9) to the food cues then less hungry children (M = 55.4, SD = 41.5), while hungry children who played the non-food game had a lower number of ﬁxations (M = 46.2, SD = 24.5) to the non-food cues than less hungry children (M = 69.8, SD = 33.2), see Fig. 5. Finally, hungry children who played the energy-dense advergame (M = 6326.5 ms, SD = 21773.9 ms) had a faster initial ﬁxation to the food cues than less hungry children (M = 14360.9 ms, SD = 25148.7 ms), while hungry children (M = 15317.8 ms, SD = 24185.2 ms) who played the non-food advergame had a slower initial ﬁxation to the non-food cues than less hungry children (M = 8204.5 ms, SD = 12485.9 ms). No eﬀects were found for sex and age (P > 0.05) Fig. 6.. Although the post hoc analyses showed no signiﬁcant diﬀerences between the individual cells (P < 0.05), it is important to show the results of these analyses, see also Fig. 4. 5. Discussion The incentive-sensitization model proposes that obesity is asso- ciated with anomalous responsitivity of reward circuitry to food. In line with this theory, hypersensitivity to motivational stimuli with high incentive salience (i.e. food cues in the advergame) produce a bias in attentional processing toward food-related cues, triggering the release of dopamine and driving attention towards food-related cues and con- sumption (Yokum et al., 2012a). Overweight is a trait that is strongly linked to attentional biases for food cues (Field & Cox, 2008). This makes it more diﬃcult for overweight children to lose weight, or maintain a healthy eating pattern, than for normal weight children. Fig. 4. Gaze duration between conditions for hunger levels. Fig. 5. Number of ﬁxations between conditions for hunger levels. Furthermore, attentional bias theory states that if people have an increased motivation to receive or avoid a rewarding substance, they will show automatically increased attention towards cues that are linked to that substance. The current results show that hungrier chil- dren have this increased motivation. Hunger is a state which is strongly related to attention for food cues. From an evolutionary perspective this is very adaptive, because a selective detection of energy-dense foods seems a very eﬀective survival characteristic of humans and animals (Brownell & Gold, 2014). But in an “obesogenic” society, where energy- dense food is omnipresent and designed to attract attention, this can have deteriorating eﬀects, because hungrier children are more suscep- tible to the overwhelming amount of food-related cues and may therefore choose more often to eat energy-dense snack (Folkvord et al., 2014). This leads to unhealthy eating patterns and, subsequently, to overweight. The results from this study add important knowledge to the existing literature about the eﬀects of food marketing. The results can be used as an explanation for the ﬁndings that overweight and hungrier children are more susceptible to the impact of food advertisements (Forman et al., 2009; Halford et al., 2004). Assessing the inﬂuence of industry- developed food cues is considered a central factor of understanding the environment and its relationship with the obesity rates nowadays (Brownell & Gold, 2014). While people are evolutionary equipped for times of scarcity, we are constantly surrounded by plenty images and videos of high caloric and tasty snacks, especially in food advertise- ments on television and internet. 5. Discussion Based on attentional bias theory (Field & Cox, 2008) and the Based on attentional bias theory (Field & Cox, 2008) and the Table 4 Results from the third multivariate analysis of covariance, with attentional bias measures as dependent variables.1 Gaze duration (ms) Number of ﬁxations Latency (ms) Sex (boy = 1, girl = 0) F (1, 88) = 0.001 F (1, 88) = 0.098 F (1, 88) = 0.540 Age (y) F (1, 88) = 0.049 F (1, 88) = 2.387 F (1, 88) = 0.179 Hunger on VAS4 (cm) F (1, 88) = 0.002 F (1, 88) = 2.057 F (1, 88) = 0.001 BMI F (1, 88) = 6.6422 F (1, 88) = 1.907 F (1, 88) = 0.319 Advergames F (1, 88) = 1.370 F (1, 88) = 0.003 F (1, 88) = 0.071 HungerAdvergames F (1, 88) = 4.2043 F (1, 88) = 3.3373 F (1, 88) = 3.0193 Explained variance (%) 13.1 11.0 4.2 1 n = 95. 2 p < 0.01. 3 p < 0.05. 4 VAS, visual analog scale. Table 4 Results from the third multivariate analysis of covariance, with attentional Table 4 Results from the third multivariate analysis of covariance, with attentional bias measures as dependen 5 Fig. 4. Gaze duration between conditions for hunger levels. F. Folkvord, et al. Food Quality and Preference 79 (2020) 103792 F. Folkvord, et al. levels and type of advergame for gaze duration, number of ﬁxation and latency of initial ﬁxation were found. Hungrier children who played the energy-dense advergame had a higher gaze duration, a higher number of ﬁxations and a faster initial ﬁxation to the food cues than less hungry children, while opposite results were found for the hungry children in the non-food advergame. The ﬁndings from the interaction analyses support H3 and refute H4. Previous studies have shown that adver- games promoting unhealthy foods have an eﬀect on pester power (Dixon, Scully, & Parkinson, 2006), food choice (Dias & Agante, 2011), food liking, nutritional knowledge Esmaeilpour, Heidarzadeh, Mansourian, & Khounsiavash, 2018), and actual eating behavior (see for an overview on the eﬀects of advergames Folkvord & van‘t Riet, 2018). Considering that A great number of studies have shown that advertising knowledge does not results in increased counterarguments against advertising eﬀects (An, Jin, & Park, 2014; Brucks, Armstrong, & Goldberg, 1988; Folkvord et al., 2016, 2017; Rozendaal, Lapierre, Van Reijmersdal, & Buijzen, 2011). References An, S., Jin, H. S., & Park, E. H. (2014). Children's advertising literacy for advergames: Perception of the game as advertising. Journal of Advertising, 43(1), 63–72. Bannon, K., & Schwartz, M. B. (2006). Impact of nutrition messages on children's food choice: Pilot study. Appetite, 46, 124e129. Berridge, K. C. (2009). “Liking” and “wanting” food rewards: Brain substrates and roles in eating disorders. Physiology & Behavior, 97, 537–550. Borzekowski, D. L., & Robinson, T. N. (2001). The 30-second eﬀect: An experiment re- vealing the impact of television commercials on food preferences of preschoolers. Journal of the American Dietetic Association, 101(1), 42–46. The ﬁrst strength of this study is that it examines attentional bias in a food advertisement that is highly comparable to advertisements that are used by food companies, thereby increasing the external validity of the ﬁndings. The second strength is that the development of an atten- tional bias for food cues is developing at a young age and it was ex- amined in a real-life situation. One limitation of this study is the rela- tively small group of overweight children, thereby possibly creating false-positive results (Simmons, Nelson, & Simonsohn, 2011). However, this study is the ﬁrst to test whether overweight children have more attention for food cues in a food advertisement. In addition, previous explorative studies have also used small numbers (Bannon & Schwartz, 2006). Therefore, it is interesting to use these results for further re- search with larger groups of overweight children. Another limitation is that we did not assess medium and long-term eﬀects of advertising. Future research should examine how repeated exposure to food cues in advertising aﬀects attentional bias to food cues among diﬀerent groups of children (Folkvord, 2019; Folkvord et al., 2016). Next, the interac- tion eﬀect between hunger and type of advergame is partly driven by the increased attention for non-food-related cues by less hungry chil- dren. Boyland, E. J., Nolan, S., Kelly, B., Tudur-Smith, C., Jones, A., Halford, J. C., et al. (2016). Advertising as a cue to consume: A systematic review and meta-analysis of the eﬀects of acute exposure to unhealthy food and nonalcoholic beverage advertising on intake in children and adults, 2. The American Journal of Clinical Nutrition, 103(2), 519–533. Brownell, K. D., & Gold, M. S. (2014). Food and addiction: A comprehensive handbook (1st ed.). UK: Oxford University Press. Bruce, A. S., Lepping, R. J., Bruce, J. M., Cherry, B. Disclosure statement Folkvord, F., Lupiáñez-Villanueva, F., Codagnone, C., Bogliacino, F., Veltri, G., & Gaskell, G. (2017). Does a ‘protective’message reduce the impact of an advergame promoting unhealthy foods to children? An experimental study in Spain and The Netherlands. Appetite, 112, 117–123. No potential conﬂict of interest was reported by the authors. Acknowledgements Folkvord, F., & van‘t Riet, J. (2018). The persuasive eﬀect of advergames promoting unhealthy foods among children: A meta-analysis. Appetite, 129, 245–251. lf d C G S d ll & ll (2009) d Forman, J., Halford, J. C. G., Summe, H., Macdougall, M., & Keller, K. L. (2009). Food branding inﬂuences ad libitum intake diﬀerently in children depending on weight status. Results of a pilot study. Appetite, 53, 76–83. The Behavioural Science Institute, Radboud University Nijmegen, the Netherlands, funded the research. p y pp , , Halford, J. C., Boyland, E. J., Hughes, G., Oliveira, L. P., & Dovey, T. M. (2007). Beyond- brand eﬀect of television (TV) food advertisements/commercials on caloric intake p y pp , , Halford, J. C., Boyland, E. J., Hughes, G., Oliveira, L. P., & Dovey, T. M. (2007). Beyond- Halford, J. C., Boyland, E. J., Hughes, G., Oliveira, L. P., & Dovey, T. M. (2007). Beyond- brand eﬀect of television (TV) food advertisements/commercials on caloric intake and food choice of 5–7-year-old children. Appetite, 49(1), 263–267. Appendix A. Supplementary data methodological approaches to measure attentional bias, and the impact of diﬀerent factors such as hunger levels, energy–density of visual food cues and level of restrained eating (Doolan, Breslin, Hanna, Murphy, & Gallagher, 2014). Based on these ﬁndings, it might be too early to consider attention to food cues as an important factor related to over- weight and obesity (Doolan et al., 2014; Field et al., 2016). However, both theoretical understandings and a great amount of empirical evi- dence highlight the importance of using the most appropriate metho- dology to use when assessing attentional bias and eating behavior or weight status. New computing methods have been developed that showed that the variability of attentional bias for food stimuli sig- niﬁcantly predict the variance in BMI, showing that large variability of attentional bias for food cues exists in obese children (Liu et al., 2019). Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodqual.2019.103792. 5. Discussion A possible explanation why an eﬀect of BMI was found on gaze duration to the food cues, and not for number of ﬁxations and latency of the initial ﬁxation to the food cues, might be that overweight children have a higher attention maintenance bias than normal weight children (Weierich, Treat, & Hollingworth, 2008). After the food cues have triggered the attention of children, overweight children cannot take their eyes oﬀthe food related cues, compared to normal weight children. It might be that overweight children have not (yet) developed strategies to direct their attention towards something else, instead of the attractive food cues. Furthermore, empirical evi- dence for attentional bias for food in overweight children, adolescents and adults is contradictory (Dürrschmid, Joechl, & Danner, 2013; Field et al., 2016; Folkvord et al., 2015; Liu, Roefs, Werthmann, & Nederkoorn, 2019). This may partly be explained by the use of diﬀerent Fig. 5. Number of ﬁxations between conditions for hunger levels. Fig. 6. Latency of initial ﬁxation between conditions for hunger levels. Fig. 6. Latency of initial ﬁxation between conditions for hunger levels. Fig. 6. Latency of initial ﬁxation between conditions for hunger levels. 6 Food Quality and Preference 79 (2020) 103792 F. Folkvord, et al. 6. Conclusion The psychology of food marketing and overeating. Routledge. F lk d F A hü D J B l d E K ll B & B ij M (2016) F d d Folkvord, F., Anschütz, D. J., Boyland, E., Kelly, B., & Buijzen, M. (2016). Food adver- tising and eating behavior in children. Current Opinion in Behavioral Sciences, 9, 26–31. Folkvord, F., Anschütz, D. J., Buijzen, M., & Valkenburg, P. M. (2013). The eﬀect of playing advergames that promote energy-dense snacks or fruit on actual food intake among children. American Journal of Clinical Nutrition, 97, 239–245. Folkvord, F., Anschütz, D. J., Nederkoorn, C., Westerik, H., & Buijzen, M. (2014). Impulsivity, “Advergames”, and food intake. Pediatrics, 133, 1007–1012. Folkvord F Anschütz D J Wiers R W & Buijzen M (2015) The role of attentional Folkvord, F., Anschütz, D. J., Wiers, R. W., & Buijzen, M. (2015). The role of attentional bias in the eﬀect of food advertising on actual food intake among children. Appetite, 84, 251–258. References C., Martin, L. E., Davis, A. M., et al. (2013). Brain responses to food logos in obese and healthy weight children. Journal of Pediatrics, 162, 759–764. Brucks, M., Armstrong, G. M., & Goldberg, M. E. (1988). Children's use of cognitive de- fenses against television advertising: A cognitive response approach. Journal of Consumer Research, 14(4), 471–482. Buijzen, M. A., & Valkenburg, P. M. (2002). Appeals in television advertising: A content analysis of commercials aimed at childeren and teenagers. Communications: The European Journal of Communication Research, 27, 349–364. Castellanos, E. H., Charboneau, E., Dietrich, M. S., Park, S., Bradley, B. P., Mogg, K., et al. (2009). Obese adults have visual attention bias for food cue images: Evidence for altered reward system function. International Journal of Obesity, 33(9), 1063. Cole, T. J., Bellizzi, M. C., Flegal, K. M., & Dietz, W. H. (2000). Establishing a standard deﬁnition for child overweight and obesity worldwide: International survey. BMJ, 320, 1240–1243. Dias, M., & Agante, L. (2011). Can advergames boost children's healthier eating habits? A comparison between healthy and non-healthy food. Journal of Consumer Behaviour, 10(3), 152–160. Dixon, H., Scully, M., & Parkinson, K. (2006). Pester power: Snackfoods displayed at supermarket checkouts in Melbourne, Australia. Health Promotion Journal of Australia, 17(2), 124–127. 6. Conclusion Doolan, K. J., Breslin, G., Hanna, D., Murphy, K., & Gallagher, A. M. (2014). Visual at- tention to food cues in obesity: An eye-tracking study. Obesity, 22(12), 2501–2507. Doolan, K. J., Breslin, G., Hanna, D., Murphy, K., & Gallagher, A. M. (2014). Visual at- tention to food cues in obesity: An eye-tracking study. Obesity, 22(12), 2501–2507. Dürrschmid, K., Joechl, M., & Danner, L. (2013). Eye-Tracking the Obese – BMI has little eﬀect on Attention Bias. Ernährung/Nutrition. 37, 149–158. Diﬀerent scholars suggest that attention bias and craving for a substance are reciprocally related (Field & Cox, 2008; Nijs, Franken, et al., 2010), but it is not exactly clear how this mechanism works with regard to palatable food (Field et al., 2016; Folkvord et al., 2016). It is important to examine the susceptibility to food and food-related cues among children in real-life contexts, such as food marketing, and un- ravel whether overweight children have developed an attentional bias for food cues, or that the children have become overweight due to the attentional bias for food cues. Furthermore, the question remains whether repeated pairings of reward from energy-dense food intake and cues that predict future snack intake create a stronger attentional bias among children, making it therefore harder to resist these palatable snacks. These are important questions that should be studied in future research. Dürrschmid, K., Joechl, M., & Danner, L. (2013). Eye-Tracking the Obese – BMI has little eﬀect on Attention Bias. Ernährung/Nutrition. 37, 149–158. Esmaeilpour, F., Heidarzadeh, Hanzaee K., Mansourian, Y., & Khounsiavash, M. (2018). Children’s food choice: Advertised food type, health knowledge and entertainment. 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W2783512207.txt	https://repositorio.unican.es/xmlui/bitstream/10902/15982/1/NewInsightsInto_nv_articulo_5711.pdf	en		New Insights into the Study of Paleolithic Rock Art: Dismantling the “Basque Country Void”	Journal of anthropological research	2,018	cc-by	10,654	New Insights into the Study of Paleolithic Rock Art Dismantling the “Basque Country Void” D I E G O G A R A T E M A I D A G A N , The Cantabria International Institute for Prehistoric Research (IIIPC), Edif. Interfacultativo, University of Cantabria. Avda. Los Castros s/n., 39005 Santander (Spain). Email: garatemaidagandiego@gmail.com The Vasco-Cantabrian region of northern Spain, together with southwestern France, is one of the richest areas in terms of Paleolithic cave art, but, until recently, by far the highest concentration of sites had been in the central-western sector: the provinces of Cantabria and Asturias. In contrast, the eastern sector, the Basque Country—between the Asón River and the Pyrenees—was thought to have a signiﬁcantly lower density of cave art loci, and few of them were major “sanctuaries.” The density of cave art sites seemed to increase again in the central French Pyrenees. This unequal distribution was difﬁcult to explain given the homogeneity in the distribution of undecorated (i.e., habitation) Upper Paleolithic sites, as well as the lack of major lithological or orographic differences between the eastern and central-western sectors of the region. In addition, the important geostrategic position of the Basque Country between southwestern France and northwestern Iberia seemed to contradict the marginal role traditionally suggested by the parietal art record. During the past decade, however, research projects aiming to address the issue of the so-called Basque rock art void have led to the tripling of the number of known decorated caves in the eastern sector. Some of the very recent discoveries, notably in Atxurra, Armintxe, and Aitzbitarte IV, fall into the category of major cave art sites. The evidence presented here contributes to a fuller understanding of artistic production, human connections, and settlement dynamics during the entire Upper Paleolithic among the Cantabrian, Pyrenean, and Aquitaine regions, underlining the importance of the Basque Country record. Keywords: cave art, recent discoveries, Upper Paleolithic, Basque Country, northern Spain, southwest France THE BASQUE COUNTRY AS A UNIT OF ANALYSIS FOR THE UPPER PALEOLITHIC The study of prehistory in the Basque Country throughout most of the twentieth century has been carried out in the tradition of the work of the late Father Jose Miguel de Barandiarán. The spatial unit of analysis established by Barandiarán continues to be a Submitted January 19, 2017; accepted March 22, 2017; published online January 8, 2018. Journal of Anthropological Research (Summer 2018). © 2018 by The University of New Mexico. All rights reserved. 0091-7710/2018/7402-0003$10.00 168 NEW INSIGHTS INTO THE STUDY OF PALEOLITHIC ROCK ART \| 169 valid analytical framework and is still in use. The current study area is bounded to the northeast by the Adour River at the southern edge of the sand-covered French region of Les Landes; to the east by the valley of the Nervión River, at whose mouth is the city of Bilbao (Vizcaya); to the south by the Atlantic-Mediterranean watershed divide along the crest of the eastern Cantabrian (Basque) Cordillera; to the east by the Central Pyrenees; and to the north by the Bay of Biscay (Cantabrian Sea). It is the space historically linked to the core area of modern Basque speakers, except to the west of the Nervión where the landscape is more akin to that of the historically Castilian province of Cantabria. The territory allows for relatively good communication with its neighboring regions (Figure 1) and therefore constitutes a corridor between France and the Iberian Peninsula, between the Ebro Valley and the Cantabrian coast, as well as between the Pyrenees and the Cantabrian mountains (among them the Picos de Europa), which were traversed with varying intensity by people carrying objects and ideas during all historical periods (Arrizabalaga 2007). This transit area is made up of mostly similar landscapes: abrupt and steep in the Atlantic and pre-Pyrenean valleys, with rolling plains in certain coastal sectors, especially south of the Adour (Chalosse) and in the Mediterranean watershed in Alava and Navarra. The access points between these areas are characterized by low-elevation mountain passes that allowed ﬂuid transit even during times of harsh climatic conditions. The earliest archaeological evidence for the presence of anatomically modern humans in this Basque region is from the Proto-Aurignacian technocomplex at sites such as Gatzarria, Isturitz, and Labeko Koba, and this presence extends throughout the Upper Paleolithic. The Aurignacian occupations at Gatzarria and Isturitz were particularly intensive and have yielded very diverse assemblages. The Gravettian, which appears quite early on, is mostly of the Noailles type and is well represented at sites such as Isturitz and Aitzbitarte III; several open-air settlements of this period have also recently been found. The Solutrean record is much more limited, centering mostly on its ﬁnal phases, and is found at such sites as Antoliña, Arlanpe, Amalda. Aitzbitarte IV, Azkonzilo, Ermittia, and Isturitz. Lastly, the Basque Country is very rich in Magdalenian assemblages, with a more intensive presence in such sites as Ekain, Urtiaga, Santimamiñe, and most especially, Isturitz. In its earlier phases, the period is best represented in the Peninsular (Spanish) area (Erralla, Bolinkoba, Urtiaga, Ermittia, Santimamiñe, etc.), whereas Middle Magdalenian occupations are more abundant on the northern Pyrenean (French) side (Isturitz, Brassempouy, Espalungue, Saint-Michel, etc.). Toward the end of the Magdalenian, the number of sites increases considerably throughout the territory. In sum, the area under study is characterized by continuous settlement during all Upper Paleolithic periods with the possible exception of the initial phases of the Solutrean, and an especially dense occupation during the Tardiglacial (Garate et al. 2014). Until a decade ago, in terms of Paleolithic artistic production, research in the area focused on two matters. On the one hand, specialists wondered about the small number of rock art sites relative to the considerable density of Upper Paleolithic habitation Figure 1. The Basque Region “crossroads” between the Iberian Peninsula and continental Europe (yellow: Les Landes sand-covered region; black arrows: passes through the Basque Mountains). NEW INSIGHTS INTO THE STUDY OF PALEOLITHIC ROCK ART \| 171 sites. This fact was even more striking given the nature of this area as a corridor, located as it is between the two European regions with the richest and most numerous rock art records: the French Central Pyrenees and the Périgord (including Quércy), and Cantabria and Asturias. On the other hand, the distribution of caves containing art appeared not to be homogeneous in temporal distribution either, given the almost complete lack of artistic activity up until the very beginning of the Magdalenian, at which time great decorated sanctuaries appear. Therefore, traditionally the idea had been that the number of decorated caves in the Basque territory was smaller than in the central/western Cantabrian region and the central/eastern Pyrenees, and especially in relation to the relatively dense peopling of the area during the various phases of the Upper Paleolithic. RESEARCH HISTORY: JOSE MIGUEL DE BARANDIARÁN AND THE GREAT BASQUE SANCTUARIES The discovery and study of the decorated Paleolithic caves of the western Pyrenees— between the basins of the Nervión and Adour rivers—was different from that of the neighboring regions in that it entailed little involvement from international researchers in favor of local specialists, and especially Jose Miguel de Barandiarán. He was a storied ethnologist, folklorist, and archaeologist who worked in the Basque region from the time when rock art was ﬁrst discovered at the cave site of Santimamiñe near Guernica (Vizcaya) in 1916 all the way to the discovery and study of Ekain cave near San Sebastián (Guipúzcoa), a half a century later in 1968. Following recognition by the international community of the existence of Paleolithic rock art—whose authenticity at the Cantabrian site of Altamira and in a few French sites was ﬁnally accepted at the Congress of the Association Française pour l’Avancement des Sciences in 1902—a vertiginous increase in the number of discoveries took place during the ﬁrst decade and a half of the twentieth century, primarily along the Cantabrian coast. This process was headed by international researchers, mainly H. Breuil and H. Obermaier in very signiﬁcant collaboration with local scholars L. Sierra and H. Alcalde del Río, whose seminal study of sites in Cantabria (then called Santander Province) and Asturias, Les Cavernes de la Région Cantabrique (Alcalde de Rio et al. 1911), presented descriptions of the decorated caves known at that time (followed by La Caverne de la Pasiega—also in Cantabria; Breuil et al. 1913). The lack of sites from the Basque region in these publications is striking. Not until the ﬁrst archaeological excavation at the cave site of Isturitz in 1913 by E. Passemard was evidence of artistic representations found, in this case in the form of a series of animal bas-reliefs engraved onto a pillar of calcite at the center of the Grande Salle. This same pillar would be restudied by I. Barandiarán and G. Laplace (and reported in Laplace 1984), whose reevaluation was signiﬁcantly different from the original study. A century after its discovery, Isturitz’s rock art was once again studied following the fortuitous discovery of red stains on and objects stuck into its walls (Normand and Turq 2003; Labarge 2011) and later, as part of a comprehensive re- 172 \| JOURNAL OF ANTHROPOLOGICAL RESEARCH SUMMER 2018 study of the rock art at the site (Garate, Labarge et al. 2013; Garate, Rivero et al. 2016b). In 1916, a group of children found the paintings at Santimamiñe cave, a discovery that had a great impact on the local populace (Arribas 2006). The excavation of the site and the study of its art was carried out by a team headed by T. Aranzadi, J. M. de Barandiarán, and E. Eguren (1925), true pioneers of scientiﬁc Basque prehistoric research. Almost in parallel, Cuadra Salcedo and Alcalá Galiano (1918) carried out another study of the cave art. The monograph compiled by Aranzadi, Eguren, and de Barandiarán was updated by de Barandiarán in various publications until 1976. A few additional, more modest discoveries and overviews were made by others in the mid-twentieth century (Apellániz 1971; Fernández García de Diego 1971; Goicoechea 1966; Nolte 1968). Santimamiñe cave was most recently studied by Gorrotxategi (2000), González Sainz (2009), and González Sainz and Ruiz Idarraga (2010). In 1930, the famous French speleologist N. Casteret (1933) discovered a small assemblage of engraved rock art at the cave of Alkerdi 1 in the pre-Pyrenean Mountains of northern Navarra, although it was not studied in depth until a few decades later, by I. Barandiarán Maestu (1974b). Recently, in a lateral gallery, a large number of engravings, mostly depicting bison and horses, have been found, and these offer a very different perspective on the art of this cave (Garate and Rivero 2015). The ﬁrst overall study of Basque Paleolithic art dates to 1934, as part of J. M. de Barandiarán’s El Hombre Primitivo en el País Vasco (Primitive Man in the Basque Country), reported in more detail a year later (1935), as well as through two publications concerning mobile art (Aranzadi and Barandiarán 1927, 1934). On the one hand, this work assigns a geostrategic role to the territory, which is a very modern idea as outlined above. On the other hand, following H. Obermaier, it insisted on the theory of hunting magic as a way of interpreting the cave art phenomenon, linking it to Basque mythology (the goddess Mari) to defend the idea of supposed Basque population continuity from the Paleolithic to the present day. J. M. de Barandiarán (1946) found a horse image engraved on the wall of the cave site of Uriogaina in 1940, a drawing of which was published by the Abbé Glory (1964), with whom he had excavated the site; they determined that the oldest habitation levels were Mesolithic. A restudy carried out by D. Garate in 2010 concluded that the engraved grooves and the style itself do not correspond to the Paleolithic. In sum, during the ﬁrst half century of research on Paleolithic rock art in Europe, the information uncovered in the Basque territory was scant, as attested by Quatre Cents Siècles d’Art Pariétal, published by H. Breuil in 1952, in which only Santimamiñe acts as the link between two artistic regions—Cantabria and the Pyrenees—separated by 400 km of otherwise near-absolute emptiness. Shortly before this seminal work on the entirety of known Paleolithic rock art was published, in 1950, the French speleologist P. Boucher discovered paintings in the caves of Etxeberri and Sasiziloaga in the Arbaila Pyrenean massif of the French Basque NEW INSIGHTS INTO THE STUDY OF PALEOLITHIC ROCK ART \| 173 Country. These were immediately studied by G. Laplace (1952) and most recently revisited by Garate and colleagues (Garate 2012a, 2012b; Garate and Bourrillon 2009, 2011; Garate et al. 2012). In 1955, in the middle level of the karstic system of the Gaztelu (Isturitz) hill, the presence of paintings and engravings at the far end of the cave of Oxocelhaya was revealed, although they were not studied until many years later (Labarge 2003, 2011; Laplace 1960; Larribau 1982, 2011); the site’s art is currently being restudied along with that of Isturitz. At both caves, the expert opinion of J. M. de Barandiarán had been relied upon to validate the authenticity of the discoveries. In 1958, this same notable prehistorian shared his thoughts on the state of knowledge of Basque region rock art with the Regional Assembly of Speleologists (Barandiarán 1959), closing out the ﬁrst half-century of the limited cave art discoveries in the Spanish and the French Basque Country (known in Basque as Euskalherria). Early in the second half of the twentieth century, interest in Paleolithic rock art was reactivated by momentous discoveries, notably those of the decorated caves of Altxerri and Ekain in Guipúzcoa, and the ﬁrst map of known rock art was put together by J. M. de Barandiarán. At the cave of Atxuri, during the 1960 excavation season, led by J. M. de Barandiarán, several faded red traces were found (Fernández García de Diego 1971), but they were destroyed by quarrying before they could be veriﬁed or studied. The poorly preserved stratigraphic sequence was not helpful when attempts were made to establish the cultural context of the (lost) rock paintings. Two years later, during the excavation of Goikolau cave, J. M. de Barandiarán (1964a) found a series of engravings attributed partly to the Paleolithic and partly to the Iron Age. Subsequent studies (Apellániz 1982; Basas 2000–2002) have ruled out the Paleolithic origin of some of the paintings, which is why we do not include the site in Figure 1. However, a study of the assemblage is currently being carried out by J. C. López Quintana (personal communication, 2017), and it appears to conﬁrm the Paleolithic origin of at least some of the art. The engravings and paintings at Altxerri were found by three speleologists from the Aranzadi Science Society in 1962 and immediately studied by J. M. de Barandiarán (1964b). Given the number of images and the wide range of themes depicted, this represented the most signiﬁcant ﬁnd since Santimamiñe. Different interpretations have been published since then (Beltrán 1966; González Sierra 1993), along with previously unpublished ﬁgures (Altuna 1996). The assemblage as a whole has been studied on two more occasions (Altuna and Apellániz 1976; Ruiz Redondo 2014). The discovery of the upper gallery of Altxerri B took place shortly after that of the intermediate gallery and was ﬁrst noted by F. Fernández García de Diego (1966); the main panel would later be restudied (Altuna and Mariezkurrena 2010). A new, comprehensive study (González Sainz et al. 2013; Ruiz Redondo et al. 2017) has located the original entrance, separate from the lower gallery, and has also identiﬁed new painted motifs and reﬁned the chronology of the art. Also in the mid-1960s, the ﬁrst assessment of 174 \| JOURNAL OF ANTHROPOLOGICAL RESEARCH SUMMER 2018 Paleolithic rock art in the Basque Country was published (Barandiarán Maestu 1966a, 1966b). Shortly afterwards, in 1969, paintings and engravings were found in Ekain cave by two members of the Antxieta archaeology group of Azpeitia, and, once again, it was J. M. de Barandiarán who directed their study (Barandiarán and Altuna 1969). The spectacular bichrome horses would later be studied more speciﬁcally by Barandiarán Maestu (1974a) and the whole of the cave restudied by J. Altuna and J. M. Apellániz (1978). More recently, some previously unseen ﬁgures have been published (Altuna and Mariezkurrena 2008; Gónzalez Sainz et al. 1999; Fano et al. 2012; Garate, Rivero et al. 2014) as well as an analysis of the pigments (Chalmin et al. 2002). The small painting assemblage at Sinhikole was found in 1971 by three speleologists from the Société Spéléologique et Préhistorique de Bordeaux and three years later was featured in a short publication by Séronie-Vivien (1974). Like the rest of the caves in the Arbaila massif, Sinhikole would later be reexamined (Garate and Bourrillon 2009). Lastly, in the still-active, lower karstic level of the Gaztelu (Isturitz) hill, engravings and paintings were found in 1975 in Erberua cave by speleologist J. D. Larribau. These would not be studied until a few years later (Larribau and Prudhomme 1983, 1989) given the difﬁculty of access to the site. It has recently been the subject of a monograph based on the ﬁeldwork carried out in the 1970s and 1980s (Larribau 2013). In short, between 1950 and 1975 a considerable number of discoveries were made, some of which were particularly important, namely those of Altxerri, Ekain, and Erberua (Figure 2). During the remaining quarter of the twentieth century, no such discoveries took place (Table 1). The main contribution to the subject of rock art in the late twentieth century was the publication of general summaries of Basque Paleolithic cave art (Altuna 1994; Apellániz 1989, 1990; Barandiarán Maestu 1988) and the “authorship” studies by J. M. Apellániz (1980, 1982). The Zubialde affair—the scandal of a forged cave art site in the interior Basque province of Alava—cannot be ignored, nor its consequences for the study of rock art in the Basque Country (Altuna et al. 1992; Apellániz 1995, 2003; Barandiarán Maestu 1995). NEW RESEARCH ADVANCES: DISMANTLING THE “BASQUE COUNTRY VOID” The scant research activity that took place in the eastern Cantabrian region during the last decades of the twentieth century coincided with an increase in the number of discoveries in neighboring regions to the west. Because of this, the difference in density of decorated caves between those sectors substantially increased. Researchers even coined the term “Basque void” to refer to the scarcity of decorated caves in this area (Garate 2009). Explanations for these different densities were sought, for example, in the smaller amount of limestone bedrock in the Basque territory—and therefore the presence of fewer caves; in an aggressive anthropogenic impact on the environment and thus Figure 2. Decorated caves between the Nervión and Adour rivers discovered between 1900 and 1999 (black: Magdalenian; red: PreMagdalenian). 176 \| JOURNAL OF ANTHROPOLOGICAL RESEARCH SUMMER 2018 Table 1. History of the discovery of decorated caves in the study area Decade N 1900–1909 – 1910–1919 2 1920–1929 – 1930–1939 1 1940–1949 – 1950–1959 3 1960–1969 4 1970–1979 2 1980–1989 – 1990–1999 – 2000–2009 3 2010–2017 17 destruction of sites from mining, quarrying, and industrialization in the Basque Country; in marine regression—a more distant and now-ﬂooded Last Glacial coastline in the eastern sector than off Cantabria and Asturias; in a supposedly later settlement of the territory, with a smaller percentage of sites antedating the Magdalenian (Solutrean, Gravettian, or Aurignacian) than in the western sector; or in an unbalanced prehistoric research tradition, centered mostly around the study of Neolithic megaliths in the Basque provinces. It would not be until 2001, a decade after the death of J. M. de Barandiarán, that the search for decorated caves was resumed in the Basque Country. At ﬁrst these discoveries took place purely by chance during new excavation projects at cave sites, and only later, from 2011 onward, as a result of survey projects speciﬁcally aimed at ﬁnding decorated caves. Projects developed since then by the team led by D. Garate (primarily along with O. Rivero and J. Rios-Garaizar) and by the Antxieta archaeology group reactivated research in the territory. During the 2001 excavation at Antoliña (Vizcaya) by M. Aguirre, a red, possible ﬁngerprint dot was found on the ceiling at the end of the north gallery (2002). Shortly afterward, during the excavation at Praile Aitz (Guipúzcoa), directed by X. Peñalver (Peñalver 2007; García Díez et al. 2012), a series of red stains was identiﬁed in 2006, and three years later at the cave of Astigarraga (Guipúzcoa), during an excavation project directed by J. A. Mujika, traces of red paired with bones embedded into the wall were found (Alberdi et al. 2010; García Díez et al. 2011). Over the past decade there has been an increase in the number of ﬁnds as a result of projects speciﬁcally aimed at surveying rock art, ﬁrst by archaeologists and later by speleologists. As a result of this work, between 2011 and 2017 seventeen new decorated NEW INSIGHTS INTO THE STUDY OF PALEOLITHIC ROCK ART \| 177 assemblages have been found both in already-excavated caves as well as at sites not previously studied archaeologically. In 2011 a series of horses painted in red and a bone inserted into the wall were found at Askondo in Vizcaya (Garate and Rios-Garaizar 2011, 2012a, 2013). The cave, which has been studied archaeologically since the beginning of the twentieth century, had been listed as a “destroyed site” (Marcos 1982). A year later, a series of red paintings—including two bison and a horse—was found on a large block detached from the ceiling at Lumentxa in coastal Vizcaya (Garate and Rios-Garaizar 2012b; Garate, Rios-Garaizar, and Ruiz-Redondo 2013). This site is one of the best-known in the territory and was the focus of many excavations during the beginning, middle, and latter part of the twentieth century, but no archaeologist had ever taken notice of the art even though it is adjacent to areas that were excavated near the cave mouth. That same year and within the framework of systematic survey conducted by the discoverers of the art at the aforementioned caves, red stains were found inside the very long-studied and excavated Aitzbitarte IV cave near San Sebastián; one of them may pertain to a now-lost zoomorphic representation (Garate, Rios-Garaizar et al. 2013). The role of speleologists in surveys and in the discovery of Paleolithic rock art is not new—it is worth recalling their role in the ﬁndings in Ekain and Altxerri, as well as all the “recently” identiﬁed cave art sites in the French Basque Country—but their participation has increased over the past few years. In 2014, speleologists from the Asociación Deportiva Espeleológica Saguzarrak (ADES) identiﬁed a series of red paintings at Morgota (Vizcaya). The cave, although known, was yet to be fully assessed archaeologically. As a result of this collaboration, it has been possible to excavate a site containing an ochre-processing deposit (López Quintana et al. 2015) associated with a panel with three animal ﬁgures and a dozen paint stains and dots (Garate et al. 2015a, 2015b). That same year, and close by, ADES noted some red stains at the cave of Ondaro, on a rock platform, accompanied by archaeological stratigraphy at the foot of the panel (Garate, González Sainz et al. 2016). Prospection had already been carried out at the site by J. M. de Barandiarán in 1921, but little information was gathered from this work (Aranzadi et al. 1925). Also in 2014, the Antxieta Jakintza Taldea archaeological group found a series of very faded paintings at Danbolinzulo (Guipúzcoa), where the archaeological site had been known since 1980. The paintings, currently under study, are close to the entrance and represent red deer hinds and ibex, painted in red using lines and dots (Abaunza et al. 2016). At the start of 2015, this same group revealed the engravings they found in the cave of Erlaitz (Guipúzcoa): close to a dozen ﬁgures of horses, hinds, and aurochs. Currently under study, the archaeological status of the cave had been known since 1978. The year 2015 proved to be especially proliﬁc in terms of the discovery of decorated caves. Under the aegis of systematic surveys carried out in the basin of the Lea River, a 178 \| JOURNAL OF ANTHROPOLOGICAL RESEARCH SUMMER 2018 series of red stains were found in the hall of Abittaga (Vizcaya) (Garate, Libano, and Vega 2016). The cavity had previously been excavated by J. M. de Barandiarán in 1965 and 1966 and by J. M. Apellániz in 1970. The joint work carried out by archaeologists and speleologists enabled D. Garate accompanied by J. Rios-Garaizar and four members of the Felix Ugarte Elkartea (FUE) speleological group to discover a new rock art assemblage at the site of Aitzbitarte V. The assemblage consists of various bison engraved in the “Pyrenean style,” characteristic of the Middle Magdalenian. A few days later, the speleologists shared the discoveries they had made in the caves of Aitzbitarte III and IX of animal engravings in the Gravettian style, perfectly comparable to the mobile art of Isturitz and Gargas in the French Pyrenees, and to the French rock art assemblages of Cussac, Cosquer, Roucadour, and Gargas, but until then unknown in the Cantabrian region (Garate, Rios-Garaizar et al. 2016). Simultaneously, during the second excavation season at the long-known cave of Atxurra (Vizcaya), directed by D. Garate together with speleologist I. Intxaurbe from ADES, an exceptional assemblage was discovered. It contains approximately 100 Magdalenian engravings and paintings on a dozen parietal sets in the ﬁnal sector of the cavity, which also has intact ﬂoors with hearths and archaeological material at the foot of the art panels (Garate, Rivero et al. 2016a). A century after the discovery of Santimamiñe, the search for Paleolithic rock art assemblages continues. This systematic work led to the staggering discovery by ADES speleologists of a large, boldly engraved panel with images of lions, bison, horses, and ibex in Armintxe cave, under an apartment building in the town of Lekeitio (coastal Vizcaya) in late 2016. This unique ﬁnd for the Cantabrian region reﬂects themes (felines), conventions (use of scraping to show fur), and signs (P-shaped claviforms) that are all more typical of French Pyrenean art than that of northern Spain. This is another good indication of signiﬁcant contact between the Basque region and southwestern France during the late Magdalenian, the period to which the highly realistic art of Armintxe clearly pertains. The Antxieta group recently made the modest but valuable discovery of Astuigaña, and a team from the Aranzadi Science Society composed of J. Tapia and D. Garate along with speleologists from Satorrak reported on the ﬁnding of Alkerdi 2 (Garate et al. 2017). In 2017, the discoveries continue. In the long-known Balzola cave (Vizcaya), I veriﬁed some red stains found by I. Intxaurbe from ADES the year before. The Antxieta group continues the surveys in the Deba/Urola valleys, where some engravings have been detected at Arbil V. Finally and most spectacularly, the FUE speleological group has just discovered a notable assemblage of modeled clay images (including a visually striking bison) in an upper gallery of Aitzbitarte IV. As a result of these major discoveries of rock art (Figure 3), new summaries have been published (Altuna and Mariezkurrena 2014; Garate 2009) and the wider geographical framework is beginning to be known (Garate, Ruiz-Redondo et al. 2014; Figure 3. Decorated caves between the Nervión and Adour rivers discovered between 2000 and 2017 (black: Magdalenian; red: PreMagdalenian). 180 \| JOURNAL OF ANTHROPOLOGICAL RESEARCH SUMMER 2018 Garate, Rivero et al. 2014; Garate, Rivero et al. 2015; Ochoa and García-Díez 2014). At present, the Basque archaeological record, in terms of its Paleolithic rock art, is very different from that which J. M. de Barandiarán knew in the last quarter of the twentieth century (Figure 2). The advances made have been very signiﬁcant: 20 new rock art assemblages were discovered between 2001 and 2017, whereas only 13 had been found during the whole of the twentieth century (Table 1). In addition, some of these later discoveries, such as Atxurra, Armintxe, and Aitzbitarte IV, can be placed in the category of “great Basque sanctuaries,” alongside Santimamiñe, Ekain, Altxerri, and Erberua. The projects speciﬁcally aimed at locating and studying this kind of archaeological evidence has led to a threefold increase in the number of decorated caves known in the Basque territory and, as a result, has caused us to revise and reevaluate our knowledge of the origin and development of cave art in this region and in southern France/northern Spain more generally. The survey work carried out by both archaeologists (teams led mainly by D. Garate and the Antxieta group) and speleologists (mainly ADES, FUE, and Satorrak) has played a particularly important role in this process. TOWARD A NEW VIEW OF BASQUE REGION CAVE ART The data obtained over the past few years represent an important shift in our knowledge of the archaeological record. As a matter of fact, we have gone from a nearly empty space, especially for art probably done prior to the Magdalenian, to an area with evidence of dense artistic activity that also indicates a long process of development during the whole of the Upper Paleolithic, with its own characteristics and evolutionary trajectory. These themes and techniques sometimes placed it at the margins of the Cantabrian or Pyrenean art orbits but at other times fully integrated it within the stylistic circulation axes of much more extended artistic forms. All these novelties have modiﬁed the image of the artistic activity developed in this area during the Upper Paleolithic signiﬁcantly. Of the 20 new assemblages, 16 provide considerable information; they include ﬁgurative representations—especially Atxurra and Armintxe, with more than 100 and 50 each, respectively. The remaining four (Praile Aitz, Abittaga, Ondaro, and Antoliña) are not as informative, given the small number, overall lack of diagnostics, and poor state of preservation of their rock art. The supposed scarcity of assemblages in the Basque Country in terms of the rest of the Cantabrian region has been disproved with the considerable increase—nearly triple—in the number of known rock art sites (Figure 4). On the other hand, what was thought to be a total absence of small assemblages with no ﬁgurative representations, which are so common in the rest of the Cantabrian area, is no longer true after the discovery of simple rock art motifs in Antoliña, Astigarraga, Praile Aitz, Ondaro, and Abittaga, just to name a few. Lastly, the near-absence of assemblages from before the Magdalenian period has been reevaluated now that more than a dozen sites can probably be assigned to this period, some discovered recently (Aitzbitarte III and IX, Figure 4. All the now-known decorated caves between the Nervión and Adour rivers (black: Magdalenian; red: Pre-Magdalenian; black symbols: pre-2000; white symbols: post-2000). 182 \| JOURNAL OF ANTHROPOLOGICAL RESEARCH SUMMER 2018 Askondo, Astigarraga, Alkerdi 2, Danbolinzulo, and Erlaitz) and others restudied (Altxerri B and Erberua, ﬁrst phase). These data enable us to outline the development of artistic activity during the Upper Paleolithic in the Basque Country in relation to its neighboring regions. Initial Upper Paleolithic Information about the earliest rock art in the Iberian Peninsula is scarce and to a large extent inconclusive. The Basque Country is home to one of the most reliable pieces of evidence of Aurignacian-age rock and ﬁgurative art based on archaeological context. Aurignacian art is found in the form of pictorial motifs at Altxerri B. The assemblage is in a closed gallery, with a collapsed access and reactivated speleothems, and is independent from the intermediate gallery, where the Magdalenian assemblage is located. A main panel containing representations of an exceptionally large bison (4 m), a feline, and a bear stands out, as well as paired traces and the remains of less legible ﬁgures, all in red and maroon. Charcoal, ochre, and bones (some burnt) were found at the foot of the panel. Three radiocarbon dates obtained from bones (34,195±1,235, 29,940±745, and 34,370±180 bp) suggest an age for the archaeological assemblage of around 39,000 cal. bp (Altuna 1994; González Sainz et al. 2013). The paintings are, from a formal point of view, more closely linked with French sites such as Chauvet (Ardèche) than with those of Cantabria or Asturias, where there are no known parallels, making the art at Altxerri B an isolated ﬁnd in the northern Spanish context. Later, during the Gravettian, Basque Country rock art exhibits greater parallels with the Cantabrian art traditions, and with other motifs more widely distributed throughout southwestern Europe. The assemblage from Askondo (Figure 5) is an example, with hand imprints and horse heads in the “duckbill” style, as deﬁned by the Abbé Henri Breuil and widely recognized throughout southwestern Europe. However, a connection with the artistic activity of Cantabria can also be noted: the horses are closely linked with ones in Altamira and La Pasiega B, also large in format and composed in pairs, located close to the entrance, and using dotting only occasionally. A bone embedded in a crack in the wall next to the Askondo panels was radiocarbon dated to 23,760±110 bp. Starting from the probable contemporaneity of the art and embedding the bone, and given the level—based on the hand imprints—from which a standing human could produce the paintings, together with their style, a Gravettian origin is most probable (Garate and Rios-Garaizar 2012a). The cave of Danbolinzulo, mainly decorated with small ibex in red made with the dot technique, has direct parallels in central/western Cantabria (Garate 2010)—the so-called Ramales School of J. M. Apellániz—and widens the perceived area of inﬂuence for this artistic tradition all the way to eastern Cantabria, where it had been unknown until now. In the case of Balzola, the red claviform signs had only been known before in Cantabria per se (Fortea 2005), but not to the east. At Erberua, the ﬁve negative hands in red and black (Larribau 2013) and a number of engraved ﬁgures with parallels in the mobile art of adjacent Isturitz and the rock art NEW INSIGHTS INTO THE STUDY OF PALEOLITHIC ROCK ART \| 183 Figure 5. Red hand stencil in Askondo cave (© D. Garate). of Gargas and Cussac all point toward a ﬁrst Gravettian phase of decoration of the cave (Rivero and Garate 2014), albeit still to be conﬁrmed. The engravings at Aitzbitarte III and IX (Figure 6) correspond to the scheme of the Gravettian caves of the Pyrenees and Périgord, and they represent the only such examples known from the Iberian Peninsula at this time (Garate, Rios-Garaizar et al. 2016) (Figure 7). Recently, the discovery of rock art very similar in style to that of Alkerdi 2 (Figure 8) and its direct dating via AMS-C14 have placed it alongside the previous discoveries at Aitzbitarte (Álvarez et al. 2016). The newly discovered caves ﬁll the previous void for the beginnings of Upper Paleolithic art in the Basque region. The westernmost ones (Askondo, Danbolinzulo, and Balzola) connect directly with the art known in the greater Cantabrian region, while those from the eastern sector (Aitzbitarte III-IX and Alkerdi 2) are markedly similar to assemblages in France. This is to say, the study area was a boundary between two different artistic territories at this time. Middle Upper Paleolithic The panel with paired traces at the cave of Astigarraga also exhibits clear links to Cantabria and Asturias, with similar art in caves such as La Garma, El Castillo, Cudón, Fuente del Salín, Chufín, Tito Bustillo, and La Lloseta. These kinds of motifs (hands, dots, discs, etc.) are known from the earliest phases of the Upper Paleolithic, although their precise chronology is unknown (González Sainz 1999). 184 \| JOURNAL OF ANTHROPOLOGICAL RESEARCH SUMMER 2018 Figure 6. Engraved Gravettian bison in Aitzbitarte IX cave (© D. Garate). Another modest assemblage is that of Erlaitz, comprising half a dozen engraved animals of which the closest parallels can be found during the Cantabrian Gravettian and Solutrean periods. At present, before a complete study, it is difﬁcult to provide a more speciﬁc attribution. Figure 7. “Gargas style” Gravettian bison engravings, including those of Aitzbitarte III and IX, the only ones in the Iberian Peninsula (1, D. Garate; 2, 4 after Aujoulat et al. 2001; 3 after Barrière 1976; 5 after Rivero and Garate 2014). NEW INSIGHTS INTO THE STUDY OF PALEOLITHIC ROCK ART \| 185 Figure 8. Tracing of the engraved Gravettian bison in Alkerdi 2 cave (© D. Garate). Final Upper Paleolithic This period saw the spectacular development of artistic activity in Cantabria, the Pyrenees, and the Périgord. Around 14,500 bp, this increase took place alongside a thematic and stylistic homogeneity previously unseen. The new ﬁnds from the Basque Country, including especially relevant assemblages such as those of Atxurra, Armintxe, and Aitzbitarte IV, and the numerous revisions of other, previously known sites, clearly show this conﬂuence of inﬂuences. The recent restudy of the “Pilier Gravé” of Isturitz has highlighted how little was really known about this art assemblage (Garate, Labarge et al. 2013). At the time of excavation, the pillar was covered by levels F1 and E, the top of the former attributed to the Upper Magdalenian and the bottom to the Solutrean, and the middle and upper parts of the latter level to the Early and Middle Magdalenian (Passemard 1944), although postdepositional processes may have given rise to this overlap (Garate, Rivero et al. 2016b). The study of the engravings on the pillar has enabled them to be placed in relation to the engraved slabs with reindeer images found during the early excavations of the site (differential relief, double chest line, cloven hooves, muscle insertions, etc.) that belong to the levels assigned to the Middle Magdalenian. The recent review of the assemblage from Etxeberri provides new information on its chronology. In the Salle des Peintures, at the foot of the panel containing horses traced using clay, and next to a bichrome (black and red) horse, and various red stains, lumps of processed ochre can be seen on the surface. A small prospection led to the recovery of various ﬂint artifacts and burnt bones associated with the ochre. Two dates were ob- 186 \| JOURNAL OF ANTHROPOLOGICAL RESEARCH SUMMER 2018 tained from bones: ca. 13,800–13,400 uncal bp. Considering the extremely difﬁcult access to the decorated area and the complementarity of the documented activities, it is reasonable to link these dates to the rock art (Garate et al. 2012). Other full (Altxerri A, Santimamiñe, Oxocelhaya, Sinhikole, Sasiziloaga) or partial (Ekain) restudies have also enabled us to reinterpret their rock art and these cave art loci in the regional context. In the case of Alkerdi 1 (Figure 9), the discovery of a previously unpublished decorated gallery, with representations of bison and horses in the Pyrenean style, provides further knowledge (Garate and Rivero 2015). In terms of the newly discovered decorated caves, some offer modest but very representative examples of the Middle/Upper Magdalenian, as at Aitzbitarte V (Garate, Rios-Garaizar et al. 2016) (Figure 10) or even Lumentxa (Garate et al. 2012) (Figure 11), with clear links to Pyrenean caves (Figure 12). The caves of Astuigaña and Arbil V could probably also be attributed to this period. What are most surprising, however, are the very recent discoveries in the upper gallery from Aitzbitarte IV of ﬁgures modeled in clay and the two large Paleolithic art assemblages in the lower basin of the River Lea, Atxurra and Armintxe. Although yet to be studied in detail, they are similar to what are known as the “great Basque sanctuaries”: Santimamiñe, Ekain, Altxerri A, and Erberua. The ﬁrst, Aitzbitarte IV, is a very well-known Paleolithic site at which excavations began 125 years ago. The Felix Ugarte speleological group leads the current explorations in collaboration with the archaeological group directed by D. Garate. They found Figure 9. Engraved Magdalenian bison in Alkerdi 1 cave (after Garate and Rivero 2015). NEW INSIGHTS INTO THE STUDY OF PALEOLITHIC ROCK ART \| 187 Figure 10. Engraved Magdalenian bison in Aitzbitarte V cave covered with grafﬁti (© D. Garate). a unique ensemble of rock art in an upper chamber including at least 15 engraved ﬁgures (bison, horses, and vulvas), some of them modeled in the clay (Figure 13). This technique had not been detected before in the Cantabrian region and is closely related to Middle Magdalenian decorated caves in Ariège (the Central Pyrenees of France), such as le Tuc d’Audoubert, Montespan, and Bédeilhac. In the Lea Valley, archaeological work at the well-known cave of Atxurra resumed in 2014 (Garate, Rivero et al. 2016a). In the deep part of the cavity, and mainly on projections and high, difﬁcult-to-access areas, more than a dozen decorated sectors were found, composed mainly of engraved animal ﬁgures along a 200-m-long stretch. Three sectors stand out given their large numbers of representations—dozens in each case—and because of the presence of archaeological material on the surface (charcoals, ﬂint tools, etc.) at the foot of the engraved panels (Figure 14). More than 100 animals are painted in black and/or engraved, mainly bison, horses, goats, reindeer, and red deer hinds. The double-line mane of the horses and the frontal perspective of the ibex would suggest an Upper Magdalenian chronology, which can be corroborated by radiocarbon dating of the archaeological remains. Also in the same watershed, the partly destroyed cave of Armintxe in the town of Lekeitio was found thanks to the clearing of an inﬁlled gallery by a team of speleologists from the ADES group. The main panel consists of around 20 engravings, among which two felines surrounded by various ibex, horses, and bison stand out (Figure 15). Alongside these animal ﬁgures is a series of P-type claviform signs with clear parallels 188 \| JOURNAL OF ANTHROPOLOGICAL RESEARCH SUMMER 2018 Figure 11. Large red bison found in Lumentxa cave covered with grafﬁti (after Garate, RiosGaraizar, and Ruiz-Redondo 2013). in the caves of Trois Frères and especially Tuc d’Audoubert (Ariège, France). The felines also exhibit parallels to Trois Frères. These similarities suggest a Middle Magdalenian chronology for this assemblage. Lastly, although its state of preservation is extremely poor, some stylistic indicators in the assemblage of red paintings from the cave of Morgota point to a pre-Magdalenian Figure 12. “Pyrenean style” Magdalenian engraved bisons, including those found in Alkerdi 1 and Aitzbitarte V (1 after Garate and Rivero 2015; 2 after Bégouën et al. 1958; 3, D. Garate; 4 after Fritz and Tosello 2004; 5 after Vialou 1986; 6 after Clottes 1995). 190 \| JOURNAL OF ANTHROPOLOGICAL RESEARCH SUMMER 2018 Figure 13. Magdalenian-style bison modeled in clay in Aitzbitarte IV (© D. Garate). chronology (Garate et al. 2015b). The excavation of the immediate archaeological context, including the processing of ochre that took place at the foot of the panel, however, suggests a link with activities that may date to the Upper Magdalenian (López Quintana et al. 2015). Figure 14. Engraved Magdalenian horses in Atxurra covered with grafﬁti (© D. Garate). Figure 15. Engraved Magdalenian animals in Armintxe (© Diputación Foral de Bizkaia, used with permission) 192 \| JOURNAL OF ANTHROPOLOGICAL RESEARCH SUMMER 2018 Figure 16. Timeline of the decorated caves in the Basque Country based on various dating methods (© D. Garate). The new ﬁnds discussed here enable us to review regional inﬂuences during the Magdalenian. “Pyrenean” characteristics are very much present in such caves as Aitzbitarte V and Atxurra. In in the case of Armintxe they include speciﬁc signs such as the “P” claviforms, and in Aitzbitarte IV the bison modeled in clay, both previously unattested in the Cantabrian region. These markings are also present at Erberua, in the same territory where the absence of “complex” signs had been traditionally asserted— NEW INSIGHTS INTO THE STUDY OF PALEOLITHIC ROCK ART \| 193 an idea disproven by the new discoveries. Contrary to the Cantabrian or even the Pyrenean situation, the assemblages dominated by horses (Ekain, Armintxe, Erberua, Oxocelhaya, Etxeberri) are still in the majority compared with the bison-dominated panels (Altxerri and Santimamiñe). In addition, the exaggerated hindquarters of the horses remains a strong convention in this area, especially at Ekain cave. Further studies will give us a deeper understanding of Magdalenian style and symbolism. CONCLUSIONS: NOW WHAT? During the past ﬁfteen years, our knowledge of the earliest art of the Basque Country has changed substantially. The inception of speciﬁc projects aimed at pinpointing and studying this kind of archaeological evidence has led to a tripling of the number of known decorated caves in the Basque Country and, as a result, has led to the reﬁnement and reevaluation of the approaches used to study the origin and development of Paleolithic artistic activity. The systematic survey work carried out by both archaeologists and speleologists has been a key part of this process. Conclusions drawn from the record known by J. M. de Barandiarán and up to the end of the twentieth century have become totally obsolete. The “Basque void” in cave art in this geographically strategic area between the central-western Cantabrian, Pyrenean, and Aquitaine regions is no longer true. In sum, the data newly obtained from decorated caves in the Basque Country show an area rich in artistic production, with a very prolonged trajectory of development throughout the Upper Paleolithic (Figure 16). It can now be seen as having its own characteristics and evolution, which at times place it on the margins of the Cantabrian or Pyrenean art styles and, at others, fully integrated in the orbits of much more extensive artistic traditions. The fast rate of discovery in the past few years—six decorated caves found in 2015 alone, and several more, including major sanctuaries in 2015, 2016, and 2017—and the training of speleologists now fully involved in this new research dynamic lead me to believe that more discoveries will follow in the years to come, adding to those currently underway that are brieﬂy reviewed here. The “gap” is closed and the two “wings” of the Franco-Cantabrian Upper Paleolithic culture area are now ﬁrmly bound together by the record of both habitation and cave art sites in the Basque Country, although the nature and intensity of the human contacts across the western end of the Pyrenees certainly changed throughout the ca. 30,000 years of the Last Glacial. NOTE This article was written while the author was at the Arkeologi Museum in Bilbao, Spain. The author thanks all the archaeologists, especially Olivia Rivero, Joseba Ríos, and Iñaki Intxaurbe; the speleology groups for their crucial work, especially Felix Ugarte Elkartea (FUE), Satorrak, Asociación Deportiva Espeleológica Saguzarrak (ADES), Burnia Espeleologia Taldea, Grupo Espeleológico Matiena (GEMA), and Aloña Mendi Taldea (AMET); and the Antxieta archaeology group for ﬂuid exchange of information. Thanks also go to the Diputación Foral of Vizcaya, which funded the research at Askondo, 194 \| JOURNAL OF ANTHROPOLOGICAL RESEARCH SUMMER 2018 Lumentxa, Morgota, Ondaro, Atxurra and Balzola; to the Diputación Foral of Guipuzkoa, which funded the research at Aitzbitarte III, IV, V, and IX; to the Comunidad Foral of Navarra, which funded the research at Alkerdi; to the Aranzadi Science Society, which is in charge of the work at Alkerdi 2; to the Archaeological Museum of Biscay for their support; and to the Nouvelle-Aquitaine region, which funded the research at Isturitz/Oxocelhaya and Etxeberri, Sinhikole, and Sasiziloaga. Finally, I want to thank the editor of JAR and the reviewers for their encouragement and recommendations, and the editor for extensive correction of my English. R E FE R E N C E S C I T E D Abaunza, A., A. Arrizabalaga, M. García-Diez, M. J. Iriarte, B. Ochoa, I. Vigiola-Toña, and Antxieta Arkeologi Taldea. 2016. Cueva de Danbolinzulo. 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Edited by André Leroi-Gourhan, pp. 280–82. Paris: Imprimerie Nationale. Larribau, J. D. 1982. Découverte de nouveaux ensembles graphiques dans la grotte d’Oxocelhaya: Note préliminaire. Bulletin de la Société Préhistorique Française 79:133–36. ———. 2011. La grotte d’Oxocelhaya: Synthèse des découvertes. Art pariétal préhistorique du pays Basque (Isturitz-Oxocelhaya-Erberua). Orthez. ———. 2013. La grotte Erberua. Art pariétal préhistorique du pays Basque (Isturitz-OxocelhayaErberua). Orthez. Larribau, J. D., and S. Prudhomme. 1983. La grotte ornée d’Erberua (Pyrénées-Atlantiques): Note préliminaire. Bulletin de la Société Préhistorique Française 80:280–84. ———. 1989. Étude préliminaire de la grotte d’Erbérua (Pyrénées-Atlantiques). L’Anthropologie 93(2):475–93. López Quintana, J. C., A. Guenaga, D. Garate, C. González Sainz, P. Castaños, A. García, and G. Aranzabal. 2015. “Evaluación arqueológica preliminar de Morgotako koba (Kortezubi, Vizcaya). Apuntes cronoestratigráﬁcos,” in La cueva de Morgota (Kortezubi, Vizcaya): Ocupación humana y arte parietal. Edited by J. C. López Quintana, pp. 5–56. Kobie (Excavaciones Arqueológicas en Vizcaya) 5. Bilbao: Diputación Foral de Vizcaya. Marcos J. L. 1982. Carta arqueológica de Vizcaya. Primera parte: Yacimientos en cueva. Universidad de Deusto. Bilbao: Diputación Foral de Vizcaya. Nolte, E. 1968. Catálogo de simas y cuevas de la Provincia de Vizcaya. Bilbao: Diputación de Vizcaya. Normand, C., and A. Turq. 2003. “Bilan des recherches 1995–1998 dans la grotte d’Isturitz (communes d’Isturitz et de Saint-Martin- d’Arberoue, Pyrénées-Atlantiques),” in Préhistoire du bassin de l’Adour. Edited by C. Chauchat, pp. 69–100. Baigorri: Izpegi de Navarre. Ochoa, B., and M. García-Díez. 2014. Chronology of western Pyrenean Paleolithic cave art: A critical examination. Quaternary International 364:272–82. 200 \| JOURNAL OF ANTHROPOLOGICAL RESEARCH SUMMER 2018 Passemard, E. 1913. Découvertes de bas-reliefs et de peintures dans la grotte d’Isturitz. Bulletin de la Société Préhistorique Française 10:532. ———. 1944. La caverne d’Isturitz en pays Basque. Paris: Presses Universitaires de France. Peñalver, X. 2007. Cueva de Praile Aitz I (Deba): VI campaña. Arkeoikuska: Investigación Arqueológica 2001:468–69. Rivero, O., and D. Garate. 2014. L’art mobilier gravettien de la grotte d’Isturitz (fouilles Saint-Perier): Une collection redécouverte. Paléo 25:247–76. Ruiz-Redondo, A. 2014. Entre el Cantábrico y los Pirineos: El conjunto de Altxerri en el contexto de la actividad graﬁca magdaleniense. Santander: Nadir. Ruiz-Redondo, A., C. González Sainz, and D. Garate. 2017. Back to the past: Symbolism and archaeology in Altxerri B (Gipuzkoa, Northern Spain). Quaternary International 432(B): 66–76. Séronie-Vivien, M. R. 1974. Découverte d’une nouvelle grotte ornée en pays Basque: La grotte du Sinhikole-ko-karbia (Camou-Cihige, Pyrénées-Atlantiques). Bulletin de la Société Préhistorique Française 71:40–44. Vialou, D. 1986. L’art des grottes en Ariège magdalénienne. XXII Supplément à Gallia Préhistoire. Paris.
https://openalex.org/W4313815952	https://jurnal.iain-bone.ac.id/index.php/alsyakhshiyyah/article/download/3132/pdf	Indonesian	null	KEDUDUKAN BARANG TITIPAN SEBAGAI AGUNAN PADA PERBANKAN SYARIAH	Al-Syakhshiyyah	2,022	cc-by-sa	4,027	Abstract This study discusses the implementation of deposited goods that are used as collateral in Islamic banking, in this case trying to find the right ways to solve problems if the goods deposited are used as collateral in Islamic banking. The methodology in this study is a literature study that is narrative in nature, with a historical approach as well as looking for juridical aspects in the settlement if there is a problem with goods deposited as collateral in the transaction process carried out in Islamic banking. In this study, it is shown that the resolution of a problem can be done through deliberation and through the courts. Those who do not trust will be given a fine in the form of compensation. AL-SYAKHSHIYYAH: Jurnal Hukum Keluarga Islam dan Kemanusiaan p-ISSN 2685-3248; e-ISSN 2685-5887 Vol. 4; No. 2; Desember 2022 AL-SYAKHSHIYYAH: Jurnal Hukum Keluarga Islam dan Kemanusiaan p-ISSN 2685-3248; e-ISSN 2685-5887 Vol. 4; No. 2; Desember 2022 KEDUDUKAN BARANG TITIPAN SEBAGAI AGUNAN PADA PERBANKAN SYARIAH Oleh. Muspita Sari1, A.Nurramadhani2, Muh.Fadli Mangenre3 123Institut Agama Islam Negeri Bone, Indonesia Email. muspita.sari@iain-bone.ac.id1, andi01nurramadhani1125@gmail.com2, Fadlimuhammad680@gmail.com3 2Hariman Surya Siregar, M. Ag., Koko Khaeruddin, M.Pd.I. Fikih Muamalah Teori dan Implementasinya, (Bandung : Remaja Rosdakarya, 2019), h. 2. 3Abu Azam Al-Hadi. Fikih Muamalah Kontemporer. (Depok : Raja Grafindo Persada, 2017), h. 180. Abstrak Kajian ini membahas mengenai implementasi barang titipan yang dijadikan sebagai agunan pada perbankan syariah, sebagai upaya dalam menemukan cara- cara yang tepat dalam menyelesaikan permasalahan, apabila barang titipan dijadikan sebagai agunan pada perbankan syariah. Metodologi dalam kajian ini adalah studi pustaka yang bersifat naratif, dengan pendekatan historis serta mencari aspek yuridis dalam penyelesaian apabila terjadi permasalahan barang titipan yang dijadikan sebagai agunan dalam proses transaksi yang dilakukan pada perbankan syariah. Dalam kajian ini, menujukkan bahwa penyelesaian suatu permasalahan dilakukan bisa dengan musyawarah dan melalui pengadilan. Pihak yang tidak amanah akan diberikan denda berupa ganti rugi. Kata Kunci : Titipan; Jaminan; Rahn; Perbankan Syariah; Wadi’ah. 188 Kedudukan Barang Titipan Sebagai Aunan… Muspitasari; A. Nurramadhani; Muh. Fadli M. 1Nyimas Lidya Putri. Jurnal Hukum dan Ekonomi Syariah. (Lampung : IAIN Metro, 2019), h. 280. Nyimas Lidya Putri. Jurnal Hukum dan Ekonomi Syariah. (Lampung : IAIN Metro, 2019), A. PENDAHULUAN Dalam kehidupan masyarakat tentunya akan saling berhubungan antara masyarakat yang satu dengan yang lain, disadari atau tidak ini dilakukan untuk memenuhi kebutuhan-kebutuhan hidup mereka.1 Hubungan-hubungan yang dilakukan dinamakan dengan muamalah. Yang dimaksud dengan muamalah yaitu hukum-hukum yang mengatur hubungan antara manusia yang satu dengan manusia yang lain serta hubungannya dengan alam sekitar untuk memenuhi kebutuhan hidupnya.2 Sebagaimana prinsip muamalah setiap orang pasti membutuhkan orang lain dalam melakukan suatu hal. Ini dilakukan agar kehidupan seseorang bisa lebih mudah, selain itu interaksi yang dilakukan juga akan lebih mendekatkan seseorang. Saling membantu ini juga diwujudkan dengan melakukan banyak hal, salah satunya yaitu wadi’ah (titipan). Wadi’ah secara garis besar berarti titipan dari seseorang kepada pihak lain yang dianggap mampu dan memenuhi syarat- syarat sebagaimana yang telah ditetapkan.3 Wadi’ah secara tersirat mengajarkan kepada orang-orang yang dititipkan sesuatu agar senantiasa bertanggung jawab dan bersikap hati-hati karena memelihara dan menjaga sesuatu yang bukan miliknya. Wadi’ah juga merupakan salah satu sarana yang digunakan untuk membantu orang lain. Misalnya dengan meminjamkan kendaraan kita kepada orang lain, kita akan membantu transportasi mereka. Begitu pula dengan rumah atau alat elektronik lainnya. Hal ini sangat sesuai dengan pengertian fiqih muamalah yaitu aturan-aturan Allah yang mengatur hubungan manusia dengan manusia lainnya dalam memperoleh dan mengembangkan harta benda. Barang titipan mencakup semua barang yang berwujud dan bernilai. Barang ini bisa berupa kendaraan, rumah, barang-barang elektronik, emas, dan 189 AL-SYAKHSHIYYAH: Jurnal Hukum Keluarga Islam dan Kemanusiaan p-ISSN 2685-3248; e-ISSN 2685-5887 Vol. 4; No. 2; Desember 2022 AL-SYAKHSHIYYAH: Jurnal Hukum Keluarga Islam dan Kemanusiaan p-ISSN 2685-3248; e-ISSN 2685-5887 Vol. 4; No. 2; Desember 2022 uang. Setiap barang yang dititipkan bisa digunakan oleh orang yang dititipi atau hanya disimpan. Barang yang bisa digunakan oleh orang yang dititipi misalnya motor. Apabila pihak penitip menitipkan motornya kepada seseorang, kemudian diberikan izin untuk menggunakan motor tersebut saat diperlukan, maka barang yang dititipkan bisa digunakan oleh orang tersebut, sampai orang yang mempunyai motor dapat meminta kembali motornya. Sedangkan barang yang hanya disimpan, misalnya emas. Apabila pihak yang menitipkan emasnya untuk disimpan dengan alasan ditakutkan akan hilang, maka pihak yang dititipi sama sekali tidak boleh menjual emas tersebut. Dia hanya harus menyimpannyaa ditempat yang aman sehingga tidak akan terjadi kerusakan dan kehilangan emas tersebut. Selain wadi’ah juga ada transaksi rahn. 7R. Bogdan dan Steven Taylor, Introduction to Qualitative Research Methods (John Wiley & Sons, 1984), 42. A. PENDAHULUAN Rahn berarti menyanderakan sejumlah harta yang diserahkan kepada pihak tertentu dimana barang ini kemudian dijadikan jaminan, secara hak dapat dimiliki dan kembali dimiliki setelah melakukan tebusan.4 Dengan semakin berkembangnya masyarakat pada masa kini istilah rahn juga dikenal dengan sebutan gadai yang secara bahasa berarti jaminan, agunan, dan rungguhan.5 6Soekanto dan Mamudji, Penelitian Hukum Normatif Suatu Tinjauan Singkat, (Jakarta: Rajawali, 2003), 23-24. 5Abu Azam Al-Hadi. Fikih Muamalah Kontemporer. (Depok : RajaGrafindo Persada, 2017), . 160. 4Zainuddin Ali. Hukum Gadai Syariah. (Jakarta : Sinar Grafika, 2008), h. 1. 8Andri Soemitra, Hukum Ekonomi Syariah Dan Fiqh Muamalah (Jakarta : Prenamedia Group, 2019), h. 156. 9 Abu Azam Al-Hadi. Fikih Muamalah Kontemporer, h. 180. 9 Abu Azam Al-Hadi. Fikih Muamalah Kontemporer, h. 180. B. METODE PENELITIAN Jenis penelitian ini adalah penelitian normatif (pustaka) yang bersifat naratif, dengan pendekatan historis serta pendekatan yuridis. Oleh karena itu data dalam penelitian ini adalah data sekunder, yang terdiri dari bahan hukum primer dan bahan hukum sekunder, yang bersumber dari berbagai literatur.6 Penelitian ini disebut juga sebagai penelitian kualitatif.7 Karena analisis data memakai teknik deskriptif kualitatif dalam menjelaskan kedudukan barang titipan sebagai agunan pada bank syariah. 190 Kedudukan Barang Titipan Sebagai Aunan… Muspitasari; A. Nurramadhani; Muh. Fadli M. Muspitasari; A. Nurramadhani; Muh. Fadli M. 11Andri Soemitra, Hukum Ekonomi Syariah Dan Fiqh Muamalah…, h. 157. a. Pengertian Wadi’ah a. Pengertian Wadi’ah Kata wadi’ah berasal dari bahasa Arab yaitu dari kata Wada’a – Yada’u- Wad’an yang diartikan membiarkan atau meninggalkan sesuatu pada orang yang menerima titipan. Jadi, wadi’ah berarti menitipkan sesuatu.8 Sedangkan menurut istilah terdapat perbedaan pendapat dari ulama besar. Pertama, menurut ulama Hanafiyah, wadi’ah (titipan) adalah mengikutsertakan orang lain dalam memelihara harta, baik dengan ungkapan jelas, melalui tindakan, maupun melalui syarat. Kedua, menurut ulama Malikiyah, Syafi’iyah, dan Hanabilah, wadi’ah adalah mewakilkan orang lain untuk memelihara harta tertentu dengan cara tertentu juga. Wadi’ah secara garis besar berarti titipan dari seseorang kepada pihak lain yang dianggap mampu dan memenuhi persyaratan sebagaimana yang telah ditentukan.9 Pemberian titipan, dilakukan tanpa ada paksaan dari masing-masing pihak yang bersangkutan. Apabila selama proses penitipan terdapat kerusakan, maka pihak yang diberikan titipan haruslah menggantinya. Penggantian ini dilakukan saat pihak yang dititipi melakukan kelalaian atau ketidaksengajaan sehingga merusak atau mengurangi nilai suatu barang. Adapun yang menjadi dasar hukum Wa’diah, sebagaimana Allah berfirman dalam QS. al- Baqarah:2/283: َ نِوَ بَعۡضُ كُم َ ِدُواْ َكَ تِبٗا فَرِهَ ٰوٞ نَّقۡبُوضَ ةٞۖٞ فَإِنۡ أ ۞ِإَون كُنتُمۡ لَعَ َ ٰ سَفَرٖ وَلَمۡ َتَ ِّبَعۡضٗ ا فَلۡيُؤَد َّلَّ ِي ٱ ٱ َؤۡتُهِو ُمَ ٰنَتَه َ أ ۥ ِوَۡلۡ َتَّق ٱ َ َّللّ ُرَبَّه ۥ Terjemahnya : Terjemahnya : “Jika kamu dalam perjalanan (dan bermu’amalah tidak secara tunai dengan kamu tidak memperoleh seorang penulis, maka hendaklah ada barang tanggungan yang dipegang (oleh yang berpiutang). Akan tetapi jika sebagian kamu mempercayai sebagian yang lain, maka hendaklah 191 AL-SYAKHSHIYYAH: Jurnal Hukum Keluarga Islam dan Kemanusiaan p-ISSN 2685-3248; e-ISSN 2685-5887 Vol. 4; No. 2; Desember 2022 AL-SYAKHSHIYYAH: Jurnal Hukum Keluarga Islam dan Kemanusiaan p-ISSN 2685-3248; e-ISSN 2685-5887 Vol. 4; No. 2; Desember 2022 yang dipercayai itu menunaikan amanatnya (hutangnya) dan hendaklah ia bertakwa kepada Allah Tuhannya… yang dipercayai itu menunaikan amanatnya (hutangnya) dan hendaklah ia bertakwa kepada Allah Tuhannya… 10 Abu Azam Al-Hadi. Fikih Muamalah Kontemporer…, h. 181. b. Rukun Wadi’ah Dalam mengimplementasikan wadi’ah haruslah dilakukan dengan menyesuaikan rukun dari wadi’ah itu sendiri. Menurut ulama Hanafiyah rukun wadi’ah hanya pelafalan ijab dan qabul, selain itu hanya dianggap sebagai syarat dan tidak termasuk rukun. Menurut ulama Hanafiyah, ijab dan qabul hanya bisa dianggap sah apabila dilakukan dengan perkataan yang jelas atau dengan perkataan sindiran/samaran (kinayah).10 Secara umum, rukun wadi’ah ada empat, yaitu : Orang yang menitipkan (al-mudi’ atau muwaddi); Orang yang dititipi (al- muda’ atau mustauda); Barang titipan (wadi’ah); dan, Sighat (ijab qabul)11 c. Syarat dari Rukun Wadi’ah 1) Orang yang menitipkan dan orang yang dititipkan 1) Orang yang menitipkan dan orang yang dititipkan Menurut ulama Hanafiyah bahwa yang menjadi syarat bagi orang yang melakukan wadi’ah adalah orang yang berakal sehat. Penetapan umur juga ditentukan dengan balighnya seseorang, hal ini digunakan untuk menentukan mampu tidaknya seseorang dalam melaksanakan wadi’ah. Tapi, apabila seorang anak belum cukup umur akan tetapi telah mendapat izin dari walinya untuk melaksanakan wadi’ah maka itu dianggap sah. Persyaratanya juga haruslah orang yang cerdas, karena di dalam transaksi wadi’ah kadang terjadi penipuan, maka dari itu, baik orang yang akan bertindak sebagai penitip atau orang yang dititipi haruslah memiliki kecerdasan yang baik. 3) Shigat (Ijab qabul) 13Oleh Mustafa, “AL-HÂJAT AL-AŞLIYYAH DAN HARTA TERBEBAS HUTANG SEBAGAI SYARAT WAJIB ZAKAT MÂL,” AL-KHARAJ 1, no. 2 (5 November 2021): 142–50, https://doi.org/10.30863/ALKHARAJ.V1I2.1961. 1) Wadi’ah Yad al-Amanah Karakteristiknya, yaitu : Orang yang menjadi penerima titipan tidak diperkenankan menggunakan harta ataupun benda yang dititipkan kepadanya; Penerima titipan memegang amanah agar mampu menjaga dan memelihara harta atau benda yang dititipkan, maka dari itu orang yang diamanahkan untuk menerima titipan tidak diperkenankan menggunakan ataupun mengambil manfaat atas harta atau benda tersebut; Orang yang menitipkan harta atau benda harus memberikan biaya sebagai kompensasi penerima titipan; Mengingat harta atau benda yang dijadikan titipan tidak diperbolehkan utnuk dipergunakan atau dimanfaatkan, maka aplikasi perbankan yang sesuai dengan hal ini adalah jasa penitipan atau save deposit box. 2) Barang yang dititipkan 2) Barang yang dititipkan Barang yang menjadi objek titipan harus berupa harta yang bisa disimpan, diserahterimakan serta memiliki nilai. Dalam bentuk penitipan, identitas barang haruslah jelas diketahui. Jenis dan jumlah dari barang itu haruslah pasti, untuk menghindari terjadinya kesalahpahaman di kemudian hari. 3) Shigat (Ijab qabul) 3) Shigat (Ijab qabul) 192 Kedudukan Barang Titipan Sebagai Aunan… Muspitasari; A. Nurramadhani; Muh. Fadli M. Ijab harus dinyatakan dengan ucapan dan perbuatan. Ucapan bisa sarih (jelas) ataupun kinayah (sindiran). Menurut ulama Mashab Maliki, lafal kinayah harus disertai dengan niat.12 d. Pembagian Wadi’ah 1) Wadi’ah Yad al-Amanah 12Abu Azam Al-Hadi. Fikih Muamalah Kontemporer…, h. 181-182. 12Abu Azam Al-Hadi. Fikih Muamalah Kontemporer…, h. 181-182. 2) Wadi’ah Yad al-Dhamanah Karakteristik Wadi’ah Yad al-Dhamanah, yaitu : Penerima titipan boleh menggunakan harta atau benda yang dititipkan kepadanya; Penerima titipan boleh menggunakan atau memanfaatkan harta atau benda yang dititipkan.13 Apabila berkehendak maka penerima titipan boleh memberikan sebagian hasilnya kepada pemilik titipan sebagai bonus; Produk perbankan yang sesuai dengan wadi’ah ini yakni giro dan tabungan yang dikelola dengan sistem syariah Islam; Pihak perbankan syariah tidak menyatakan akan memberikan bonus kepada pemilik titipan di dalam kontrak ataupun perjanjian, tetapi apabila harta atau benda titipan digunakan, pihak bank bisa memberikan bonus sebagai tanda terima kasih; Jumlah bonus yang diberikan sesuai dengan kewenangan yang diberikan oleh pihak bank syariah, pihak pemberi titipan tidak boleh ikut campur dalam urusan tersebut; Tabungan bisa dikatakan sebagai salah satu produk yang menggunakan akad 193 AL-SYAKHSHIYYAH: Jurnal Hukum Keluarga Islam dan Kemanusiaan p-ISSN 2685-3248; e-ISSN 2685-5887 Vol. 4; No. 2; Desember 2022 AL-SYAKHSHIYYAH: Jurnal Hukum Keluarga Islam dan Kemanusiaan p-ISSN 2685-3248; e-ISSN 2685-5887 Vol. 4; No. 2; Desember 2022 wadi’ah karena sesuai prinsipnya hampir mirip dengan giro. Perbedaannya hanya terletak pada tabungan tidak bisa ditarik dengan cek atau alat lain yang semacam itu.14 16Abdul Ghofur Anshori. Hukum Perjanjian Islam di Indonesia (Yogyakarta : Gadjah Mada University Press, 2010), h. 123. 19Nasrun Haroen, Fiqh Muamalah. (Jakarta : Gaya Media Pratama, 2000), h. 254. 14Abu Azam Al-Hadi. Fikih Muamalah Kontemporer…, h. 191-193. 17Abu Azam Al-Hadi, Fikih Muamalah Kontemporer…, h. 160. 18Zainuddin Ali, Hukum Gadai Syariah…, h. 1. a. Pengertian Rahn Dalam bahasa arab rahn dapat pula diartikan sama maknanya dengan al- hubsu. Secara etimologi rahn diartikan kekal, lama dan tetap.15 Sedangkan al- habsu diartikan penahanan suatu barang dengan hak yang dimilikinya sehingga dapat dijadikan sebagai pengganti pembayaran dari suatu barang.16 Dengan semakin berkembangnya masyarakat pada masa kini istilah rahn juga dikenal dengan sebutan gadai yang secara bahasa berarti jaminan, agunan, dan rungguhan.17 Secara terminologi berarti menyanderakan sejumlah harta yang diserahkan kepada pihak tertentu dimana barang ini kemudian dijadikan jaminan, secara hak dapat dimiliki dan dan kembali dimiliki setelah melakukan tebusan.18 b. Rukun Rahn 15Zainuddin Ali. Hukum Gadai Syariah…, h. 1. 15Zainuddin Ali. Hukum Gadai Syariah…, h. 1. b. Rukun Rahn Menurut jumhur ulama rukun rahn ada empat, yaitu : Shigat (lafal ijab dan qabul); Orang yang berakad (yang menggadaikan [al-rahin] dan yang menerima gadai (al-murtahin); Harta yang dijadikan agunan (al-marhun); Utang (marhun bih).19 c. Syarat-Syarat Rahn 1) Shigat 14Abu Azam Al-Hadi. Fikih Muamalah Kontemporer…, h. 191-193. 15Zainuddin Ali. Hukum Gadai Syariah…, h. 1. 16Abdul Ghofur Anshori. Hukum Perjanjian Islam di Indonesia (Yogyakarta : Gadjah Mada University Press, 2010), h. 123. 17Abu Azam Al-Hadi, Fikih Muamalah Kontemporer…, h. 160. 18Z i ddi Ali H k G d i S i h h 1 14Abu Azam Al-Hadi. Fikih Muamalah Kontemporer…, h. 191-193. 15Zainuddin Ali. Hukum Gadai Syariah…, h. 1. 16Abdul Ghofur Anshori. Hukum Perjanjian Islam di Indonesia (Yogyakarta : Gadjah Mada University Press, 2010), h. 123. 17Abu Azam Al-Hadi, Fikih Muamalah Kontemporer…, h. 160. 18Zainuddin Ali, Hukum Gadai Syariah…, h. 1. 19Nasrun Haroen, Fiqh Muamalah. (Jakarta : Gaya Media Pratama, 2000), h. 254. 21 Mardani, Hukum Sistem Ekonomi Islam (Depok : Raja Grafindo Persada, 2017), h. 248. 22 Andri Soemitra, Hukum Ekonomi Syariah Dan Fiqh Muamalah…, h. 158. 20 Abu Azam Al-Hadi, Fikih Muamalah Kontemporer…, h. 161-162. 2) Orang yang Berakad (Rahin dan Murtahin) Syarat untuk orang yang melakukan akad adalah orang yang cakap. Menurut jumhur ulama berupa orang-orang yang mampu bertindak secara hukum, yaitu orang yang telah baligh dan berakal sehat. Menurut ulama Hanafiyah, kedua belah pihak boleh belum baligh, asalkan berakal sehat dan telah mendapatkan izin dari walinya. 20 Abu Azam Al-Hadi, Fikih Muamalah Kontemporer…, h. 161-162. 1) Shigat Shigat bisa dilakukan baik secara lisan maupun tulisan asalkan di dalam perbuatan tersebut sudah terkandung adanya kalimat yang menyatakan adanya rahn. Apabila di dalam perjanjian disertakan syarat atau dikaitkan dengan masa 194 yang akan datang, maka syaratnya dianggap tidak sah, tetapi rahn tetap bisa dilakukan. Menurut ulama Malikiyah, Syafi’iyah, dan Hanabilah apabila syarat yang diajukan demi untuk kelancaran perjanjiaan itu diperbolehkan, tetapi apabila tidak sesuai maka syaratnya harus dibatalkan. 3) Marhun (harta yang dijadikan agunan) 3) Marhun (harta yang dijadikan agunan) Barang yang dijadikan jaminan boleh dijual sesuai dengan nilai utang dan juga persetujuan dari pemilik awal, tetapi harus dilakukan setelah waktu jatuh tempo pengembalian barang. Barang yaang dijadikan jaminan haruslah barang- barang yang bermanfaat, sehingga bisa digunakan oleh orang yang memberi gadai; Barang yang digadaikan harus jelas identitasnya; Barang yang digadaikan sudah milik sah orang yang menggadaikannya; Barang yang digadaikaan bukanlah milik orang lain; Barang yang digadaikan boleh diserahkan berupa benda atau bukti surat kepemilikannya saja; Marhun bih (utang); Orang yang menggadaikan barang harus dengan utang yang pasti.20 Utang tersebut sudah tetap; Utang itu sudah diketahui dengan jelas jumlah dan sifat-sifatnya.21 3. Barang Titipan yang dijadikan sebagai Agunan pada Perbankan Syariah 3. Barang Titipan yang dijadikan sebagai Agunan pada Perbankan Syariah 3. Barang Titipan yang dijadikan sebagai Agunan pada Perbankan Syariah Dalam melakukan transaksi haruslah sesuai dengan rukun dan syarat- syarat yang telah ditetapkan. Apabila salah satu rukun tidak ada ataupun syarat yang diajukan tidak terlaksana, tidak sesuai serta melanggar perjanjian maka transaksi yang dilakukan akan dianggap tidak sah atau batal.22 195 AL-SYAKHSHIYYAH: Jurnal Hukum Keluarga Islam dan Kemanusiaan p-ISSN 2685-3248; e-ISSN 2685-5887 Vol. 4; No. 2; Desember 2022 AL-SYAKHSHIYYAH: Jurnal Hukum Keluarga Islam dan Kemanusiaan p-ISSN 2685-3248; e-ISSN 2685-5887 Vol. 4; No. 2; Desember 2022 Dalam melaksanakan transaksi wadi’ah tentunya juga harus dilakukan oleh dua pihak, baik itu antara seseorang dengan orang lain atau antara seseorang dengan pihak bank. Transaksi yang dilakukan terhadap seseorang biasanya akadnya hanya dilakukan dengan ucapan serah terima, apalagi yang biasanya melakukan transaksi ini hanyalah orang yang dianggap dekat saja. Sedangkan dengan pihak bank, transaksi yang dilakukan lebih memperhatikan detail tertentu. Dengan begitu, diharapkan tidak akan terjadi kesalahpahaman ataupun ingkar janji dikemudian hari. Akan tetapi, tidak menutup kemungkinan ada salah seorang pihak yang tidak amanah. Konsep wadi’ah menegaskan bahwa transaksi yang dilakukan hanya berupa menitipkan harta benda yang dimiliki kepada orang lain, yang sewaktu-waktu apabila diperlukan maka harta benda itu dikembalikan kepada pemiliknya. Rahn atau jaminan atau lebih dikenal dengan sebutan gadai, juga banyak dilakukan masyarakat saat ini. Rahn menjaminkan sebuah benda kepada seorang pihak untuk dianggapkan sebagai utang, dengan imbalan pemberi gadai akan memberikan sejumlah uang. Barang yang digadaikan akan dijadikan sebagai jaminan. Apabila pihak yang menggadaikan tidak mampu untuk melunasinya, maka barang tersebut akan berubah kepemilikan menjadi milik pemberi gadai. Dalam permasalahan barang titipan yang dijadikan sebagai agunan dalam perbankan syariah ini bisa saja dianggap melanggar prinsip amanah dalam wadi’ah, yaitu: Apabila terjadi kerusakan terhadap barang yang dititipkan oleh orang lain, sedangkan orang yang diamanahkan untuk menjaga barang tersebut bisa mengcegahnya tapi tidak melakukannya, maka dia berkewajiban mengganti barang tersebut. Atas kesalahan ini orang tersebut wajib melakukan ganti rugi (al- daman). Apabila barang titipan berada di tangan orang ketiga dan terjadi kerusakan, maka orang kedua bertanggung jawab untuk melakukan ganti rugi; Apabila barang yang dititipkan kemudian digunakan oleh orang yang dititipkan dan mengalami kerusakan, maka dia harus melakukan ganti rugi. 196 Kedudukan Barang Titipan Sebagai Aunan… Muspitasari; A. Nurramadhani; Muh. Fadli M. Kedudukan Barang Titipan Sebagai Aunan… Muspitasari; A. Nurramadhani; Muh. Fadli M. 24I Putu Gede Parwata, Made Nurmawati. Akibat Hukum Perjanjian Gadai yang dilakukan dengan Jaminan Barang Bergerak Bukan Milik Debitur. h. 9. 23 Nasrun Haroen. Fiqh Muamalah, h. 248. 23 Nasrun Haroen. Fiqh Muamalah, h. 248. 3. Barang Titipan yang dijadikan sebagai Agunan pada Perbankan Syariah Apabila orang yang dititipkan barang berkhianat, dengan tidak mengakui bahwa mendapat titipan atas suatu barang tersebut dan menyembunyikannya disuatu tempat, serta dia mampu untuk mengganti barang tersebut maka dia harus melakukan ganti rugi. Apabila barang yang dititipkan tercampur dengan barang pribadi orang yang dititipkan. Dalam hal ini terjadi perbedaan pendapat ulama, apabila barang tersebut sulit untuk dipisahkan, maka orang tersebut harus melakukan ganti rugi. Akan tetapi apabila bisa dipisahkan, maka penitip boleh mengambil barangnya. Apabila orang yang dititipkan barang melanggar syarat-syarat yang telah disepakati pada saat melakukan akad, maka orang tersebut harus melakukan ganti rugi; Apabila barang titipan dibawa bepergian dalam waktu yang lama, maka pihak pemberi titipan bisa meminta ganti rugi.23 Dari uraian diatas, diketahui hal-hal apa saja yaang bisa membatalkan wadi’ah. Sebelumnya telah dibahas mengenai pembagian wadi’ah, yaitu Wadi’ah Yad al-Amanah dan Wadi’ah Yad al-Dhamanah. Secara singkat, Wadi’ah Yad al- Amanah membahas mengenai barang titipan yang hanya bisa dilakukan diantara dua pihak. Maksudnya, pihak pemberi titipan hanya menitipkan barang titipannnya kepada penerima titipan. Penerima titipan haruslah menjaga serta memelihara barang titipan tersebut. Barang titipan juga hanya boleh disimpan tanpa digunakan. Permasalahan tentang barang titipan yang dijadikan agunan pada perbankan syariah merupakan suatu perbuatan yang melanggar undang-undang sehingga wadi’ah yang dilakukan bisa dikatakan batal demi hukum. Ini dikarenakan tidak terpenuhinya salah satu unsur rahn yaitu barang yang digadaikan bukanlah milik orang lain.24 Adapun jenis aset yang bisa dijadikan agunan di perbankan syariah, yaitu : a. Agunan berwujud terbagi atas agunan bergerak dan agunan yang tidak bergerak. Contoh agunan bergerak berupa kendaraan seperti pesawat, 197 AL-SYAKHSHIYYAH: Jurnal Hukum Keluarga Islam dan Kemanusiaan p-ISSN 2685-3248; e-ISSN 2685-5887 Vol. 4; No. 2; Desember 2022 kapal, kereta, mobil, dan motor. Sedangkan agunan yang tidak bergerak berupa tanah dan mesin pabrik. kapal, kereta, mobil, dan motor. Sedangkan agunan yang tidak bergerak berupa tanah dan mesin pabrik. b. Agunan tidak berwujud dapat berupa hak paten, surat berharga, dan deposito.25 b. Agunan tidak berwujud dapat berupa hak paten, surat berharga, dan deposito.25 b. Agunan tidak berwujud dapat berupa hak paten, surat berharga, dan deposito.25 Pada saat hendak melakukan penjaminan di bank syariah setiap orang haruslah membuktikan dirinya memiliki bukti milik dari barang tersebut. Pihak bank sendiri harus melakukan analisis terlebih dahulu sebelum melakukan transaksi rahn. Ini dilakukan sesuai dengan asas-asas perkreditan yang sehat, salah satunya yaitu saat memberikan jaminan pihak bank syariah harus melakukan suatu perjanjian tertulis. 25Pratama Akhdi Mardin. 2021. “Ini Jenis Aset yang Bisa dijadikan Aset di Bank” dari https://money.kompas.com/read/2021/06/11/210000226/ini-jenis-aset-yang-bisa-dijadikan-agunan- ke-bank. Diakses pada tanggal 05 April 2022 pukul 08.25. p gg p p 26I Putu Gede Parwata, Made Nurmawati. Akibat Hukum Perjanjian Gadai yang dilakukan dengan Jaminan Barang Bergerak Bukan Milik Debitur, h. 10. 3. Barang Titipan yang dijadikan sebagai Agunan pada Perbankan Syariah Karena didasarkan pada prinsip bagi hasil, pihak bank syariah tentunya juga akan melakukan pemeriksaan latar belakang dari pihak yang melakukaan transaksi. Ini semua dilakukan agar tidak terjadi kesalahpahaman di kemudian hari. Surat-surat yang dijadikan jaminan pada pihak bank syariah juga harus ditanda tangani oleh nama yang tertera pada sertifikat. Misalnya, apabila hendak menjaminkan BPKB motor atau mobil maka yang bertanda tangan pada surat jaminan adalah orang yang tertera namanya pada surat tersebut, begitu pula dengan sertifikat tanah, rumah, atau gedung. Dengan banyaknya persyaratan yang terjadi pada saat hendak melakukan akad rahn pada perbankan syariah tentunya semakin sulit untuk seseorang bisa melakukan penipuan dengan menjadikan sebuah harta atau benda milik orang lain sebagai agunan dalam melakukan transaksi pada perbankan syariah. Akan tetapi, apabila terjadi hal demikian maka pihak yang menerima titipan sekaligus sebagai pihak yang melakukan transaksi rahn diperbankan syariah sebagaimana yang dinyatakan dalam Pasal 1365 KUHPerdata menyatakan bahwa semua perbuatan yang melanggar hukum serta merugikan orang lain, harus memberikan ganti rugi kepada pihak-pihak yang dirugikan.26 198 Kedudukan Barang Titipan Sebagai Aunan… Muspitasari; A. Nurramadhani; Muh. Fadli M. Kedudukan Barang Titipan Sebagai Aunan… Muspitasari; A. Nurramadhani; Muh. Fadli M. Pengembalian barang titipan dari pihak perbankan syariah juga dilakukan dengan dua cara, yaitu dengan musyawarah atau melakukan penyelesaian di pengadilan. Tapi, pihak yang barang miliknya dijadikan titipan diperbankan syariah juga harus bisa membuktikan bahwa harta benda tersebut benar-benar adalah miliknya. Sedangkan dalam Wadi’ah Yad al-Dhamanah yaitu membahas mengenai barang titipan yang selain untuk dijaga dan dipelihara dengan baik, juga bisa dimanfaatkaan oleh penerima titipan apabila dibutuhkan. Pihak pemberi titipan dan penerima titipan pada awal permulaan akad sudah melakukan perjanjian terlebih dahulu, sehingga pihak penerima titipan bisa menggunakan harta atau benda tersebut. Dalam permasalahan benda tersebut dijadikan agunan dalam perbanksan syariah tentunya tidak akan terjadi hal-hal sebagaimana dalam Wadi’ah Yad al- Amanah. Ini dikarenakan apabila barang titipan hendak dijadikan agunan dalam perbankan syariah tentunya telah mendapat izin dari pemilik barang titipan. Selain itu, dalam melakukan transaksi di perbankan syariah pihak pemberi titipan ikut serta dalam proses transaksi, karena tanda tangan dari pemilik barang diperlukan. D PENUTUP D. PENUTUP Dalam permasalahan barang titipan yang dijadikan sebagai agunan pada perbankan syariah bisa dinyatakan sebuah tindak tidak amanah dari pihak penerima titipan. Apabila hal ini terjadi maka akad wadi’ah ataupun rahn bisa dikatakan batal demi hukum. penyelesaian sengketa harta atau benda tersebut bisa dilakukan dengan melakukan musyawarah atau melalui jalur pengadilan. Dan pihak yang dinyatakan bersalah harus membayar ganti rugi kepada pihak-pihak yang dirugikannya. 199 AL-SYAKHSHIYYAH: Jurnal Hukum Keluarga Islam dan Kemanusiaan p-ISSN 2685-3248; e-ISSN 2685-5887 Vol. 4; No. 2; Desember 2022 Buku-Buku Siregar, Hariman Surya, Koko Khoeruddin. Fikih Muamalah dan Implementasinya. Bandung : Remaja Rosdakarya, 2019 Al Hadi, Abu Azam. Fikih Muamalah Kontemporer. Depok : Raja Grafindo Persada. 2017. Andri Soemitra. Hukum Ekonomi Syariah Dan Fiqh Muamalah. (Jakarta: Prenamedia Group, 2019. Mardani. Hukum Sistem Ekonomi Islam. Depok : Raja Grafindo Persada, 2017. Ali, Zainuddin. Hukum Gadai Syariah. Jakarta : Sinar Grafika, 2008. Anshori, Abdul Ghofur. Hukum Perjanjian Islam di Indonesia. Yogyakarta: Gadjah Mada University Press, 2010 Haroen Nasrun, Fiqh Muamalah. Jakarta : Gaya Media Pratama, 2000. Marzuki, Peter Mahmud. Penelitian Hukum. Jakarta : Kencana Pernada Media Group, 2010. R. Bogdan dan Steven Taylor, Introduction to Qualitative Research Methods. John Wiley & Sons, 1984. Soekanto dan Mamudji. Penelitian Hukum Normatif Suatu Tinjauan Singkat. Jakarta: Rajawali, 2003. Artikel Jurnal: Mustafa, Oleh. “AL-HÂJAT AL-AŞLIYYAH DAN HARTA TERBEBAS HUTANG SEBAGAI SYARAT WAJIB ZAKAT MÂL.” AL-KHARAJ 1, no. 2 (5 November 2021): 142–50. https://doi.org/10.30863/ALKHARAJ.V1I2.1961. Putri, Nyimas Lidya. Jurnal Hukum dan Ekonomi Syariah Aplikasi Gadai Syariah dalam Perbankan Syariah. Vol. VII. No. 2. September 2019 Parwata, I Putu Gede, Made Nurmawati. Jurnal Akibat Hukum Perjanjian Gadai yang dilakukan dengan Jaminan Barang Bergerak Bukan Milik Debitur. Bali : Fakultas Hukum Universsitas Udayana Pratama Akhdi Mardin. 2021. “Ini Jenis Aset yang Bisa dijadikan Aset di Bank” dari https://money.kompas.com/read/2021/06/11/210000226/ini-jenis- aset-yang-bisa-dijadikan-agunan-ke-bank. Diakses pada tanggal 05 April 2022 pukul 08.25. 200
W3199009659.txt	https://www.mdpi.com/2304-8158/10/9/2201/pdf?version=1631868452	en		Supercritical CO2 Extraction of Bioactive Compounds from Mango (Mangifera indica L.) Peel and Pulp	Foods	2,021	cc-by	13,490	foods Article Supercritical CO2 Extraction of Bioactive Compounds from Mango (Mangifera indica L.) Peel and Pulp José Villacís-Chiriboga 1,2,3 , Stefan Voorspoels 1 , Maarten Uyttebroek 1 , Jenny Ruales 3 , John Van Camp 2 , Edwin Vera 3 and Kathy Elst 1, * 1 2 3 * Citation: Villacís-Chiriboga, J.; Voorspoels, S.; Uyttebroek, M.; Ruales, J.; Van Camp, J.; Vera, E.; Elst, K. Supercritical CO2 Extraction of Bioactive Compounds from Mango (Mangifera indica L.) Peel and Pulp. Foods 2021, 10, 2201. https:// Flemish Institute for Technological Research (VITO), Business Unit Separation and Conversion Technology, Boeretang 200, 2400 Mol, Belgium; jose.villacis-chiriboga@vito.be (J.V.-C.); stefan.voorspoels@vito.be (S.V.); Maarten.Uyttebroek@flandersfood.com (M.U.) Department of Food Technology, Safety and Health, Ghent University, Coupure Links 653, 9000 Ghent, Belgium; John.VanCamp@UGent.be Department of Food Science and Biotechnology, Escuela Politécnica Nacional, Ecuador, Campus Rubén Orellana, Ladrón de Guevara E11-253, Quito P.O. Box 17 012759, Ecuador; jenny.ruales@epn.edu.ec (J.R.); edwin.vera@epn.edu.ec (E.V.) Correspondence: kathy.elst@vito.be; Tel.: +32-14335617 Abstract: The potential of supercritical CO2 (SC-CO2 ) for the extraction of bioactive compounds from mango by-products was assessed. Carotenoid extraction was optimized using a design of experiments based on temperature (35, 55 and 70 ◦ C), pressure (10 and 35 MPa) and co-solvent addition (0%, 10% and 20% of ethanol or acetone). Moreover, the co-extraction of phenolic acids, flavonoids and xanthonoids was evaluated in a subset of parameters. Finally, a comparison was made between SC-CO2 and a two-step organic solvent extraction of the bioactive compounds from the pulp and peel fractions of two Ecuadorian varieties. The optimal extraction temperature was found to be dependent on the bioactive type, with phenolics requiring higher temperature than carotenoids. The optimal overall conditions, focused on maximal carotenoids recovery, were found to be 55 ◦ C, 35 MPa and 20% of ethanol. The main carotenoid was β-carotene, while phenolics differed among the varieties. The bioactive content of the peel was up to 4.1-fold higher than in the pulp fraction. Higher antioxidant activity was found in the extracts obtained with organic solvents. SC-CO2 is a promising technology for the isolation of valuable compounds from mango by-products. doi.org/10.3390/foods10092201 Keywords: carotenoids; phenolics; antioxidant; mango; supercritical CO2 ; response surface optimization Academic Editor: Danijela Bursać Kovačević Received: 9 August 2021 Accepted: 15 September 2021 Published: 17 September 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1. Introduction Mango (Mangifera indica L.) is among the most traded tropical fruits in the world with around 48 thousand tons produced in 2017. Nevertheless, large amounts of peels (7–24%) and stones (45–85%) are discarded after industrial processing of the fruit. Such by-products represent a significant material source of compounds with potentially high commercial value and applicability for various purposes. In this line, the concept of biorefinery aims to produce valuable chemicals, food, feed and energy by using biomass as feedstock through various transformation steps. More specifically for mango, several studies have stated that its by-products could potentially be used for the extraction of macro molecules such as pectins, oils and starch, but also bioactive compounds such as carotenoids and phenolics [1]. Carotenoids and phenolic compounds are among the major bioactive compounds of mango. Studies have indicated the positive effects of carotenoids on human health, i.e., radical scavenging, cancer prevention, cardiovascular diseases, cataracts and neural tube defects [2]. Similarly, phenolic compounds exert various biological activities, including antimicrobial, anti-cardiovascular, anti-obesity, antioxidant, anti-hyperglycemic and anticancer activities [3]. Moreover, polyphenols of mango have been shown to exert chemopreventive and anti-inflammatory activities [4]. Such activities make of mango by-products a potential source of compounds with application in various industrial sectors. Foods 2021, 10, 2201. https://doi.org/10.3390/foods10092201 https://www.mdpi.com/journal/foods Foods 2021, 10, 2201 2 of 22 Bioactive compounds are traditionally extracted from vegetable matrices using organic solvents, which can leave potentially toxic residues and be harmful when released into the environment. In contrast, supercritical CO2 (SC-CO2 ) provides a sustainable, environmentally friendly and cost-effective alternative solvent that offers the possibility to tailor the extraction process by changing its pressure or altering its polarity by the addition of low concentrations of organic solvents. It is especially appropriate for the isolation of lipophilic compounds. Moreover, it allows one to avoid thermal damage of labile bioactive compounds, given the low working temperature for reaching the supercritical state (T = 31.1 ◦ C and P = 7.38 MPa) [5]. Given the crescent interest of SC-CO2 as an extraction solvent of carotenoids, in the last few years, several matrices have been extracted with this methodology, such as microalgae [6] and carrot peels [7]. Nevertheless, as far as mango is concerned, the few scientific reports available focus on the tree leaves as main sources of bioactive compounds. To the best of our knowledge, the research on the application of SC-CO2 is still limited. Garcia-Mendoza et al. (2015) [8], determined the total extraction yield of carotenoids, phenols and flavonoids from one variety under a single SC-CO2 extraction condition, followed by a pressurized EtOH extraction. In a similar study, Sánchez-Camargo et al. (2019) [9] optimized the conditions of SC-CO2 extraction of carotenoids from mango peels of the variety “Sugar”. The authors evaluated the total carotenoid content as a function of the drying techniques (freeze drying and air drying). Furthermore, the carotenoid extract obtained under the best working parameters was evaluated in terms of its carotenoids profile and as protector of the oxidation of sunflower oil. Since both carotenoids and polyphenols are composed of different molecules with different polarity and weight, it is expected that the extraction yield of individual compounds will depend on the conditions applied. Given the fact that the individual molecules may vary among the different mango fractions and varieties as well as the storage/processing conditions, it is important to understand their individual behavior as a function of the conditions used for extraction. For this purpose, response surface methodology (RSM) provides a powerful statistical tool to model and predict the outcome of an extraction in function of the conditions applied based on a limited amount of experiments [10]. The present work aims to investigate the use of SC-CO2 for the extraction of bioactive compounds from the peel and pulp of mango. To obtain a better knowledge on the effect of temperature, pressure and co-solvent on the extraction of carotenoids, the extraction of the individual carotenoids is analyzed and modelled by the RSM methodology. Moreover, the extracts of a subset of the most promising extraction conditions are characterized in detail for their composition in the individual phenolics. Based on the modelling results, optimized conditions are proposed and used for the supercritical extraction of bioactive compounds from the pulp and peel of different mango varieties. The results are compared to a two-step organic solvent extraction. 2. Materials and Methods 2.1. Sample Preparation In this study, two batches of mango were selected. For the first batch, the dejuiced whole fruit, with no discrimination between the pulp and peel, was used in order to have a maximal amount of a representative mixed material required for the optimization process. The variety “Kasturi”, grown in Israel, was purchased on a local market in Belgium. The selected mangoes, which were commercially ripe, similar in size and free of mechanical or biological damages, were immediately taken to the laboratory and kept at 8 ◦ C. The mangoes were manually washed, and the seeds were manually removed from the fruits. The remaining pulp (entire pulp after seed removal) and peel fraction was juiced with an Angel Juicer 8500s (Angelia, Sasang-gu, Busan, Korea) and the juice and pulp were frozen with liquid nitrogen, freeze dried, milled and sieved to reject the material with a diameter greater than 500 µm. The average particle size was 496 ± 71 µm (weight basis), measured Foods 2021, 10, 2201 3 of 22 by laser diffraction. The samples were then stored in vacuum bags away from light and kept at −20 ◦ C until further analysis. The second type of samples was extracted at the optimal conditions and the samples were used to study local Ecuadorian varieties and the effect of the separation of pulp and peel. The varieties “Kent” and “Haden” were obtained from local growers in the province of Imbabura—Ecuador (0◦ 270 06.400 N; 77◦ 590 22.600 W). The mangoes were selected based on an appropriate maturity degree, uniform shape and size and no damages of biological or mechanical origin. In the laboratory, the mangoes were manually washed and the three fractions—peel, pulp and stones—were manually separated. The pulp was juiced with an industrial juicer machine and the pulp resulting from this step, along with the peel, was freeze dried, milled and the process continued as for the first batch of mango. 2.2. Extraction with Supercritical CO2 The tests were performed with a laboratory scale Jasco SFE system from JASCO Isogen Life Science [11]. For each extraction, 2.0 g of freeze-dried powder pulp was loaded in a 10 mL capacity extractor vessel, and the empty space was filled with glass wool. CO2 constantly flowed at 1 mL·min−1 at a pressure of 10 or 35 MPa, at a temperature of 35, 55 or 70 ◦ C and with pure CO2 or a co-solvent percentage of 10% or 20% ethanol (EtOH); alternatively, 10% or 20% acetone was used. All the experiments were performed randomly and in duplicate. A total extraction time of 3 h was applied, but the extract was collected in different collection vials as a function of time (10 min per vial). Amber-colored glass collection vials were used to avoid light degradation of the extracted molecules. As explained above, a modifier solvent was added at the depressurization point to avoid precipitation. The same solvent was used as the co-solvent of the experiment, except that additionally butylhydroxytoluene BHT (0.1%) was added to preserve the extract until the analysis. The modifier flow was set to obtain a total solvent flow (co-solvent + modifier) of 0.5 mL·min−1 in order to always have the same volume of collected extract in the vials. To correct for the differences in BHT content in the final extract (as this was uniquely dosed through the modifier solvent), additional BHT was directly added to the collection vials, always to keep its final concentration at 0.1%. After the extraction, the solvents were evaporated with a N2 flux and the dry extracts were stored under −18 ◦ C. For the samples that were used for measuring the antioxidant activity, no BHT was added to the vial nor to the modifier. In between successive extractions, the tubing of the equipment was extensively rinsed with isopropanol to avoid any contamination for remnants. 2.3. Solvent Extraction The solvents system used were previously optimized after performing tests with several organic solvents and their mixtures. Hence, two successive extractions were applied. A combination of acetone:methanol (70:30; v/v) was first applied, then an extraction with dichloromethane:methanol (50:50; v/v) was carried out in an ultrasound bath Branson 5510 (Branson ultrasonics corporation, Danbury, CT, USA) at 4 ◦ C. Both solvent systems contained 0.1% BHT. The ratio of material to solvent was 1:10 (w/v), and the time was set at 45 min for each extraction. At the end, both extracts were mixed together and separated in aliquots. The solvents were evaporated and the dried extracts were stored at −18 ◦ C. As explained above, the samples used for the antioxidant activity measurement were not stabilized with BHT. 2.4. Determination of Dry Matter Content The amount of dry matter was assessed by weight difference. For this, 5.0 g of dry sample were dried in an oven for 24 h at 105 ◦ C, the difference in mass was calculated and used for correcting the values obtained in the experiments. Foods 2021, 10, 2201 4 of 22 2.5. Determination of the Total Carotenoid Content One aliquot of 2 mL of the extract was dried and before the analysis it was dispersed in EtOH (95%). Then, the absorbance of an appropriately diluted sample was analyzed in a spectrophotometer (Tecan, Infinite, M200 Pro) using ethanol (95%) as a blank at 470, 649 and 664 nm. The total carotenoid concentrations were calculated in duplicate from the different absorbances using the equations of Lichtenthaler and Buschmann (2001) [12]. 2.6. Determination of the Individual Carotenoids The protocol was based on the work of Bijttebier et al. (2013) [13]. One µL of extract was injected on a 2.1 mm × 100 mm, 1.8 µm HSS C18 SB column (Waters, Milford, MA, USA) at 35 ◦ C in a Waters Acquity UPLC® . Mobile phase A consisted of water +5 mM ammonium acetate:methanol:acetonitrile:ethylacetate (50/22.5/22.5/5 v/v/v/v) and mobile phase B consisted of acetonitrile:ethylacetate (50/50 v/v) with a flow rate of 500 µL·min−1 . The following gradient (%/min B) was applied: 10/0; 10/0.1; 30/0.8; 91/20.0; 100/20.1; 100/20.4; 10/20.5; 10/23. For detection, an orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) operating with an APCI was used. A full scan of the ions was acquired at m/z range of 200 to 1400 at a resolution of 70,000 full width at half-maximum. As the internal standard, 36.25 ng·mL−1 of trans-β-Apo-80 -carotenal was used. The concentration of the standards (α-carotene, antheraxanthin, β-cryptoxanthin, β-carotene, fucoxanthin, trans-lutein, trans-violaxanthin, zeaxanthin) ranged between 0.034 and 26.55 µg·mL−1 (average). In addition, 3 quality controls (corresponding to the standard 5) were placed in between the injections of the samples. The lowest calibration point was set as the limit of quantification. The relative standard deviation of these controls was on average 6.35 ± 4.21%. 2.7. Determination of Individual Phenolic Compounds The analytical method was based on the work of De Paepe et al. (2013) [14]. Five µL of extract were injected on a 3.0 mm × 150 mm, 1.7 µm BEH SHIELD RP18 column (Waters, Milford, MA, USA) at 40 ◦ C in a Waters Acquity UPLC® . Mobile phase A consisted of water +0.1% formic acid and mobile phase B constituted acetonitrile + 0.1% formic acid flowing at 500 µL·min−1 at the following gradient (%/min B): 0–26/9.91; 65/18.51; 100/18.76–25.76. An orbitrap mass spectrometer (Thermo Fisher Scientific) coupled with an ESI source was used for identification. All ions were identified in a m/z range of 70–10,500 at a mass resolving power of 70,000 full width at half-maximum. Daidzein was included as internal standard in a concentration of 50 ng·mL−1 in all the extracts. The concentration of the standards (aromadendrin, astragalin, avicularin, caffeic acid, catechin, chlorogenic acid, cyanoroside, epicatechin, ferulic acid, gallic acid, hyperin, isoquercitrin, kaempferol, mangiferin, naringenin, p-coumaric acid, phlorizin, protocatechuic acid, quercetin, quercitrin, taxifolin) ranged between 3.19 and 9848.47 µg·L−1 (average). Additionally, 7 quality controls (corresponding to the standard 3) were placed in between the injections of the samples. The lowest calibration point was set as the limit of quantification. The relative standard deviation of these controls was on average 6.13 ± 1.48%. 2.8. Determination of Antioxidant Activity To assess the antioxidant properties of the samples, the 2,2-diphenyl-1-picrylhydrazyl (DPPH·) test, which monitors the neutralization of the stable DPPH· radical, was carried out. Additionally, the FRAP method was applied. This method consists of measuring the increase in absorbance at 593 nm of the blue reaction when the TPTZ-Fe+3 complex is reduced to TPTZ-Fe+2 . The DPPH· method was performed in duplicate per extraction, following the procedure of Brand-Williams et al. (1995) [15] with modifications. To prepare the stock solution, 3.0 mg of DPPH· was dissolved to a final concentration of 20 mM. In a next step, the Foods 2021, 10, 2201 5 of 22 stock solution was diluted with MeOH to a concentration of 200 µM. The samples were prepared as follows. First, 2 mL of extract was taken, evaporated and re-dissolved with 2 mL dimethyl sulfoxide (DMSO). A series of at least 6 different aliquots was taken from this DMSO solution, with a volume ranging between 0.5 and 300 µL, which was adjusted in volume to 500 µL with MeOH. For each test, 100 µL of the DPPH· working solution and 100 µL of each aliquot were mixed in a 96-well plate. The DPPH· working solution mixed with MeOH and pure MeOH was used as the blank. The mixtures were allowed to react for 30 min away from light. The absorbance was taken at 517 nm. Results were expressed as IC50 . The final value was related to the biomass intake during the extraction (in DM) and expressed as µg biomass on dry matter base per mL (µg·mL−1 ). The FRAP method was done according to the modified method of Benzie and Strain (1996) [16]. To prepare the FRAP solution, buffer acetate of 300 mM, 2,4,6-Tris-(2-pyridyl)-striazine (0.156 g in 50 mL of EtOH) and Fe2 Cl3 (0.5404 g in 2 mL of HCl 37% and 98 mL of water) were mixed in a concentration of 40:4:4 mL. A stock solution of 25 mM Trolox was prepared and successively diluted in a concentration between 200 and 10 µM. The solutions of the sample were prepared as explained for the DPPH· method. Next, 2700 µL of the FRAP solution and 300 µL of each concentration were mixed and allowed to react for 4 min. Then the absorbance was monitored at 593 nm. The results were related to the biomass intake during the extraction (in DM) and expressed as µmol Trolox per g biomass on dry matter base (µmol Trolox·g−1 ). 2.9. Calculations The mass yield of the extraction processes was expressed as the ratio between the amount of extracted material and the biomass loaded in the extraction vessel on the dry matter base. In addition, the yield of carotenoids and phenolic compounds was determined. The carotenoids were treated individually. To ease the interpretation on the phenolic compounds, the different compounds were grouped in four classes: hydroxycinnamic acids (p-coumaric acids, caffeic, ferulic, chlorogenic), flavonoids (epicatechin, kaempferol, quercetin, quercitrin, aromadendrin, astragalin, avicularin, catechin, cynaroside, hyperin, isoquercitrin, naringenin, phlorizin, taxifolin), xanthonoids (mangiferin) and hydroxybenzoic acids (gallic and protocatechuic acids). All results were expressed as mass yield per g of biomass treated on the dry matter base. 2.10. Statistical Analysis Most of the statistical analysis was performed by a design of experiments, using response surface methodology (RSM). At the start, a quadratic response model was considered for co-solvent concentration and temperature, whereas a linear response model was selected for the pressure, also including all interaction parameters. All values were subjected to analysis of variance (ANOVA) with a 95% confidence level. The final model (Equation (1)) was derived by removing all insignificant terms and used to determine the optimal conditions. For the final model, the values of the determination coefficient (R2 ) were also determined as an indication of the goodness of fit. 3 3 i =1 i =1 3 Y = β o + ∑ β i Xi + ∑ β ii Xi2 + ∑ 3 ∑ β ij Xi X j (1) i =1 j = i +1 where Y is the dependent variable to be modelled (total carotenoids or individual carotenoids); Xi and X j are the independent variables (temperature, pressure and co-solvent) and β 0 is the intercept; β i is the coefficient of linear effect; β ii and β ij are the coefficients of quadratic and interaction effects, respectively; and 3 is the number of variables. As explained, in this study, several models were developed for optimizing the extraction of total carotenoids as well as individual carotenoids. Additionally, surface plots from the fitted equations were generated in order to evaluate the effects of independent parameters and their mutual interaction. Foods 2021, 10, 2201 6 of 22 To determine the significance of the differences between extraction conditions, the least significant difference (LSD) was calculated for each of the classes. The ANOVA was performed with at a 95% of confidence level. For the optimization study and the study on the different varieties, each extraction type was performed in duplicate, and the extracts were analyzed using the methodology as described above. In the figures, the average of both extractions is shown, whereby the difference calculated based on the percentage difference between the two replicate extractions is detailed in the legend. All statistical tests were conducted using STATISTICA XII, 2015 (Dell Inc., Tulsa, OK, USA). 3. Results and Discussion In this research, the impact of pressure, temperature, type (acetone/ethanol) and concentration of the co-solvent was assessed on the extraction yield of bioactive compounds from mango by-products using SC-CO2 as the main solvent. In the first instance, the extraction procedure was optimized by maximizing the extraction of carotenoids through an RSM approach. Not only the total, but also the main individual carotenoids were assessed. The model was validated and, thereafter, the concentration of phenolics was evaluated for the selected conditions. In a second stage, the bioactive compounds of two mango varieties and two types of by-products, i.e., pulp and peel, were extracted at the optimized conditions and compared against a two-step reference extraction method using organic solvents. 3.1. Extraction as Function of Time First, the extraction of the total carotenoids was analyzed as a function of time. The cumulative extraction yield was determined as a function of time for a temperature of 55 ◦ C, a pressure of 35 MPa and using pure SC-CO2 as well as SC-CO2 modified with ethanol or acetone at different concentrations. In all cases, the extraction rate was found to decrease steadily with time (Supplementary Material SS.1). The declining concentration of extract in the outflowing solvent may be due to depletion in the extractable material or diffusional limitations due to mass transfer processes. After three hours, the steady state was reached, and the extraction was terminated in order to avoid degradation and isomerization due to temperature effects. This time was taken as a reference time for all further extractions. 3.2. Optimization of the Total Carotenoids Extraction via RSM Modelling The influence of the co-solvent percentage, temperature and pressure on the overall carotenoid extraction yield was evaluated and optimized through a design of experiments, where temperature and co-solvent addition was studied at three levels and pressure at two levels. In order to have a representative concentration of carotenoids for the further experiments, a mixture of peel and pulp of the Kasturi variety was used. In total, 30 experiments were done, of which the conditions and their corresponding extracted total carotenoids content are given in Supplementary Material SS.2. As explained above, the effects of three main variables on SC-CO2 extraction of carotenoids from mango were simultaneously evaluated using the RSM methodology to construct a model that allows for the selection of the best combination of conditions: temperature (X1 ), pressure (X2 ) and co-solvent percentage (X3 ). For each of the co-solvents, ethanol and acetone, a separate model was made. The carotenoid extraction (response) was mathematically modelled to predict the relationships between the working parameters and the extraction yield; however, all insignificant variables (95% level of significance) were removed. The regression models are shown below. As can be seen, the lack of fit for each Foods 2021, 10, 2201 7 of 22 case was found to be not significant. Moreover, the statistical analysis of the models is displayed in Supplementary Material SS.3: 1, 10, x FOR PEER REVIEW 7 of 24 Total carotenoids using EtOH as co-solvent [µg/gDW] = −25.115 + X1 +0.467 0.881 × 2 2 2 × X2 −0.243 × X3 −0.008 × X1 +0.006 × X2 × X3 +0.015 × X3 R = 0.97 As explainedTotal above, the effectsusing of three mainasvariables on[µg/gDW SC-CO2 extraction carotenoids acetone co-solvent ] = −6.242of+ca0.031 × X1 +0.670 rotenoids from mango were simultaneously evaluated using the RSM methodology 2 × X2 +0.079 × X3 −0.003 × X1 × X2 −0.008 × X2 × X3 R = 0.91 to construct a model that allows for the selection of the best combination of conditions: temperature (X1), pressure (Xthree-dimensional 2) and co-solvent percentage (X3). For each of the co-solvents, The surface plots provide a more comprehensive representation of ethanol and acetone, a separate model was made. The carotenoid (response) the interaction between the working variables and extraction the experimental response. Likewise, was mathematically modelled to predict the relationships between the working paramethese figures facilitate the selection of the area within the graph where the optimal conditers and the extraction yield; however, all insignificant (95%that level of significance) tions are located. Hence, in Figure 1 itvariables can be seen when EtOH is used as co-solvent, ◦ C, similarly were removed. The are shown As can as bewhen seen, the of fit for were performed lowregression extractionsmodels are obtained at 35below. the lack experiments ◦ each case was found to be not significant. statistical thean models at a temperature of 70 C.Moreover, Similarly,the high pressureanalysis allowedoffor extraction of a higher is displayed in Supplementary Material SS.3: yield, as compared to the yield obtained at 10 MPa. It should be mentioned that different Total carotenoids using EtOH co-solvent [µg/g DW]and = −25.115 + 0.881 𝑋 + 0.467used as co-solvents. behaviors wereasobtained when EtOH acetone were ×separately × 𝑋 − 0.243 × 𝑋 Overall, − 0.008 more × 𝑋 +carotenoids 0.006 × 𝑋 ×were 𝑋 +comparatively 0.015 × 𝑋 (R2extracted = 0.97) with the use of EtOH as co-solvent than with the useasofco-solvent acetone. [µg/g DW] = −6.242 + 0.031 × 𝑋 + 0.670 Total carotenoids using acetone Figure×2𝑋depicts the× Pareto of the remaining linear, interaction and quadratic × 𝑋 + 0.079 × 𝑋 − 0.003×𝑋 − 0.008 𝑋 × 𝑋 charts (R2 = 0.91) effects of the variables the total yield. Overall, it can be observed that pressure The three-dimensional surface plotson provide a more comprehensive representation and co-solvent addition are decisive in the concentration of total carotenoids recovered of the interaction between the working variables and the experimental response. Likewise, from mango. these figures facilitate the selection of the area within the graph where the optimal condiIn the with ethanol a co-solvent (Figure 2a), the linear effects of tions are located. Hence, in extraction Figure 1 itperformed can be seen that when as EtOH is used as co-solvent, pressure (standardized effect of 28.1) and co-solvent addition (standardized low extractions are obtained at 35 °C, similarly as when the experiments were performedeffect of 6.9) were a majorhigh and beneficial of carotenoids. at a temperaturefound of 70 to °C.have Similarly, pressure influence allowed on forthe an recovery extraction of a higher The temperature dependence was mostly dominated the quadratic effect (−that 5.09),different resulting in an optimum yield, as compared to the yield obtained at 10 MPa. by It should be mentioned ◦ C. As shown in Figure 2a, other terms, such as the quadratic effect temperature around 50–55 behaviors were obtained when EtOH and acetone were separately used as co-solvents. of concentration ethanol and the interaction between of ethanol, Overall, more carotenoids were of comparatively extracted with the usepressure of EtOHand as concentration co-solexerted a significant, though minor effect on the carotenoids’ extraction. vent than with the use of acetone. Fitted Surface for total carotenoids extraction with SC-CO2 [ug/g DW] MS Residual=1.983 36 34 32 30 Pressure [MPa] 28 26 24 22 20 18 16 14 12 10 8 30 35 40 45 50 55 Temperature[C] (a) Figure 1. Cont. 60 65 70 75 > < < < < < 14 13 9 5 1 -3 Foods 2021, 10, 2201 2021, 10, x FOR PEER REVIEW 8 of 24 8 of 22 Fitted Surface for total carotenoids extraction with SC-CO2, modified with ethanol [ug/g DW] MS Residual=1.983 36 34 32 30 28 Pressure [MPa] 26 24 22 20 18 16 14 > < < < < < < 12 10 8 30 35 40 45 50 55 60 65 70 75 Temperature[C] 20 19 15 11 7 3 -1 (b) Fitted Surface for total carotenoids extraction w ith SC-CO2 modified w ith acetone [ug/g DW] MS Residual=3.505 36 34 32 30 Pressure [MPa] 28 26 24 22 20 18 16 14 12 10 8 30 35 40 45 50 55 Temperature[C] 60 65 70 75 > 14 < 13 < 11 <9 <7 <5 <3 <1 < -1 (c) Figure RSM for total carotenoids with 0% (a),(b) 20% EtOH and 20% acetone (c). Figure 1. RSM for total1.carotenoids extracted withextracted 0% co-solvent (a),co-solvent 20% EtOH and 20% (b) acetone (c). However, when the SC-CO2 was modified with acetone (Figure 2b), it was seen that only thePareto linearcharts effect of of the pressure (standardized effect of and 17.0)quadratic positively influenced Figure 2 depicts the remaining linear, interaction the recovery of carotenoids. Conversely, co-solvent concentration (standardized effect of effects of the variables on the total yield. Overall, it can be observed that pressure and co− 2.6), temperature (standardized effect − 1.95) and the separate conjunctions of pressure solvent addition are decisive in the concentration of total carotenoids recovered from and co-solvent and temperature and pressure (−2.59 and −1.88, respectively) exerted a mango. significant but negative these results2a), show pressure In the extraction performed with influence. ethanol as Overall, a co-solvent (Figure the that linear effects stands as an important factor for the extraction of compounds with SC-CO modified 2 of pressure (standardized effect of 28.1) and co-solvent addition (standardized effect ofwith acetone, where increasing temperature negatively influences on the overall yield. 6.9) were found to have a major and beneficial influence on the recovery of carotenoids. The positive effect with pressure is well-known and associated with The temperature dependence was mostly dominated by the quadratic effect (−5.09),the re-higher density of the SC-CO2 [6]. The optimum temperature at intermediate values (55 ◦ C), on the other sulting in an optimum temperature around 50–55 °C. As shown in Figure 2a, other terms, hand, can be explained by the presence of two opposing mechanisms. It is well-known that such as the quadratic effect of concentration of ethanol and the interaction between presmass transfer is improved with temperature because of the increase in the vapor pressure sure and concentration of ethanol, exerted a significant, though minor effect on the carotof the analytes present, facilitating their solvation [17]. The solubility, on the other hand, is enoids’ extraction. diminished with temperature because of the decreasing density of the CO affecting the However, when the SC-CO2 was modified with acetone (Figure 2b), it◦was seen that2 solvating power. The reduction observed in the yield above 55 C is therefore likely caused only the linear effect of pressure (standardized effect of 17.0) positively influenced the by the fact that a decrease in solubility of the solvent has a greater effect compared to the recovery of carotenoids. Conversely, co-solvent concentration (standardized effect of increase in vapor pressure of the component. −2.6), temperature (standardized effect −1.95) and the separate conjunctions of pressure and co-solvent and temperature and pressure (−2.59 and −1.88, respectively) exerted a sig- Foods 2021, 10, x FOR PEER REVIEW Foods 2021, 10, 2201 9 of 24 nificant but negative influence. Overall, these results show that pressure stands as an important factor for the extraction of compounds with SC-CO2 modified with acetone, where increasing temperature negatively influences on the overall yield. 9 of 22 Pareto Chart of Standardized Effects for total carotenoids extraction with SC-CO2 modified with ethanol [ug /g DW] MS Residual=1.983 28.110 (2)Pressure [MPa](L) 6.867 (3)Ethanol [%](L) - 5.099 Temperature[C](Q) 3.036 Ethanol [%](Q) 2.665 2Lby3L (1)Temperature[C](L) - 0.655 p=,05 Standardized Effect Estimate (Absolute Value) (a) Pareto Chart of Standardized Effects for total carotenoids extraction with SC-CO2 modified with acetone [ug /g DW] MS Residual=3.439 (2)Pressure [MPa](L) 17.026 2Lby3L - 2.592 (3)Acetone [%](L) - 2.588 (1)Temperature[C](L) - 1.956 1Lby2L - 1.881 p=,05 Standardized Effect Estimate (Absolute Value) (b) Figure 2. Pareto for total carotenoids extracted with ethanol (a) and acetone(a) (b). Figurechart 2. Pareto chart for total carotenoids extracted with ethanol and acetone (b). The positive with pressure is well-known and associated with the higher den-maxima of the Theeffect conditions to maximize the response were determined by the ◦ C,°C), sity of the SC-CO2 [6]. The optimum temperature at intermediate values on theof 35 MPa and generated plots (Figure 1a–c). By applying a temperature of 55(55 pressure other hand, can (v/v) be explained by the of two opposing mechanisms. It is wellat 20% of ethanol thepresence highest extraction yield was obtained. With the application known that massconditions, transfer is improved with temperature because of the were increase in the of such the predicted yield and the real yield 19.52 µg·g−1 DW and −1analytes vapor pressure of·g the present, facilitating their solvation Theobtained solubility,by onthe model and 20.56 µg DW, respectively. The ratio between the[17]. values the otherexperimental hand, is diminished with temperature because of the decreasing density of the value prediction was 94.9%. CO2 affecting the solvating power. The reduction observed in the yield above 55 °C is 3.3. Optimization of the Extraction Yield of the Individual Carotenoids via RSM Modelling To obtain a comprehensive knowledge for each of the carotenoids the influence of the extraction conditions was also assessed on each of the individual carotenoids in a similar way as described above for the total carotenoids (Table 1). In the extracts α and β-carotene, β-cryptoxanthin and phytoene were found to be the most abundant. Additionally, lower concentrations of cantaxanthin, capsanthin, lutein, violaxanthin and zeaxanthin were observed. For the most abundant carotenoids, an RSM model was constructed, whereby the insignificant parameters (confidence level 95%) were removed. The trends of the resulting Foods 2021, 10, 2201 10 of 22 models are shown in Figure 3. For all the compounds, pressure was found to be the most important factor, independent of the temperature, concentration or type of co-solvent, whose linear and/or quadratic effects had a lower but still significant influence on the extraction. The correlation (R2 ) was 0.81, 0.92 and 0.64 for β-cryptoxanthin, β-carotene and phytoene, respectively. As can be seen in Table 1, β-carotene is the most abundant carotenoid, and as expected, follows a similar behavior as reported above for the total carotenoids. Pressure and co-solvent are the major impacting parameters, whereby ethanol is more efficient than acetone (Figure 3(a1,b1,c1)). The presence of ethanol positively influences the amount of extracted β-carotene, leading to values up to 18.74 µg·g−1 DW under the optimal conditions. This increase can be explained based on molecular interactions between the solvent and the carotenoids via the Hansen solubility parameters (HSP). Tirado and Calvo (2019) [18], calculated that the HSP of β-carotene and SC-CO2 modified with ethanol are very similar, explaining the suitability of the latter as an extracting medium. Specifically, the distance (Ra) between the HSP of the mixture CO2 + EtOH and those of β-carotene when extracted at 40 and 50 ◦ C were smaller when compared with acetone and other organic solvents, specifically 12.14 and 13.57 for each temperature, respectively. It must be noted that this model only considers the affinity between the solute and solvent and does not consider the competitive interactions that exist in the matrix. As for the other carotenoids, β-cryptoxanthin had a very similar behavior as βcarotene, as can be seen in Figure 3(a2,b2). Phytoene, on the other hand, had a different behavior, since their highest extraction yield was obtained at 35 ◦ C. This different behavior is likely due to a higher sensitivity to isomerization or oxidative degradation with increasing temperature [19]. At a temperature of 55 ◦ C and with EtOH as co-solvent, both β-carotene and β-cryptoxanthin have their highest yield of 18.74 and 2.23 µg·g−1 DW, respectively. These values are very close to the predicted 17.45 µg·g−1 DW and 1.55 µg·g−1 DW, respectively. Nevertheless, if extracts rich in phytoene are targeted, a lower operating temperature of 35 ◦ C is better suited, resulting in a yield of 0.26 µg·g−1 DW, which is comparable to the predicted value of 0.23 µg·g−1 DW in the model. 3.4. Validation of the Model for Carotenoids Extraction and Behavior of the Phenolic Compounds As the phenolic compounds are also valuable constituents that may be co-extracted, their presence was also evaluated in the extracts obtained at this subset of conditions. To validate the model obtained, the extractions at the optimum as well as at a subset of conditions with higher carotenoid yield were repeated. As a pressure of 35 MPa resulted in good carotenoid extraction, the subset was focused on variation of temperature and co-solvent, while keeping the pressure mostly constant. The results obtained for phenolics are described in Figure 4. The modification of SC-CO2 with 20% ethanol enhanced the extraction yield of phenolics when compared with pure CO2 and acetone. On the other hand, the temperature and the extraction yield of phenolics were directly proportional. The higher yield was obtained at 70 ◦ C (608.13 µg·g−1 DW). The values presented in this study are higher than those provided by Garcia-Mendoza et al. (2015) [8]. Moreover, Meneses et al. (2015) [20], indicated that environmental factors like UV radiation, factors inherent to the process like storage and the cultivar itself affect the phenolic content of mango. The higher yield obtained with SC-CO2 modified with organic solvents can likely be attributed to the predominance of the vapor pressure effect over the density effect. The solvent’s viscosity and the surface tension decrease at higher temperatures; thus, its penetration into the matrix is enhanced. Moreover, the molecular interactions (e.g., solute-matrix, in the form of sorption isotherm/isobar models) are broken; therefore, the desorption energy of the target compounds is reduced [21]. Moreover, it is possible that the particle cell walls are damaged, which can result in an increment in the mass transfer [22]. It should also be indicated that certain phenolic compounds like gallic and vanillic acids are relatively stable, while catechin is the most unstable when exposed to temperatures from 80 to 100 ◦ C, so that degradation effects are expected to be less important as in the case of carotenoids [23]. Foods 2021, 10, 2201 11 of 22 Table 1. Quantification of the amount of carotenoids extracted with SC-CO2 in the optimization set of experiments per amount of biomass extracted from the mango variety Kasturi. 10 MPa 35 ◦ C; 100% CO2 35 ◦ C; 10% EtOH 35 ◦ C; 20% EtOH 35 ◦ C; 10% Ac 35 ◦ C; 20% Ac 55 ◦ C; 100% CO2 55 ◦ C; 10% EtOH 55 ◦ C; 20% EtOH 55 ◦ C; 10% Ac 55 ◦ C; 20% Ac 70 ◦ C; 100% CO2 70 ◦ C; 10% EtOH 70 ◦ C; 20% EtOH 70 ◦ C; 10% Ac 70 ◦ C; 20% Ac 35 MPa α-Carotene [µg·g−1 DW] β-Cryptoxantin [µg·g−1 DW] β-Carotene [µg·g−1 DW] Phytoene [µg·g−1 DW] α-Carotene [µg·g−1 DW] β-Cryptoxantin [µg·g−1 DW] β-Carotene [µg·g−1 DW] Phytoene [µg·g−1 DW] <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 0.24 ± 0.01 d 0.14 ± 0.07 cd 0.91 ± 0.08 e 0.03 ± 0.00 a 0.02 ± 0.00 a 0.04 ± 0.01 a 0.07 ± 0.00 ab 0.19 ± 0.01 de 0.10 ± 0.02 bc 0.13 ± 0.00 c 0.07 ± 0.04 ab 0.07 ± 0.01 ab 0.11 ± 0.00 bc 0.11 ± 0.01 bc 0.07 ± 0.01 ab 0.31 ± 0.02 ab 0.17 ± 0.02 a 1.52 ± 0.08 c 0.25 ± 0.08 ab 0.13 ± 0.00 a 0.11 ± 0.02 a 1.44 ± 0.17 c 3.70 ± 0.87 d 0.11 ± 0.02 a 0.22 ± 0.00 ab <LOD 0.01 ± 0.00 a 0.61 ± 0.05 b 0.02 ± 0.01 a 0.03 ± 0.00 a 0.16 ± 0.04 ef <LOD 0.11 ± 0.02 d 0.08 ± 0.02 cd 0.04 ± 0.02 bc 0.05 ± 0.01 bc 0.06 ± 0.02 bc 0.15 ± 0.02 e 0.09 ± 0.02 d 0.03 ± 0.00 ab 0.05 ± 0.04 bc 0.05 ± 0.00 bc 0.02 ± 0.00 ab 0.04 ± 0.00 b 0.19 ± 0.02 g 1.03 ± 0.07 ef 1.24 ± 0.11 g 1.51± 0.09 h 0.26 ± 0.03 b 0.39 ± 0.01 c 1.32 ± 0.02 g 1.01 ± 0.12 e 1.13 ± 0.14 f 0.39 ± 0.08 c 0.27 ± 0.02 b 0.41 ± 0.04 cd 0.52 ± 0.00 d 0.38 ± 0.05 c 0.05 ± 0.00 a 0.04 ± 0.00 a 0.65 ± 0.01 abc 1.52 ± 0.27 ef 1.49 ± 0.17 ef 0.33 ± 0.07 a 0.39 ± 0.01 ab 1.68 ± 0.03 f 2.23 ± 0.14 g 1.13 ± 0.04 de 1.52 ± 0.04 ef 0.24 ± 0.18 a 0.81 ± 0.09 cd 1.29 ± 0.10 ef 1.13 ± 0.00 de 0.83 ± 0.49 cd 0.74 ± 0.07 bcd 8.96 ± 0.23 d 6.81 ± 0.19 c 15.28 ± 0.92 h 11.36 ± 0.37 e 12.16 ± 0.38 f 11.63 ± 1.14 ef 13.41 ± 0.42 g 18.74 ± 0.21 i 5.81 ± 0.23 b 3.35 ± 0.34 a 6.23 ± 0.52 bc 5.80 ± 0.57 b 11.08 ± 0.20 e 8.65 ± 0.03 d 6.46 ± 0.25 bc 0.13 ± 0.00 abc 0.19 ± 0.03 cde 0.19 ± 0.01 de 0.26 ± 0.03 e 0.23 ± 0.01 de 0.14 ± 0.04 bcd 0.07 ± 0.00 ab 0.10 ± 0.07 ab 0.09 ± 0.02 ab 0.07 ± 0.06 ab 0.06 ± 0.02 a 0.08 ± 0.01 ab 0.19 ± 0.04 cde 0.07 ± 0.00 ab 0.13 ± 0.08 bcd Values are mean ± relative difference (n = 2). LOD = limit of quantification. Means with different letters indicate statistically significant differences between extraction conditions and the specific compound in the mango matrix (p < 0.05). Foods 2021, 10, 2201 Foods 2021, 10, x FOR PEER REVIEW 12 of 22 12 of 24 β-carotene β-cryptoxanthin Phytoene (a2) (a3) (a1) Fitted Surf ace; Variable: β-cry ptoxanthin Fitted Surf ace; Variable: β-carotene 34 30 28 26 Pressure [MPa] Pressure [MPa] 100% CO2 32 24 22 20 18 16 14 12 10 35 40 45 50 55 60 65 70 75 Temperature [C] > < < < < < < < 10 10 8 6 4 2 0 -2 36 34 34 32 32 30 30 28 28 26 26 24 22 20 18 22 20 18 16 14 14 12 > < < < < < 10 8 30 35 40 45 50 55 60 65 70 75 Temperature [C] 1.2 1.1 0.7 0.3 -0.1 -0.5 12 10 8 30 Fitted Surf ace; Variable: β-carotene Fitted Surf ace; Variable: β-cry ptoxanthin MS Residual=1.079 MS Residual=0.002 34 32 32 30 30 30 28 28 28 26 26 26 22 20 18 16 Pressure [MPa] 34 32 24 24 22 20 18 16 14 12 10 8 50 55 Temperature [C] 60 65 70 75 > < < < < < < 18 17 13 9 5 1 -3 14 12 10 8 30 35 40 45 50 55 Temperature [C] Figure 3. Cont. 55 60 65 70 75 MS Residual=0.003 34 45 50 0.16 0.16 0.14 0.12 0.1 0.08 0.06 0.04 0.02 Fitted Surf ace; Variable: Phy toene 36 40 45 > < < < < < < < < (b3) 36 35 40 Temperature [C] 36 30 35 (b2) Pressure [MPa] Pressure [MPa] 24 16 (b1) 20% EtOH MS Residual=0.001 36 Pressure [MPa] 36 8 30 Fitted Surf ace; Variable: Phy toene MS Residual=0.102 MS Residual=1.820 60 65 70 75 > < < < < < < < < 1.6 1.5 1.3 1.1 0.9 0.7 0.5 0.3 0.1 24 22 20 18 16 14 12 10 8 30 35 40 45 50 55 Temperature [C] 60 65 70 75 > < < < < < < < 0.18 0.17 0.15 0.13 0.11 0.09 0.07 0.05 Foods 2021, 10, 2201 Foods 2021, 10, x FOR PEER REVIEW 13 of 22 13 of 24 (c1) (c2) (c3) Fitted Surf ace; Variable: β-cry ptoxanthin Fitted Surf ace; Variable: β-carotene 36 34 32 30 26 24 22 20 18 16 14 12 10 35 40 45 50 55 Temperature [C] 60 65 70 75 > < < < < < < < < < 14 14 12 10 8 6 4 2 0 -2 Pressure [MPa] 28 32 30 30 28 28 26 26 Pressure [MPa] 32 Pressure [MPa] 20% Acetone MS Residual=0.001 36 34 34 8 30 Fitted Surf ace; Variable: Phy toene MS Residual=0.019 36 MS Residual=3.098 24 22 20 18 24 22 20 18 16 16 14 14 12 12 10 8 30 35 40 45 50 55 Temperature [C] 60 65 70 75 > < < < < < 0.8 0.75 0.55 0.35 0.15 -0.05 10 8 30 35 40 45 50 55 Temperature [C] 60 65 70 75 > < < < < < < < 0.31 0.3 0.25 0.2 0.15 0.1 0.05 0 Figure RSM β-caroteneextracted extractedwith with100% 100%CO CO2(a1), (a1),20% 20% EtOH EtOH (b (b1)) and 20% Acetone (c1); β-cryptoxanthin extracted with 100% CO2 (a2), 20% EtOH (b2) and 20% Acetone (c2) Figure 3. 3. RSM forfor β-carotene 1 and 20% Acetone (c1); β-cryptoxanthin extracted with 100% CO2 (a2), 20% EtOH (b2) and 20% Acetone (c2) 2 and phytoene extracted with 100% CO2 (a3), 20% EtOH (b3) and 20% Acetone (c3). and phytoene extracted with 100% CO2 (a3), 20% EtOH (b3) and 20% Acetone (c3). at higher temperatures; thus, its penetration into the matrix is enhanced. Moreover, the molecular interactions (e.g., solute-matrix, in the form of sorption isotherm/isobar models) are broken; therefore, the desorption energy of the target compounds is reduced [21]. Moreover, it is possible that the particle cell walls are damaged, which can result in an increment in the mass transfer [22]. It should also be indicated that certain phenolic com14 of 22 pounds like gallic and vanillic acids are relatively stable, while catechin is the most unstable when exposed to temperatures from 80 to 100 °C, so that degradation effects are expected to be less important as in the case of carotenoids [23]. Foods 2021, 10, 2201 400 Flavonoid Xanthonoids Dihydroxybenzoic acids k Hydroxycinnamic acids j 300 i 200 j ij 250 h h 150 g 100 h e b ab b d g c fg h f g e cd e f bc c ef b b ab c f c ab b de a a a a ab b fg d d 50 e f g Phenolic compounnds [ug/g DW] 350 20% EtOH – 35 MPa – 70 C 100% CO2 – 35 MPa – 70 C 20% Acet – 35 MPa – 55 C 20% EtOH – 35 MPa – 55 C 100%CO2 - 35 Mpa - 55 C 20% Acet – 35 MPa – 35 C 20% EtOH – 35 MPa – 35 C 100% CO2 – 35 MPa – 35 C 20% Acet – 10 MPa – 55 C 20% EtOH – 10 MPa – 55C 100%CO2 - 10 Mpa - 55 C 100% CO2 – 10 MPa – 35 C 0 Figure 4. 4. Total Total phenolic (evaluated as as sum sum of of individual individual compounds) compounds) obtained obtained in in the the confirmation confirmation data data set. set. Figure phenolic compounds compounds (evaluated Values are mean ± relative difference (n = 2). Means with different letters indicate statistically significant differences Values are mean ± relative difference (n = 2). Means with different letters indicate statistically significant differences bebetween extraction conditions and concentration phenolics same class < 0.05). tween extraction conditions and thethe concentration of of phenolics of of thethe same class (p (p < 0.05). As stated, the addition of co-solvents significantly improved the extraction yield of phenolics in comparison to the extraction performed with pure CO2 . In this line, higher yields were obtained with EtOH than with acetone. This enhancement could be likely explained by the higher hydrogen donor strength of ethanol than acetone (0.83 vs. 0.08, respectively) [24], which is related to the extraction yield. Besides the processing parameters, Molino et al. (2020) [6] explained there are inherent factors that affect the solubility of the compounds in SC-CO2 , like the molecular mass and structure of the compounds, since the complexity of the molecule produces a reduction in the solubility. For a better understanding of the behavior of different phenolic classes, the individual compounds were separated in four groups: dihydroxybenzoic acids, flavonoids, hydroxycinnamic acids and xanthonoids (see Figure 4). Dihydroxybenzoic acids were quantified between 2.90 and 353.81 µg·g−1 DW. Flavonoids had a total yield ranging from 1.07 to 188.39 µg·g−1 DW. Within this group, quercetin was the most concentrated one, representing between 13 and 55 wt. % of the total class. With respect to hydroxycinnamic acids, ferulic acid represented between 28 and 86 wt. % of the total class. The overall yield of hydroxycinnamic acids was found to be between 0.15 µg·g−1 DW and 37.78 µg·g−1 DW. Finally, mangiferin was the only representative of the xanthones group, whose yield was between 1.1 and 28.15 µg·g−1 DW. The profile and the principal compounds found in this study were similar to those reported for mango by Schieber et al. (2000) and AgatonovicKustrin et al. (2018) [25,26] (chromatograms of the analysis of phenolics and carotenoids are displayed in Supplementary Material SS.4). Foods 2021, 10, 2201 15 of 22 3.5. Extraction of Carotenoids and Phenolic Compounds at Optimized Conditions on By-Products Obtained from Different Ecuadorian Mango Varieties and Assessment of the Antioxidant Activities After optimizing the extraction conditions, the concentration of carotenoids and phenolics was compared in extracts performed with SC-CO2 and organic solvents in the peel and pulp of two different mango varieties: “Kent” and “Haden”. Moreover, the DPPH· and FRAP methods were applied to assess the antioxidant activity of the extracts. 3.5.1. Carotenoids Given that carotenoids are secondary metabolites, their presence and concentration can be different depending on the variety and the environmental conditions of the vegetal matrix. Therefore, the effect of extraction conditions (SC-CO2 or organic solvent extraction) was evaluated in the variability of the carotenoids profile in the by-products of two different mango varieties. In this study, it was observed that the concentration of the carotenoids was influenced by the varieties of mango and the type of by-products analyzed. Overall, the concentration of total carotenoids on peels of the different varieties were between 44.71 and 65.66 µg·g−1 DW on the samples extracted with organic solvents, while for the extracts performed with SC-CO2 , the concentration was between 43.12 and 53.87 µg·g−1 DW. Lower concentrations were registered in the pulp, with 17.35 to 32.23 µg·g−1 DW and 13.09 to 21.99 µg·g−1 DW in the samples extracted with organic solvents and SC-CO2 , respectively. The varietal differences in the concentration of carotenoids in mango have been previously reported. Mercadante and Rodriguez-Amaya (1998) [27], found a concentration of β-carotene of 5.8 µg·g−1 DW in the “Tommy” pulp variety, and of 15 µg·g−1 DW in the “Keitt” pulp variety, which is significantly lower than the concentration reported in the present study for the by-products of all varieties analyzed. Similarly, Pott et al. (2003) [28] reported variations of the carotenoids present in the flesh from three cultivars of mangoes. The concentration of β-carotene in the “Kent” and “Tommy” variety was 46 and 37 µg·g−1 DW, respectively, similar to those obtained in the varieties utilized in this study. These variations could be due to both genotypic [29] and environmental factors [30]. It was also found that the carotenoid concentration in the peels was between 1.3- and 4.1-fold higher than in the pulp. This could be due to the active role of carotenoids in photosystem assembly where they act as a photoprotector. The higher exposure to sunlight induces an increase in carotenogenesis, leading to higher concentrations of carotenoids in the peels [30], making the peels a valuable resource for bioactive compound recovery. As can be seen in Table 2, the use of organic solvents allowed for a higher extraction yield of total carotenoids (between 6% and 46%). The concentration of total carotenoids determined for the extracts performed with SC-CO2 in all the varieties in this study was similar to the range reported by Haque et al. (2015) [31] in different mango varieties from Bangladesh extracted with n-hexane, but lower than the results reported by GarciaMendoza et al. (2015) [8] in mango peels with non-modified SC-CO2 . The concentration of β-carotene, β-cryptoxanthin and lutein (Table 2) was lower than the concentration reported in mango “Ataulfo” [32]. In addition, the solid remnants after extraction with SC-CO2 could be used for several applications. In another study, de Andrade Lima et al. (2018) [33] found that carbohydrates, proteins and lipids are retained within the biomass after SFE. Consequently, the extracted biomass could potentially be used as source of such macronutrients for different uses (e.g., stabilizers or emulsifiers), and in a subsequent process for transformation into platform or biofuels. Foods 2021, 10, 2201 16 of 22 Table 2. Amount of the various carotenoids extracted with CE and SFE from the pulp and peel of different mango varieties. Total Carotenoids [µg·g−1 DW] α-Carotene [µg·g−1 DW] β-Kryptoxanthin [µg·g−1 DW] β-Carotene [µg·g−1 DW] Lutein [µg·g−1 DW] Violaxanthin [µg·g−1 DW] Phytoene [µg·g−1 DW] Phytofluene [µg·g−1 DW] Zeaxanthin [µg·g−1 DW] Kent peel CE * SFE ** 44.71 ± 0.02 a 43.12 ± 6.53 b 0.99 ± 0.01 a 1.45 ± 0.59 b 0.28 ± 0.06 a 0.26 ± 0.01 a 34.38 ± 1.89 a 35.52 ± 4.09 a 1.26 ± 0.03 a 1.69 ± 0.12 b 0.08 ± 0.01 b 0.17 ± 0.02 c 0.43 ± 0.02 a 0.57 ± 0.04 b 0.76 ± 0.01 b 0.92 ± 0.02 c 0.26 ± 0.06 a 0.33 ± 0.02 b Kent pulp CE SFE 32.23 ± 0.99 a 22.00 ± 0.12 b 1.36 ± 0.34 a 0.38 ± 0.19 b 0.76 ± 0.06 a 0.33 ± 0.01 b 24.02 ± 0.49 a 17.87 ± 1.88 b 0.94 ± 0.14 a <LOQ <LOQ <LOQ 0.01 ± 0.02 a 0.14 ± 0.02 b <LOQ <LOQ 0.24 ± 0.03 a <LOQ Haden peel CE SFE 65.66 ± 4.45 a 53.87 ± 0.87 b 7.09 ± 0.59 a 2.52 ± 0.04 b 1.50 ± 0.21 a 1.12 ± 0.09 b 46.47 ± 1.13 a 33.30 ± 4.16 b 1.29 ± 0.00 a 1.05 ± 0.07 b 0.45 ± 0.13 b 0.14 ± 0.03 b 0.69 ± 0.14 b 0.20 ± 0.06 a 0.78 ± 0.01 b 0.19 ± 0.00 c 0.14 ± 0.01 a 0.12 ± 0.02 b Haden pulp CE SFE 17.35 ± 0.33 a 13.09 ± 0.91 b 0.16 ± 0.05 a 0.17 ± 0.06 a 0.24 ± 0.03 a 0.26 ± 0.08 a 11.33 ± 8.45 a 8.72 ± 0.22 a 0.07 ± 0.00 a 0.04 ± 0.00 a 0.03 ± 0.00 b 0.01 ± 0.00 c 0.28 ± 0.01 a 0.19 ± 0.02 a 0.23 ± 0.01 b 0.24 ± 0.00 b 0.03 ± 0.01 a 0.01 ± 0.01 b * CE = conventional extraction. ** SFE = supercritical fluid extraction (20% EtOH, 55 ◦ C, 35 MPa). Values are mean ± relative difference (n = 2). LOQ = limit of quantification. Means with different letters in the same row indicate statistically significant differences between the concentration of the compounds as function of the extraction method (p < 0.05). Foods 2021, 10, x FOR PEER REVIEW Foods 2021, 10, 2201 18 of 24 17 of 22 3.5.2. Phenolic Compounds 3.5.2. Phenolic Compounds The health-promoting activities of phenolic compounds are of interest for researchers The health-promoting activities of phenolics phenolic compounds are of interest for researchers and industry. Similar to carotenoids, are secondary metabolites whose profile and industry. Similar to carotenoids, phenolics are secondary metabolites whose profile can vary depending on the extraction technique. Hence, SC-CO2 and solvent extraction can vary depending on the extraction technique. Hence, SC-CO2 and solvent extraction were compared in terms of concentration of individual phenolics in extracts obtained from were compared in terms of concentration of individual phenolics in extracts obtained from mango by-products, firstpriority priorityfocused focused carotenoids. mango by-products,with with the the first onon carotenoids. InIn total, 21 different phenolic compounds were found in mango the mango matrix, whereby total, 21 different phenolic compounds were found in the matrix, whereby thethe flavonoids and phenolic acids were the most important ones (see Figure 5). flavonoids and phenolic acids were the most important ones (see Figure 5). Neverthe-Nevertheless, theprofile profile of the individual compounds significantly, only less, the of the individual compounds variedvaried significantly, not only not among theamong dif- the ferent extraction techniques, butbut alsoalso among the fraction assessed (Table 3). different extraction techniques, among the fraction assessed (Table 3). 120 Hydroxycinnamic acids Xanthonoids Dihydroxybenzoic acids g 100 Phenolic compounds [ug/g DW] Flavonoids f e 80 60 d 40 c d c f 20 ab a c a a a e c b b c d c b a a bc a a a a a b c Haden pulp_ SFE Haden pulp_ CE Haden peel_ SFE Haden peel_ CE Kent pulp_ SFE Kent pulp_ CE Kent peel_ SFE Kent peel_ CE 0 . Figure 5. Total phenolic compounds (evaluated as sum of individual compounds) obtained in the different mango varieties. Figure 5. Total phenolic compounds (evaluated as sum of individual compounds) obtained in the different mango varieValuesties. areValues meanare ± mean relative difference (n = 2). Means with different letters indicate statistically significant differences ± relative difference (n = 2). Means with different letters indicate statistically significant differences between extraction method and phenolics in same the same fraction and of variety (p =< 0.05). between extraction method andthe theconcentration concentration ofofphenolics in the fraction and variety mangoof (p mango < 0.05). CE conventional extraction. SFE = supercritical fluidfluid extraction. CE = conventional extraction. SFE = supercritical extraction. Table 3. Main phenolics identified in the peel and pulp of the mango varieties Kent and Haden. Flavonoids Hydroxycinnamic acids Kent Peel Caffeic acid Kent Pulp Haden Peel Haden Pulp CE * SFE ** CE SFE CE SFE CE SFE 0.19 ± 1.03 b 0.27 ± 4.82 c 0.16 ± 6.90 a 0.22 ± 13.26 b 0.39 ± 0.22 e 0.24 ± 0.05 b 0.30 ± 1.46 d 0.41 ± 3.53 e <LOQ 0.05 ± 0.33 a 0.43 ± 6.79 c <LOQ 0.08 ± 9.50 b 0.57 ± 9.39 d 0.17 ± 1.36 b 0.25 ± 0.95 f 0.66 ± 1.59 e 0.02 ± 8.97 a 0.21 ± 1.73 e 0.76 ± 2.65 g <LOQ 0.15 ± 5.99 c 0.22 ± 5.08 a <LOQ 0.27 ± 8.88 g 0.35 ± 1.87 b <LOQ <LOQ 0.01 ± 10.95 c 0.27 ± 18.11 a 0.11 ± 18.29 a 1.40 ± 17.65 b 0.01 ± 12.79 a 0.10 ± 3.04 c 0.01 ± 0.67 a 10.97 ± 4.16 d 0.56 ± 4.29 b 27.98 ± 4.31 e <LOQ <LOQ <LOQ 0.01 ± 1.51 a <LOQ 0.07 ± 3.22 a <LOQ 0.02 ± 3.66 a <LOQ 0.01 ± 6.28 a 0.26 ± 40.31 a 0.11 ± 24.90 a 1.58 ± 2.31 b Chlorogenic acid Ferulic acid p-Coumaric acid <LOQ 0.14 ± 16.09 c 0.46 ± 8.79 c Cymaroside Aromadendrin Avicularin Astragalin Hyperin Phlorizin Taxifolin Naringenin Epicathecin Kaempferol Isoquercitrin Quercetin Quercitrin Catechin 0.06 ± 4.63 b 0.02 ± 4.04 e 10.53 ± 3.18 c 0.86 ± 2.50 d 26.57 ± 4.89 e 0.18 ± 3.88 d 0.37 ± 1.64 e 0.15 ± 12.59 e 0.14 ± 1.49 d 0.04 ± 78.95 a 27.66 ± 1.50 g 0.99 ± 13.54 c 5.03 ± 5.75 e 21.28 ± 0.99 e <LOQ 0.18 ± 3.77 d 0.71 ± 3.63 f 0.02 ± 4.00 a 0.03 ± 4.08 f 10.41 ± 1.26 c 1.94 ± 4.04 e 23.97 ± 4.19 d 0.16 ± 2.82 c 0.77 ± 2.68 f 0.15 ± 4.21 e 0.06 ± 4.69 c 0.07 ± 18.32 b 26.81 ± 1.95 f 1.67 ± 5.37 d 4.34 ± 3.12 d 10.13 ± 1.43 d 0.01 ± 13.20 d 0.02 ± 65.27 a <LOQ <LOQ <LOQ 0.03 ± 7.59 a 0.02 ± 7.15 a 0.03 ± 4.99 b <LOQ 0.11 ± 31.36 b <LOQ 0.01 ± 26.32 a 3.67 ± 3.75 c 0.06 ± 16.93 b 0.03 ± 14.16 c 0.03 ± 11.95 b <LOQ 1.11 ± 17.34 c 0.29 ± 38.82 a 0.08 ± 21.53 b 3.31 ± 8.64 c 0.18 ± 0.05 d 0.15 ± 1.33 c 0.16 ± 2.79 d 0.14 ± 3.86 d 0.07 ± 1.24 b 27.47 ± 2.01 f 2.78 ± 0.71 f 5.01 ± 1.27 e 21.06 ± 3.95 e 0.02 ± 1.82 b 4.49 ± 4.05 b 0.64 ± 3.96 c 10.73 ± 2.05 c 0.06 ± 3.75 b 0.27 ± 0.75 d 0.14 ± 3.79 b 0.01 ± 1.16 a 0.09 ± 1.56 c 11.11 ± 0.60 e 1.92 ± 5.28 e 1.70 ± 0.68 c 1.45 ± 4.50 b <LOQ 0.01 ± 15.69 a <LOQ 0.08 ± 6.35 a <LOQ <LOQ <LOQ <LOQ 0.03 ± 5.55 a 0.01 ± 7.85 a <LOQ 0.07 ± 28.42 b 1.26 ± 2.51 d 0.53 ± 9.01 b 0.08 ± 25.13 b 0.35 ± 1.20 a Foods 2021, 10, 2201 18 of 22 Table 3. Cont. Dihydroxybenzoic acids Xanthonoids Kent Peel Kent Pulp Haden Peel Haden Pulp CE * SFE ** CE SFE CE SFE CE SFE Mangiferin 22.19 ± 1.44 h 9.47 ± 4.54 g 0.78 ± 16.39 c 1.30 ± 15.86 d 5.90 ± 3.45 f 0.48 ± 10.95 b 0.34 ± 0.87 a 1.96 ± 23.30 e Gallic acid 29.70 ± 8.11 e 30.45 ± 0.99 e 11.55 ± 7.74 a 18.98 ± 13.52 c 48.21 ± 1.15 f 13.13 ± 7.87 b 13.21 ± 8.33 b 21.15 ± 11.89 d Protocatechuic acid 0.72 ± 70.58 d 1.18 ± 2.53 f 0.22 ± 14.14 a 0.94 ± 16.33 e 0.29 ± 5.50 b 0.55 ± 60.96 c 0.56 ± 18.65 c 1.01 ± 27.49 f * CE = conventional extraction. ** SFE = supercritical fluid extraction (20% EtOH, 55 ◦ C, 35 MPa). Values are mean ± relative difference (n = 2). LOQ = limit of quantification. Means with different letters in the same row indicate statistically significant differences in the concentration of the compounds as function of the extraction method (p < 0.05). Overall, the two-step organic solvent extraction resulted in a higher yield of phenolics. The total yield of phenolics, determined as the sum of the individual compounds, ranged between approximately 147.28 and 152.52 µg·g−1 DW in the peel extracts, and between 15.42 µg·g−1 DW and 17.06 µg·g−1 DW in the pulp extracts. In general, gallic acid was the most abundant compound, in line with other studies [34]. The extraction of the peels resulted in a yield of 29.70 µg·g−1 DW for the variety “Kent” and 48.21 µg·g−1 DW (for the variety “Haden”), while the yields of the corresponding pulps were 11.55 and 13.21 µg·g−1 DW for the “Kent” and “Haden” varieties, respectively. The results obtained in the peels are similar to those reported by Yao et al. (2020) [35], who found between 84.86 and 147.99 µg·g−1 DW of mango pulp (variety “Sensation”). The hydroxybenzoic acids were quantified up to 48.5 µg·g−1 DW in the peels of the variety “Haden”, and the pulp was characterized by a yield of 13.68 µg·g−1 DW. The flavonoids were the second group of phenolic compounds present in the mango by-products. The extraction of the peels of the “Haden” variety resulted in a yield of 96.65 µg·g−1 DW, as compared to 93.89 µg·g−1 DW for the “Kent” variety. In the pulp, up to 3.89 µg·g−1 DW was quantified, which is lower than reported by Rumainum et al. (2018) [36] in the pulp of different mango cultivars from Thailand (up to 670 µg·g−1 DW). Finally, the yield of mangiferin (5.90 to 22.19 µg·g−1 DW and 0.33 to 0.78 µg·g−1 DW in the peels and pulps, respectively), a xanthone glycoside with possible applications in auto-immune diseases (e.g., psoriasis, rheumatoid arthritis and dermatitis) [37], was lower than the results presented by Vithana et al. (2019) [34] in the pulp (13 µg·g−1 DW) and in the peel (194 µg·g−1 ) of the mango “Kensington Pride” in the extracts obtained with 80% methanol. Overall, it could be observed that the variety “Kent” exceled in the concentration of mangiferin as compared with the other varieties. As expected, the extraction technique affected the phenolic profile of the mango fractions, in which higher contents of phenolic compounds were determined in extracts performed with organic solvents than the content in extracts performed with SC-CO2 . Hence, SC-CO2 extraction led to a significantly lower yield of phenolics. The total compounds were between 48.01 and 122.76 µg·g−1 DW in the peel extracts, while for the pulp this yield was between 28.80 µg·g−1 DW and 29.45 µg·g−1 DW. In general, the quantification of phenolics in peels was between 16% and 68% lower when SC-CO2 was used as solvent, while in pulp the extraction with SC-CO2 it was significantly higher (40% to 47%). The trends in the different extracts remained similar to the ones obtained in the extracts obtained with organic solvents. Moreover, no specificity for the compounds was found towards the extraction method used. According to de O. Silva et al. (2019) [38], the differences evidenced in the yield between the two extraction methods were mainly due to the polarity of the extraction media, meaning that higher amounts of phenolics were dragged out to the solvent. Moreover, the high phenolic contents obtained with organic solvents may be due to the better interaction, in the form of hydrogen bonds, on the polar sites of the phenolics with the solvent. Additionally, it has been shown that interferences could be avoided by additional sample purification procedures for the exclusion of non-target substances (e.g., terpenes, Foods 2021, 10, 2201 19 of 22 vitamins, pigments and fats) [39]. In general, data on the phenolic composition of mango extracts obtained by SC-CO2 are limited. 3.5.3. Antioxidant Activity As can be seen in Table 4, the antioxidant activity of the extracts, as determined by the DPPH· and FRAP essays, is influenced by both the type of extraction as well as the by-product considered. The two assays show an opposite response, which relates to their methodology. With increasing antioxidant activity, in DPPH·, more radicals are neutralized, resulting in a lower absorbance. Conversely, in FRAP, more Fe3+ is reduced to the colored Fe2+ complex, giving a higher absorbance. Table 4. Antioxidant activity determined by the DPPH· and FRAP methods. DPPH [µg·mL−1 ] FRAP [µmol Trolox g−1 ] Kent peel CE * SFE ** 0.63 ± 0.00 a 5.68 ± 0.08 b 10.89 ± 0.27 a 2.46 ± 0.15 b Kent pulp CE SFE 7.97 ± 0.24 a 3.68 ± 0.08 b 2.60 ± 0.15 a 2.06 ± 0.02 b Haden peel CE SFE 0.64 ± 0.02 a 1.34 ± 0.03 b 17.45 ± 0.13 a 4.25 ± 0.16 b Haden pulp CE SFE 6.11 ± 0.27 a 17.36 ± 0.63 b 2.09 ± 0.03 a 0.91 ± 0.02 b * CE = conventional extraction. ** SFE = supercritical fluid extraction (20% EtOH, 55 ◦ C, 35 MPa). Values are mean ± relative difference (n = 2). Means with different letters in the same column indicate statistically significant differences in the antioxidant activity as function of the extraction method measured by the same technique (p < 0.05). Both methods indicate a higher antioxidant activity in peel extracts, with a value between 0.63 and 5.68 µg·mL−1 for DPPH, and 2.46 and 17.45 µmol Trolox·g−1 for FRAP, depending on the extraction methodology. The pulp extracts possessed systematically lower antioxidant activities with values ranging between 3.68 and 17.36 µg·mL−1 for DPPH, and 0.91 µmol and 2.60 µmol Trolox·g−1 for FRAP. The antioxidant activity was systematically higher for “Haden” as compared to the “Kent” extracts, which can be attributed to their higher concentration in phenolics and carotenoids (Table 2, Figure 5). Studies have demonstrated that the antioxidant activity of vegetal materials is due to the interaction of carotenoids and polyphenols [40]. As discussed above, catechin, gallic acid and isoquercitrin were found to be the main polyphenols in the two mango varieties. Regression analysis between their concentration and the observed antioxidant activity showed a correlation of 0.88, 0.82 and 0.71, respectively, suggesting their importance to the observed antioxidant activity. This is in agreement with other studies where the antioxidant activity of mango was primarily attributed to the presence of gallic acid and flavonoids [41,42]. Through their chelating properties, these compounds likely reduce the availability of metal ions by the formation of metal complexes that otherwise would be involved in the formation of radicals [43]. As for carotenoids, mostly β-carotene in the mango extracts, their scavenging activity is attributed to the double bonds on the molecular structure that are expected to act as effective quenchers [44]. SFE extracts have in general lower antioxidant activity as compared to the extracts obtained via the more complex solvent extraction system. The latter is based on two successive steps involving solvents with a range of different polarities, possibly reducing biomass interactions, (e.g., the weak non-covalent interaction between plant proteins and phenolic compounds) [45], leading to a more complete extraction. Moreover, other compounds with antioxidant activity could have been co-extracted. However, the large solvent use and associated complex solvent recovery makes the conventional extraction, as it was performed in this research, not suitable for upscaling. Conversely, the more Foods 2021, 10, 2201 20 of 22 environmentally friendly SFE based on a single step with integrated SC-CO2 recovery has been shown to be scalable for the extraction of plant-origin bioactive compounds and is considered to be a promising alternative. 4. Conclusions Through the results delivered in the present study, it was possible to evaluate the effect of process parameters on the extraction of carotenoids and phenolics from mango byproducts via supercritical CO2 extraction. The modeling of the extraction behavior for the main carotenoids was presented as part of the overall assessment for a complete biorefinery process of the by-products. As expected, β-carotene was the main carotenoid in the mango matrix, while the phenolic composition was diverse depending on the conditions applied for the extraction. The modification of CO2 with 20% ethanol and with mild and high temperatures resulted in a higher extraction yield of carotenoids and phenolics, respectively. Nevertheless, the extraction yield and antioxidant activity were lower when compared with conventional extraction techniques with organic solvents. Overall, the outcomes of this study show the potential of SC-CO2 modified with ethanol for the recovery of bioactive compounds from mango by-products as a first step of a complete green biorefinery process. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/foods10092201/s1, material SS.1: Cumulative SC-CO2 extraction kinetics of carotenoids from mango at 55 ◦ C and 35 MPa with ethanol and acetone as co-solvents, material SS.2: Extraction yield obtained under the different experimental combinations performed for the optimization process, material SS.3: Statistical analysis of the models applied for the optimized extraction of carotenoids from mango with SC-CO2 modified with EtOH or acetone, material SS.4: Carotenoid analysis via UPLC. Author Contributions: M.U., J.V.C. and E.V.; Data curation, S.V. and K.E.; Formal analysis, S.V.; Funding acquisition, J.V.C.; Investigation, J.V.-C.; Methodology, M.U.; Project administration, J.R., J.V.C. and K.E.; Supervision, K.E.; Validation, J.V.-C.; Writing—original draft, J.V.-C.; Writing—review and editing, J.R., J.V.C. and K.E. All authors have read and agreed to the published version of the manuscript. Funding: This work was performed within the frame of the TEAM EC2017TEA442A103 VLIR-UOS project “Improving Ecuadorian child nutrition by using mango by-products as potential sources of bioactive compounds” and the PIMI 15-05 from EPN “Evaluación de la biorefinería de subproductos del procesamiento de alimentos para la obtención de compuestos de interés industrial”. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this article are available upon reasonable request, from the corresponding author. Acknowledgments: J.V.-C. wants to acknowledge the quality technical support of Samara Fernández de Souza and Miranda Maesen from VITO. Conflicts of Interest: The authors declare no conflict of interest. References 1. 2. 3. Villacís-Chiriboga, J.; Elst, K.; Van Camp, J.; Vera, E.; Ruales, J. 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https://openalex.org/W4392930886	https://ejurnal.seminar-id.com/index.php/josyc/article/download/4921/2596	Indonesian	null	IOT Rancang Bangun Alat Pengusir Hama Burung pada Padi Sawah Petani Berbasis Internet of Things (IoT)	Journal of Computer System and Informatics/Journal of Computer System and Informatics (JoSYC)	2,024	cc-by	6,623	Rancang Bangun Alat Pengusir Hama Burung pada Padi Sawah Petani Berbasis Internet of Things (IoT) Imam Sufaat, Juliandri Sains dan Teknologi, Sistem Komputer, Universitas Pembangunan Panca Budi, Medan, Indonesia Email: 1,imamsufaat01@gmail.com, 2andri@dosen.pancabudi.ac.id Correspondence Author Email: imamsufaat01@gmail.com Submitted: 07/02/2024; Accepted: 19/02/2024; Published: 20/02/2024 Abstrak−Penelitian ini bertujuan untuk mengembangkan alat pengusir hama burung yang efektif dan efisien pada sawah petani dengan memanfaatkan teknologi Internet of Things (IoT). Hama burung seringkali menjadi ancaman serius bagi hasil pertanian, terutama pada lahan sawah. Melalui penerapan IoT, alat ini dapat memantau secara real-time aktivitas burung di sekitar area pertanian dan mengambil tindakan pencegahan yang tepat.Alat ini dirancang dengan memanfaatkan sensor gerak atau sesnsor pir, kamera pengintai, dan sistem pengiriman sinyal suara atau visual untuk mengusir burung yang mendekati area pertanian. Sensor PIR banyak digunakan dalam berbagai aplikasi, termasuk sistem keamanan rumah,lampu sensor gerak,danotomatisasi bangunan, Keuntungan utama dari sensor Pir adalah konsumsi daya yang rendah dan kemampuan untuk memberikan respon yang cepat terhadap gerakan, menjadikannya pilihan yang efesien dan efektif untuk banyak aplikasi deteksi gerak, Data yang terkumpul dari sensor dan kamera akan dikirim ke platform IoT dengan aplikasi Telegram dan yang terhubung melalui koneksi internet, memungkinkan petani untuk memantau dan mengendalikan alat secara jarak jauh melalui perangkat pintar seperti smartphone atau komputer. Keunggulan utama dari alat ini adalah kemampuannya untuk memberikan respons cepat terhadap kehadiran hama burung, mengurangi kerugian hasil pertanian dan meningkatkan produktivitas tanaman. Selain itu, integrasi dengan IoT memberikan fleksibilitas dan keterhubungan yang tinggi, memungkinkan pengoptimalan sistem secara terus-menerus. perancangan alat ini melibatkan tahap desain perangkat keras dan perangkat lunak. Perangkat keras terdiri dari sensor pir, mikrokontroler esp 32 cam, dan pengeras suara dan servo. Sementara perangkat lunak terdiri dari arduino ide, blynk dan telegram. Setelah desain, alat ini diuji di lapangan untuk kinerjanya. Hasil pengujian menunjukkan bahwa alat ini mampu mendeteksi kehadiran burung dengan akurasi baik, memberikan respon yang cepat dalam mengusir mereka, telegram mampu berkomunikasi dengan dapat memeberi perintah dan menerima gambar, blynk dapat memberikan informasi keadaan alat saat online dan memeberi perintah untuk mengaktifkan servo, suara mauapun keduanya. Dengan tingkat keberhasilan pengusiran yang signifikan, alat ini berhasil mengurangi kerugian yang disebabkan oleh serangan burung dalam pertanian. Selain itu, alat ini memiliki konsumsi daya yang rendah dan dapat terintegrasi dengan platform IoT yang sudah ada untuk pemantauan jarak jauh dan analisis data. Penelitian ini diharapkan dapat memberikan kontribusi positif dalam upaya pengembangan teknologi pertanian yang berkelanjutan dan meningkatkan kesejahteraan petani melalui perlindungan hasil pertanian dari serangan hama burung. Kata Kunci: Hama Burung; IoT (Internet of Things); Blynk; Telegram; Sensor Pir Kata Kunci: Hama Burung; IoT (Internet of Things); Blynk; Telegram; Sensor Pir Abstract−This research aims to develop an effective and efficient bird pest repellent tool in farmers' rice fields by utilizing Internet of Things (IoT) technology. Bird pests often pose a serious threat to agricultural products, especially in rice fields. Through the application of IoT, this tool can monitor real-time bird activity around agricultural areas and take appropriate preventive measures. This tool is designed to utilize motion sensors or pir sensors, surveillance cameras, and a sound or visual signal delivery system to chase away birds. approaching agricultural areas. PIR sensors are widely used in a variety of applications, including home security systems, motion sensor lighting, and building automation. The main advantages of PIR sensors are their low power consumption and ability to provide fast response to movement, making them an efficient and effective choice for many detection applications. motion, data collected from sensors and cameras will be sent to the IoT platform with the Telegram application and which is connected via an internet connection, allowing farmers to monitor and control tools remotely via smart devices such as smartphones or computers. The main advantage of this tool is its ability to provide a quick response to the presence of bird pests, reduce agricultural yield losses and increase crop productivity. Additionally, integration with IoT provides high flexibility and connectivity, enabling continuous system optimization. designing this tool involves the hardware and software design stages. The hardware consists of a pir sensor, an esp 32 cam microcontroller, and a speaker and servo. While the software consists of Arduino Ide, Blynk and Telegram. After design, the tool is field tested for performance. Test results show that this tool is able to detect the presence of birds with good accuracy, provides a fast response in chasing them away, Telegram is able to communicate by giving commands and receiving images, Blynk can provide information on the condition of the tool when it is online and give commands to activate the servo, sound or both of them. With a significant removal success rate, this tool is successful in reducing losses caused by bird strikes in agriculture. Additionally, it has low power consumption and can be integrated with existing IoT platforms for remote monitoring and data analysis. Kata Kunci: Hama Burung; IoT (Internet of Things); Blynk; Telegram; Sensor Pir It is hoped that this research can make a positive contribution to efforts to develop sustainable agricultural technology and improve farmers' welfare by protecting agricultural products from bird pest attacks. Thus, it is hoped that the implementation of this tool in the field can provide an innovative and efficient solution in controlling bird pests in farmers' rice fields. Keywords: Bird Pests; IoT (Internet of Things); Blynk; Telegram; Pir Sensor Keywords: Bird Pests; IoT (Internet of Things); Blynk; Telegram; Pir Sensor Journal of Computer System and Informatics (JoSYC) ISSN 2714-8912 (media online), ISSN 2714-7150 (media cetak) Volume 5, No. 2, February 2024, Page 306-314 https://ejurnal.seminar-id.com/index.php/josyc DOI 10.47065/josyc.v5i2.4921 Journal of Computer System and Informatics (JoSYC) ISSN 2714-8912 (media online), ISSN 2714-7150 (media cetak) Volume 5, No. 2, February 2024, Page 306-314 https://ejurnal.seminar-id.com/index.php/josyc DOI 10.47065/josyc.v5i2.4921 Keywords: Bird Pests; IoT (Internet of Things); Blynk; Telegram; Pir Sensor Rancang Bangun Alat Pengusir Hama Burung pada Padi Sawah Petani Berbasis Internet of Things (IoT) Dengan demikian, implementasi alat ini di lapangan diharapkan dapat memberikan solusi inovatif dan efisien dalam pengendalian hama burung pada sawah petani. Kata Kunci: Hama Burung; IoT (Internet of Things); Blynk; Telegram; Sensor Pir 1. PENDAHULUAN Dalam konteks ini, penting untuk memahami bahwa alat pengusir hama burung berbasis IoT tidak hanya bersifat proaktif dalam mendeteksi dan mengusir burung[15], tetapi juga dapat memberikan informasi secara real-time kepada petani. Melalui pemanfaatan sensor gerak dan kamera pengintai atau sensor pir, alat ini dapat memonitor aktivitas burung di sekitar lahan pertanian secara akurat[16]. Data yang terkumpul dapat diakses oleh petani melalui perangkat pintar mereka, memungkinkan pengawasan yang efisien dan pengambilan keputusan yang cepat[17]. Sensor PIR (Passive Infrared) adalah perangkat deteksi yang mendeteksi perubahan radiasi inframerah di sekitarnya. Radiasi inframerah ini dipancarkan oleh objek atau tubuh yang memiliki suhu di atas suhu mutlak nol (0 Kelvin), yang berarti semua objek dengan suhu di atas nol mutlak dapat memancarkan energi inframerah. Sensor PIR bekerja berdasarkan prinsip ini untuk mendeteksi perubahan suhu yang disebabkan oleh gerakan. Sensor pir memiliki lensa khusus yang memfokuskan radiasi inframerah dari objek di area pemantauan ke elemen dektor di dalam sensor, Ketika adanya perubahan suhu dan gerak. Dan sensor PIR biasanya memiliki dua elemen dektor atau yang terbuat dari bahan piroelektrik, Elemen ini menghasilkan tegangan saat terjadi perubahan suhu[18], setelah elemen detektor mendeteksi perubahan suhu , sinyalnya diolah oleh rangkaian elektronik di dalam sensor, Pemprosesan membantu mengindentifikasikan pola gerak dan memastikan bahwa hanya gerak yang relevan yang di anggap sebagai input yang valid, Sensor PIR banyak digunakan dalam berbagai aplikasi, termasuk sistem keamanan rumah,lampu sensor gerak,danotomatisasi bangunan[19], Keuntungan utama dari sensor Pir adalah konsumsi daya yang rendah dan kemampuan untuk memberikan respon yang cepat terhadap gerakan, menjadikannya pilihan yang efesien dan efektif untuk banyak aplikasi deteksi gerak. Beberapa penelitian sebelumnya telah menggunakan sensor gerak dan suara untuk medeteksi kehadiran burung dan mengusirnya[20]. Namun, penelitian ini merupakan pengembangan teknologi dengan penggunaan teknologi sensor di kombinasikan dengan perangkat lunak yang mengoptimalkan fungsi alat, baik secara otomatis dan dapat di control secara realtime dengan penggunaan jaringan internet. Penggunaan teknologi IoT dalam konteks pertanian tidak hanya menghadirkan manfaat dalam peningkatan produktivitas, tetapi juga memberikan dampak positif terhadap keberlanjutan lingkungan. Dengan kemampuan alat pengusir hama burung berbasis IoT untuk mengoptimalkan penggunaan sumber daya, seperti air dan pupuk, dapat membantu mengurangi jejak lingkungan dan meningkatkan efisiensi pengelolaan lahan pertanian[21]. Pentingnya pengembangan merupakan solusi yang dapat diandalkan dan mudah diimplementasikan oleh petani menjadi fokus utama dalam penelitian ini. Alat yang dirancang harus dapat diintegrasikan dengan mudah ke dalam praktik pertanian sehari-hari tanpa menimbulkan kompleksitas atau biaya yang tinggi. 1. PENDAHULUAN Pertanian sebagai tulang punggung perekonomian banyak negara seringkali dihadapkan pada berbagai Copyright © 2024 the auhor, Page 306 This Journal is licensed under a Creative Commons Attribution 4.0 International License Journal of Computer System and Informatics (JoSYC) ISSN 2714-8912 (media online), ISSN 2714-7150 (media cetak) Volume 5, No. 2, February 2024, Page 306-314 https://ejurnal.seminar-id.com/index.php/josyc DOI 10.47065/josyc.v5i2.4921 tantangan[1], salah satunya adalah permasalahan serangan hama burung yang dapat merugikan hasil panen petani[2]. Sawah sebagai lahan pertanian utama, sering menjadi target empuk bagi ribuan burung yang mencari makanan di antara tanaman yang subur[3]. Serangan ini dapat mengakibatkan kerugian besar bagi para petani, menyebabkan penurunan hasil panen dan dampak ekonomi yang signifikan[4]. Laporan kegagalan panen di Mojokerto tahun 2019 hampir mencapai ratusan kilogram (Surya Co.id,2019). Di Kota Banjar, puluhan hektar padi gagal panen akibat serangan burung pipit (Supendi 2019)[5]. Puluhan hektare tanaman padi di kawasan tersebut rusak akibat serangan hama burung pipit, bahkan ada yang sampai gagal panen dan harus diganti dengan tanaman lain. (ANTARA FOTO/Siswowidodo 2022) Dalam upaya untuk mengatasi permasalahan ini, muncul sebuah inovasi yang menjanjikan: "Rancangan Bangun Alat Pengusir Hama Burung pada Sawah Petani Berbasis Internet of Things (IoT)[6]." Internet of Things (IoT) adalah istilah yang baru-baru ini banyak ditemukan tetapi hanya sedikit yang mengerti arti istilah ini[7]. Internet of Things secara umum dapat diartikan sebagai benda di sekitar kita yang dapat berkomunikasi satu sama lain melalui internet[8]. Internet of Things memiliki konsep memperluas manfaat yang terhubung dalam koneksi internet terus menerus[9] .IoT merupakan konsep di mana objek atau perangkat di sekitar kita dapat saling terhubung dan bertukar informasi melalui jaringan internet[10]. Saat ini, dunia tengah menyaksikan lonjakan penggunaan teknologi tingkat tinggi dalam berbagai sektor kehidupan, dan pertanian bukanlah pengecualian. Perkembangan teknologi Internet of Things (IoT) menjadi kunci untuk meningkatkan efisiensi dan produktivitas dalam berbagai aspek pertanian[11], termasuk pengendalian hama burung. Penerapan teknologi IoT dalam rancangan alat pengusir hama burung bertujuan untuk memberikan solusi yang lebih cerdas dan responsif terhadap tantangan yang dihadapi oleh petani. Hama burung bukan hanya merupakan ancaman terhadap hasil panen, tetapi juga menjadi faktor yang dapat menghancurkan mata pencaharian petani[12]. Dengan demikian, pengembangan alat pengusir hama burung yang cerdas dan berbasis teknologi menjadi suatu kebutuhan mendesak untuk meningkatkan ketahanan pertanian[13]. Penelitian ini menggali potensi solusi inovatif yang dapat memberikan solusi yang lebih efektif dan efisien dalam mengatasi permasalahan serangan hama burung di sawah petani[14]. Copyright © 2024 the auhor, Page 307 This Journal is licensed under a Creative Commons Attribution 4.0 International License 2.1 Tahapan Penelitian Penelitian ini akan dilaksanakan dengan mengikuti serangkaian metode penelitian yang cermat dan sistematis untuk dengan konsep Internet of Things (IoT) menjadi inti dari seluruh proses. IoT merujuk pada jaringan perangkat fisik yang terhubung ke internet dan dapat saling berkomunikasi dan berinteraksi. Tahap Penelitian ini mencakup beberapa tahapan yang akan dilakukan secara berurutan seperti terlihat pada gambar 1 berikut. Gambar 1. Kerangka Tahapan Penelitian Gambar 1. Kerangka Tahapan Penelitian Dari gambar 1 tersebut dapat di jelaskan sebagai berikut: a. Studi Literatur Penelitian: Akan dimulain dengan studi literatur untuk mendapatkan pemahaman yang mendalam tentang perkembangan terkini dalam teknologi Internet Of Things (IOT) yang dapt di terapkan dalam Pertanian, khususnya dalam pengendalian hama Burung. Informasi yang diperoleh dari literatur akan menjadi dasar bagi perancangan alat yang inofatif dan sesuai dengan kebutuhan petani. j g p g y g g p b. Analisis kebutuhan dan Kendala: Indentifikasi kebutuhan dan kendala dari perfektif petani akanmenjadi langkah selanjutnya. Survei dan wawancara dengan petani lokal akan dilakukan untuk memahami masalah seputar serangan hama burung dan kendala yang mereka hadapi dalam penggunaan teknologi. b. Analisis kebutuhan dan Kendala: Indentifikasi kebutuhan dan kendala dari perfektif petani akanmenjadi langkah selanjutnya. Survei dan wawancara dengan petani lokal akan dilakukan untuk memahami masalah seputar serangan hama burung dan kendala yang mereka hadapi dalam penggunaan teknologi. c. Perancangan Konsep: berdasarkan literatur dan analisis kebutuhan, konsep alat pengusir hama burung yang akan dirancang. Ini mencakup sensor-sensor yang sesuai,sistem pemantauan, dan mekanisme pengusiran yang efektif. Konsep akan disusun dan mempertimbangkan aspek ketersediaan sumber daya dan kemudahan implamentasi di lapangan. c. Perancangan Konsep: berdasarkan literatur dan analisis kebutuhan, konsep alat pengusir hama burung yang akan dirancang. Ini mencakup sensor-sensor yang sesuai,sistem pemantauan, dan mekanisme pengusiran yang efektif. Konsep akan disusun dan mempertimbangkan aspek ketersediaan sumber daya dan kemudahan implamentasi di lapangan. d. Pengembangan Prototipe: Prototipe dari alat pengusir hama burung akan dikembangkan berdasarkan konsep yang telah dirancang. Pengembangan ini melibatkanprogram perangkat lunak, Pengintegrasian sensor-sensor IoT, dan perakitan peragkat keras, Prototipe ini akan diuji secara berkala untuk memastikan kinerjanya sesuai dengan harapan. d. Pengembangan Prototipe: Prototipe dari alat pengusir hama burung akan dikembangkan berdasarkan konsep yang telah dirancang. Pengembangan ini melibatkanprogram perangkat lunak, Pengintegrasian sensor-sensor IoT, dan perakitan peragkat keras, Prototipe ini akan diuji secara berkala untuk memastikan kinerjanya sesuai dengan harapan. e. Uji Coba Lapangan: Setelah prototipe berhasil, alat akan di ujicobakan secara langsung dilapangan sawah petani. 2.1 Tahapan Penelitian Uji coba ini akan memberikan pemahaman tentang efektivitas alat dalam mengusir hama burung, daya tahan terhadap kondisi lingkungan pertanian, dan kemudahan penggunaan oleh petani. f. Analisis Data: Data yang diperoleh dari uji coba lapangan akan dianalisis untuk mengevaluasi kin Hal yang akan mencakup efektivitas pengusiran burung, respon waktu, dan keandalan sistem keselur g. Pengembangan Lanjutan: berdasarkan hasil analisis, prototipe akan perbaiki dan ditingkatkan untuk memenuhi standart yang diinginkan. Langkah ini dapat melibatkan perubahan disain, penambahan fitur, atau umpan balik dari petani. p p h. Penyusunan Laporan Penelitian: seluruh proses penelitian dan hasilnya akan disusun dalam bentuk laporan penelitian. Laporan ini akan mencakup deksripsi rinci tentang Metodologi , Temuan, dan Implementasi dari rancangan dari alat pengusir hama burung berbasis IoT. h. Penyusunan Laporan Penelitian: seluruh proses penelitian dan hasilnya akan disusun dalam bentuk laporan penelitian. Laporan ini akan mencakup deksripsi rinci tentang Metodologi , Temuan, dan Implementasi dari rancangan dari alat pengusir hama burung berbasis IoT. Dengan mengikuti serangkaian metode penelitian ini, diharapkan penelitian ini akan memberikan kontribusi nyata terhadap pengembangan teknologi pertanian yang berkelanjutan dan membantu petani mengatasi masalah hama burung yang ada di sawah mereka. p p p p g g , , p rancangan dari alat pengusir hama burung berbasis IoT. Dengan mengikuti serangkaian metode penelitian ini, diharapkan penelitian ini akan memberikan kontribusi nyata terhadap pengembangan teknologi pertanian yang berkelanjutan dan membantu petani mengatasi masalah hama burung yang ada di sawah mereka. g p g g Dengan mengikuti serangkaian metode penelitian ini, diharapkan penelitian ini akan memberikan kontribusi nyata terhadap pengembangan teknologi pertanian yang berkelanjutan dan membantu petani mengatasi masalah hama burung yang ada di sawah mereka. Dalam setiap tahap pengembangan solusi IoT, peran dan konsep IoT sangat penting karena sistem ini memungkinkan koneksi dan komunikasi antara perangkat fisik dan sistem terpusat. Hal ini memungkinkan untuk mendapatkan wawasan yang berharga dari data yang dikumpulkan oleh perangkat IoT dan mengambil tindakan yang diperlukan berdasarkan informasi tersebut. 1. PENDAHULUAN Oleh karena itu, penelitian ini berupaya memberikan kontribusi nyata dalam menciptakan solusi praktis yang dapat meningkatkan kesejahteraan petani dan keberlanjutan pertanian dengan menggunakan sensor gerak atau sensor pir. Dengan latar belakang tersebut, penelitian ini bertujuan untuk menjembatani kesenjangan antara perkembangan teknologi tinggi dan kebutuhan petani di lapangan. Melalui penerapan alat pengusir hama burung berbasis IoT, diharapkan dapat diciptakan lingkungan pertanian yang lebih aman, produktif, dan berkelanjutan. Dengan begitu, penelitian ini tidak hanya menjadi sumbangan ilmiah, tetapi juga menjadi jawaban atas tantangan Copyright © 2024 the auhor, Page 307 This Journal is licensed under a Creative Commons Attribution 4.0 International License Journal of Computer System and Informatics (JoSYC) ISSN 2714-8912 (media online), ISSN 2714-7150 (media cetak) Volume 5, No. 2, February 2024, Page 306-314 https://ejurnal.seminar-id.com/index.php/josyc DOI 10.47065/josyc.v5i2.4921 Journal of Computer System and Informatics (JoSYC) ISSN 2714-8912 (media online), ISSN 2714-7150 (media cetak) Volume 5, No. 2, February 2024, Page 306-314 https://ejurnal.seminar-id.com/index.php/josyc DOI 10.47065/josyc.v5i2.4921 nyata yang dihadapi oleh para petani dalam menjaga ketahanan pangan dan keberlanjutan pertanian di masa depan. py g , g This Journal is licensed under a Creative Commons Attribution 4.0 International License 2.2 Diagram Blok Dalam penelitian ini, alat pengusir hama burung pada padi sawah petani menggunakan berbasis internet of things secara keseluruhan diperlihatkan pada gambar 2. Copyright © 2024 the auhor, Page 308 py g g This Journal is licensed under a Creative Commons Attribution 4.0 International License Journal of Computer System and Informatics (JoSYC) ISSN 2714-8912 (media online), ISSN 2714-7150 (media cetak) Volume 5, No. 2, February 2024, Page 306-314 https://ejurnal.seminar-id.com/index.php/josyc DOI 10.47065/josyc.v5i2.4921 Gambar 2. Diagram Blok Journal of Computer System and Informatics (JoSYC) ISSN 2714-8912 (media online), ISSN 2714-7150 (media cetak) Volume 5, No. 2, February 2024, Page 306-314 https://ejurnal.seminar-id.com/index.php/josyc DOI 10.47065/josyc.v5i2.4921 Journal of Computer System and Informatics (JoSYC) ISSN 2714-8912 (media online), ISSN 2714-7150 (media cetak) Volume 5, No. 2, February 2024, Page 306-314 https://ejurnal.seminar-id.com/index.php/josyc DOI 10.47065/josyc.v5i2.4921 Journal of Computer System and Informatics (JoSYC) l of Computer System and Informatics (JoSY p y ISSN 2714-8912 (media online), ISSN 2714-7150 (m Volume 5, No. 2, February 2024, Page 306-314 https://ejurnal.seminar-id.com/index.php/josyc DOI 10.47065/josyc.v5i2.4921 Gambar 2. Diagram Blok Dengan gambar 2 diagram blok, dapat melihat setiap komponen saling berhubungan dalam sistem, bagaimana informasi dan instruksi aplikasi blynk atau telegram mengalir dari internet ke mikrokontroler, kemudian ke sensor pir, dan akhirnya menuju ke perangkat fisik seperti servo dan speaker untuk menghasilkan respons yang diinginkan. Metode penelitian dan pengembangan dipahami sebagai metode penelitian yang digunakan untuk membuat alat tertentu dan melakukan percobaan untuk melihat efektivitas alat yang dipelajari. Untuk dapat menciptakan alat yang diinginkan digunakan penelitian yaitu analisis kebutuhan dan evaluasi efektivitas alat agar efektif dan bermanfaat bagi masyarakat. 3. HASIL DAN PEMBAHASAN Copyright © 2024 the auhor, Page 309 This Journal is licensed under a Creative Commons Attribution 4.0 International License 3. HASIL DAN PEMBAHASAN gkat Keras kaian alat ditunjukkan pada Gambar 3. Gambar 3. Rangkaian 2.3 Index serangan hama burung pada padi Waktu serangan hama burung pada tanaman padi dapat bervariasi tergantung pada beberapa faktor seperti jenis burung, Hama burung menyerang mulai pagi hingga jam 10.00 WIB, selanjutnya pukul 15.00 WIB sampai matahari tenggelam. Pada siang hari cenderung cuacanya panas, sehinga burung relatif tidak ada yang menyerang tanaman padi. fase pertumbuhan tanaman padi, musim, dan wilayah geografis. Beberapa burung dapat menjadi hama pada berbagai tahap pertumbuhan tanaman padi. Berikut adalah beberapa contoh waktu umum ketika serangan burung dapat terjadi: 1. Fase Pertumbuhan Vegetatif: Beberapa burung, seperti burung padi (Estrildidae), dapat menyerang tanaman padi pada fase pertumbuhan vegetatif. Pada saat ini, burung dapat mencari biji-bijian atau bagian tanaman yang lezat. g Beberapa burung, seperti burung padi (Estrildidae), dapat menyerang tanaman padi pada fase pertu vegetatif. Pada saat ini, burung dapat mencari biji-bijian atau bagian tanaman yang lezat. g rapa burung, seperti burung padi (Estrildidae), dapat menyerang tanaman padi pada fase pertumbuhan atif. Pada saat ini, burung dapat mencari biji-bijian atau bagian tanaman yang lezat. 2. Fase Pembentukan Malai dan Bulir: Serangan burung pada fase ini dapat menjadi lebih signifikan karena burung dapat tertarik pada biji-bijian yang mulai terbentuk. 2. Fase Pembentukan Malai dan Bulir: Serangan burung pada fase ini dapat menjadi lebih signifikan karena burung dapat tertarik pada biji-bijian yang mulai terbentuk. 3 Pematangan Bulir dan Panen: 3. Pematangan Bulir dan Panen: 3. Pematangan Bulir dan Panen: Pada tahap ini, burung dapat merusak tanaman padi untuk mencapai biji-bijian yang sudah matang. 4 Musim Tanam dan Musim Panen: g Pada tahap ini, burung dapat merusak tanaman padi untuk mencapai biji-bijian yang sudah matang. 4. Musim Tanam dan Musim Panen: 4. Musim Tanam dan Musim Panen: Aktivitas burung pada tanaman padi dapat meningkat selama musim tanam dan musim panen ketika tanaman dan biji-bijian menjadi lebih mudah diakses. Aktivitas burung pada tanaman padi dapat meningkat selama musim tanam dan musim panen ketika tanaman dan biji-bijian menjadi lebih mudah diakses. 3.2 Perakitan Alat Hubungkan mikrokontroler Esp 32 Cam pada Sensor Pir, Motor Servo, Speaker, masukkan komponen kedalam box penempatan alat, box di pasang dengan baut pada tiang setinggi 1,2 meter atau lebih tinggi dari padi sawah, hubungkan motor servo pada tali yang di ikatkan pada kaleng bekas .Yang terahir hubungkan ke Baterai. Gambar 4. Alat Setelah di rakit Gambar 4. Alat Setelah di rakit Pada gambar 4 dapat di lihat bahwa seluruh komponen di pasang pada tiang tripod 3 kaki yang menyesuaikan pada tinggi padi sawah dan keadaan sawah agar dapat berdiri tegak dan seluruh dapat alat bekerja dengan baik 3.1 Perancangan Perangkat Keras Dalam penelitian ini rangkaian alat ditunjukkan pada Gambar 3. Gambar 3. Rangkaian py g g This Journal is licensed under a Creative Commons Attribution 4.0 International License Journal of Computer System and Informatics (JoSYC) ISSN 2714-8912 (media online), ISSN 2714-7150 (media cetak) Volume 5, No. 2, February 2024, Page 306-314 https://ejurnal.seminar-id.com/index.php/josyc DOI 10.47065/josyc.v5i2.4921 Pada gambar 3 rangkaian di atas dapat di lihat bawha Sensor pir sebagai pendeteksi keberadaan burung, motor servo sebagai pergerakan tali yang diikatkan ke kaleng bekas, ESP 32 CAM Sabagai Mikrokontroler dan untuk mengambi gambar, baterai Sebagai sumber daya listrik pada komponen, DF player untuk memutar audio dari SD Card ke Loudspeaker / Toa sebagai pengeras suara. Ada beberapa hardware atau perangkat keras yang dimafaatkan dalam penelitian ini, yaitu: Sensor PIR, Esp 32 Cam, Servo motor, DF Player, Speaker, dan Baterai. Sensor PIR yang akan ditaruh pada alat dan menghadap kepada padi. Gunanya peletakan ini adalah mendeteksi hama dan akan dihubungkan pada pin-pin Esp 32 Cam. Pada esp 32 cam sebagai mikrokontroller yang memperoleh informasi dari sensor PIR, kemudian diteruskan ke servo motor dan Speaker yang akan menjadi arus jalannya sistem. Dan terakhir perancangan pada servo motor dan speaker yang juga akan diletakkan pada kaki penyangga. Motor servo akan bergerak menarik tali yang di ikat pada benang yang membuat gerakan menghalau hama, dan speaker mengeluarkan suara sebagai penakut hama burung. 3.3 Perancangan Perangkat Lunak Ada beberapa software atau perangkat lunak yang digunakan dalam dalam peneliatian ini, yaitu Arduino Ide, Telegram dan Blynk. Arduino IDE di gunakan untuk memberikan program sesuai keinginan pada mikrokontroler esp 32 cam, telegram di gunakan untuk memberikan perintah foto, menerima gambar dan memberikan informasi waktu kepada klien saat hama burung tiba, Blynk di gunakan untuk memberikan infomasi tentang keadaan alat online atau tidak, dan dapat memberikan perintah seperti menghidupkan motor servo, speaker dan keduanya seara bersamaan. 3.4 Implementasi Alat Implementasi alat pengusir hama burung dapat melibatkan penggunaan teknologi IoT (Internet of Things) untuk mendeteksi dan mengusir burung. Berikut adalah implementasi alat tersebut: a. Pemrograman ESP 32 CAM, pastikan user telah mengunduh dan instal Arduino IDE dari situs resmi Arduino. Dibutuhkan juga software tambahan seperti Blynk dan Telegram yang dapat di hubungkan ke arduino sebagai media komunikasi Internet Of Things. a. Pemrograman ESP 32 CAM, pastikan user telah mengunduh dan instal Arduino IDE dari situs resmi Arduino. Dibutuhkan juga software tambahan seperti Blynk dan Telegram yang dapat di hubungkan ke arduino sebagai media komunikasi Internet Of Things. b. Network Configuration g 1. Wifi, pastikan bahwa modul WiFi sudah terhubung dengan Arduino. Setelah berhasil terhubung, alat dapat mengakses internet atau melakukan tindakan lain yang memerlukan koneksi internet. g Wifi, pastikan bahwa modul WiFi sudah terhubung dengan Arduino. Setelah berhasil terhubung, maka at dapat mengakses internet atau melakukan tindakan lain yang memerlukan koneksi internet. 2. Blynk, buat proyek baru. Tambahkan widget yang sesuai dengan kebutuhan proyek (seperti tombol, slider, grafik, dll.). Salin token autentikasi proyek yang dibuat. 2. Blynk, buat proyek baru. Tambahkan widget yang sesuai dengan kebutuhan proyek (seperti tombol, slider, grafik, dll.). Salin token autentikasi proyek yang dibuat. Copyright © 2024 the auhor, Page 310 Journal of Computer System and Informatics (JoSYC) ISSN 2714-8912 (media online), ISSN 2714-7150 (media cetak) Volume 5, No. 2, February 2024, Page 306-314 https://ejurnal.seminar-id.com/index.php/josyc DOI 10.47065/josyc.v5i2.4921 Gambar 5. Tampilan BLYNK Journal of Computer System and Informatics (JoSYC) ISSN 2714-8912 (media online), ISSN 2714-7150 (media cetak) Volume 5, No. 2, February 2024, Page 306-314 https://ejurnal.seminar-id.com/index.php/josyc DOI 10.47065/josyc.v5i2.4921 Journal of Computer System and Informatics (JoSYC) ISSN 2714-8912 (media online), ISSN 2714-7150 (media cetak) Volume 5, No. 2, February 2024, Page 306-314 https://ejurnal.seminar-id.com/index.php/josyc DOI 10.47065/josyc.v5i2.4921 l of Computer System and Informatics (JoSY Gambar 5. Tampilan BLYNK Gambar 5. Tampilan BLYNK Pada gambar 5 merupakan tampilan pada aplikasi blynk saat terhubung terhadap system sehingga menampilan status online warna hijau, lalu klien dapat memberikan perintah untuk menghidupkan motor servo atau suara maupun bersamaan. p TELEGRAM. Menggunakan Arduino IDE memberikan program kepada mikrokontoler Esp 32 Ca Token telegram klien melalui bootfahter dan chat id. Gambar 6. Tampilan Telegram Mengirim Foto Pada gambar 6 tersebut telegram terhubung dan mengirimkan kata kepada klien, menyediakan menu perintah sehingga klien bisa meminta kepada sistem untuk mengirimkan foto. Gambar 6. 3.4 Implementasi Alat Tampilan Telegram Mengirim Foto Pada gambar 6 tersebut telegram terhubung dan mengirimkan kata kepada klien, menyediakan men perintah sehingga klien bisa meminta kepada sistem untuk mengirimkan foto. Pada gambar 6 tersebut telegram terhubung dan mengirimkan kata kepada klien, menyediakan menu perintah sehingga klien bisa meminta kepada sistem untuk mengirimkan foto. Pada gambar 6 tersebut telegram terhubung dan mengirimkan kata kepada klien, menyediakan menu perintah sehingga klien bisa meminta kepada sistem untuk mengirimkan foto. p gg p g 4. Pengujian Sensor PIR Uji fungsional pada sensor PIR dilakukan serupa cara menempatkan objek rupa burung ditempatkan depan sensor PIR. Sensor tersebut akan mendetek objek muncul pada zona tersebut pada radius sensor. Pada pengujian jarak yang dipakai berbeda, bermula mendekati pada sensor hingga jauh sensor. 4. Pengujian Sensor PIR Uji fungsional pada sensor PIR dilakukan serupa cara menempatkan objek rupa burung ditempatkan depan sensor PIR. Sensor tersebut akan mendetek objek muncul pada zona tersebut pada radius sensor. Pada pengujian jarak yang dipakai berbeda, bermula mendekati pada sensor hingga jauh sensor. 4. Pengujian Sensor PIR Uji fungsional pada sensor PIR dilakukan serupa cara menempatkan objek rupa burung ditempatkan depan sensor PIR. Sensor tersebut akan mendetek objek muncul pada zona tersebut pada radius sensor. Pada pengujian jarak yang dipakai berbeda, bermula mendekati pada sensor hingga jauh sensor. Gambar 7. Pengujian Sensor PIR menggunakan meteran Gambar 7. Pengujian Sensor PIR menggunakan meteran Copyright © 2024 the auhor, Page 311 Copyright © 2024 the auhor, Page 311 This Journal is licensed under a Creative Commons Attribution 4.0 International License Copyright © 2024 the auhor, Page 311 This Journal is licensed under a Creative Commons Attribution 4.0 International License Journal of Computer System and Informatics (JoSYC) ISSN 2714-8912 (media online), ISSN 2714-7150 (media cetak) Volume 5, No. 2, February 2024, Page 306-314 https://ejurnal.seminar-id.com/index.php/josyc DOI 10.47065/josyc.v5i2.4921 Journal of Computer System and Informatics (JoSYC) ISSN 2714-8912 (media online), ISSN 2714-7150 (media cetak) Volume 5, No. 2, February 2024, Page 306-314 https://ejurnal.seminar-id.com/index.php/josyc DOI 10.47065/josyc.v5i2.4921 Gambar 7 terlihat sensor PIR bisa mendeteksi objek dari 1 sampai 5 meter. Ketika objek ditempatkan didepan sensor dari jarak sekitar 6 sampai 10 meter, sensor tidak dapat mendetek objek burung. Ketika sensor mendetek objek, maka motor servo hidup. Ketika sensor tidak mendetek maka motor servo mati. Berikut tabel 1 merupakan hasil pengujian sensor PIR. Tabel 1. Hasil Pengujian Sensor Pir Tabel 1. 3.4 Implementasi Alat Hasil Pengujian Sensor Pir No Jarak (cm) Keterangan 1 100 Terdeteksi 2 200 Terdeteksi 3 300 Terdeteksi 4 500 Terdeteksi 5 600 Tidak Terdeteksi 6 700 Tidak Terdeteksi 7 750 Tidak Terdeteksi 8 800 Tidak Terdeteksi c. Lokasi/Lahan Untuk Pengujian Alat Kriteria percobaan alat ini ialah tanaman padi yang beumur 60-90 hari. Saat tanaman padi mulai berbuah dan menguning, kemungkinan besar akan diserang oleh hama burung pipit. Dan setelah dilakukan survey di beberapa lokasi di precut sei tuan, maka didapatkan lahan sawah dengan tanaman padi yang berumur 60-90 hari yang dimiliki oleh Ibu Sutiyem. Setelah melakukan observasi, di lahan tersebut terdapat hama burung pipit. Dalam kasus ini, banyak biji-bijian padi yang hilang karena dimakan oleh hama burung pipit. Luas lahan 4800m2 menggunakan cara tradisional pembasmian hama burung yaitu penggunaan suara menggunakan kaleng bekas yang disusun dengan tali yang diikat ke tiang, kemudian petani menariknya dengan tali panjang. Yang mana cara ini sangat menguras tenaga dan membuang banyak waktu. Ibu Sutiyem beserta anaknnya bergantian untuk menjaga lahan mereka dari serangan hama burung pipit dari pagi hari sampai sore hari terkadang jika merka kelelahan mereka membayar orang untuk menjaga lahan sebesar Rp.40.000 per harinya. Ada juga yang menggunakan jaring yang tentunya banyak mengeluarkan biaya juga. Gambar 8 berikut merupakan foto hasil dari observasi lahan. c. Lokasi/Lahan Untuk Pengujian Alat Kriteria percobaan alat ini ialah tanaman padi yang beumur 60-90 hari. Saat tanaman padi mulai berbuah dan menguning, kemungkinan besar akan diserang oleh hama burung pipit. Dan setelah dilakukan survey di beberapa lokasi di precut sei tuan, maka didapatkan lahan sawah dengan tanaman padi yang berumur 60-90 hari yang dimiliki oleh Ibu Sutiyem. Setelah melakukan observasi, di lahan tersebut terdapat hama burung pipit. Dalam kasus ini, banyak biji-bijian padi yang hilang karena dimakan oleh hama burung pipit. Luas lahan 4800m2 menggunakan cara tradisional pembasmian hama burung yaitu penggunaan suara menggunakan kaleng bekas yang disusun dengan tali yang diikat ke tiang, kemudian petani menariknya dengan tali panjang. Yang mana cara ini sangat menguras tenaga dan membuang banyak waktu. Ibu Sutiyem beserta anaknnya bergantian untuk menjaga lahan mereka dari serangan hama burung pipit dari pagi hari sampai sore hari terkadang jika merka kelelahan mereka membayar orang untuk menjaga lahan sebesar Rp.40.000 per harinya. Ada juga yang menggunakan jaring yang tentunya banyak mengeluarkan biaya juga. Gambar 8 berikut merupakan foto hasil dari observasi lahan. Gambar 8. Observasi Lahan Gambar 8. Observasi Lahan 4. KESIMPULAN Dengan demikian, alat pengusir hama burung berbasis IoT ini tidak hanya efektif dalam mengurangi kerugian pertanian, tetapi juga membantu dalam menjaga keseimbangan ekosistem dan mempromosikan pertanian yang berkelanjutan. Meskipun demikian, penelitian dan pengembangan lebih lanjut masih diperlukan untuk meningkatkan kinerja dan fungsionalitas alat ini. Integrasi dengan teknologi baru seperti kecerdasan buatan atau analisis data yang lebih canggih dapat meningkatkan efisiensi dan efektivitas pengusiran burung. Selain itu, adaptasi terhadap kondisi lingkungan yang berbeda dan pemahaman yang lebih baik tentang perilaku burung juga perlu dipertimbangkan untuk mengoptimalkan kinerja alat ini dalam berbagai konteks aplikasi. 3.5 Data Hasil Pengujian Pengujian alat pengusir hama burung pipit pada tanaman padi dikerjakan pada siang hari hingga sore hari dilahan tempat pengujian. Pengujian alat mulai dari jam 12.00-17.00 hingga selesai. Pengujian alat ini terfokus kepada fungsi alat dan pengaruh gelombang suara terhadap burung pipit. Selanjutnya ialah menguji fungi semua alat apakah alat yang selesai dirakit berguna dengan baik atau ada kendala dan bagaimana perilaku burung pipit ketika diberikan gelombang suara. Tingkah lakunya terbagi menjadi tiga, yaitu, tidak diganggu, sedikit diganggu, dan burung meninggalkan lahan sawah. Berikut tabel 2 merupakan rangkuman dari hasil uji lapangan. Copyright © 2024 the auhor, Page 312 This Journal is licensed under a Creative Commons Attribution 4.0 International License Tabel 2. Hasil Uji Lapangan NO Komponen Harapan Hasil Kesimpulan 1. SENSOR PIR Dapat menampilkan kondisi sesuai dengan input dari sensor Sensor PIR dapat mendeteksi adanya pergerakan Selesai 2. ESP 32 CAM Dapat memfoto dan mengirimkan ke telegram. Alat dapat bekerja dengan fungsinya Selesai. Copyright © 2024 the auhor, Page 312 Journal of Computer System and Informatics (JoSYC) ISSN 2714-8912 (media online), ISSN 2714-7150 (media cetak) Volume 5, No. 2, February 2024, Page 306-314 https://ejurnal.seminar-id.com/index.php/josyc DOI 10.47065/josyc.v5i2.4921 NO Komponen Harapan Hasil Kesimpulan 3. Motor Servo Dapat menggerakkan tali yang dihubungkan ke kaleng bekas Alat dapat bekerja dengan fungsinya Selesai 4. Telegram Dapat Terhubung ke jaringan. Telegram terhubung dapat memberikan perintah, menerima foto, dan memberikan informasi waktu. Selesai 5. BLYNK Dapat Terhubung ke jaringan. BLYNK Terhubung dapat memberikan perintah hidupkan motor dan speaker. Selesai 6. Speaker Dapat memberikan gelombang suara. Speaker mengeluarkan suara rekaman dan mengusir hama. Selesai Journal of Computer System and Informatics (JoSYC) ISSN 2714-8912 (media online), ISSN 2714-7150 (media cetak) Volume 5, No. 2, February 2024, Page 306-314 https://ejurnal.seminar-id.com/index.php/josyc DOI 10.47065/josyc.v5i2.4921 Journal of Computer System and Informatics (JoSYC) Journal of Computer System and Informatics (JoSYC) ISSN 2714-8912 (media online), ISSN 2714-7150 (media cetak) Journal of Computer System and Informatics (JoSYC) ISSN 2714-8912 (media online), ISSN 2714-7150 (media cetak) Volume 5, No. 2, February 2024, Page 306-314 htt // j l i id /i d h /j 4. KESIMPULAN Dalam kesimpulan, alat pengusir hama burung berbasis IoT telah terbukti menjadi solusi yang efektif dalam mengatasi masalah serangan burung pada pertanian dan lingkungan lainnya. Dengan memanfaatkan teknologi sensor gerak, mikrokontroler, dan konektivitas internet, alat ini mampu mendeteksi kehadiran burung secara akurat dan memberikan respons yang cepat dalam mengusir mereka. Hasil pengujian menunjukkan bahwa alat ini dapat mengurangi kerugian yang disebabkan oleh serangan burung secara signifikan, sehingga membantu petani dan pemilik tanaman untuk menjaga hasil panen mereka. Selain itu, integrasi dengan platform IoT dan aplikasi pengendalian jarak jauh seperti Blynk atau Telegram memberikan kemudahan dalam pengoperasian dan pemantauan alat dari jarak jauh. Hal ini memungkinkan pengguna untuk mengontrol alat dan menerima notifikasi secara real-time, sehingga mempermudah manajemen dan pengawasan terhadap keadaan di lapangan. Keunggulan lain dari alat ini adalah konsumsi daya yang rendah, yang membuatnya ramah lingkungan dan ekonomis dalam jangka panjang. Dengan demikian, alat pengusir hama burung berbasis IoT ini tidak hanya efektif dalam mengurangi kerugian pertanian, tetapi juga membantu dalam menjaga keseimbangan ekosistem dan mempromosikan pertanian yang berkelanjutan. Meskipun demikian, penelitian dan pengembangan lebih lanjut masih diperlukan untuk meningkatkan kinerja dan fungsionalitas alat ini. Integrasi dengan teknologi baru seperti kecerdasan buatan atau analisis data yang lebih canggih dapat meningkatkan efisiensi dan efektivitas pengusiran burung. Selain itu, adaptasi terhadap kondisi lingkungan yang berbeda dan pemahaman yang lebih baik tentang perilaku burung juga perlu dipertimbangkan untuk mengoptimalkan kinerja alat ini dalam berbagai konteks aplikasi. Dalam kesimpulan, alat pengusir hama burung berbasis IoT telah terbukti menjadi solusi yang efektif dalam mengatasi masalah serangan burung pada pertanian dan lingkungan lainnya. Dengan memanfaatkan teknologi sensor gerak, mikrokontroler, dan konektivitas internet, alat ini mampu mendeteksi kehadiran burung secara akurat dan memberikan respons yang cepat dalam mengusir mereka. Hasil pengujian menunjukkan bahwa alat ini dapat mengurangi kerugian yang disebabkan oleh serangan burung secara signifikan, sehingga membantu petani dan pemilik tanaman untuk menjaga hasil panen mereka. Selain itu, integrasi dengan platform IoT dan aplikasi pengendalian jarak jauh seperti Blynk atau Telegram memberikan kemudahan dalam pengoperasian dan pemantauan alat dari jarak jauh. Hal ini memungkinkan pengguna untuk mengontrol alat dan menerima notifikasi secara real-time, sehingga mempermudah manajemen dan pengawasan terhadap keadaan di lapangan. gg p j p g p p g Keunggulan lain dari alat ini adalah konsumsi daya yang rendah, yang membuatnya ramah lingkungan dan ekonomis dalam jangka panjang. REFERENCES 2, February 2024, Page 306-314 https://ejurnal.seminar-id.com/index.php/josyc DOI 10.47065/josyc.v5i2.4921 PENGEMBANGAN USAHA TANI PADI DAN KONTRIBUSINYA TERHADAP KESEJAHT PETANI DI DESA PENDEM KOTA BATU,” J. Darma Agung, vol. 31, no. 1, pp. 345–356, 2023. g g pp [13] A. M. 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https://openalex.org/W2946289400	https://strathprints.strath.ac.uk/69054/1/Trotter_etal_BioMetals_2019_The_interaction_of_silver_II_complexes_with_biological_macromolecules.pdf	English	null	The interaction of silver(II) complexes with biological macromolecules and antioxidants	BioMetals	2,019	cc-by	8,502	Biometals (2019) 32:627–640 https://doi.org/10.1007/s10534-019-00198-0 (0123456789().,-volV)( 01234567 89().,-volV) The interaction of silver(II) complexes with biological macromolecules and antioxidants Katherine D. Trotter . Olawale Owojaiye . Stuart P. Meredith . Pat E. Keating . Mark D. Spicer . John Reglinski . Corinne M. Spickett Introduction The antiseptic and oligodynamic behaviour of silver has been appreciated for more than 200 years (Marx and Barillo 2014; Mijnendonckx et al. 2013). How- ever, even in recent years its application as a biocide has been limited to the use of a few relatively simple preparations (Azo´car et al. 2014). Many medical items such as catheters and prostheses are coated with the elemental form of silver, which prevents colonization of their surface by pathogens (Hetrick and Schoenﬁsch 2006). The release of silver cations through slow dissolution of silver halide is an effective method of disinfecting water for domestic consumption (Sil- vestry-Rodriguez et al. 2007). Since the late 1960s, silver sulfadiazine (SSD, or Flamazine) has been common in anti-bacterial preparations for the treat- ment of burns (Atiyeh et al. 2007). Even this compound is a simple preparation comprising an antimicrobial cation and anion formulated together as a weakly bonded complex (Cook and Turner 1975). Although there has been robust interest in developing new silver-based anti-infective agents such as silver nanoparticles (Ag/Ag2O), novel silver complexes and silver impregnated fabrics (Ag0/Ag?) (Fromm 2013; Konop et al. 2016; Le Ouay and Stellacci 2015; Simoncic and Klemencic 2016; Singh et al. 2015), these are mainly reformulations of silver in its commonest Ag0/Ag? forms. In addition to Ag0 and Ag?, silver also has two higher oxidation states that are potentially extremely powerful oxidants: Ag2? (Ag2? ? e- ? Ag?; E0 = 2.0 V) and Ag3? (Ag3? ? e- ? Ag2?; Eo unknown) (Weast 1979). Biocidal silver compounds such as SSD are all based on compounds of silver in its less reactive, lower oxidation states (Ag0/Ag?). This choice, in part, was due to an inability to stabilize and control silver in its higher oxidation states in early synthetic studies. However, many of these issues have been resolved and routes are available for the produc- tion of a wide range of silver(II) compounds and a more limited range of silver(III) species (Levason and Spicer 1987). This opens the door for development of novel, high oxidation state silver compounds for antimicrobial disinfection. Increasing the redox potential of the silver agent is an effective method of enhancing biocidal activity, as it limits the effectiveness of antioxidant defence. Katherine D. Trotter . Olawale Owojaiye . Stuart P. Meredith . Pat E. Keating . Mark D. Spicer . John Reglinski . Corinne M. Spickett 2015; Vindigni and Surawicz 2015), development of more powerful formulations of silver with higher oxidizing potential would be desirable, especially for external use in disinfection and cleansing. Katherine D. Trotter . Olawale Owojaiye . Stuart P. Meredith . Pat E. Keating . Mark D. Spicer . John Reglinski . Corinne M. Spickett Received: 15 February 2019 / Accepted: 30 April 2019 / Published online: 16 May 2019 The Author(s) 2019 them, as well as determining the reaction products. Spectrophotometric analysis showed that Ag2,6P was rapidly reduced by the antioxidants glutathione, ascorbic acid and vitamin E; the unsaturated lipids arachidonic and linoleic acids, model carbohydrate b- cyclodextrin, and protein cytochrome c also reacted readily. Analysis of the reaction with glutathione by NMR and electrospray mass spectrometry conﬁrmed that the glutathione was oxidized to the disulﬁde form. Mass spectrometry also clearly showed the addition of multiple oxygen atoms to the unsaturated fatty acids, suggesting a radical mechanism, and cross-linking of linoleic acid was observed. The seven hydroxyl groups of b-cyclodextrin were found to be completely oxidized to the corresponding carboxylates. Treatment of cytochrome c with Ag2,6P led to protein aggrega- tion and fragmentation, and dose-dependent oxidative damage was demonstrated by oxyblotting. Thus Ag2,6P was found to be highly oxidizing to a wide variety of polar and nonpolar biological molecules. Abstract Silver is widely used for its antimicrobial properties, but microbial resistance to heavy metals is increasing. Silver(II) compounds are more oxidizing and therefore have the potential to overcome resis- tance via extensive attack on cellular components, but have traditionally been hard to stabilize for biological applications. Here, the high oxidation state cation was stabilised using pyridinecarboxylate ligands, of which the 2,6-dicarboxypyridine Ag(II) complex (Ag2,6P) was found to have the best tractability. This complex was found to be more stable in phosphate buffer than DMSO, allowing studies of its interaction with water soluble antioxidants and biological macromolecules, with the aim of demonstrating its potential to oxidize Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10534-019-00198-0) con- tains supplementary material, which is available to authorized users. K. D. Trotter O. Owojaiye P. E. Keating M. D. Spicer J. Reglinski Department of Pure & Applied Chemistry, Strathclyde University, 295 Cathedral Street, Glasgow G1 1XL, UK Keywords Ag(II) 2,6-dicarboxypyridine Antimicrobial metal Glutathione Lipid peroxidation Oxidative stress S. P. Meredith C. M. Spickett School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, UK C. M. Spickett (&) Strathclyde Institute of Pharmacy and Biomedical Sciences, Strathclyde University, 161 Cathedral Street, Glasgow G4 0NR, UK e-mail: c.m.spickett@aston.ac.uk 123 628 Biometals (2019) 32:627–640 Heiman et al. Introduction Powerful oxidants such as silver(II) can be expected to irreversibly chemically oxidize a wide range of functional (sulfhydryl, vicinal diols) and structural components (unsaturated lipid, proteins, carbohy- drates) on the surface and inside the microbial cell. However, a change in oxidation state not only increases the redox potential, it also changes the preferred shape of the metal complex. Silver(I) has a marked preference for tetrahedral geometry, whereas d9 silver(II) predominantly adopts square planar geometry. It is known that the geometry a metal complex adopts can affect its biological activity; for example, the ability of platinum compounds to interact with DNA (Rosenberg et al. 1969) and the antimicro- bial and anticancer activities of various metal com- plexes (Malik et al. 2018). Metal complexes can be transported across membranes by passive and active mechanisms (Martinho et al. 2018), and it has been reported that speciﬁc coordination structures may occur during active transport; for example, in N-MBD Cu?-ATPases Cu? adopts a trigonal planar form (Arguello et al. 2012). Thus, biocides based on silver(II) could allow an enhanced oxidative attack and, depending on their geometry, might exert diverse effects on biological systems. There is a wide range of The mechanisms of antimicrobial action of these silver formulations are still not completely under- stood, but the consensus is that they can act on a variety of targets, including interactions with bacterial cell membranes, binding to and inhibition of thiol- containing proteins, and release of reactive oxygen species through processes still to be fully elucidated (Konop et al. 2016). As Ag? is a moderate oxidizing agent (Ag? ? e- ? Ag; E0 = 0.80 V), only the most sensitive redox sites are affected by it. Although it has sometimes been suggested that the multimodal mech- anism of Ag0/Ag? formulations make resistance less likely to develop, resistance to heavy metals in general is well known (Pal et al. 2014), and reports of resistance to silver compounds are on the increase (Hanczvikkel et al. 2018; Panacek et al. 2018; Percival et al. 2005). In view of the fact that antimicrobial resistance is a major problem worldwide and aggres- sive microbial species such as E. coli 0157, MRSA, and C. difﬁcile are on the increase (Brandt et al. Introduction 2014; 12 123 Biometals (2019) 32:627–640 629 simple ligands which can be used to stabilize silver(II), but the pyridinecarboxylates are an excel- lent initial choice, as they have been synthesized previously and mostly form planar complexes (Drew et al. 1970; Drew et al. 1971; Fowles et al. 1968), although the silver(II) complex with 2,6-dicar- boxypyridine has been reported to be octahedral (Drew et al. 1969). However, their ability to react with biological molecules and cause oxidative damage has not been studied previously. Consequently, sil- ver(II) complexes were prepared using pyridinecar- boxylates as ligands, and the stability of these complexes was investigated. The aim of the study was to determine the effectiveness of the silver(II) complexes for oxidizing biological antioxidants, lipids and proteins. solvent. The resulting solution was subjected imme- diately to spectrophotometric analysis and the con- centration of Ag2,6P in solution calculated retrospectively using molar extinction coefﬁcient obtained from the reference sample. Thus although all the experiments were carried out in duplicate or triplicate, the difﬁculty of producing completely dry complex meant that it was impossible to generate solutions containing exactly the same concentration of reagents. Consequently, the data shown are derived from representative experiments. The stability of bis-(2,6- dicarboxypyridyl)silver(II) in solution The stability of bis-(2,6- A solution of bis-(2,6-dicarboxypyridyl)silver(II) was prepared either in phosphate buffer (0.1 M KH2PO4, pH 7.0) at 4.47 mM or in DMSO at 18.4 mM. Aliquots of solutions were transferred immediately to a cuvette and the absorbance (400–1000 nm) was monitored over a 2 h period. Reaction of bis-(2,6-dicarboxypyridyl)silver(II) with glutathione An 8.5 mM solution of Ag2,6P in phosphate buffer was prepared and 2.25 mL was transferred to a cuvette and the visible spectrum (400–900 nm) recorded. Aliquots (20 lL) of reduced glutathione (GSH) solu- tion (84.3 mM) in phosphate buffer were added to the cuvette and the spectrum re-recorded after each addition until the band (kmax 570 nm) attributed to silver(II) disappeared (* 120 lL). Experimental All reagents were obtained commercially. UV–Vis spectra were recorded on an Agilent Technologies Cary 60 UV–Vis spectrophotometer. NMR analysis was carried out on a Bruker AMX 400 operating at 400 MHz for 1H. Solid reﬂectance spectra (400–900 nm) were recorded on a Photonics CCD array UV–Vis spectrophotometer. Silver(II) com- plexes of 2-carboxypyridine, 2,3-dicarboxypyridine, 2,4-dicarboxypyridine, 2,5-dicarboxypyridine and 2,6-dicarboxypyridine (Ag2,6P) as were prepared using literature methods (Drew et al. 1970; Drew et al. 1971; Fowles et al. 1968). Reaction of bis-(2,6-dicarboxypyridyl)silver(II) with vitamin E (a-tocopherol) Reaction of bis-(2,6-dicarboxypyridyl)silver(II) with vitamin E (a-tocopherol) The visible spectrum (400–900 nm) of 2.25 mL of 9.1 mM bis-(2,6-dicarboxypyridyl)silver(II) in DMSO was recorded before and after addition of 10 lL aliquots of 54.3 mM a-tocopherol in DMSO, with loss of absorbance at 620 nm after addition of 70 lL. Due to the competition between the concurrent reactions of Ag2,6P with a-tocopherol and Ag2,6P with DMSO, a deﬁnite end point cannot be given for the reaction of Ag2,6P with a-tocopherol (vide infra). Reaction of bis-(2,6-dicarboxypyridyl)silver(II) with linoleic and arachidonic acid Reaction of bis-(2,6-dicarboxypyridyl)silver(II) with linoleic and arachidonic acid A 2.25 mL aliquot of 19.5 mM bis-(2,6-dicar- boxypyridyl)silver(II) in DMSO in a cuvette was reacted sequentially with 10 lL aliquots of 91.1 mM sodium linoleate or 98.7 mM sodium arachidonate, both prepared in DMSO. The reaction was monitored by recording the visible spectrum until the band (kmax 620 nm) had disappeared. No deﬁnite end point could be given due to the competing reaction with DMSO occurring. Reaction of bis-(2,6-dicarboxypyridyl)silver(II) with b-cyclodextrin before Fourier transformation to reduce the effect of the baseline noise. before Fourier transformation to reduce the effect of the baseline noise. The reaction with b-cyclodextrin (25 mmol/L) with Ag2,6P was tested essentially as for ascorbic acid described above, except that the reaction was carried out in a sample bottle and 200 lL aliquots of b- cyclodextrin were added sequentially until the band at 570 nm had been extinguished (after addition of 2.0 mL). The protocol for handling Ag2,6P in solution The protocol for handling Ag2,6P in solution To investigate the products of the reaction using NMR, three solutions of reduced glutathione (6.1 mg in 1 mL of D2O, 20 mmol) were treated with 8.2 mg (18.5 mmol), 16.6 mg (36 mmol) or 24.4 mg (53 mmol) of Ag2,6P respectively. The solutions were allowed to react overnight and then ﬁltered into a 5 mm NMR tube. 1H NMR spectra were obtained using a Bruker AVANCE 3 spectrometer operating at 400.12 MHz. Samples were maintained at 300 K during spectral acquisition. The NMR spectra were collected using a standard pulse sequence. The free induction decay was generated by a 3.13 ls pulse width corresponding to a 30o pulse. Each data set (4 k scans; no water suppression) was collected in 32 k of memory. A 1 Hz line broadening function was applied A reference sample of Ag2,6P was prepared using published methods (Fowles et al. 1968). The sample was subjected to elemental analysis (found: C 32.77, H 2.68, N 5.85%: expected for Ag2,6P4H2O: C 32.97, H 2.77, N 5.49%), which conﬁrmed the hydration state. This reference sample was used to calculate the molar extinction coefﬁcient of Ag2,6P in water (e570, 252/M/ cm; e890, 207/M/cm) and DMSO (e600, 88.6/M/cm). Ag2,6P was found to decompose slowly with the natural green/black colour giving way to a white product. Hence small batches of Ag2,6P were pre- pared immediately prior to use to avoid problems associated with degradation, and given amounts of Ag2,6P were quickly dissolved in a given amount of 12 23 Biometals (2019) 32:627–640 630 Reaction of bis-(2,6-dicarboxypyridyl)silver(II) with b-cyclodextrin Reaction of bis-(2,6-dicarboxypyridyl)silver(II) with ascorbic acid The reaction of bis-(2,6-dicarboxypyridyl)silver(II) (7.6 mM) with ascorbic acid (144 mM) was investi- gated essentially as described for glutathione. Aliquots (20 lL) of the ascorbate solution were added to a cuvette containing 2.25 mL of Ag2,6P and the spec- trum re-recorded until the band (kmax 570 nm) disap- peared, which corresponded to the addition of 80 lL. For analysis by mass spectrometry, aqueous solu- tions (2 mL) of b-cyclodextrin containing 100 mg (88 lmoles) were treated with Ag2,6P (200 mg, 0.44 mmol). The solutions were allowed to react overnight and then ﬁltered before analysis as described below. Reaction of bis-(2,6-dicarboxypyridyl)silver(II) with cytochrome-c Aqueous solutions (2 mL) of cytochrome c (16.5 mg, 54 lmoles) were incubated with 20 mg (44 lmoles), 40 mg (88 lmoles) or 60 mg (131 lmoles) of Ag2,6P. The solutions were allowed to react overnight and then ﬁltered before analysis as described below. For analysis by mass spectrometry, 2 mL of aqueous suspensions of the sodium salts of the fatty acids (sodium linoleate; 16.5 mg, 54 lmoles or arachidonic acid; 16.5 mg, 50 lmoles) were reacted with 20 mg (45 lmoles), 40 mg (91 lmoles) or 60 mg (136 lmoles) of Ag2,6P. The solutions were allowed to react overnight and then ﬁltered before analysis as described below. Analysis of protein oxidation by oxyblotting for DNP-carbonyl adducts Electrospray mass spectrometric analysis of small molecules Electrospray mass spectra were recorded using an Agilent 6130 (dual source). Samples of glutathione, vitamin E, linoleic acid, and arachidonic acid prepared as described above were diluted in methanol, while b- cyclodextrin was diluted in 50:50 acetonitrile con- taining 0.1% formic acid:water and introduced into the instrument with an infusion rate of 0.2 mL/min using methanol. Spectra were acquired with the following parameters: ionization mode, MM-ES ? APCI, -ve ionization; source temperature, 300 C; Voltage, 4000 V; Curtain gas ﬂow rate, 12 L/min; m/z range 50–2000. Spectra were typically acquired for 30 s and averaged. The MS data were analysed using Agilent Chemstation. Analysis of protein oxidation by oxyblotting for DNP-carbonyl adducts Aliquots of the samples (10 lL; * 75 lg protein) were resolved by SDS-PAGE with a 12% resolving gel 12 3 631 Biometals (2019) 32:627–640 (Sambrook and Russell 2006) and then either stained with InstantBlue stain (Sigma-Aldrich, UK) or trans- ferred onto PVDF membrane for oxyblotting as described previously (Shacter 2000). After washing, the membrane was acidiﬁed with 2 N HCl and labelled with 10 mM dinitrophenylhydrazine (DNPH) (Sigma- Aldrich, UK) for 5 min. After further washing and blocking the membrane was incubated in blocking buffer containing monoclonal primary antibodies rabbit anti-dinitrophenylhydrazone (anti-DNP) (D9656, Sigma-Aldrich, UK) at a working dilution of 1:1000 overnight at 4 C. The secondary antibody was HRP-linked goat anti-mouse (6154, Sigma- Aldrich, UK) antibody (working dilution 1:1000) for 2 h at room temperature. The membrane was washed again as described above and HRP-linked anti-mouse was detected using enhanced chemiluminescence (ECL kit 34078, Thermo Fisher Scientiﬁc, Hemel Hempstead, UK) according to the manufacturer’s instructions. The membrane was scanned using a G:BOX system (Syngene, Cambridge, UK) running the GeneSys software (Syngene, Cambridge, UK). cell membrane transport, in the case of Ag(II) their potential may be limited by their lack of solubility. p y y y In view of the high redox potential of silver(II) (equation 2; E0 = 2.0 V NHE (Weast 1979)), the silver (II) compounds were expected to be quite reactive even to the extent of potentially oxidizing water. Therefore, the ﬁrst step was to investigate the lifetime of Ag2,6P in aqueous buffer and DMSO, by monitoring the visible absorbance spectrum (Suppl. Fig. 1a). In aqueous solution a small but manageable degradation (* 10%) of Ag2,6P was observed over a 2 h period. The proﬁle of the degradation process was linear within the lifetime of the experiment, suggest- ing that decomposition does not occur via an SN2 displacement of the axial carboxylates by water or phosphate. In contrast, the stability of Ag2,6P in DMSO was poor, having a half life of only 25 min (Suppl. Fig. 1b). DMSO can be oxidized to dimethyl- sulphone (Me2SO2; E0 = 1.54 V vs. NHE) and the silver(II) complex studied here is therefore theoreti- cally capable of driving this reaction (Krtil et al. 1996). Solid reﬂectance spectrophotometry indicated that the solid and DMSO solution phase structures of Ag2,6P are similar (kmax 600 nm), suggesting that decomposition occurs via electron transfer rather than ligand exchange. Analysis of protein oxidation by oxyblotting for DNP-carbonyl adducts The reaction of the silver complex with DMSO limits the interpretation of reactions with other compounds carried out in this solvent, but in some cases there was no feasible alternative. To obviate problems with the slow decomposition of Ag2,6P in solution, fresh solutions were prepared immediately before the start of each experiment. Solubility and stability of silver(II) complexes Silver(II) complexes of 2-carboxypyridine (Ag2P), 2,3-dicarboxypyridine (Ag2,3P), 2,4-dicar- boxypyridine (Ag2,4P), 2,5-dicarboxypyridine (Ag2,5P) and 2,6-dicarboxypyridine (Ag2,6P) were prepared using literature methods (Drew et al. 1970; Drew et al. 1971; Fowles et al. 1968). Ag2P, Ag2,3P, Ag2,4P and Ag2,5P had limited solubility in both water and DMSO, and were not studied further owing to the limited relevance to biological environments. In contrast, Ag2,6P was observed to be reasonably soluble in both water (* 10 mM) and DMSO (* 20 mM). The solubility proﬁle of the compounds most likely arises from their solid state structures: Ag2P and Ag2,3P are planar species and prone to p- stacking in the solid state (Fowles et al. 1968), which is known to affect solubility detrimentally. In contrast, Ag2,6P adopts an octahedral geometry in the solid state and is unable to p-stack, which lowers the lattice energy and promotes its solubility in polar solvents (Drew et al. 1970). The structures are shown in Fig. 1. Thus, despite the desirability of planar compounds for Results and discussion Solubility and stability of silver(II) complexes Reaction with antioxidants Biological systems utilize a number of species as co- factors and reducing agents (e.g. glutathione, ascorbic acid, a-tocopherol), and depletion or oxidation of antioxidants and structural biological molecules is an early stage in the stress leading to the toxic effects of oxidizing compounds (Halliwell and Gutteridge 1998). Consequently, the reactions of Ag2,6P with these three antioxidants was investigated. The reaction with an antioxidant can readily be inferred by spectrophotometric titrations in which the stepwise reduction of the coloured Ag2,6P to its colourless silver(I) product is observed. The reactions of glu- tathione and ascorbate were carried out in aqueous solution, whereas the reaction of a-tocopherol was 12 3 3 632 Biometals (2019) 32:627–640 Fig. 1 Structures of the silver (II) complexes synthesized. 2,3-dicarboxypyridine (Ag2,3P), 2,4-dicarboxypyridine (Ag2,4P), 2,5- dicarboxypyridine (Ag2,5P) and 2,6-dicarboxypyridine (Ag2,6P) Fig. 1 Structures of the silver (II) complexes synthesized. 2,3-dicarboxypyridine (Ag2,3P), 2,4-dicarboxypyridine (Ag2,4P), 2,5- dicarboxypyridine (Ag2,5P) and 2,6-dicarboxypyridine (Ag2,6P) Fig. 1 Structures of the silver (II) complexes synthesized. 2,3-dicarboxypyridine (Ag2,3P), 2,4-dicarboxy dicarboxypyridine (Ag2,5P) and 2,6-dicarboxypyridine (Ag2,6P) carried out in DMSO. Using the diminution of the band at 570 nm it was possible to titrate Ag2,6P with glutathione (Fig. 2), and the molar ratio at the end point was calculated to be slightly greater than 2:1 Ag2,6P:GSH. This would be generally consistent with the 2-electron oxidation of GSH to GSSG, assuming that the Ag2,6P undergoes a 1-electron reduction to Ag?, as the appearance of metallic silver was not observed. Fig. 2), which clearly showed that treatment of GSH (m/z 306.1) with Ag2,6P resulted in conversion to GSSG (m/z 611.1). carried out in DMSO. Using the diminution of the band at 570 nm it was possible to titrate Ag2,6P with glutathione (Fig. 2), and the molar ratio at the end point was calculated to be slightly greater than 2:1 Ag2,6P:GSH. This would be generally consistent with the 2-electron oxidation of GSH to GSSG, assuming that the Ag2,6P undergoes a 1-electron reduction to Ag?, as the appearance of metallic silver was not observed. The reaction of Ag2,6P with ascorbic acid was monitored spectrophotometrically in the same way as that of glutathione, and it could clearly be seen that addition of ascorbic acid resulted in loss of absorbance at 570 nm (Suppl. Fig. 3). The molar ratio at the end point was calculated to be approx. 2:1 Ag2,6P:ascor- bate, consistent with the 2-electron oxidation of ascorbate to dehydroascorbate. Reaction with antioxidants There was also evidence of addition of a single oxygen atom at m/z 295, and another signal at m/z 293, which was most probably due to loss of water from the species at m/z 311, suggesting that it may be a bis-hydroxide rather than a hydroperoxide (Spickett and Pitt 2015). Interestingly, a peak was observed at m/z 557, which was consistent with the formation of a cross-linked dimer of linoleic acid (loss of 3H but singly charged) and there were also dimers containing 2, 3 and 4 additional oxygens. Crosslinking of oxidized fatty acyl chains under highly oxidizing conditions has been The reaction of a-tocopherol with Ag2,6P in DMSO was also discernable using spectrophotometry (Suppl. Fig. 4). However, due to the competing interaction of Ag2,6P with DMSO discussed above, it was not possible to obtain an accurate end point for the titration. ESI-mass spectrometry was used to monitor the reaction of Ag2,6P with the unsaturated fatty acid linoleic acid (Fig. 4). A small amount of adventitious oxidation of the control sample is apparent in Fig. 4a, but the major signal is the native fatty acid at m/z 279 ([M–H]-). There was also a strong signal at m/z 325, which was identiﬁed as the formate adduct of linoleic acid ([M ? CHO2]-). At the lower treatment concen- tration (Fig. 4b) the signal of the native lipid was greatly reduced and the major signal was at m/z 311, corresponding to the addition of O2 (? 32 Da). There was also evidence of addition of a single oxygen atom at m/z 295, and another signal at m/z 293, which was most probably due to loss of water from the species at m/z 311, suggesting that it may be a bis-hydroxide rather than a hydroperoxide (Spickett and Pitt 2015). Interestingly, a peak was observed at m/z 557, which was consistent with the formation of a cross-linked dimer of linoleic acid (loss of 3H but singly charged) and there were also dimers containing 2, 3 and 4 additional oxygens. Crosslinking of oxidized fatty acyl chains under highly oxidizing conditions has been Reaction with antioxidants ESI-mass spectrometry was used to monitor the reaction of Ag2,6P with the unsaturated fatty acid linoleic acid (Fig. 4). A small amount of adventitious oxidation of the control sample is apparent in Fig. 4a, but the major signal is the native fatty acid at m/z 279 ([M–H]-). There was also a strong signal at m/z 325, which was identiﬁed as the formate adduct of linoleic acid ([M ? CHO2]-). At the lower treatment concen- tration (Fig. 4b) the signal of the native lipid was greatly reduced and the major signal was at m/z 311, corresponding to the addition of O2 (? 32 Da). There was also evidence of addition of a single oxygen atom at m/z 295, and another signal at m/z 293, which was most probably due to loss of water from the species at m/z 311, suggesting that it may be a bis-hydroxide rather than a hydroperoxide (Spickett and Pitt 2015). Interestingly, a peak was observed at m/z 557, which was consistent with the formation of a cross-linked dimer of linoleic acid (loss of 3H but singly charged) and there were also dimers containing 2, 3 and 4 additional oxygens. Crosslinking of oxidized fatty Fig. 3 1H-NMR analysis of the reaction between Ag2,6P and glutathione. 27 lmols of glutathione (GSH) was reacted with increasing amounts of Ag2,6P as indicated on the spectra. The spectrum of commercial glutathione disulﬁde is shown at the bottom for comparison Biometals (2019) 32:627–640 633 Biometals (2019) 32:627–640 633 f P e Fig. 3 1H-NMR analysis of the reaction between Ag2,6P and glutathione. 27 lmols of glutathione (GSH) was reacted with increasing amounts of Ag2,6P as indicated on the spectra. The spectrum of commercial glutathione disulﬁde is shown at the bottom for comparison ESI-mass spectrometry was used to monitor the reaction of Ag2,6P with the unsaturated fatty acid linoleic acid (Fig. 4). A small amount of adventitious oxidation of the control sample is apparent in Fig. 4a, but the major signal is the native fatty acid at m/z 279 ([M–H]-). There was also a strong signal at m/z 325, which was identiﬁed as the formate adduct of linoleic acid ([M ? CHO2]-). At the lower treatment concen- tration (Fig. 4b) the signal of the native lipid was greatly reduced and the major signal was at m/z 311, corresponding to the addition of O2 (? 32 Da). Reaction with antioxidants However, this reaction was not investigated further due to the lack of stability of dehydroascorbate in the presence of redox metals; metal-mediated ascorbic acid oxidation and redox cycling is a facile process involving low as well as high valent metals (Halliwell and Gutteridge 1998; Skov and Vonderschmitt 1975). To investigate the nature of the oxidation in more depth, the reaction of GSH was monitored using 1H- NMR. Figure 3 clearly demonstrates that Ag2,6P oxidized GSH to the disulﬁde form (GSSG), with increasing Ag2,6P amounts correlating with increased loss of the GSH triplet signals at * 2.95 ppm and appearance of the GSSG pairs of doublets at * 3.05 and 3.3 ppm. This ﬁnding was supported by negative ion electrospray mass spectrometry analysis (Suppl Fig. 2 Spectrophotometric analysis of the reaction between Ag2,6P and glutathione. The titration of 8.5 mM Ag2,6P (18.7 lmoles in 2.2 mL) in 0.1 M KH2PO4, pH 7.0 with glutathione (GSH). The glutathione (84.3 mM) was added in 20 uL aliquots (a total of 6) and the corresponding lmoles of GSH are indicated on the right-hand side of the traces 123 Fig. 2 Spectrophotometric analysis of the reaction between Ag2,6P and glutathione. The titration of 8.5 mM Ag2,6P (18.7 lmoles in 2.2 mL) in 0.1 M KH2PO4, pH 7.0 with glutathione (GSH). The glutathione (84.3 mM) was added in 20 uL aliquots (a total of 6) and the corresponding lmoles of GSH are indicated on the right-hand side of the traces 12 3 The reaction of a-tocopherol with Ag2,6P in DMSO was also discernable using spectrophotometry (Suppl. Fig. 4). However, due to the competing interaction of Ag2,6P with DMSO discussed above, it was not possible to obtain an accurate end point for the titration. ESI–MS analysis of the reaction with fatty acids The unsaturated fatty acids linoleic acid and arachi- donic acid were used as models to investigate the ability of Ag2,6P to oxidize lipid. Preliminary studies by spectrophotometric analysis indicated that reac- tions occurred, but the reactions were so fast that kinetic analysis was not possible; moreover the experiments were carried out in DMSO as the fatty acid salts were sparingly soluble in aqueous solution, and therefore were limited by the issues with this solvent mentioned above. The focus of these studies was therefore the analysis of oxidation products of the biomolecules. ESI–MS analysis of the reaction with fatty acids The unsaturated fatty acids linoleic acid and arachi- donic acid were used as models to investigate the ability of Ag2,6P to oxidize lipid. Preliminary studies by spectrophotometric analysis indicated that reac- tions occurred, but the reactions were so fast that kinetic analysis was not possible; moreover the experiments were carried out in DMSO as the fatty acid salts were sparingly soluble in aqueous solution, and therefore were limited by the issues with this solvent mentioned above. The focus of these studies was therefore the analysis of oxidation products of the biomolecules. 12 3 3 Biometals (2019) 32:627–640 634 Fig. 4 The reaction of linoleic acid with Ag2,6P studied by ESI-mass spectrometry in negative ion mode. a Untreated linoleic acid. b 29 mM Linoleic acid treated with 37 mM Ag2,6P and c 29 mM linoleic acid treated with 110 mM Ag2,6P reported previously (Muizebelt and Nielen 1996; Schroter et al. 2016; Tolvanen et al. 2008). At the higher treatment concentration the native lipid was further depleted, probably resulting from degradation of the lipid to small, non-ionized breakdown products, but otherwise the oxidation pattern was comparable. Treatment of arachidonic acid with Ag2,6P also clearly showed the occurrence of oxidation (Fig. 5). As with linoleic acid, there was some adventitious Fig. 4 The reaction of linoleic acid with Ag2,6P studied by ESI-mass spectrometry in negative ion mode. a Untreated linoleic acid. b 29 mM Linoleic acid treated with 37 mM Ag2,6P and c 29 mM linoleic acid treated with 110 mM Ag2,6P reported previously (Muizebelt and Nielen 1996; Schroter et al. 2016; Tolvanen et al. 2008). At the higher treatment concentration the native lipid was further depleted, probably resulting from degradation of the lipid to small, non-ionized breakdown products, but otherwise the oxidation pattern was comparable. Treatment of arachidonic acid with Ag2,6P also clearly showed the occurrence of oxidation (Fig. 5). As with linoleic acid, there was some adventitious reported previously (Muizebelt and Nielen 1996; Schroter et al. 2016; Tolvanen et al. 2008). At the higher treatment concentration the native lipid was further depleted, probably resulting from degradation of the lipid to small, non-ionized breakdown products, but otherwise the oxidation pattern was comparable. Treatment of arachidonic acid with Ag2,6P also clearly showed the occurrence of oxidation (Fig. 5). As with linoleic acid, there was some adventitious Treatment of arachidonic acid with Ag2,6P also clearly showed the occurrence of oxidation (Fig. 5). ESI–MS analysis of the reaction with fatty acids 7.6 mM Ag2,6P (17.2 lmol in 2.2 mL) in 0.1 M KH2PO4, pH 7.0 was titrated against b- cyclodextrin (b-CD). The b- cyclodextrin (25.6 mM) was added in 200 lL aliquots (9 10). The mole ratio at the end point was calculated to be 1:3 Ag2,6P: b-CD mol:mol 636 previously (Fraschini and Vignon 2000). Spectropho- tometric titrations in aqueous solution (400–900 nm) clearly showed evidence of a reaction of Ag2,6P with b-cyclodextrin (Fig. 6). An end point was obtained at approximately 3:1 Ag2,6P: b-CD, which suggests that multiple oxidations might be occurring. Analysis by mass spectrometry also suggested that oxidation had occurred (Fig. 7). Figure 7a shows untreated b-cy- clodextrin, which was the major species in the sample at m/z 1133.1 and therefore 100% relative intensity. As the samples were prepared in solvent containing formate, there was also a signiﬁcant formate adduct at 1179.0. Treatment of the b-cyclodextrin with a 5-fold excess of Ag2,6-P led to free 2,6-picolinate presenting the strongest signal (100%) at m/z 166.1 (data not shown), while the b-cyclodextrin signal was substantially depleted and a signal at m/z 1231.1 appeared, corresponding to the oxidation of all of the hydroxyl groups into carboxylic acid to form a hepta- carboxylate b-cyclodextrin (plus 7 9 14 Da). A 2,6- dicarboxypyridine adduct of cyclodextrin at m/z 1300.3 was also observed, probably reﬂecting the high level of the free ligand present in the sample after reduction of Ag2,6-P by the carbohydrate. In contrast to the treatment of b-cyclodextrin with HOCl reported previously [20], there was no clear evidence of intermediate oxidation products, such as tris and hexakis carboxylate species, presumably owing to the highly oxidizing nature of the Ag2? complex. oxidation in the untreated sample, reﬂecting the susceptibility of this polyunsaturated fatty acid to autoxidation (Fig. 5a), but even at the low treatment concentration (Fig. 5b) the incorporation of oxygen was increased, with signals at m/z 319 (1 O), m/z 335 (2 O), m/z 351 (3 O) and m/z 367 (4 O). A strong signal at 349.2 was also observed, corresponding to ? 46 Da; in view of its appearance only in treated samples, this is likely to be a dehydration product following the addition of 4 oxygens such as an epoxyisoprostane, which are known as relatively stable products of arachidonic acid (Spickett and Pitt 2015). At the higher treatment concentration the native signal at m/z/303 was almost abolished (Fig. Reaction with the carbohydrate beta-cyclodextrin Reaction with the carbohydrate beta-cyclodextrin ESI–MS analysis of the reaction with fatty acids As with linoleic acid, there was some adventitious 12 123 635 Biometals (2019) 32:627–640 Fig. 5 The reaction of arachidonic acid with Ag2,6P studied by negative ion ESI-mass spectrometry. a ESI-mass spectra of untreated arachidonic acid. b arachidonic acid (27 mM) treated with Ag26P (22.5 mM) and c arachidonic acid (27 mM) treated with Ag26P (68 mM). All samples were diluted equivalently in methanol prior to infusion into the instrument. The signal at m/z 349 is probably a formate adduct, while the one at 333 appears to be a contaminant egative ometry. of c acid. 27 mM) 27 mM) es were in usion The robably hile the o be a 3 Biometals (2019) 32:627–640 636 oxidation in the untreated sample, reﬂecting susceptibility of this polyunsaturated fatty acid autoxidation (Fig. 5a), but even at the low treatm concentration (Fig. 5b) the incorporation of oxyg was increased, with signals at m/z 319 (1 O), m/z 3 (2 O), m/z 351 (3 O) and m/z 367 (4 O). A strong sig at 349.2 was also observed, corresponding ? 46 Da; in view of its appearance only in trea samples, this is likely to be a dehydration prod following the addition of 4 oxygens such as epoxyisoprostane, which are known as relativ stable products of arachidonic acid (Spickett and P 2015). At the higher treatment concentration native signal at m/z/303 was almost abolish (Fig. 5c), and the fatty acid was more highly oxidiz with the strongest signal at m/z 335.1 and all oth products at higher masses and levels of oxidation. contrast, there was no evidence of dimers of arac donic acid analogous to those observed with linol acid, which should have occurred at m/z 605 (data n shown), although the ions that appeared between m 515–553 were not identiﬁed. Comparing the reactio of linoleic and arachidonic acids with Ag2,6P, it w clear that higher concentrations of the silver w required to deplete the more unsaturated fatty ac reﬂecting its greater capacity for oxidat modiﬁcation. Reaction with the carbohydrate beta-cyclodextrin To investigate the effects of Ag2,6P on carbohydrat b-cyclodextrin was used as a model, as it can readily observed by mass spectrometry ([M-H]- at m/z 113 and moreover its oxidation by HOCl has been stud 0.0 2.0 4.0 6.0 8.0 10.0 12.0 0.0 2 [Ag26P] mmol/L Fig. 6 Spectrophotometric analysis of the reaction of b- cyclodextrin with Ag2,6P. ESI–MS analysis of the reaction with fatty acids 5c), and the fatty acid was more highly oxidized with the strongest signal at m/z 335.1 and all other products at higher masses and levels of oxidation. In contrast, there was no evidence of dimers of arachi- donic acid analogous to those observed with linoleic acid, which should have occurred at m/z 605 (data not shown), although the ions that appeared between m/z 515–553 were not identiﬁed. Comparing the reactions of linoleic and arachidonic acids with Ag2,6P, it was clear that higher concentrations of the silver were required to deplete the more unsaturated fatty acid, reﬂecting its greater capacity for oxidative modiﬁcation. Reaction with cytochrome c 46 Da; b b-cyclodextrin (100 mg, 88 lmoles) treated with a 5-fold excess of Ag2,6P (200 mg, 0.44 mmol); and c structure of b-cyclodextrin together with its formula. Note that the y-axis scale is relative signal intensity, where in a the native b- cyclodextrin is the major species and therefore 100%, whereas in b 2,6-picolinate at m/z 166.1 was the major species at 100% disruption of cell signaling, so the effect of Ag2,6P on cytochrome C as a model ubiquitous protein was studied. Preliminary studies by spectrophotometry showed that Ag2,6P was consumed by relatively small amounts of protein (data not shown), which is consistent with the presence of multiple oxidation sites on the polypeptides. To conﬁrm the oxidative action of Ag2,6P on the protein, the formation of protein carbonyls was investigated, as these are well- established oxidation products (Domingues et al. 2013; Shacter 2000). Carbonyl formation may occur by oxidative deamination of lysines, or radical attack and fragmentation of various other residues (Davies 2016). The samples were ﬁrst separated on denaturing polyacrylamide gels and visualized by staining with disruption of cell signaling, so the effect of Ag2,6P on cytochrome C as a model ubiquitous protein was studied. Preliminary studies by spectrophotometry showed that Ag2,6P was consumed by relatively small amounts of protein (data not shown), which is consistent with the presence of multiple oxidation sites on the polypeptides. To conﬁrm the oxidative action of Ag2,6P on the protein, the formation of protein carbonyls was investigated, as these are well- established oxidation products (Domingues et al. 2013; Shacter 2000). Carbonyl formation may occur by oxidative deamination of lysines, or radical attack and fragmentation of various other residues (Davies 2016). The samples were ﬁrst separated on denaturing polyacrylamide gels and visualized by staining with coomassie blue; then labeling of carbonyl groups with DNPH followed by western blotting with antibody to the DNP-adduct (commonly known as oxy-blotting) was carried out. Figure 8 shows that even low levels of Ag2,6P resulted in loss of the cytochrome c band at 12.3 kDa and appearance of high molecular weight aggregates that were retained at the top of the resolving gel, while higher concentrations led to more aggregates and additionally some degradation prod- ucts observed at the bottom of the gel (Fig. 8a). Reaction with cytochrome c To investigate the effects of Ag2,6P on carbohydrates, b-cyclodextrin was used as a model, as it can readily be observed by mass spectrometry ([M-H]- at m/z 1133), and moreover its oxidation by HOCl has been studied Many adverse effects of oxidizing antimicrobial agents are mediated by protein oxidation and 0.0 2.0 4.0 6.0 8.0 10.0 12.0 0.0 2.0 4.0 6.0 8.0 10.0 12.0 [cyclodextrin] mmol/L [Ag26P] mmol/L Fig. 6 Spectrophotometric analysis of the reaction of b- cyclodextrin with Ag2,6P. 7.6 mM Ag2,6P (17.2 lmol in 2.2 mL) in 0.1 M KH2PO4, pH 7.0 was titrated against b- cyclodextrin (b-CD). The b- cyclodextrin (25.6 mM) was added in 200 lL aliquots (9 10). The mole ratio at the end point was calculated to be 1:3 Ag2,6P: b-CD mol:mol 123 0.0 2.0 4.0 6.0 8.0 10.0 12.0 0.0 2.0 4.0 6.0 8.0 10.0 12.0 [cyclodextrin] mmol/L [Ag26P] mmol/L 12 Biometals (2019) 32:627–640 637 disruption of cell signaling, so the effect of Ag2,6P on cytochrome C as a model ubiquitous protein was studied. Preliminary studies by spectrophotometry showed that Ag2,6P was consumed by relatively small amounts of protein (data not shown), which is consistent with the presence of multiple oxidation coomassie blue; then labeling of carbonyl groups with DNPH followed by western blotting with antibody to the DNP-adduct (commonly known as oxy-blotting) was carried out. Figure 8 shows that even low levels of Ag2,6P resulted in loss of the cytochrome c band at 12 3 kDa and appearance of high molecular weight Fig. 7 Electrospray mass spectrometry analysis of the reaction of Ag2,6P with b- cyclodextrin a the negative ion ESI-mass spectra of b- cyclodextrin, also showing a formate adduct at ? 46 Da; b b-cyclodextrin (100 mg, 88 lmoles) treated with a 5-fold excess of Ag2,6P (200 mg, 0.44 mmol); and c structure of b-cyclodextrin together with its formula. Note that the y-axis scale is relative signal intensity, where in a the native b- cyclodextrin is the major species and therefore 100%, whereas in b 2,6-picolinate at m/z 166.1 was the major species at 100% Fig. 7 Electrospray mass spectrometry analysis of the reaction of Ag2,6P with b- cyclodextrin a the negative ion ESI-mass spectra of b- cyclodextrin, also showing a formate adduct at ? Reaction with cytochrome c Oxyblotting for oxidative damage to the protein showed the presence of increased carbonyls with increasing severity of the Ag2,6P treatment, initially in the cytochrome c band but this was lost at higher Ag2,6P concentrations and staining of the high 123 12 3 3 638 Biometals (2019) 32:627–640 Fig. 8 The effect of Ag2,6P treatment on Cytochrome c. Protein (8.25 mg/mL) was treated with 0, 63, 126, 189 or 252 lM Ag2,6P overnight. Molecular weights markers (MWM) are shown on the lefthandside with numbers in kDa; the molecular weight of horse heart Cytc is 12,384 Da. a Coomassie blue-stained 12% SDS-PAGE reducing gel. b Oxyblot of a comparable gel using an anti-DNPH primary antibody to determine the formation of carbonyl groups on cytochrome c as a marker of oxidative damage blue-stained 12% SDS-PAGE reducing gel. b Oxyblot of a comparable gel using an anti-DNPH primary antibody to determine the formation of carbonyl groups on cytochrome c as a marker of oxidative damage blue-stained 12% SDS-PAGE reducing gel. b Oxyblot of a comparable gel using an anti-DNPH primary antibody to determine the formation of carbonyl groups on cytochrome c as a marker of oxidative damage Fig. 8 The effect of Ag2,6P treatment on Cytochrome c. Protein (8.25 mg/mL) was treated with 0, 63, 126, 189 or 252 lM Ag2,6P overnight. Molecular weights markers (MWM) are shown on the lefthandside with numbers in kDa; the molecular weight of horse heart Cytc is 12,384 Da. a Coomassie intermediate action via oxidation of the solvents H2O or DMSO, leading to production of hydroxyl radicals (HO) or other radical species. molecular weight aggregates predominated (Fig. 8b). This clearly conﬁrmed that extensive protein oxida- tion occurred following Ag2,6P treatment of the cytochrome c. The reactivity of Ag2,6P with a range of different biomolecules suggests that it is likely to have signif- icant toxicity to bacterial cells and therefore good biocidal potential. Its solubility in the organic solvent DMSO implies that it may be able to penetrate lipid membranes and thus gain access to the intracellular environment, as lipophilicity is known to be a factor in biological transport of metal complexes (Oldﬁeld et al. 2007). 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https://openalex.org/W2345783572	https://europepmc.org/articles/pmc4864917?pdf=render	English	null	Assessment of refractive astigmatism and simulated therapeutic refractive surgery strategies in coma-like-aberrations-dominant corneal optics	Eye and vision	2,016	cc-by	7,672	© 2016 Zhou et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhou et al. Eye and Vision (2016) 3:13 DOI 10.1186/s40662-016-0044-8 Zhou et al. Eye and Vision (2016) 3:13 DOI 10.1186/s40662-016-0044-8 METHODOLOGY Open Access Assessment of refractive astigmatism and simulated therapeutic refractive surgery strategies in coma-like-aberrations- dominant corneal optics Assessment of refractive astigmatism and simulated therapeutic refractive surgery strategies in coma-like-aberrations- dominant corneal optics Wen Zhou1,2, Aleksandar Stojanovic1,3 and Tor Paaske Utheim4,5,6 Abstract Background: The aim of the study is to raise the awareness of the influence of coma-like higher-order aberrations (HOAs) on power and orientation of refractive astigmatism (RA) and to explore how to account for that influence in the planning of topography-guided refractive surgery in eyes with coma-like-aberrations-dominant corneal optics. Methods: Eleven eyes with coma-like-aberrations-dominant corneal optics and with low lenticular astigmatism (LA) were selected for astigmatism analysis and for treatment simulations with topography-guided custom ablation. Vector analysis was used to evaluate the contribution of coma-like corneal HOAs to RA. Two different strategies were used for simulated treatments aiming to regularize irregular corneal optics: With both strategies correction of anterior corneal surface irregularities (corneal HOAs) were intended. Correction of total corneal astigmatism (TCA) and RA was intended as well with strategies 1 and 2, respectively. Results: Axis of discrepant astigmatism (RA minus TCA minus LA) correlated strongly with axis of coma. Vertical coma influenced RA by canceling the effect of the with-the-rule astigmatism and increasing the effect of the against-the-rule astigmatism. After simulated correction of anterior corneal HOAs along with TCA and RA (strategies 1 and 2), only a small amount of anterior corneal astigmatism (ACA) and no TCA remained after strategy 1, while considerable amount of ACA and TCA remained after strategy 2. Conclusions: Coma-like corneal aberrations seem to contribute a considerable astigmatic component to RA in eyes with coma-like-aberrations dominant corneal optics. If topography-guided ablation is programmed to correct the corneal HOAs and RA, the astigmatic component caused by the coma-like corneal HOAs will be treated twice and will result in induced astigmatism. Disregarding RA and treating TCA along with the corneal HOAs is recommended instead. Keywords: Coma, Higher-order-aberrations, Corneal irregular astigmatism, Topography-guided ablation Correspondence: wen@synslaser.no 1SynsLaser Kirurgi AS, Tromsø, Troms, Norway 2School of Ophthalmology and Optometry, Wenzhou Medical University, Wenzhou, China Full list of author information is available at the end of the article Background aberrations. It occurs in keratoconus [1, 2], cornel ecta- sia after laser in situ keratomileusis (LASIK) [3], decen- tered refractive surgery [4] and may also occur after any type of incisional corneal surgery, after pterygium- surgery [5], as well as after scarring due to corneal injur- ies or keratitis. In these conditions visual distortions and decrease in visual acuity occur irrespective of spherocy- lindrical error and its correction [6, 7]. Based on a com- putational model, it has been estimated that the refractive effect of a root-mean-square (RMS) HOA of 0.43 μm or greater is equivalent to at least 0.50 D of spherical error for a 5 mm pupil aperture [8, 9]. Wei Orthogonally asymmetric corneas have either different power amplitudes on the opposite sides of their astig- matic hemi-meridians or the hemi-meridians are not aligned along the same axis. This results in irregular op- tics, dominated by odd-order, higher-order aberrations (HOAs), most of which are coma and coma-like * Correspondence: wen@synslaser.no 1SynsLaser Kirurgi AS, Tromsø, Troms, Norway 2School of Ophthalmology and Optometry, Wenzhou Medical University, Wenzhou, China Full list of author information is available at the end of the article Page 2 of 11 Page 2 of 11 Zhou et al. Eye and Vision (2016) 3:13 and colleagues reported that the coma-like HOAs and trefoil contribute to (subjective) refractive astigmatism (RA) and that the amount of RA was directly influenced by the amount of horizontal coma and trefoil [10]. Alpins et al. also found that in the absence of lenticular astigmatism (LA), there was a difference between RA and the astigmatism measured by corneal topography [11]. This was ascribed to the contribution of the irregu- lar astigmatism component i.e., the asymmetry of topo- graphic hemi-meridians. During subjective refraction the patient is systematically presented with combinations of spherical and cylindrical lenses in the search for the one that forms the retinal images with least diffusion and distortion. Thus, the subjective refraction can neither determine the amount or type of HOAs nor their contri- bution to the resulting spherocylindrical refraction. This has a major impact in ablation planning in therapeutic refractive surgery cases with irregular astigmatism. If custom ablation, which treats HOAs is programmed to also treat RA, we would be treating the coma itself and its astigmatic effect within RA at the same time, result- ing in a “double treatment” (Fig. 1a, b). Background measurements that could be used in software simula- tions of topography-guided custom ablation, as it seems to be the most reasonable current approach in treatment of highly aberrated corneas [12, 13]. The magnitude and orientation of the TCA, measured by Scheimpflug topo/tomography (Precisio; iVIS Tech- nology, Taranto, Italy) was calculated by ray tracing in- dependent of the HOAs. Internal astigmatism (IA), representing the astigmatism originating from the structures from the posterior corneal surface to the retina, was measured by OPD scan-II (NIDEK Co Ltd, Gamagori, Japan), which integrates automatic retinoscopy-based wavefront aberrometry and placido-based corneal topography. The difference between the total ocular astigmatism (which does not include the influence from HOAs) obtained by wavefront measurement and the anterior corneal astigmatism (ACA) obtained by corneal topography, gave the IA, consisting of the sum of the posterior corneal astig- matism (PCA) and the LA. The ACA was calculated using Snell’s law, using 1.3760 as the corneal refract- ive index and then translated to Zernicke polynomials after being adjusted for recentering from the corneal vertex to the line-of-sight, for the sake of compatibility with the ocular wavefront measurements. The IA differs from the ocular residual astigmatism as described by Alpins [14], which is calculated using manifest refraction and consequently includes the neural processing compo- nent as well. The goal of this study is to raise awareness of the con- tribution of coma-like HOAs to the amount and orienta- tion of RA in eyes with coma-like-dominant corneal optics and to explore how to account for that contribu- tion in the planning of therapeutic refractive surgery using topography-guided custom ablation. Methods PCA, measured by Precisio, was calculated using the equation (n´-n)/R, with 1.336(n') for refractive indexes for aqueous and 1.376(n) for cornea, and R as the pos- terior corneal curvature radius. From the population of patients referred for therapeutic refractive surgery at the eye department of the Univer- sity Hospital of North Norway, 11 eyes with coma-like- aberration-dominant optics due to keratoconus (eight eyes), LASIK flap complication (one eye), corneal scar- ring after photorefractive keratectomy (PRK) (one eye) and after keratitis (one eye) (Table 1) were selected for evaluation of contribution of their coma-like HOAs to their RA, as well as for the simulations of therapeutic topography-guided ablation aimed at regularization of cor- neal optics. Inclusion criteria were: 1) anterior corneal topography with orthogonal asymmetric power along any meridian ≥2 D, or misalignment of axis between principal hemi-meridians exceeding 10°; 2) vector difference be- tween total corneal astigmatism (TCA) and subjective RA ≥1.5D; and 3) LA representing ≤30 % of the vector difference between total corneal and RA (Table 2). LA was calculated as the vector difference between IA and the PCA. RA was obtained from non-cycloplegic manifest re- fraction. RA was first converted to cross-cylinder nota- tion then transferred from spectacle plane to corneal plane using the vertex distance of 12 mm for direct comparison with the corneal astigmatism. Discrepant astigmatism (DA) represents the discrep- ancy between RA and the sum of TCA and LA. Since the sum of TCA and LA represents the “pure” astigma- tism excluding the effect of the coma-like HOAs. DA is normally negligible in eyes with normal optics but can become significant when coma-like HOAs refract as astigmatism. DA was calculated by vector analysis. The orientation of astigmatism in the current article was presented as axis of the corrective cylinder (using minus cylinder for display in the tables to be accordant with usual practice, but using plus cylinder for double angle plot in the figures). RMS values of coma-like HOAs measured by wavefront aberrometry (OPD scan- II), were defined as square root of the sum of c3 1, c3 −1, c5 1 Astigmatism and coma-like aberration measurements Astigmatism and coma-like aberration measurements Since our main goal was to study the contribution of coma-like HOAs on RA in highly irregular corneas, measurements of corneal astigmatism and HOAs were obtained using different technologies to most objectively highlight the two components. We also chose the Page 3 of 11 Zhou et al. Eye and Vision (2016) 3:13 Fig. 1 Schematic figure showing astigmatic components and effect of different treatment strategies. a. Different astigmatic components and coma-like HOAs. b. Simulated effect after strategy 2 (treating t along with coma-like HOAs), resulting in double correction of the astigmatic contribution of coma-like HOAs. c. Simulated effect after strategy 1 (treating total corneal astigmatism along with coma-like HOAs), resulting in uncorrected lenticular astigmatism. d. Simulated effect after correction of ocular astigmatism along with coma-like HOAs, resulting in full correction Fig. 1 Schematic figure showing astigmatic components and effect of different treatment strategies. a. Different astigmatic components and coma-like HOAs. b. Simulated effect after strategy 2 (treating t along with coma-like HOAs), resulting in double correction of the astigmatic contribution of coma-like HOAs. c. Simulated effect after strategy 1 (treating total corneal astigmatism along with coma-like HOAs), resulting in uncorrected lenticular astigmatism. d. Simulated effect after correction of ocular astigmatism along with coma-like HOAs, resulting in full correction coma. Coma with other orientations was defined as oblique coma. and c5 −1. Individual pupil size, which is an average of photopic and scotopic pupil, was used as the diameter at which astigmatism and coma-like HOAs for each case were analyzed. Simulations The axis of the corneal coma was defined as the axis passing through both corneal vertex and the midpoint of the specific elevated or depressed area on the anterior corneal elevation topography, using the aconic fitting (Fig. 2). Coma with axis oriented at 90° ± 30° was defined as vertical coma, and coma with axis oriented at 180° ± 30° was defined as horizontal Anterior corneal elevation maps obtained by Precisio were used as the basis for customized ablation design with Corneal Interactive Programmed Topographic Ablation software (CIPTA; Ligi, Taranto, Italy). CIPTA cal- culates simulated postoperative topography by subtracting the ablation map from the imported preoperative anterior topography map. The simulated postoperative topography Zhou et al. Eye and Vision (2016) 3:13 Page 4 of 11 Table 1 Demographic data for 11 cases No. Age Eye Gender CDVA Pupil (mm)a Coma (μm)b Diagnosis Visual symptoms 1 33 os Female 0.9 6.4 0.8 Keratoconus Double images, starburst 2 33 os Male 0.8 6.9 1.7 Keratoconus Multiple images, haloes 3 44 od Male 0.9 4.9 0.4 Keratoconus Multiple images, starburst 4 44 os Male 0.6 4.7 0.6 Keratoconus Double images, starburst 5 49 os Male 0.3 4.6 0.7 Keratoconus Multiple images, starburst 6 27 od Male 0.9 6.2 1.0 Keratoconus Haloes, starburst 7 36 od Male 1.0 6.0 0.7 scarring post PRK Double images, starburst 8 60 os Male 0.8 4.6 0.8 LASIK flap complication Double images, starburst 9 20 os Male 0.8 3.9 0.4 Keratoconus Double images, starburst 10 24 od Male 1.0 5.5 1.1 Keratoconus Starburst 11 27 os Male 1.0 4.8 0.5 Keratitis Double images, starburst CDVA = corrected distance visual acuity asize of average of photopic and scotopic pupil bRMS coma-like aberrations within individual pupil size, including primary coma from 3rd order and secondary coma from 5th order shows the amount and the orientation of the postoperative corneal astigmatism (ACA, PCA and TCA). influence of subjective sphere and spherical-aberration- compensation were not specifically analyzed, as they were outside the scope of the simulations used for the current study and since the simulations were not used for the actual treatments. Simulations The simulations comprised corneal vertex fitting with two different aconic surfaces of 6-mm in diameter, de- fined as the targeted surfaces for two different strategies: Strategy 1 aimed to correct TCA, along with the anterior corneal surface irregularities (the source of anterior cor- neal HOAs) i.e., using TCA and corneal topography as the basis for treatment of lower- and HOAs, respect- ively. Strategy 2 was aimed at correcting RA along with the anterior corneal surface irregularities i.e., using sub- jective refraction and corneal topography as the basis for treatment of lower- and higher-order corneal surface ab- errations, respectively. In both cases the tissue between the existing anterior corneal surface and the targeted regular surface within the 6 mm would be ablated. The TCA = total corneal astigmatism, RA = refractive astigmatism, IA = internal astigmatism, PCA = posterior corneal astigmatism, LA = lentic discrepant astigmatism, OA = ocular astigmatism TCA = total corneal astigmatism, RA = refractive astigmatism, IA = internal astigmatism, PCA = posterior corneal astigmatism, LA = lenticular astigmatism, DA = discrepant astigmatism OA = ocular astigmatism Vector analysis Vector analysis was performed according to the method outlined by Jack T. Holladay [15, 16]: Each astigmatic value was converted to x and y Cartesian values (x = \|Astigma- tism\| * Cos (2 * axis), y = \|Astigmatism\| * Sin (2 * axis)), and displayed using doubled- angle plots as positive cylin- der notation. The difference between RA and TCA (RA – TCA) and the difference between IA and PCA (IA – PCA) = LA, were calculated using vectors: Table 2 Deviation of different types of astigmatism and amount of coma-like aberration for 11 cases No. TCA RA IA PCA LA LA/(RA-TCA) DA OA (TCA + LA) 1 −2.20/4 −0.5/95 −0.71/87 −0.16/113 −0.62/81 0.23 −2.16/98 −1.66/9 2 −3.40/176 −2.11/90 −1.45/95 −0.94/86 −0.63/109 0.11 −5.05/85 −2.99/172 3 −0.12/43 −2.95/95 −0.89/104 −0.17/80 −0.79/109 0.26 −2.29/92 −0.71/105 4 −0.37/170 −4.09/85 −1.29/88 −0.23/99 −1.08/86 0.24 −3.38/94 −0.72/89 5 −1.12/106 −6.36/110 −1.20/46 −0.51/40 −0.71/50 0.13 −5.65/114 −1.09/87 6 −1.94/121 −3.68/103 −0.75/96 −0.35/60 −0.67/110 0.28 −1.95/82 −2.58/118 7 −2.06/171 −1.51/46 −0.60/86 −0.30/142 −0.76/75 0.26 −2.22/64 −1.32/174 8 −0.43/31 −2.19/55 −0.27/39 −0.47/88 −0.57/12 0.30 −2.07/68 −0.95/20 9 −2.2/172 −0.50/80 −1.35/83 −0.57/84 −0.78/82 0.29 −1.92/81 −1.42/172 10 −1.14/142 −2.04/68 −1.00/90 −0.26/71 −0.81/96 0.24 −3.10/55 −1.59/127 11 −2.49/19 −0.96/20 −0.29/179 −0.08/174 −0.21/0.9 0.14 −1.71/106 −2.66/18 TCA = total corneal astigmatism, RA = refractive astigmatism, IA = internal astigmatism, PCA = posterior corneal astigmatism, LA = lenticular astigmatism, DA = discrepant astigmatism, OA = ocular astigmatism Page 5 of 11 Zhou et al. Eye and Vision (2016) 3:13 Fig. 2 Measurement of axis of coma. Measurement of axis of coma on anterior corneal topography fitting by aconic surface The distributions of TCA, RA, IA, and PCA for the 11 eyes are shown in Table 2 and Figs. 3–4. The axis of DA and axis of coma for each case is displayed in Table 4 and Fig. 5 and the amplitude of DA and RMS value of coma is shown in Fig. 6. Concerning the patients’ re- fractive error, only the astigmatism, including the re- fractive and all the objective measurements were analyzed and shown in Table 2. The simulated postoperative ACA and TCA after using our two simulation strategies are presented in Table 3 and Figs. 7–8. Vector analysis After simulated correction of an- terior corneal HOAs along with TCA and RA (strategies 1 and 2, respectively), only a small amount of ACA and no TCA remained after strategy 1, while considerable amounts of ACA and TCA remained after strategy 2. DA ¼ RA‐ðTCA þ LAÞ ¼ RA‐ðTCA þ IA‐PCAÞ In the current study, we chose cases with relatively in- significant LA, manifested as LA representing ≤30 % of the vector difference between RA and TCA (Table 2). The calculation is shown as following: LA= RA‐TCA ð Þ < 30% LA= RA‐TCA ð Þ < 30% The current study was approved by the Norwegian data protection authority and was granted exemption from the regional ethics committee (REK-NOR). Manifest refraction, a common way to assess subject- ive sphere, astigmatism and visual acuity, is influenced by the amount, type and spatial distribution of corneal HOAs. It has been reported that spherical aberrations refract as hyperopia or myopia [8], while comas refract as astigmatism [10]. During phoropter testing the result- ant subjectively refracted cylinder power and axis will be a vector sum of two components: One caused by the “real astigmatism” i.e., ocular astigmatism (the second order optical aberration with frequency of 2, originated Discussion RA is mostly correlated to corneal astigmatism due to the mere fact that the cornea contributes to more than 70 % of the total ocular refractive power [17]. Being the second most important refractive element, the crystalline lens may also contribute to RA. However, a significant discrepancy between RA and objectively measured ocu- lar astigmatism is known to occur in conjunction with corneal pathologies resulting in HOAs-dominated optics as well. In the current study, we chose to analyze the cases that had coma-like-aberration-dominant optics with a difference between RA and TCA ≥1.5 D and with a relatively insignificant LA, in order to diminish the in- fluence of LA as a source of DA. Fig. 2 Measurement of axis of coma. Measurement of axis of coma on anterior corneal topography fitting by aconic surface Fig. 2 Measurement of axis of coma. Measurement of axis of coma on anterior corneal topography fitting by aconic surface RA‐TCA ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ xRA −xTCA ð Þ2 þ yRA −yTCA ð Þ2 q LA ¼ IA‐PCA ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ ðxIA −xPCAÞ2 þ ðyIA −yPCAÞ2 q For most of the normal corneas, RA mainly originates from both TCA and LA i.e., RA-TCA = LA. However, for the corneas which are dominated by coma-like HOAs, RA would also be influenced by this irregularity [8–11]. This influence was defined as DA in the study, representing the discrepancy between RA and the total of TCA and LA. DA was calculated using the vectors as follows: Before the “wavefront” terminology was common, the term irregular astigmatism as introduced by Alpins [11] was used to describe corneal optics dominated by HOAs. Coma has been found to be the dominant HOA in asymmetric corneas [18], in decentered laser ablations [4], and in Keratoconus [1, 2, 9], where the corneal morphology changes are displaced from the optical cen- ter of the cornea, and form a coma-type wavefront aber- ration. Hence, the corneal morphological coma and the optical wavefront coma are highly associated especially in cases with no internal ocular coma. In the current study, we measured the location of coma and coma-like HOAs at the most asymmetric part of the corneal anter- ior elevation topography. Results All eyes had above 0.3 μm RMS coma-like HOAs along with decreased corrected distance visual acuity (CDVA) and/or visual disturbances such as double/multiple im- ages/contours, starburst, or haloes, not correctable by sphere and cylinder (Table 1). Page 6 of 11 Zhou et al. Eye and Vision (2016) 3:13 Fig. 3 Total corneal astigmatism and refractive astigmatism. A double-angle, plus cylinder power plot of an 11-case set for total corneal astigmatism and refractive astigmatism from both cornea and lens) and the other caused by coma subjectively refracting as cylinder (Fig. 1a). The coma-like HOAs were calculated within the “individual pupil diameter” (a mean value between photopic and scotopic pupil) in order to match the situation of non- cycloplegic clinical examination under which the RA is measured and to estimate the influence of the coma-like HOAs on that measurement. the sum. In one case the vector sum of TCA and LA was oriented WTR, with RA also WTR but of lower magnitude than the sum. The effect of coma residing along an oblique axis with respect to the RA, as in the last three cases, was less obvious and would require a more complex analysis. The results above show that vertical coma is influen- cing the RA by canceling the effect of the WTR ocular astigmatism and increasing the effect of the ATR ocular astigmatism. In other words, vertical coma is refracted as ATR astigmatism. In the same manner, one could as- sume that the horizontal coma would be refracted as WTR astigmatism i.e., enhancing the effect of the WTR ocular astigmatism, and cancelling the effect of the ATR ocular astigmatism (Fig. 9). Our results show that the axis of the DA and the axis of coma correlated well (Table 4, Fig. 5). In three cases the difference in axes was slightly above 30° (cases 8, 9 and 11), but this difference may be ascribed to the influ- ence of the concurrent trefoil present in those cases, as the trefoil was described to also influence the RA (Fig. 7) [10]. Table 4 shows that three cases had their vector sum of TCA and LA oriented with the rule (WTR), while their RA was against the rule (ATR), presumably due to the presence of vertical coma that influenced the RA. Also in the presence of vertical coma, four cases had their vector sum of TCA and LA oriented ATR. Results Their RA was also ATR, but of higher magnitude than The mechanism of how coma-like HOAs affect the sub- jective refraction has not been explored in the literature. For the 11 cases in the current study, there is no apparent correlation between the amplitude of coma-like HOAs and DA (Fig. 6). We would assume that the change in RA orientation is influenced by the location of the coma, but Page 7 of 11 Zhou et al. Eye and Vision (2016) 3:13 Fig. 4 Internal astigmatism and posterior corneal astigmatism. A double-angle, plus cylinder power plot of an 11-case set for internal astigmatism and posterior corneal astigmatism planning rather than the wavefront information from the entire optical system of the eye, since the former more closely represents the source of aberrations. Further- more, the pupil-diameter limited operating principle of the ocular wavefront aberrometry may lead to unreliable results in corneas with distorted optics resulting from the pathologies residing outside the pupil, as in keratoconus and after pterygium, or incisional surgery. Meanwhile, precise (Scheimpflug-based) elevation topography por- trays accurate corneal morphology thus resulting in more accurate corneal optics calculations. In addition, it pro- vides the ability to map the posterior cornea and produce an accurate pachymetric and total corneal optics map. The primary measured elevation data is more accurate in determining the corneal morphology compared with wavefront aberrometry or even with Placido-technology- based curvature data, because the primary elevation measurements are not based on any assumed axis and therefore will not be influenced by displaced cor- neal apex [12, 13]. Depending on the calculation method and on how narrowly the corneal astigmatism is defined with respect to symmetry and alignment of estimating the contribution from coma-like HOAs to the magnitude of RA seems to be more complex, involving pupil size during the manifest refraction examination, as well as the individual patient’s neural image processing. In eyes with normal corneas, correcting refractive sphere and cylinder as measured by manifest refraction and using a non-customized laser ablation will lead to good postoperative visual outcomes in most cases. How- ever, approaching the cases with aberrated corneal optics the same way will not include the treatment of HOAs and will most likely not result in improvement of the quality of the eye optics. Results If an ablation to treat regular astigmatism is performed based on the magnitude and axis orientation decided by subjective refraction i.e., not corresponding to the corneal astigmatism, new surface ir- regularities resulting in new HOAs will likely be induced. Why topography guided and not wavefront guided ablation? When visual disturbances can be ascribed to corneal op- tical irregularities, it is more appropriate to use corneal topography as the source of data for custom ablation Page 8 of 11 Zhou et al. Eye and Vision (2016) 3:13 Fig. 5 A double-angle plot of an 11-case set for orientation of coma-like aberrations and discrepant astigmatism current study defines TCA (anterior plus posterior) by ray tracing and wavefront error estimation and is supposed to provide astigmatism measurements not influenced by coexisting HOAs. its hemi-meridians, more or less of coma-like HOAs would be included in the value denominating astig- matism as measured by different instruments [19]. This may be an important reason for discrepancies in measurements of the astigmatism between different systems, rather than the differences in the raw data. The Precisio Scheimpflug topographer system used in the Why is the topography-guided strategy where TCA (along with the corneal HOAs) is treated preferable to treating RA (along with the corneal HOAs)? RA in the presence of coma-like HOAs and low LA rep- resents a vector sum of HOAs refracting as astigmatism and TCA (Fig. 1a). When the corneal HOAs and the TCA are treated, all the sources of RA, except for LA, are addressed (Fig. 1c). However, when corneal HOAs and RA are treated, then the corneal coma itself, as a part of the corneal HOAs and its effect on RA are both treated. This amounts to a double treatment of the subjective astig- matic component i.e., removal of its cause and at the same time as the treatment of its effect (Fig. 1b). Why not use the total ocular astigmatism acquired by aberrometry? Using total ocular astigmatism acquired by aberrometry would clearly have been the most elegant choice to determine the magnitude and orientation of the astigma- tism to be treated (Fig. 1d), but the quality of the aberro- metry data in highly aberrated corneas is often insufficient and cannot be used in topography-guided ablation with- out compatibility issues. Fig. 7 Simulated ablation maps and resultant postoperative anterior corneal elevation maps. Preoperative Scheimpflug based anterior corneal elevation maps fitting by sphere and aconic surface (column A and column B, respectively), simulated ablation maps based on total corneal astigmatism correction and resultant postoperative anterior corneal elevation maps fitting by sphere (column C and column D, respectively), simulated ablation maps based on refractive astigmatism correction and resultant postoperative anterior corneal elevation maps fitting by sphere (column E and column F, respectively) Fig. 7 Simulated ablation maps and resultant postoperative anterior corneal elevation maps. Preoperative Scheimpflug based anterior corneal elevation maps fitting by sphere and aconic surface (column A and column B, respectively), simulated ablation maps based on total corneal astigmatism correction and resultant postoperative anterior corneal elevation maps fitting by sphere (column C and column D, respectively), simulated ablation maps based on refractive astigmatism correction and resultant postoperative anterior corneal elevation maps fitting by sphere (column E and column F, respectively) Why total corneal astigmatism and not anterior corneal astigmatism? Simulated residual total corneal astigmatism after strategy 2 (correction of refractive astigmatism) lower visual performance were seen in keratoconic eyes upon correction with rigid gas-permeable lenses [23, 24]. The simulation in the current study based on strategy 1 recognizes this issue and uses the TCA as the basis for corneal optical regularization. It showed that simulated ablation aimed at correction of TCA and corneal HOAs results in a regularized anterior corneal surface with a low level residual ACA compensating the PCA (Table 3), while its ablation map reflects correction of the morphological coma and TCA. As expected, a better symmetry of the simulated postoperative anterior corneal topography using strategy 1 was also achieved compared to the symmetry of the simulated postoperative topography after the ablation based on strategy 2 (aimed at correction of RA and cor- neal HOAs) (Fig. 7). Fig. 7 Simulated ablation maps and resultant postoperative anterior corneal elevation maps. Preoperative Scheimpflug based anterior corneal elevation maps fitting by sphere and aconic surface (column A and column B, respectively), simulated ablation maps based on total corneal astigmatism correction and resultant postoperative anterior corneal elevation maps fitting by sphere (column C and column D, respectively), simulated ablation maps based on refractive astigmatism correction and resultant postoperative anterior corneal elevation maps fitting by sphere (column E and column F, respectively) Why total corneal astigmatism and not anterior corneal astigmatism? Total corneal power has traditionally been calculated on the basis of the anterior corneal curvature, using the ker- atometric refractive index of 1.3375, to compensate for the effect of the posterior surface. Neglecting the real influence of the posterior corneal surface in vision- correction planning may not result in errors if the profiles of anterior and posterior surfaces follow each other. However, if there is a discrepancy between the two, the influence of the posterior surface on the total corneal refraction can be significant [20, 21]. In kerato- conus or irregular astigmatism secondary to incisional refractive surgery, major morphological changes originate from the posterior surface and are optically balanced by less pronounced changes of similar profile on the anterior surface due to epithelial remodeling [22]. In such cases, regularization of the anterior surface via topography- guided ablation may break that balance and lead to the manifestation of the latent refractive errors originating from the posterior surface. This has been shown in some cases in which significantly increased ocular HOAs and Fig. 6 Scattergram showing the magnitude of coma-like HOAs and discrepant astigmatism. DA: Discrepant astigmatism. RMS: root mean square. HOAs: higher order aberrations Fig. 6 Scattergram showing the magnitude of coma-like HOAs and discrepant astigmatism. DA: Discrepant astigmatism. RMS: root mean square. HOAs: higher order aberrations Zhou et al. Eye and Vision (2016) 3:13 Page 9 of 11 Table 3 Simulated residual astigmatism for different strategies 1 2 3 4 5 6 7 8 9 10 11 A. −0.75@173 −0.59@136 −0.17@20 −0.36@146 −0.08@101 −0.22@20 −0.28@28 −0.45@178 −0.43@171 −0.48@4 −0.08@173 B. −2.15@177 −4.03@179 −3.12@8 −4.07@173 −4.62@21 −2.75@4 −2.10@135 −1.90@152 −2.90@173 −3.04@152 −1.80@18 C. −2.67@176 −3.70@174 −2.96@9 −3.88@171 −4.07@21 −2.65@5 −1.66@138 −2.01@157 −3.03@173 −2.97@156 −1.67@17 A. Simulated residual anterior corneal astigmatism after strategy 1 (correction of total corneal astigmatism) B. Simulated residual anterior corneal astigmatism after strategy 2 (correction of refractive astigmatism) C. Simulated residual total corneal astigmatism after strategy 2 (correction of refractive astigmatism) A. Simulated residual anterior corneal astigmatism after strategy 1 (correction of total corneal astigmatism) B. Simulated residual anterior corneal astigmatism after strategy 2 (correction of refractive astigmatism) C. Simulated residual total corneal astigmatism after strategy 2 (correction of refractive astigmatism) A. Simulated residual anterior corneal astigmatism after strategy 1 (correction of total corneal astigmatism) B. Simulated residual anterior corneal astigmatism after strategy 2 (correction of refractive astigmatism) C. Shortcomings of the study The main shortcoming of the study is a possible error due to combination of the data acquired by two separate in- struments using three different types of technologies (Scheimpflug- and Placido-based topography along with Page 10 of 11 Zhou et al. Eye and Vision (2016) 3:13 Fig. 8 Postoperative astigmatism. A double-angle, plus cylinder power plot of an 11-case set for simulated postoperative astigmatism based on total corneal astigmatism correction and refractive astigmatism correction Fig. 8 Postoperative astigmatism. A double-angle, plus cylinder power plot of an 11-case set for simulated postoperative astigmatism based on total corneal astigmatism correction and refractive astigmatism correction Table 4 Influence of coma on orientation of refractive astigmatism Axis of Coma Axis of DA TCL + LA Coma RA 1 94 98 WTR Vertical ATR 2 84 85 WTR Vertical ATR 3 97 92 ATR Vertical ATR↑ 4 93 84 ATR Vertical ATR↑ 5 119 114 ATR Vertical ATR↑ 6 84 82 ATR Vertical ATR↑ 7 51 64 WTR Oblique Oblique 8 34 68 WTR Oblique Oblique 9 115 81 WTR Vertical ATR 10 45 55 Oblique Oblique ATR 11 76 106 WTR Vertical WTR↓ DA = discrepant astigmatism; TCA = total corneal astigmatism, LA = lenticular astigmatism, RA = refractive astigmatism, WTR = with the rule, ATR = against the rule ↑: increased amplitude of astigmatism ↓: decreased amplitude of astigmatism Fig. 9 Schematic diagram showing the possible influence of coma on refractive astigmatism Table 4 Influence of coma on orientation of refractive astigmatism Axis of Coma Axis of DA TCL + LA Coma RA 1 94 98 WTR Vertical ATR 2 84 85 WTR Vertical ATR 3 97 92 ATR Vertical ATR↑ 4 93 84 ATR Vertical ATR↑ 5 119 114 ATR Vertical ATR↑ 6 84 82 ATR Vertical ATR↑ 7 51 64 WTR Oblique Oblique 8 34 68 WTR Oblique Oblique 9 115 81 WTR Vertical ATR 10 45 55 Oblique Oblique ATR 11 76 106 WTR Vertical WTR↓ DA = discrepant astigmatism; TCA = total corneal astigmatism, LA = lenticular astigmatism, RA = refractive astigmatism, WTR = with the rule, ATR = against the rule ↑: increased amplitude of astigmatism ↓: decreased amplitude of astigmatism Fig. 9 Schematic diagram showing the possible influence of coma on refractive astigmatism Fig. 9 Schematic diagram showing the possible influence of coma on refractive astigmatism Fig. ↑: increased amplitude of astigmatism Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit p y y p • We accept pre-submission inquiries Shortcomings of the study Corneal imaging with slit-scanning and Scheimpflug imaging techniques. Clin Exp Optom. 2011;94(1):33–42. 14. Alpins NA. New method of targeting vectors to treat astigmatism. J Cataract Refract Surg. 1997;23(1):65–75. 15. Holladay JT, Dudeja DR, Koch DD. Evaluating and reporting astigmatism for individual and aggregate data. J Cataract Refract Surg. 1998;24(1):57–65. 16. Holladay JT, Moran JR, Kezirian GM. Analysis of aggregate surgically induced refractive change, prediction error, and intraocular astigmatism. J Cataract Refract Surg. 2001;27(1):61–79. 17. Courville CB, Smolek MK, Klyce SD. Contribution of the ocular surface to visual optics. Exp Eye Res. 2004;78(3):417–25. 18. Leung TW, Lam AK, Kee CS. Ocular Aberrations and Corneal Shape in Adults with and without Astigmatism. Optom Vis Sci. 2015;92(5):604–14. 19. Roh HC, Chuck RS, Lee JK, Park CY. The effect of corneal irregularity on astigmatism measurement by automated versus ray tracing keratometry. Medicine (Baltimore). 2015;94(13):e677. 20. Ho JD, Tsai CY, Liou SW. Accuracy of corneal astigmatism estimation by neglecting the posterior corneal surface measurement. Am J Ophthalmol. 2009;147(5):788–95,795.e1-2. 21. Koch DD, Ali SF, Weikert MP, Shirayama M, Jenkins R, Wang L. Contribution of posterior corneal astigmatism to total corneal astigmatism. J Cataract Refract Surg. 2012;38(12):2080–7. 22. Reinstein DZ, Archer TJ, Gobbe M. Corneal epithelial thickness profile in the diagnosis of keratoconus. J Refract Surg. 2009;25(7):604–10. 23. Negishi K, Kumanomido T, Utsumi Y, Tsubota K. Effect of higher-order aberrations on visual function in keratoconic eyes with a rigid gas permeable contact lens. Am J Ophthalmol. 2007;144(6):924–9. 24. Marsack JD, Parker KE, Pesudovs K, Donnelly WJ 3rd, Applegate RA. Uncorrected wavefront error and visual performance during RGP wear in keratoconus. Optom Vis Sci. 2007;84(6):463–70. • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www biomedcentral com/submit Submit your next manuscript to BioMed Central and we will help you at every step: OPD-based wavefront aberrometry) for calculation of the LA. In addition to the registration error that may occur between any two separate examinations, one must take into consideration the potential error due to compatibility issues. Ideally, one instrument, using one type of technol- ogy, should be used for the measurement of the LA. 3. Piñero DP, Alió JL, Barraquer RI, Uceda-Montanes A, Murta J. Shortcomings of the study Clinical characterization of corneal ectasia after myopic laser in situ keratomileusis based on anterior corneal aberrations and internal astigmatism. J Cataract Refract Surg. 2011;37(7):1291–9. 4. McCormick GJ, Porter J, Cox IG, MacRae S. Higher-order aberrations in eyes with irregular corneas after laser refractive surgery. Ophthalmology. 2005; 112(10):1699–709. 5. Pesudovs K, Figueiredo FC. Corneal first surface wavefront aberrations before and after pterygium surgery. J Refract Surg. 2006;22(9):921–5. A support for the outlined treatment strategy by real results would have been desirable, as the real outcomes would have been influenced by the healing and biomech- anical responses, as well as epithelial remodeling, all of which cannot be accounted for in the current simula- tions. However, the eight keratoconus cases were not candidates for refractive surgery, while the other three treated cases were patients coming from abroad and therefore not being available to undertake their follow- up examinations under the conditions needed for a meaningful analysis. Finally there were too few cases for a global statistical analysis. 6. Applegate RA, Ballentine C, Gross H, Sarver EJ, Sarver CA. Visual acuity as a function of Zernike mode and level of root mean square error. Optom Vis Sci. 2003;80(2):97–105. 7. Applegate RA, Sarver EJ, Khemsara V. Are all aberrations equal? J Refract Surg. 2002;18(5):S556–62. 8. Cheng X, Bradley A, Ravikumar S, Thibos LN. Visual impact of Zernike and Seidel forms of monochromatic aberrations. Optom Vis Sci. 2010;87(5):300–12. 9. Bruce AS, Catania LJ. Clinical applications of wavefront refraction. Optom Vis Sci. 2014;91(10):1278–86. 10. Wei RH, Lim L, Chan WK, Tan DT. Higher order ocular aberrations in eyes with myopia in a Chinese population. J Refract Surg. 2006;22(7):695–702. 11. Alpins NA. Treatment of irregular astigmatism. J Cataract Refract Surg. 1998; 24(5):634–46. 12. Belin MW, Khachikian SS. An introduction to understanding elevation-based topography: how elevation data are displayed - a review. Clin Experiment Ophthalmol. 2009;37(1):14–29. 13. Oliveira CM, Ribeiro C, Franco S. Corneal imaging with slit-scanning and Scheimpflug imaging techniques. Clin Exp Optom. 2011;94(1):33–42. Authors’ contributions WZ i d th t d WZ conceived the study, performed the statistical analysis, participated in its design and coordination, and drafted the manuscript. AS participated in the study design and drafted the manuscript, and performed critical revisions. TPU participated in the study design, drafted the manuscript, and performed critical revision. All authors read and approved the final manuscript. 22. Reinstein DZ, Archer TJ, Gobbe M. Corneal epithelial thickness profile in the diagnosis of keratoconus. J Refract Surg. 2009;25(7):604–10. 23. Negishi K, Kumanomido T, Utsumi Y, Tsubota K. Effect of higher-order aberrations on visual function in keratoconic eyes with a rigid gas permeable contact lens. Am J Ophthalmol. 2007;144(6):924–9. 24. Marsack JD, Parker KE, Pesudovs K, Donnelly WJ 3rd, Applegate RA. Uncorrected wavefront error and visual performance during RGP wear in keratoconus. Optom Vis Sci. 2007;84(6):463–70. 4. McCormick GJ, Porter J, Cox IG, MacRae S. Higher-order aberrations in eyes with irregular corneas after laser refractive surgery. Ophthalmology. 2005; 112(10):1699–709. Author details 1S 1SynsLaser Kirurgi AS, Tromsø, Troms, Norway. 2School of Ophthalmology and Optometry, Wenzhou Medical University, Wenzhou, China. 3Eye Department, University Hospital North Norway, Tromsø, Norway. 4Department of Ophthalmology, Vestre Viken Hospital Trust, Drammen, Norway. 5Department of Medical Biochemistry, Oslo University Hospital, Oslo, Norway. 6Department of Oral Biology, Faculty of Dentistry, University of Oslo, Oslo, Norway. Competing interests None. Competing interests None. Competing interests None. 21. Koch DD, Ali SF, Weikert MP, Shirayama M, Jenkins R, Wang L. Contribution of posterior corneal astigmatism to total corneal astigmatism. J Cataract Refract Surg. 2012;38(12):2080–7. 9. Bruce AS, Catania LJ. Clinical applications of wavefront refraction. Optom Vis Sci. 2014;91(10):1278–86. 10. Wei RH, Lim L, Chan WK, Tan DT. Higher order ocular aberrations in eyes with myopia in a Chinese population. J Refract Surg. 2006;22(7):695–702. 7. Applegate RA, Sarver EJ, Khemsara V. Are all aberrations equal? J Refract Surg. 2002;18(5):S556–62. 6. Applegate RA, Ballentine C, Gross H, Sarver EJ, Sarver CA. Visual acuity as a function of Zernike mode and level of root mean square error. Optom Vis Sci. 2003;80(2):97–105. 5. Pesudovs K, Figueiredo FC. Corneal first surface wavefront aberrations before and after pterygium surgery. J Refract Surg. 2006;22(9):921–5. 11. Alpins NA. Treatment of irregular astigmatism. J Cataract Refract Surg. 1998; 24(5):634–46. 8. Cheng X, Bradley A, Ravikumar S, Thibos LN. Visual impact of Zernike and Seidel forms of monochromatic aberrations. Optom Vis Sci. 2010;87(5):300–12. 1. Maeda N, Fujikado T, Kuroda T, Mihashi T, Hirohara Y, Nishida K, et al. Wavefront aberrations measured with Hartmann-Shack sensor in patients with keratoconus. Ophthalmology. 2002;109(11):1996–2003. 2. Saad A, Gatinel D. Evaluation of total and corneal wavefront high order aberrations for the detection of forme fruste keratoconus. Invest Ophthalmol Vis Sci. 2012;53(6):2978–92. Shortcomings of the study 9 Schematic diagram showing the possible influence of coma on refractive astigmatism Table 4 Influence of coma on orientation of refractive astigmatism Axis of Coma Axis of DA TCL + LA Coma RA 1 94 98 WTR Vertical ATR 2 84 85 WTR Vertical ATR 3 97 92 ATR Vertical ATR↑ 4 93 84 ATR Vertical ATR↑ 5 119 114 ATR Vertical ATR↑ 6 84 82 ATR Vertical ATR↑ 7 51 64 WTR Oblique Oblique 8 34 68 WTR Oblique Oblique 9 115 81 WTR Vertical ATR 10 45 55 Oblique Oblique ATR 11 76 106 WTR Vertical WTR↓ DA = discrepant astigmatism; TCA = total corneal astigmatism, LA = lenticular astigmatism, RA = refractive astigmatism, WTR = with the rule, ATR = against the rule ↑: increased amplitude of astigmatism ↓: decreased amplitude of astigmatism Table 4 Influence of coma on orientation of refractive astigmatism ↑: increased amplitude of astigmatism Page 11 of 11 Zhou et al. Eye and Vision (2016) 3:13 Zhou et al. Eye and Vision (2016) 3:13 3. Piñero DP, Alió JL, Barraquer RI, Uceda-Montanes A, Murta J. Clinical characterization of corneal ectasia after myopic laser in situ keratomileusis based on anterior corneal aberrations and internal astigmatism. J Cataract Refract Surg. 2011;37(7):1291–9. 4. McCormick GJ, Porter J, Cox IG, MacRae S. Higher-order aberrations in eyes with irregular corneas after laser refractive surgery. Ophthalmology. 2005; 112(10):1699–709. 5. Pesudovs K, Figueiredo FC. Corneal first surface wavefront aberrations before and after pterygium surgery. J Refract Surg. 2006;22(9):921–5. 6. Applegate RA, Ballentine C, Gross H, Sarver EJ, Sarver CA. Visual acuity as a function of Zernike mode and level of root mean square error. Optom Vis Sci. 2003;80(2):97–105. 7. Applegate RA, Sarver EJ, Khemsara V. Are all aberrations equal? J Refract Surg. 2002;18(5):S556–62. 8. Cheng X, Bradley A, Ravikumar S, Thibos LN. Visual impact of Zernike and Seidel forms of monochromatic aberrations. Optom Vis Sci. 2010;87(5):300–12. 9. Bruce AS, Catania LJ. Clinical applications of wavefront refraction. Optom Vis Sci. 2014;91(10):1278–86. 10. Wei RH, Lim L, Chan WK, Tan DT. Higher order ocular aberrations in eyes with myopia in a Chinese population. J Refract Surg. 2006;22(7):695–702. 11. Alpins NA. Treatment of irregular astigmatism. J Cataract Refract Surg. 1998; 24(5):634–46. 12. Belin MW, Khachikian SS. An introduction to understanding elevation-based topography: how elevation data are displayed - a review. Clin Experiment Ophthalmol. 2009;37(1):14–29. 13. Oliveira CM, Ribeiro C, Franco S. 12. Belin MW, Khachikian SS. An introduction to understanding elevation-based topography: how elevation data are displayed - a review. Clin Experiment Ophthalmol. 2009;37(1):14–29. Conclusions 13. Oliveira CM, Ribeiro C, Franco S. Corneal imaging with slit-scanning and Scheimpflug imaging techniques. Clin Exp Optom. 2011;94(1):33–42. 14. Alpins NA. New method of targeting vectors to treat astigmatism. J Cataract Refract Surg. 1997;23(1):65–75. To our knowledge, this is the first study looking closer at the influence of coma-like HOAs on RA, as well as on the implications this issue brings to custom ablation planning in eyes with coma dominated optics. Our study shows that coma-like HOAs may have substantial influ- ence on RA, depending on its amount and orientation with respect to TCA. Topography-guided custom abla- tion which aims to correct corneal HOAs along with TCA independent on RA appears to be the preferred treatment strategy in dealing with this issue, except in cases with significant LA. Scheimpflug imaging techniques. Clin Exp Optom. 2011;94(1):33 42. 14. Alpins NA. New method of targeting vectors to treat astigmatism. J Cataract Refract Surg. 1997;23(1):65–75. 15. Holladay JT, Dudeja DR, Koch DD. Evaluating and reporting astigmatism for individual and aggregate data. J Cataract Refract Surg. 1998;24(1):57–65. 16. Holladay JT, Moran JR, Kezirian GM. Analysis of aggregate surgically induced refractive change, prediction error, and intraocular astigmatism. J Cataract Refract Surg. 2001;27(1):61–79. 16. Holladay JT, Moran JR, Kezirian GM. Analysis of aggregate surgically induced refractive change, prediction error, and intraocular astigmatism. J Cataract Refract Surg. 2001;27(1):61–79. 17. Courville CB, Smolek MK, Klyce SD. Contribution of the ocular surface to visual optics. Exp Eye Res. 2004;78(3):417–25. 17. Courville CB, Smolek MK, Klyce SD. Contribution of the ocular surface to visual optics. Exp Eye Res. 2004;78(3):417–25. 18. Leung TW, Lam AK, Kee CS. Ocular Aberrations and Corneal Shape in Adults with and without Astigmatism. Optom Vis Sci. 2015;92(5):604–14. 18. Leung TW, Lam AK, Kee CS. Ocular Aberrations and Corneal Shape in Adults with and without Astigmatism. Optom Vis Sci. 2015;92(5):604–14. 19. Roh HC, Chuck RS, Lee JK, Park CY. The effect of corneal irregularity on astigmatism measurement by automated versus ray tracing keratometry. Medicine (Baltimore). 2015;94(13):e677. 20. Ho JD, Tsai CY, Liou SW. Accuracy of corneal astigmatism estimation by neglecting the posterior corneal surface measurement. Am J Ophthalmol. 2009;147(5):788–95,795.e1-2. 2. Saad A, Gatinel D. Evaluation of total and corneal wavefront high order aberrations for the detection of forme fruste keratoconus. Invest Ophthalmol Vis Sci. 2012;53(6):2978–92. 1. Maeda N, Fujikado T, Kuroda T, Mihashi T, Hirohara Y, Nishida K, et al. Wavefront aberrations measured with Hartmann-Shack sensor in patients with keratoconus. Ophthalmology. 2002;109(11):1996–2003. References 1. Maeda N, Fujikado T, Kuroda T, Mihashi T, Hirohara Y, Nishida K, et al. Wavefront aberrations measured with Hartmann-Shack sensor in patients with keratoconus. Ophthalmology. 2002;109(11):1996–2003. 2. Saad A, Gatinel D. Evaluation of total and corneal wavefront high order aberrations for the detection of forme fruste keratoconus. Invest Ophthalmol Vis Sci. 2012;53(6):2978–92.
https://openalex.org/W2029349353	https://europepmc.org/articles/pmc3469839?pdf=render	English	null	Phytoplankton Chytridiomycosis: Fungal Parasites of Phytoplankton and Their Imprints on the Food Web Dynamics	Frontiers in microbiology	2,012	cc-by	11,999	REVIEW ARTICLE published: 12 October 2012 doi: 10.3389/fmicb.2012.00361 Reviewed by: Michael R. Twiss, Clarkson University, USA Hans-Peter Grossart, Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Germany Correspondence: Télesphore Sime-Ngando, UMR CNRS 6023, Laboratoire Microorganismes: Génome et Environnement, Clermont Université Blaise Pascal, BP 80026, 63171 Aubière Cedex, Keywords: fungi, chytrids, microbial parasites, phytoplankton hosts, food webs, microbial ecology, aquatic ecosystems Télesphore Sime-Ngando* Télesphore Sime-Ngando UMR CNRS 6023, Laboratoire Microorganismes: Génome et Environnement, Clermont Université Blaise Pascal, Clermont-Ferrand, France UMR CNRS 6023, Laboratoire Microorganismes: Génome et Environnement, Clermont Université Blaise Pascal, Clermont Edited by: Hans-Peter Grossart, Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Germany Reviewed by: Michael R. Twiss, Clarkson University, USA Hans-Peter Grossart, Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Germany Correspondence: Télesphore Sime-Ngando, UMR CNRS 6023, Laboratoire Microorganismes: Génome et Environnement, Clermont Université Blaise Pascal, BP 80026, 63171 Aubière Cedex, Clermont-Ferrand, France. e-mail: telesphore.sime- ngando@univ-bpclermont.fr Edited by: Hans-Peter Grossart, Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Germany Reviewed by: Michael R. Twiss, Clarkson University, USA Hans-Peter Grossart, Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Germany Correspondence: Télesphore Sime-Ngando, UMR CNRS 6023, Laboratoire Microorganismes: Génome et Environnement, Clermont Université Blaise Pascal, BP 80026, 63171 Aubière Cedex, Clermont-Ferrand, France. e-mail: telesphore.sime- ngando@univ-bpclermont.fr Edited by: Hans-Peter Grossart, Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Germany Reviewed by: Michael R. Twiss, Clarkson University, USA Hans-Peter Grossart, Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Germany Correspondence: Télesphore Sime-Ngando, UMR CNRS 6023, Laboratoire Microorganismes: Génome et Environnement, Clermont Université Blaise Pascal, BP 80026, 63171 Aubière Cedex, Clermont-Ferrand, France. e-mail: telesphore.sime- ngando@univ-bpclermont.fr Edited by: Parasitism is one of the earlier and common ecological interactions in the nature, occurring in almost all environments. Microbial parasites typically are characterized by their small size, short generation time, and high rates of reproduction, with simple life cycle occurring generally within a single host.They are diverse and ubiquitous in aquatic ecosystems, com- prising viruses, prokaryotes, and eukaryotes. Recently, environmental 18S rDNA surveys of microbial eukaryotes have unveiled major infecting agents in pelagic systems, consisting primarily of the fungal order of Chytridiales (chytrids). Chytrids are considered the earlier branch of the Eumycetes and produce motile, ﬂagellated zoospores, characterized by a small size (2–6 µm), and a single, posterior ﬂagellum. The existence of these dispersal propagules includes chytrids within the so-called group of zoosporic fungi, which are par- ticularly adapted to the plankton lifestyle where they infect a wide variety of hosts, including ﬁshes, eggs, zooplankton, algae, and other aquatic fungi but primarily freshwater phyto- plankton. Related ecological implications are huge because chytrids can killed their hosts, release substrates for microbial processes, and provide nutrient-rich particles as zoospores and short fragments of ﬁlamentous inedible hosts for the grazer food chain. Furthermore, based on the observation that phytoplankton chytridiomycosis preferentially impacts the larger size species, blooms of such species (e.g., ﬁlamentous cyanobacteria) may not totally represent trophic bottlenecks. Besides, chytrid epidemics represent an important driving factor in phytoplankton seasonal successions. In this review, I summarize the knowledge on the diversity, community structure, quantitative importance, and functional roles of fun- gal chytrids, primarily those who are parasites of phytoplankton, and infer the ecological implications and potentials for the food web dynamics and properties. I reach the conclu- sion that phytoplankton chytridiomycosis represents an important but as yet overlooked ecological driving force in aquatic food web dynamics and network organization. Hans-Peter Grossart, Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Germany Keywords: fungi, chytrids, microbial parasites, phytoplankton hosts, food webs, microbial ecology, aquatic ecosystems Phytoplankton chytridiomycosis: fungal parasites of phytoplankton and their imprints on the food web dynamics Télesphore Sime-Ngando UMR CNRS 6023, Laboratoire Microorganismes: Génome et Environnement, Clermont Université Blaise Pascal, Clermont-Ferrand, France INTRODUCTION (López-García et al., 2001; Lefranc et al., 2005; Lefèvre et al., 2007, 2008), adding to the other typical parasitic entities such as viruses (Sime-Ngando and Colombet, 2009). Eukaryotic parasites known from 18S rDNA diversity surveys include fungal members of the Phylum Chytridiomycota as a major water-borne group, com- prising both host-attached vegetative (i.e., sporangia) and free- swimming infective (i.e., ﬂagellated zoospores) stages (Gleason and MacArthur, 2008). The Phylum Chytridiomycota (thereafter, chytrids) occupies the basal branch of the Kingdom Fungi and because associated members are small in size and lack conspic- uous morphological features, chytrids are hardly distinguishable from many ﬂagellated protists such as the sessile choanoﬂagel- lates or bicosoecids which are bacterivores (Lefèvre et al., 2007, 2008). Chytrids exhibit different trophic strategies (i.e., para- sitism, saprotrophy) than these phagotrophic protists. In addition, chytrid propagules can represent key intermediates in the food chain (Gleason et al.,2009). Indeed,fungal zoospores have suitable Parasitism is one of the earlier known and most common eco- logical interactions in nature (Cavalier-Smith, 1993), occurring in almost all environments (Lafferty et al., 2006). Parasites have received much more attention in terrestrial than in aquatic ecosys- tems (Kuris et al., 2008), where they represent a strong forcing factor for critical evolutionary and ecological processes, e.g., pop- ulation dynamics, species successions, competition for resources, species diversiﬁcation, and energy and gene ﬂows (Hudson et al., 2006). Few attempts have been made to include parasites in the food web dynamics of aquatic systems (McCallum et al., 2004; Amundsen et al., 2009), with special emphasis on parasites of plants (Buschmann et al.,2001),invertebrates (Perkins,1993),and vertebrates (Marcogliese, 2004). Recent ecological and molecular surveys in pelagic environ- ments have revealed a high occurrence of eukaryotic puta- tive parasitoids, especially in the picoplanktonic size-fraction October 2012 \| Volume 3 \| Article 361 \| 1 www.frontiersin.org www.frontiersin.org Phytoplankton chytridiomycosis Sime-Ngando (reviewed in Jobard et al., 2010a; Wurzbacher et al., 2010, 2011). They constitute one of the last frontiers of the undiscovered biodi- versity and the related functions that challenge aquatic microbial ecology today. The number of fungi present on earth was estimated to about 1.5 million species, from which approximately 97,000 have so far been identiﬁed,including about 300 species of anamor- phic fungi and 300 species of Ascomycota but only few species of Basidiomycota and Chytridiomycota. INTRODUCTION Known fungal species cor- respond mostly to cultured specimens thriving in moist soils, lotic systems, mangroves, and wetlands, or to economically interesting pathogens of humans, plants, and animals. For example, Batra- chochytrium dendrobatidis, the chytridiomycosis agent of one of the most deadly contemporary skin diseases that drive the decline of amphibian populationsworldwidehasbeen wellstudied(Voyles et al., 2009), leading to a great deal of publicity. dimensions and represent a valuable food source for zooplank- ton. Similar to some protists (Desvilettes and Bec, 2009), fungal zoospores also contain essential fatty acids that might further upgrade the nutritional quality of food ingested by zooplankton such as Daphnia (Kagami et al., 2007a,b). Itisthusimportanttoincludeeukaryoticparasitesinthemicro- bial ecology of aquatic environments. This is timely and will help to integrate“novel”ecological perspectives and extend the concept of parasitismandtheirfunctionalpotentialtotheaquaticfoodweb dynamics (Gachon et al., 2010; Sime-Ngando and Niquil, 2011). This review focuses primarily on chytrid parasites of phytoplank- ton, and complements our recent review on eukaryotic microbial parasites in the plankton (Rasconi et al., 2011), by summarizing the knowledge on their diversity, community structure, quanti- tative importance, and ecological roles, and infer the ecological implications and potentials for the food web dynamics,properties, and overall topology. The kingdom Eumycota groups the following four divi- sions: Ascomycota, Basidiomycota, Zygomycota, and Chytrid- iomycota (James et al., 2006), but also include a particular group of microorganisms phylogenetically afﬁliated to the divisions Ascomycota and Basidiomycota (i.e., the so-called Deuteromy- cota), known as “fungi imperfecti” because only asexual forms (or anamorphs) have been observed during their life cycle. This group contains the particularly well studied members of Fungi in running waters, i.e., the hyphomycetes (Shearer et al., 2007). Zygomycota and Chytridiomycota do not emerge as mono- phyletic groups in recent phylogenetic analyses (James et al., 2000, 2006). Taxa traditionally placed in Zygomycota are distributed among Glomeromycota (Figure 1) and several subphyla incer- tae sedis, including Mucoromycotina, Entomophtoromycotina, ECOLOGICAL CONCEPTUALIZATION OF CHYTRID LIFE CYCLE AND ADAPTATION TO PELAGIC LIFESTYLE g Adaptation to dispersal in running water is typical of some fungi which have developed spores with particular morphologies (e.g., large and multiradiate conidia of hyphomycetes) allowing successfulattachmentonsubstratesinﬂowingwaters(Jobardetal., 2010a). Chytrids represent the sole true-fungi phylum with species producing ﬂagellated cells in their life cycle. These temporary swimming life zoospores are particularly well adapted to dispersal in pelagic medium where chytrids represent the best studied group of fungi, primarily in lakes where they occur mainly as phyto- plankton parasites (Ibelings et al.,2004; Rasconi et al.,2009,2012). However,these studies remain limited,mainly because of method- ological constraints and under sampling of pelagic ecosystems, where the occurrence of fungi is often considered as contamina- tion from allochthonous watershed inputs (Goh and Hyde, 1996; Jobard et al., 2010a). Chytrid species have an interesting life cycle in the context of the pelagic realm where the two main stages (i.e., sporangium and zoospore) have different effects on the food web dynamics. Most members reproduce asexually by releasing zoospores with a sin- gle posteriorly directed whiplash ﬂagellum (Sparrow, 1960; Barr, 2001). In a few species of the Neocallimastigales, zoospores are multiﬂagellate (Trinci et al., 1994) or in at least one species of the Blastocladiales (Hoffman et al., 2008) and one species in the Monoblepharidales (Ustinova et al., 2000), the spores lack ﬂagella. The thallus can be either monocentric, polycentric, or ﬁlamentous (hyphal; Sparrow, 1960) and are able to grow either on top or within substrates. In the typical life cycle, a free-living zoospore encysts to the host and expands intracellularly as a tubular rhizoid, i.e., the nutrient conveying system for the formation of fruit bod- ies (i.e., the infective sporangium) from which propagules (i.e., motile zoospores) are released into the environment. The hosts of parasitic chytrids in aquatic systems are highly diverse, including both prokaryotic (i.e., cyanobacteria) and eukaryotic phytoplank- ton, protists, invertebrates (larvae of insects, rotifers, nematodes, crustacean such as copepods, ostracods, cladocera etc. . .), ﬂower- ing plants or other fungi. Chytridiomycosis epidemics are known to produce massive amount of zoospores, now known as valu- able food source for zooplankton (Kagami et al., 2007a,b). The two main development stages of chytrids thus highlight two over- looked ecological potentials in the food web dynamics: (i) parasitic predation of host populations, most of which are inedible (i.e., unexploited by grazers), and (ii) the subsequent trophic link via the release of suitable zoospore food for zooplankton. CHYTRIDS: A DISREGARDED DIVERSITY WITHIN THE KINGDOM OF EUMYCOTA The term Fungi globally embraces all organisms that belong to the kingdom Eumycota (i.e., the so-called true-fungi), while the term fungi also includes other microorganisms (i.e., fungus-like organisms) traditionally studied by mycologists such as mem- bers of Myxomycota (Amobozoa) also called slime molds, and of Oomycota (Heterokonts) also called water molds, as well as mushrooms and other molds (Figure 1). All these organisms share similar trophic strategies, namely saprophytism, parasitism, and other symbiotic associations, and can occur in the same ecosystem Saprolegniomycedae Peronosporomycedae OOMYCOTA Dictyostelia Myxogastria Protostelia mobozoa MYXOMYCOTA Excavata Am Excavata EUMYCOTA EUMYCOTA FIGURE 1 \| Approximate position of true-Fungi (Eumycota) and fungus-like (Oomycota and Myxomycota) organisms in a schematic phylogenetic tree of eukaryotes. Modiﬁed from Jobard et al. (2010a). Excavata Excavata FIGURE 1 \| Approximate position of true-Fungi (Eumycota) and fungus-like (Oomycota and Myxomycota) organisms in a schematic phylogenetic tree of eukaryotes. Modiﬁed from Jobard et al. (2010a). FIGURE 1 \| Approximate position of true-Fungi (Eumycota) and fungus-like (Oomycota and Myxomycota) organisms in a schematic phylogenetic tree Frontiers in Microbiology \| Aquatic Microbiology October 2012 \| Volume 3 \| Article 361 \| 2 Frontiers in Microbiology \| Aquatic Microbiology October 2012 \| Volume 3 \| Article 361 \| 2 Phytoplankton chytridiomycosis Sime-Ngando marine green algae and diatoms (review in Gleason et al., 2011). Water-borne fungi have to face various difﬁculties and constraints characteristic of aquatic habitats,among which oxygen availability perhaps may be one of the most restrictive parameters. Except for Neocallimastigales in which all species are amitochondrial obligate anaerobes known from the rumen hindguts of herbivorous mam- mals where ambient temperatures approach 40˚C, all chytrids are obligate aerobes and their growth rates are greatly inhibited by low dissolved oxygen concentrations (Gleason et al., 2008). Inves- tigations of the tolerance to anoxia in the chytrids Rhizophydium sphaerotheca and Phlyctochytrium punctatum have revealed that these fungi are facultative anaerobes (Goldstein, 1960). Kickxellomycotina, and Zoopagomycotina. The Chytridiomycota is retained in a restricted sense, with Neocallimastigomycota and Blastocladiomycota representing segregate phyla of ﬂagellated Fungi, also known as zoosporic Fungi. These zoosporic true-fungi (chytrids) were described according to modes of reproduction, thallus development and ecology and, most importantly, mor- phological characteristics of the thallus such as size, shape, and ornamentation of the sporangia, presence or absence of an oper- culum,and rhizoidal arrangement (Blackwell et al.,2006). CHYTRIDS: A DISREGARDED DIVERSITY WITHIN THE KINGDOM OF EUMYCOTA Because they rely on free water phase for whole or part of their life cycle, chytrids are considered typical pelagic (i.e., ﬂoating) species. ECOLOGICAL CONCEPTUALIZATION OF CHYTRID LIFE CYCLE AND ADAPTATION TO PELAGIC LIFESTYLE In addi- tion, we have recently shown that chytrid parasitism of cells within the ﬁlaments of cyanobacteria during bloom events can result in a mechanical fragmentation of the inedible ﬁlaments into shorter-size edible ﬁlaments (Gerphagnon et al., submitted), therebyenhancedthecontributionof fungalparasitestothebloom decline (Figure 2). METHODOLOGICAL LIMITATIONS www.frontiersin.org METHODOLOGICAL LIMITATIONS October 2012 \| Volume 3 \| Article 361 \| 3 www.frontiersin.org www.frontiersin.org Sime-Ngando Phytoplankton chytridiomycosis Grazers Trophic upgrading Swimming zoospores Host fragments Enhanced trophic link ost search, ermina!on Mature sporangium Zoospores Ho ge Infected cells Host fragmenta!on Healthy cells Pelagial S di t Weakened C export Sediments Grazers Trophic upgrading Swimming zoospores Host fragments Enhanced trophic link ost search, ermina!on Mature sporangium Zoospores Ho ge Infected cells Host fragmenta!on Healthy cells Pelagial S di t Weakened C export Sediments FIGURE 2 \| Phytoplankton chytridiomycosis affects food web dynamics and properties by killing part of or the entire host ﬁlaments or free-living cells, and provide nutrient-rich particles as zoospores and short fragments of ﬁlamentous inedible hosts for the grazer (Daphnia in this case) food chain. Because chytrids preferentially attack larger size species such as the ﬁlamentous cyanobacterium Anaebaena macroscopora in this case, blooms of such species may not totally represent trophic bottleneck to be sequestered in the sediments. Part of their energy are retained in the pelagial and recycled through the trophic cascade. Source for Daphnia zooplankton micrograph is the science photo library: http://www.sciencephoto.com/media/367262/enlarge. The microphotograph of infected A. macroscopra ﬁlament with Cytox Green stained zoospores observed under confocal microscopy is a courtesy of Melanie Gerphagnon. Grazers Trophic upgrading Swimming zoospores Host fragments Enhanced trophic link Pelagial Weakened C export S di t Sediments FIGURE 2 \| Phytoplankton chytridiomycosis affects food web dynamics and properties by killing part of or the entire host ﬁlaments or free-living cells, and provide nutrient-rich particles as zoospores and short fragments of ﬁlamentous inedible hosts for the grazer (Daphnia in this case) food chain. Because chytrids preferentially attack larger size species such as the ﬁlamentous cyanobacterium Anaebaena macroscopora in this case, blooms of such species may not totally represent trophic bottleneck to be sequestered in the sediments. Part of their energy are retained in the pelagial and recycled through the trophic cascade. Source for Daphnia zooplankton micrograph is the science photo library: http://www.sciencephoto.com/media/367262/enlarge. The microphotograph of infected A. macroscopra ﬁlament with Cytox Green stained zoospores observed under confocal microscopy is a courtesy of Melanie Gerphagnon. Molecular biology techniques have allowed a thorough recon- struction of chytrid phylogenies (James et al., 2006) and are increasingly providing more speciﬁc tools, primarily oligonu- cleotide probes, for the quantitative ecological study of aquatic parasites. METHODOLOGICAL LIMITATIONS Conventional methods for analysis of zoosporic fungi to date have mainly relied on direct observation and baiting techniques, with subsequent fungal identiﬁcation using morphological char- acteristics (review in Marano et al., 2012). Earlier descriptions of chytrid parasites were based on microscopic observation of sporangia which exhibit morphological features that can allow approximate phenotypic identiﬁcation of specimens in living samples or those preserved with Lugol’s iodine (Rasconi et al., 2011). Such approaches have provided detailed descriptions of the morphological features using light or phase-contrast microscopy (Ingold, 1940; Canter, 1949, 1950, 1951). Subsequently, electron microscopy was used to describe different life stages, and a num- ber of studies describing the ultrastructural cytology of fungal zoospores and spore differentiation are available (Rasconi et al., 2011). The chytrid Blastocladiella sp. was the ﬁrst fungal model for detailed structural studies on sporogenesis (Lovett, 1963). The precise conformation of the ﬂagellar rootlets and the spa- tial distribution of organelles in zoospores have been determined, providing the basis for chytrid taxonomy (review in Gleason and Lilje, 2009). Chytrids are more commonly found growing in bodies of waters, primarily in freshwater environments, and in soils as well. In general, chytrids prefer environments with low osmotic potentials. Only three species (i.e., Rhizophydium littoreum, Tha- lassochytrium gracilariopsis, and Chytridium polysiphoniae) have been properly identiﬁed and partially characterized from brack- ish and marine ecosystems. These species are either facultative or obligate parasites of marine macroalgae and invertebrates. Also, some species of Olpidium and Rhizophydium are parasites of small Ecological investigations of the dynamics of chytrid popula- tions in natural environments have been improved more recently withepiﬂuorescencemicroscopy.Severalﬂuorochromeshavebeen used, among which the chitin stain calcoﬂuor white (CFW) pene- trates infected host cells remarkably well and is more efﬁcient for the observation and photomicrography of the complete rhizoidal system of parasites, which is an important criterion for chytrid identiﬁcation (Rasconi et al., 2009; Sime-Ngando et al., 2012a). METHODOLOGICAL LIMITATIONS Fluorescent in situ hybridization, ampliﬁed with horse- radish peroxidase activation by ﬂuorescent tyramide (also known as catalyzed reporter deposition, CARD FISH or TSA FISH), is a reliable approach to detect and count speciﬁc chytrid para- sites, especially the zoospore stages that lack a chitinaceous wall, precluding any simple use of ﬂuorochromes such as CFW (Sime- Ngando et al., 2012b). The clone-FISH approach, which was orig- inally designed for prokaryotes, was recently adapted by Jobard et al. (2010b) to the assessment of zoosporic fungi in natural sam- ples. Clone-FISH is based on the genetic modiﬁcation of a clone of Escherichia coli by inserting plasmid vector containing the tar- get 18S rDNA sequence. The main advantage of the clone-FISH method is that pure cultures which are necessary to validate the speciﬁcity and hybridization conditions of FISH or CARD FISH probing are not needed (Jobard et al., 2010b). Oligonucleotidic probes have also been used in quantitative real-time PCR (qPCR), which is an excellent tool for solving the limitations in the detec- tion of less abundant and rare species (Lefèvre et al., 2010; Sime- NgandoandJobard,2012).Furtherapplicationof PCR-basemeth- ods,primarily of next generation sequencing technologies,will not only advance our quantitative understanding of zoosporic fungal ecology and diversity, but also their function through the analy- sis of their genomes and gene expression (Monchy et al., 2011). Nevertheless, it is still necessary to complement these molecular- based approaches with cultivation-based methods in order to gain a fuller understanding of the ecological and physiological roles of zoosporic fungi (Marano et al., 2012). The methodological difﬁculties are thus increasingly being overcome, and it is becoming evident that techniques from mol- ecular biology are useful for the study of zoosporic fungi. The challenge of matching molecular sequences to microscopic phe- notypes is also on the route to be tackled (Jobard et al., 2012; Monchy et al., 2012). Furthermore, to assess the functional impact of chytrid parasites on host populations, algorithms commonly October 2012 \| Volume 3 \| Article 361 \| 4 Frontiers in Microbiology \| Aquatic Microbiology Phytoplankton chytridiomycosis Sime-Ngando Melosira spp. The genus Rhizosiphon comprised two species that are speciﬁc parasites of vegetative cells and akinetes (R. cras- sum) and of akinetes alone (R. akinetum), which correspond to different niches offer by the ﬁlamentous cyanobacterium host Anabaena macrospora in productive lakes (Gerphagnon et al., sub- mitted). SEASONAL DIVERSITY, ABUNDANCE, COMMUNITY STRUCTURE AND TROPHODYNAMICS OF CHYTRIDS Studies on pelagic chytrids started in the British lakes (Cook, 1932), and different authors have provided descriptions of mor- phological characters (Reynolds, 1940; Canter and Lund, 1948, 1953; Pongratz, 1966; Canter, 1972). Quantitative assessment of the importance of parasitism indicated that infection of diatoms, desmids, and other green algae is fairly common in freshwaters (Canter and Lund, 1948, 1968). However, most studies on parasite dynamics in natural plankton assemblages are focused on hosts or limited to the investigations of one or two fungal populations (Table 1). This makes the generalization of observed patterns dif- ﬁcult, and the seasonal dynamics of chytrids at the community level remain largely unknown. In the Rasconi and coauthor’s model (Rasconi et al., 2012), during winter, the development and activities of both chytrid parasites and their phytoplanktonic hosts were at their lowest lev- els, because of low temperature, freezing, or ice-cover. From late winter on, the environmental conditions, primarily the increase in water temperature and in mixing-derived nutrient availability, favor the development of host communities, with the dominance of k-strategists (e.g., large diatoms) toward spring. As a conse- quence, the host – parasite contact probability increases, raising the chytrid infectivity and the production of large amount of zoospores. Enhanced infection prevalence then limits and pro- vokesthedeclineof largediatoms,liberatingnichesforadiversiﬁed phytoplankton community of small size r-strategists. The abun- dance of chytrid sporangium reaches their lowest level, while the availability of food (i.e., small phytoplankton and fungal zoospores) favors the development of grazers and the establish- ment of a typical clear-water phase at the end of spring. During thesummermonths,favorableenvironmentalconditions,together with a high grazing pressure,allow the development of a diversiﬁed and complex plankton community. Small edible hosts are inhib- ited by the grazing pressure,while the availability of large size hosts favors the proliferation of different chytrid species toward the end of the summer. From here, oligotrophic lakes signiﬁcantly diverge from productive waters. In oligotrophic situations,autumnal over- turn promotes species coexistence and phytoplankton diversity leads to the association of different species of chytrid parasites of chlorophytes and diatoms, but with general low infection preva- lence due to a balanced host – parasite growth. METHODOLOGICAL LIMITATIONS Although almost all the chytrid species were observed from oligo- to eutrophic conditions, the seasonal fungal com- munity composition was largely dominated by species of the genus Rhizophidium (90% of total sporangium abundance) in oligotrophic conditions, and of the genera Rhizophidium (56%), Zygorizidium (22%), Chytridium (19%), and Rhizosiphon (14%) in eutrophic conditions (Rasconi et al., 2012). used by parasitologists such as the prevalence and the intensity of infection have been applied in ecological studies of chytridiomyco- sis (Rasconi et al., 2012). These parameters are derived from direct microscopy and are critical for assessing the community structure of parasites, interactions with hosts, and epidemiology, as well as the potential impact of fungal parasites on the food web dynamics. Understanding the environmental factors that induce epidemics can also be inferred this way (i.e., through empirical correlations) or by using epidemiological approaches such as the changes in incidence rates (i.e., the number of new cases of infections occur- ring during a given time) or the occurrence of epidemics (i.e., a widespread outbreak of an infection) within host populations (Fox, 2003). The community structure of natural chytrids is intimately linked to the availability of hosts (Ibelings et al., 2004). However, exceptthestudybyRasconietal.(2012)thathasproposedageneral empirical model on chytrid seasonality and trophodynamics (i.e., with their hosts) based on the theoretical PEG model of seasonal succession of planktonic events in freshwaters (Sommer et al., 1986),there is still no study assessing the fungal species successions in natural environments. This contrasts with the general hypothe- ses and patterns of plankton (primarily phytoplankton and zoo- plankton) successions and community structure, which are well described in temperate lakes (Sommer et al., 1986). This was recently revisited by considering a suite of overlooked ecological interactions that included parasitism (Sommer et al., 2012). These authors concluded that the effects of these “novel” interactions on plankton seasonal succession are limited in terms of seasonal biomass patterns but strong in terms of species replacements. SEASONAL DIVERSITY, ABUNDANCE, COMMUNITY STRUCTURE AND TROPHODYNAMICS OF CHYTRIDS In eutrophic lakes, nutrient conditions and persistent stratiﬁcation favor the A recent extensive seasonal study in temperate freshwater lakes (Rasconi et al., 2012), based on CFW staining of infective spo- rangia and phenotypic identiﬁcation, has identiﬁed up to 15 different chytrid species on diverse host populations, with speciﬁc biovolume ranging from 7 to 72 µm3 sporangium−1 (Table 1). Seasonal abundances increased from 0.0005 to 0.4 × 106 sporan- gia l−1 in oligotrophic conditions to 0.0 to 32 × 106 sporangia l−1 in eutrophic conditions (Table 1). In both conditions, spo- rangium abundances peaked with the development of preferential diatom hosts in spring and cyanobacteria in autumn, the autumn peak being largely higher in eutro- than in oligotrophic condi- tions, when a monospeciﬁc bloom of Anaebaena sp. occurred. Quantitative data on the seasonal abundance of zoospores are lacking. All the 15 species identiﬁed were monocentric (i.e., with one center of growth and development) and eucarpic (using part of the thallus for the fruit-body formation and with a special- ized rhizoidal system), typical of the class Chytridiomycetes, and belonged to two orders: the Rhizophidiales which contained one genus (Rhizophidium),and the Chytridiales which contained three genera (Chytridium,Zygorhizidium,and Rhizosiphon). The species of Rhizophidium spp. infected a wide diversity of hosts, including both large size (e.g., the Chlorophyta Staurastrum spp. and the diatoms Asterionella formosa, Synedra spp. and Fragilaria croto- nensis) and small size algae (e.g., the diatom Cyclotella spp. and the Chlorophyta Chodatella ciliata and Ankistrodesmus convolu- tus). The species Chitridium spp. infected the chlorophyte Oocystis sp., the diatom F. crotonensis, and the cyanobacterium Microcys- tis sp., while the species of Zygorhizidium infected the diatoms October 2012 \| Volume 3 \| Article 361 \| 5 www.frontiersin.org Sime-Ngando Phytoplankton chytridiomycosis Table 1 \| Quantitative data on fungal parasites and parasitism of phytoplankton in temperate lake ecosystems. Environment, country (trophy) Method Sampling period (depth, m) Chytrid sporangia (106 l−1) Sporangium biovolume (µm3) Prevalence of infection (% infected host cells) Intensity of infection (sporangia host cell−1) Host (Chytrid) Reference Lake Pavin, France (O-M) CFW staining and EM Feb.–Dec. 2007 (Ze) 0.005–3.7 6.7–67.4 1.5–59.1 1–2.5 Phytoplankton communities (mixed) Rasconi et al. (2012) Lake Aydat, France (E) CFW staining and EM Feb.–Dec. 2007 (Ze) 0–3.4 8.7–72.4 0–98* 1–2 Phytoplankton communities (mixed) Rasconi et al. (2012) Lake Schöhsee, Germany (M) Lugol staining and LM (Utermöhl) Mar. October 2012 \| Volume 3 \| Article 361 \| 6 ECOLOGICAL IMPLICATIONS OF PHYTOPLANKTON CHYTRIDIOMYCOSIS Based on the study by Rasconi et al. (2012), the prevalences of infection (% of infected host cells) typically average around 20%, with no signiﬁcant variation with the trophic status of freshwa- ter temperate lakes. These values increase to reach about 100% when monospeciﬁc blooms of infected hosts occurred in nat- ural conditions or when speciﬁc chytrid-host systems are targeted in controlled conditions (Table 1). Chytrid infection commonly leads to the death of their host cells (Canter and Lund, 1951; Sen, 1988b,c; Kudoh and Takahashi, 1990; Bruning et al., 1992; Holfeld,1998,2000;Ibelings et al.,2004,2011) and this is enhanced by the intensity of infection (number of parasites per host cell) which can largely exceed 1 (Table 1). Empty sporangia are cur- rently found attached on dead phytoplankton cells (Rasconi et al., 2012), which is suggestive of the lethal issue of chytrid infection. There are several evidences that parasitism inhibits the develop- ment of sensible species, and particular attention has been paid to the occurrence of fungi on diatoms, and to the effects of par- asitism on their seasonal distributions (Canter and Lund, 1948; Van Donk and Ringelberg, 1983). For example, in the oligotrophic Lake Pavin (France), the spring development of the diatoms Aste- rionella and Synedra was found to be inhibited by the chytrid Rhizophidium planktonicum. In productive Lake Aydat (France) another diatom, Fragilaria, became abundant but the prolifera- tion of their parasites, Rhizophidium fragilariae, interrupted their development (Rasconi et al., 2012). In natural phytoplankton community, the parasitized popula- tions are often replaced by others with similar ecologically require- ments, which can render unchanged the standing stocks of phyto- plankton hosts in the ecosystem, with no visible obvious damage to the total community (Reynolds, 1940). However, chytrids seem to preferentially infect large and less edible phytoplankton species, as discussed previously. Diverse environmental conditions, including temperature, tur- bulence, light, nutrient concentrations, and biotic factors such as predation but primarily the host availability, can inﬂuence the growth rate of fungal parasites (Canter and Jaworski, 1981; Brun- ing and Ringelberg, 1987; Bruning, 1991; Kagami et al., 2004). The primary factor determining the absence or the presence of a particular parasite in the environment is the availability of suit- able hosts (Ibelings et al., 2004). The host population density is frequently considered an important factor in the ecology of par- asites. SEASONAL DIVERSITY, ABUNDANCE, COMMUNITY STRUCTURE AND TROPHODYNAMICS OF CHYTRIDS bloom of ﬁlamentous cyanobacteria from the end of the summer period toward early autumn, with the development of a monospe- ciﬁc community of chytrids (i.e., Rhizosiphon spp.). The highest infection prevalence is noted, followed by the decline of cyanobac- teria – chytrid system toward the late seasonal phase (for more details, see ﬁgure number 7 in Rasconi et al., 2012). bloom of ﬁlamentous cyanobacteria from the end of the summer period toward early autumn, with the development of a monospe- ciﬁc community of chytrids (i.e., Rhizosiphon spp.). The highest infection prevalence is noted, followed by the decline of cyanobac- teria – chytrid system toward the late seasonal phase (for more details, see ﬁgure number 7 in Rasconi et al., 2012). PHYTOPLANKTON CHYTRIDIOMYCOSIS AND THE INFLUENCE OF ENVIRONMENTAL FACTORS A basis for study chytrid epidemics within phytoplankton commu- nities was recently provided for freshwater lakes where, in contrast to sporangium abundance and biomass that increased from oligo- to eutrophic conditions, the prevalence of infection is quite simi- lar in both conditions, averaging about 20% (Rasconi et al., 2012). The highest prevalence (98%) was noted for the autumn bloom of a ﬁlamentous cyanobacterium (A. macospora) facing the parasite Rhizosiphon crassum in a productive lake. The host species compo- sition and their size appeared as critical for chytrid infectivity, the larger hosts being more vulnerable, including pennate diatoms, desmids, and ﬁlamentous cyanobacteria. Such host species are apparently easier to hit because their size naturally increase the host – parasite contact rates, and are expected to excrete more attracting substances known to favor the zoospores searching of suitable hosts (Canter and Jaworski, 1981). Larger algae also con- tainmoreresourcesforthedietof parasites,andthisisthecommon explanation of why algal species with larger cell size can be heavily infected, even at lower population density (Lund, 1957; Holfeld, 1998). On the other hand, the prevalence of infection was also shown to be correlated with total phosphorus, which may be related to the productivity of the milieu that offers good substrate conditions for the growth of parasite-host systems (Rasconi et al., 2012). The abundances but also the cell volumes of hosts thus seem important features in determining the amplitude of chytrid epidemics within natural phytoplankton. These parameters also appeared to be related with the tolerance threshold of infection, i.e., the critical prevalence or the level of prevalence from which the standing stock of phytoplankton starts to decline (Bruning et al., 1992). At low host abundance, the critical infection preva- lence is generally lower that 20%, but increases with increasing host abundance. This is probably one of the mechanisms from which parasites regulate host populations. SEASONAL DIVERSITY, ABUNDANCE, COMMUNITY STRUCTURE AND TROPHODYNAMICS OF CHYTRIDS 1987–May, 1989 (0–10) ND ND 0–>90** (≤1–10% of total host volume) ND Different species within Phytoplankton communities (mixed) Holfeld (1998) Lake Maarsseveen, The Netherlands (O-M) 1978–2010 (0–10) ND ND 0–90 ND Asterionella formosa (Rhizophydium planktonicum) Ibelings et al. (2004, 2011) Lake Kinneret, Israel (O-M) LM (Utermöhl) Oct. 2000–Dec. 2003 (2 and 7) ND ND 0–83 ND Peridinium gatunense (Phlyctochytrium sp.) Alster and Zohary (2007) Lake Schöhsee, Germany (M) Lugol staining and LM (Utermöhl) 27 Jan.–7 Feb. 1989 (0–10) ND ND ∼10–80 ∼0.1–1.8 Stephanodiscus alpinus (Zygorhizidium spp.) Holfeld (2000) Lake Suwa, Japan (E) Dialysis tube cultures and LM Nov. 1986–Nov. 1987 (0–4) ND ND 2–30 ND Asterionella formosa (Rhizophydium planktonicum or Zygorhizidium afﬂuens) Kudoh and Takahashi (1990) Shearwater, UK (E) Several occasions between 1978 and 1981 (net samples) ND ND 0.2–1.4 Up to 4 Centric diatoms: Cyclotella spp., Stephanodiscus hantzschii, and Melosira spp. (mixed) Sen (1988a) Shearwater, UK (E) 10 epidemic periods between 1978 and 1980 (net samples) ND ND 15–90% ND Microcystis aeruginosa (Rhizidium microcystidis) Sen (1988b) Shearwater, UK (E) Several occasions between 1978 and 1981 (net samples) ND ND Up to 85% ND Several species of chlorophytes (mixed) Sen (1988c) Recorded during a monospeciﬁc bloom of Anabaena ﬂosaquae infected by Rhizosiphon crassum; *corresponds to the infection of Synedra acus infected by Zygorhizidium planktonicum. O, oligotrophic; M, mesotrophic; E, eutrophic or productive with recurrent cyanobacterial blooms; Ze, euphotic zone; CFW, calcoﬂuor white (cf. Rasconi et al., 2009), EM, epiﬂuorescence microscopy; LM, light microscopy; ND, not determined or not given explicitly. ata on fungal parasites and parasitism of phytoplankton in temperate lake ecosystems. Frontiers in Microbiology \| Aquatic Microbiology Phytoplankton chytridiomycosis Sime-Ngando epidemics in natural phytoplankton populations arise more eas- ily when growth conditions for hosts are worst (Reynolds, 1984). Under such conditions, the growth rate of the algae would be rel- atively slow, contrasting with their fast-growing parasites. Rasconi et al. (2012) hypothesized that there are two different phases in the parasite – host trophodynamic: a synchronous growth phase of both chytrids and algae when high availability of hosts favored the encounter between parasites and newly produced sensible host cells, followed by a second phase corresponding to the decline of host populations due to the infection, characterized by an increase in the infection prevalence. ECOLOGICAL IMPLICATIONS OF PHYTOPLANKTON CHYTRIDIOMYCOSIS Indeed, irrespective of temperature and light conditions, a minimal threshold value of the host density is required for the occurrence of epidemics (Bruning, 1991). Several studies have reported that parasites seem to grow better on healthy individ- uals within actively growing host populations (Canter and Lund, 1948; Van Donk and Ringelberg, 1983). However, this is far from being a generalization because evidence was also provided that Some examples provided in the literature suggest that large infected diatoms such as Asterionella sp. and Fragilaria sp. (mean length 50 and 70 mm, respectively) can be replaced by small cen- tric diatoms such as Stephanodiscus spp. (10 mm; Van Donk and Ringelberg, 1983; Sommer, 1987). This implies that the develop- ment of large species is inhibited by infection, while smaller algae proliferate. Active parasitism may thus act on the host standing stock in a continuum from no change to signiﬁcant changes but in all cases will affect the phytoplankton community structure. In the context of phytoplankton seasonal dynamics and species successions, this can have profound ecological implications (Van October 2012 \| Volume 3 \| Article 361 \| 7 www.frontiersin.org www.frontiersin.org Phytoplankton chytridiomycosis Sime-Ngando as amebae and ﬂagellates, detritivores, grazers such as ﬁlter feed- ing zooplankton, and benthic suspension feeders (Gleason et al., 2009; Sime-Ngando, in press). Since most of these consumers do not discriminate between food resources except by size, we would expect zoospores as well as hyphae and non-motile spores to be eaten by many of these consumers, although published records are lacking. Fungal zoospores are well within the range of a good particle size for zooplankton feeding behavior and consequently, when fed upon, transfer matter to higher trophic levels in the food chain. For example, zoospores are efﬁciently grazed by crus- tacean zooplankton such as Daphnia spp. (Kagami et al., 2007a,b, 2011; Sime-Ngando,in press),before they grow into a mature thal- lus (i.e., body). Thus zoospores may provide organic compounds containing nitrogen, phosphorus and sulfur, mineral ions, and vitamins to grazing zooplankton (Figure 2). Donk, 1989). For example, due to chytrid parasites, the phyto- planktoncommunitycanshiftfromamaturestageof development typically dominated by large, k-strategist species toward a pio- neer stage of succession that favors the development of small, r-strategist species. ECOLOGICAL IMPLICATIONS OF PHYTOPLANKTON CHYTRIDIOMYCOSIS In addition, by controlling the phytoplankton dynamics, chytrids can signiﬁcantly affect the primary production of aquatic systems, as suggested by a negative correlation between the primary production and the per sporangium biovolume of chytrids in lakes (Rasconi et al., 2012). More importantly, phytoplankton chytridiomycosis produce massive amount of propagules, i.e., zoospores. Because chytrids are small in size and lack conspicuous morphological features, a situation that makes them hardly distinguishable from many ﬂa- gellatedprotistssuchasthesessilechoanoﬂagellatesorbicosoecids, theirfunctionalroles,primarilyassaprotrophsorparasites,remain most of the time cryptic in classical microscopy studies (Lefèvre et al.,2007,2008; Sime-Ngando et al.,2011). Previously,the modes of nutrition for all heterotrophic ﬂagellates in the plankton were thought to be restricted to bacterivory, but zoosporic fungi are not bacterivores (i.e., bacterial feeders). It is now clearly evident that not all heterotrophic ﬂagellates thriving in pelagic systems are either protists or bacterivores as previously thought, and that parasitism and saprophytism from fungal ﬂagellates might rep- resent important potential functions in these ecosystems. Fungal zoospores are valuable food sources for zooplankton because cyto- plasm of chytrids contains storage carbohydrates such as glycogen, storage proteins,a wide range of fatty acids,phospholipids,sterols, and other lipids (Gleason et al., 2009; Sime-Ngando, in press). When chytrids reproduce, most of the cytoplasm is converted into zoospores which swim away to colonize new substrates or infect new hosts. Lipids are considered to be high energy compounds, some of which are important for energy storage. Indeed, lipids are present mainly in the form of endogenous reserves, often as membrane bound vesicles called lipoid globules which can eas- ily be seen in the cytoplasm of fungal zoospores with both the light and electron microscopes (Gleason and Lilje, 2009; Sime- Ngando, in press). The size and numbers of lipoid globules within zoospores varied and their ultrastructure is complex. The chem- ical composition of lipids, including both fatty acids and sterols, has been characterized in a number of genera of zoosporic fungi. These endogenous reserves are consumed during the motile phase of the zoospores. They presumably provide energy for the move- ment of ﬂagella during the motile phase which can last for up to several hours, as well as for the attachment and germination of zoospores on the appropriate substrates or hosts (Figure 2). ECOLOGICAL IMPLICATIONS OF PHYTOPLANKTON CHYTRIDIOMYCOSIS Besides, many zoosporic fungi can grow in the laboratory on minimal synthetic media containing one carbon source such as cellulose,xylan,starch,or chitin along with salts containing nitrate, sulfate, and phosphate (Gleason et al., 2008; Sime-Ngando, in press). This establishes fungi as potential competitors of bacteria and primary producers for essential minerals (Figure 2). Th th i iﬁ t f ti f th hi h More interestingly, zoospores are a particularly good food source because of their nutritional qualities. Presumably many consumers must obtain at least some essential nutrients from their food sources because these compounds cannot be pro- duced de novo. One example is found in the cladoceran Daph- nia. Recent research has shown that zoospores of the para- sitic chytrid, Zygorhizidium, are quite rich in polyunsaturated fatty acids (PUFAs) and cholesterols, which are essential nutri- ents for the growth of Daphnia (Kagami et al., 2007a,b). These zoospores are found to facilitate the trophic transfer from the inedible large diatom hosts, Asterionella sp., and the growth of Daphnia. PUFAs and cholesterol are known to promote growth and reproduction in crustacea. This phenomenon, known as the “trophic upgrading concept” (Sime-Ngando et al., 2011; Sime- Ngando, in press), is of signiﬁcant importance in the aquatic food webs because it highlights not only the quantity but also the quality of the matter being transferred via fungal zoospores (Figure 2). Frontiers in Microbiology \| Aquatic Microbiology IMPACT OF PHYTOPLANKTON CHYTRIDIOMYCOSIS ON THE FOOD WEB PROPERTIES Given that food webs are central to ecological concepts (Pascual and Dunne, 2006), it is important to establish the role of parasites in the structure and function of food webs. In theory, parasites can have a variety of effects. Lafferty et al. (2006, 2008) suggested that parasites affect food web properties and topology since they dou- ble connectance (deﬁned as the number of observed links divided by the number of possible links) and quadruple the number of links. Others have postulated that parasites drive an increase in speciesrichness,trophiclevels,andtrophicchainlengthof thefood web (Huxham et al., 1995; Thompson et al., 2005). These prop- erties may stabilize community structure (Hudson et al., 2006). However, the potential effects of parasites on food web stability is a complex and unresolved issue since the concept of stability is the center of a perhaps inﬁnite debate in community ecology (Elton,1958;May,1972;Pimm,1984;McCann,2000;Hosack et al., 2009; O’Gorman and Emmerson, 2009). Based on the ideas of May (1973), parasites should lead to a destabilized trophic net- work because they increase species diversity and the connectance. In addition, adding parasites to food webs extends the length of trophic chains, which can decrease food web stability (Williams and Martinez, 2004). However, the addition of long loops of weak There are other signiﬁcant functions for the high energy com- pounds found in fungal zoospores, especially as food resources for zooplankton and probably for many other consumers in aquatic ecosystems. Fungal spores and hyphae in general are known to be eaten by a large number of different consumers in both aquatic and soil ecosystems,including a variety of mycophagous protozoa such October 2012 \| Volume 3 \| Article 361 \| 8 Phytoplankton chytridiomycosis Sime-Ngando chytrids and the resulting ﬂows (MWOC,Model without Chytrid), as traditionally done in previous plankton food web analysis (e.g., Niquil et al., 2006). MWC and MWOC models were constructed on the basis of the same data set corresponding to the spring bloom period in Lake Pavin (i.e., March–June 2007). These models were built using the Linear Inverse Modeling procedure (LIM, Véz- ina and Platt, 1988) recently modiﬁed into the LIM-Monte Carlo Markov Chain (LIM-MCMC;Van den Meersche et al., 2009). This method allow reconstruction of missing ﬂow values and alleviates the problem of under sampling using the principle of conservation of mass, i.e., the quantity of carbon coming into each compart- ment considered as equal to the amount leaving it (Vézina and Platt, 1988). IMPACT OF PHYTOPLANKTON CHYTRIDIOMYCOSIS ON THE FOOD WEB PROPERTIES Thanks to recent development of the inverse analysis into LIM – MCMC, a probability density function covering the range of possible values was generated for each ﬂow. The results of this exercise are summarized in Figure 3 where the inclusion of the two life stages of chytrid parasites of microphytoplank- ton (>20 µm) increases the number of compartments and ﬂows. These parasites were able to short-circuit about 20% of the gross primary production, of which 15% is transferred to grazers with high throughput (Figure 3). interactions, which may be a characteristic of parasites with com- plex life cycles, might offset the destabilizing effects of increased connectance (Neutel et al., 2002). To investigate ecosystem properties and ecological theories, the application of mathematical tools, such as models, is useful and allows trophic network representation through carbon ﬂows (Figure 3). In the absence of quantiﬁcation of the ﬂows induced by fungal parasites of phytoplankton, we recently simulate their potential role in the plankton food web of the Lake Biwa, Japan (Niquil et al., 2011). The presence of this indirect pathway chan- neling microphytoplankton production to the consumers via the fungi leads to an enhancement of the trophic efﬁciency index, and a decrease of the ratio detritivory: herbivory. The results suggested that the food web relies less on the consumption of detritus, and that the transfer of carbon to higher trophic levels is higher than estimated without taking into account the parasites. Due to the lack of data quantifying carbon transfer through parasitism in pelagic ecosystems, no attempt was made to build model based on ﬁeld estimated ﬂows. Thus, the roles and ecological implications of chytrid infections of phytoplankton remain to be fully explored for aquatic microbial food webs. As a ﬁrst attempt, we recently provided such exercise for the ﬁrst time,by adding parasitic chytrids of phytoplankton as an indi- vidualized compartment in a well studied pelagic food web and quantify their impact on matter ﬂow through a trophic network (Grami et al., 2011). To describe the food web, models represen- tative of carbon ﬂows were built, including chytrid parasitism and the amount of primary production channeled in food web via chytrid infection. IMPACT OF PHYTOPLANKTON CHYTRIDIOMYCOSIS ON THE FOOD WEB PROPERTIES Carbon ﬂows between the complete food web including parasitic chytrids (MWC, Model with Chytrids) were compared to the same model that did not consider the presence of Inaddition,foreachcalculatedsetof ﬂowsgeneratedbytheLin- ear Inverse Modeling procedure, there is a set of calculated indices which allows application of statistical tests. The ﬂows obtained from the models were used for calculations of Ecological Network Analysis indices that characterize the structure of the food web, and help reveal emergent properties (Ulanowicz,1986,1997,2003; Ulanowicz et al., 2009). The use of ecological indices moreover, allows an indirect evaluation of the effects of network properties on the stability of the ecosystem, as several authors have pro- posed theoretical links between structural properties and local Phytoplankton Detritus 11%/3% Phytoplankton 262/268 Detritus 62/45 6%/4% 21%/10% 59%/31% 12.5% Microzooplankton 68/88 Sporangia 7.4 2%/2% 9.6%/10.4% 21.4% 71% 1.5% 42%/43% 2%/2% / 17.4% 5% 85% 81.4% 14% Mesozooplankton 58/92 Zoospores 6.8 7 8%/10 7% 15% 85% 1.9% l i i d i ( C 2d 1) 14% 2.5% 7.8%/10.7% Flowing primary production (mgCm-2d-1) Transfert throughput Sinking ﬂows (mgCm-2d-1) Values into each compartment are carbon biomasses (mgCm-2) FIGURE 3 \| Impact of parasitic chytrids on the microbial loop: ﬂowing and sinking carbon from the gross primary production of phytoplankton (>20 µm) during the spring diatom bloom in the oligo-mesotrophic Lake Pavin, France. The effects of infective fungal sporangia and their propagules (zoospores) are highlighted in red color. The diagram corresponds to steady state models of the euphotic zone of the lake generated from a linear inverse modeling analysis. For more details, see the main text. Modiﬁed from Grami et al. (2011). % l i i d i ( C 2d 1) % Flowing primary production (mgCm-2d-1) Transfert throughput Sinking ﬂows (mgCm-2d-1) Values into each compartment are carbon biomasses (mgCm-2) (zoospores) are highlighted in red color. The diagram corresponds to steady state models of the euphotic zone of the lake generated from a linear inverse modeling analysis. For more details, see the main text. Modiﬁed from Grami et al. (2011). FIGURE 3 \| Impact of parasitic chytrids on the microbial loop: ﬂowing and sinking carbon from the gross primary production of phytoplankton (>20 µm) during the spring diatom bloom in the oligo-mesotrophic Lake Pavin, France. The effects of infective fungal sporangia and their propagules (zoospores) are highlighted in red color. formosa on the zoospore produc- tion of its fungal parasite Rhizophy- dium planktonicum. Aquat. Ecol. 21, 49–54. CONCLUSION Chytrid symbionts and the related trophic modes, primarily par- asitism, are omnipresent in aquatic ecosystems, including marine habitats (Gleason et al., 2011). In these ecosystems, they are diver- siﬁed, with different taxa featured by different biological charac- teristics and requirements that determine their distributions in relation to environmental parameters but primarily to the sea- sonal dynamics of their phytoplankton hosts. The host abundance but also the host cell size and biomass likely establish the thresh- old limit for the critical prevalence of infection and the related decline in host communities. Related ecological implications are huge, because chytrid parasites can kill their hosts, release sub- strates for microbial processes, and provide nutrient-rich particles as zoospores and short fragments of ﬁlamentous hosts for the grazer food chain. This implies that cyanobacterial blooms, and other large size inedible phytoplankton blooms as well, may not totallyrepresenttrophicbottlenecks.Basedontheobservationthat phytoplankton fungal parasitism preferentially impacts the larger size species (i.e., characteristics of climax populations), chytrid epidemics represent an important driving factor in phytoplank- ton successions and maturation, in addition to the sole seasonal forcing. The activity of chytrid parasites of phytoplankton thus represents an important but as yet overlooked ecological driving force in aquatic food web dynamics. In addition to being able to resist adverse conditions and use different sources of carbon and IMPACT OF PHYTOPLANKTON CHYTRIDIOMYCOSIS ON THE FOOD WEB PROPERTIES The diagram corresponds to steady state models of the euphotic zone of the lake generated from a linear inverse modeling analysis. For more details, see the main text. Modiﬁed from Grami et al. (2011). October 2012 \| Volume 3 \| Article 361 \| 9 www.frontiersin.org Phytoplankton chytridiomycosis Sime-Ngando stability (cf. Ulanowicz, 2003). On these bases, the model results support recent theories on the probable impact of parasites on food web function. In the lake, during spring, when “inedible” algae (unexploited by planktonic herbivores) were the dominant primary producers, the epidemic growth of chytrid parasites sig- niﬁcantly reduced the sedimentation loss of algal carbon from 21 to 10% of gross primary production (Figure 3). Furthermore, from the review of some theories about the potential inﬂuence of parasites on ecological network properties, we argue that para- sitism contributes to longer carbon path lengths, higher levels of activity and specialization, and lower recycling. We then conclude that considering the“structural asymmetry” hypothesis as a stabi- lizing pattern,chytrids should contribute to the stability of aquatic food webs (Grami et al., 2011). nutrients, chytrid parasites can indeed affect the plankton food web functions and ecosystem properties and topology, such as sta- bility and trophic transfer efﬁciency.We are perhaps approaching a new paradigm-shift point in the development of aquatic microbial ecology (Sime-Ngando and Niquil, 2011). gy g q However, the available study on phytoplankton chytridiomy- cosis remain restricted to a few temperate lakes, and extensive studies in the world’s aquatic ecosystems, at wide geographi- cal and time scales, are needed. Besides, the identiﬁcation of chytrid species based on phenotypic features requires time and experience, and chytrid diversity provided this way probably is an underestimate. In this context, the increasing development of molecular tools is promising and will, in the near future, improves the linkage of cell identity and function, which is critical for an accurate assessment that includes microbial para- sites in the carbon ﬂows, and the related biogeochemical cycling in aquatic ecosystems (Jobard et al., 2012). Furthermore, par- asitic lifestyle is generally highly subtle and can, for example, control competition by dominant species for resources, thereby promoting species coexistence and diversity. Parasites can also form long-lived associations with hosts, reducing their ﬁtness for survival, or allowing infected hosts to remain strong com- petitors, although few models exist for microbial fungus-host systems. Blackwell, W. H., Letcher, P. M., and Powell, M. J. (2006). Thal- lus development and the system- atics of Chytridiomycota: an addi- tional developmental pattern repre- sented by Podochytrium. Mycotaxon 97, 91–109. ACKNOWLEDGMENTS I am particularly indebted to students and postdocs of mine (cf. download the list at http://www.lmge.univ-bpclermont.fr/ spip.php?rubrique100), particularly to those who have worked on the topic reviewed herein: Mélanie Gerphagnon, Boutheina Grami, Marlène Jobard, Emilie Lefèvre, Sébastien Monchy, Serena Rasconi, and Kimiko Uchii. Thanks are owed to my colleagues, particularly Frank H. Gleason and Nathalie Niquil, for fruitful discussion and inspiring collaborative papers. I am also grateful to Hans-Peter Grossart and co-editors for the invitation to contribute to this special issue in Frontiers in Aquatic Microbiology. This is a contribution to the French ANR Programme Blanc DREP – Diver- sity and Roles of Eumycetes in the Pelagial – Coordinator Télesphore Sime-Ngando, PIs: Nathalie Niquil, Eric Viscogliosi, and Urania Christaki. British chytrids, XII. Ann. Bot. 15, 129–156. REFERENCES London: Chapman & Hall. Hosack, G. R., Li, H. W., and Rossignol, P. A. (2009). Sensitivity of system stability to model structure. Ecol. Model. 220, 1054–1062. Lefranc, M., Thenot, A., Lepere, C., and Debroas, D. (2005). Genetic diversity of small eukaryotes in lakes differing by their trophic sta- tus. Appl. Environ. Microbiol. 71, 5935–5942. Kagami, M., von Elert, E., Ibelings, B. W., de Bruin, A., and van Donk, E. (2007b). The parasitic chytrid, Zygorhizidium, facilitates the growth of the cladoceran zoo- plankter, Daphnia, in cultures of the inedible alga, Asterionella. Proc. Biol. Sci. 274, 1561–1566. Fox, A. (2003). Glossary of epidemi- ological terms. Internet J. Pedi- atr. Neonatol. 3. 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C., Whitney, K. L., Aguirre-Macedo, James, T. Y., Letcher, P. M., Longcore, J. E., Mozley-Standridge Porter, D., October 2012 \| Volume 3 \| Article 361 \| 11 www.frontiersin.org Phytoplankton chytridiomycosis Sime-Ngando quantitative PCR assay for the assessment of uncultured zoosporic fungi,” in Laboratory Protocols in Fungal Biology: Current Methods in Fungal Biology, eds V. K. Gupta, M. Tuohy,A. Manimaran, K. M. Turner, and A. O’Donovan (New York: Springer). Available at: http://www. springer.com/life+sciences/plant+ sciences/book/978-1-4614-2355-3 Fournieand (London: CRC Press), 255–287. succession of planktonic events in fresh waters. Arch. Hydrobiol. 106, 433–471. Fournieand (London: CRC Press), 255–287. May, R. M. (1972). Will a large com- plex system be stable? Nature 238, 413–414. Pimm, S. L. (1984). The complexity and stability of ecosystems. Nature 307, 321–326. May, R. M. (1973). Stability and Com- plexity in Model Ecosystems. Prince- Sparrow, F. K. (1960). 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Carbon steady state model of the planktonic food web of Lake Biwa, Japan. Freshwater Biol. 8, 1570–1585. Sen, B. (1988b). Fungal parasitism of planktonic algae in shearwater IV. Parasitic occurrence of a new chytrid species on the blue-green alga. Arch. Hydrobiol. Suppl. 79, 177–184. Van den Meersche, K., Soetaert, K., and Van Oevelen, D. (2009). An R function for sampling linear inverse problems. J. Stat. Softw. 30, 1–15. Sen, B. (1988c). Fungal parasitism of planktonic algae in Shearwater V. Fungal parasites of the green algae. Arch. Hydrobiol. Suppl. 79, 185–205. Niquil, N., Kagami, M., Urabe, J., Chris- taki, U., Viscogliosi, E., and Sime- Ngando, T. (2011).“Potential role of fungi in plankton food web func- tioning and stability: a simulation analysis based on Lake Biwa inverse model,” in Disregarded Microbial Diversity and Ecological Potentials in Aquatic Systems, eds T. Sime- Ngando and N. Niquil (New York: Springer), 65–79. Sime-Ngando, T., and Niquil, N. (eds). (2011). 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October 2012 \| Volume 3 \| Article 361 \| 13 Received: 03 June 2012; paper pend- ing published: 02 July 2012; accepted: 24 September 2012; published online: 12 October 2012. Citation: Sime-Ngando T (2012) Phy- toplankton chytridiomycosis: fungal parasites of phytoplankton and their imprints on the food web dynam- ics. Front. Microbio. 3:361. doi: 10.3389/fmicb.2012.00361 REFERENCES in The Dynamical Processes of Biodiversity – Case Studies of Evolution and Spatial Distribu- tion, eds O. Grillo and G. Venora (InTech open access). Available at: http://www.intechopen.com/articles /show/title/aquatic-fungi This article was submitted to Frontiers in Aquatic Microbiology, a specialty of Frontiers in Microbiology. Copyright © 2012 Sime-Ngando. This is an open-access article distributed under the terms of the Creative Com- mons Attribution License, which per- mits use, distribution and reproduc- tion in other forums, provided the orig- inal authors and source are credited and subject to any copyright notices concerning any third-party graphics etc. could be construed as a potential conﬂict of interest. This article was submitted to Frontiers in Aquatic Microbiology, a specialty of Frontiers in Microbiology. Received: 03 June 2012; paper pend- ing published: 02 July 2012; accepted: 24 September 2012; published online: 12 October 2012. Copyright © 2012 Sime-Ngando. This is an open-access article distributed under the terms of the Creative Com- mons Attribution License, which per- mits use, distribution and reproduc- tion in other forums, provided the orig- inal authors and source are credited and subject to any copyright notices concerning any third-party graphics etc. Williams, R., and Martinez, N. D. (2004). 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https://openalex.org/W2058032884	https://europepmc.org/articles/pmc2984489?pdf=render	English	null	Peripheral Nerve Injury and TRPV1-Expressing Primary Afferent C-Fibers Cause Opening of the Blood-Brain Barrier	Molecular pain	2,010	cc-by	9,370	Beggs et al. Molecular Pain 2010, 6:74 http://www.molecularpain.com/content/6/1/74 (2 November 2010) Abstract Background: The blood-brain barrier (BBB) plays the crucial role of limiting exposure of the central nervous system (CNS) to damaging molecules and cells. Dysfunction of the BBB is critical in a broad range of CNS disorders including neurodegeneration, inflammatory or traumatic injury to the CNS, and stroke. In peripheral tissues, the vascular-tissue permeability is normally greater than BBB permeability, but vascular leakage can be induced by efferent discharge activity in primary sensory neurons leading to plasma extravasation into the extravascular space. Whether discharge activity of sensory afferents entering the CNS may open the BBB or blood-spinal cord barrier (BSCB) remains an open question. Results: Here we show that peripheral nerve injury (PNI) produced by either sciatic nerve constriction or transecting two of its main branches causes an increase in BSCB permeability, as assessed by using Evans Blue dye or horseradish peroxidase. The increase in BSCB permeability was not observed 6 hours after the PNI but was apparent 24 hours after the injury. The increase in BSCB permeability was transient, peaking about 24-48 hrs after PNI with BSCB integrity returning to normal levels by 7 days. The increase in BSCB permeability was prevented by administering the local anaesthetic lidocaine at the site of the nerve injury. BSCB permeability was also increased 24 hours after electrical stimulation of the sciatic nerve at intensity sufficient to activate C-fibers, but not when A-fibers only were activated. Likewise, BSCB permeability increased following application of capsaicin to the nerve. The increase in permeability caused by C-fiber stimulation or by PNI was not anatomically limited to the site of central termination of primary afferents from the sciatic nerve in the lumbar cord, but rather extended throughout the spinal cord and into the brain. Conclusions: We have discovered that injury to a peripheral nerve and electrical stimulation of C-fibers each cause an increase in the permeability of the BSCB and the BBB. The increase in permeability is delayed in onset, peaks at about 24 hours and is dependent upon action potential propagation. As the increase is mimicked by applying capsaicin to the nerve, the most parsimonious explanation for our findings is that the increase in permeability is mediated by activation of TRPV1-expressing primary sensory neurons. Our findings may be relevant to the development of pain and neuroplastic changes in the CNS following nerve injury. RESEARCH Open Access * Correspondence: mike.salter@utoronto.ca Program in Neurosciences & Mental Health, Hospital for Sick Children, Department of Physiology, University of Toronto, and University of Toronto Centre for the Study of Pain, Toronto, ON, Canada Abstract In addition, our findings may provide the basis for developing methods to purposefully open the BBB when needed to increase brain penetration of therapeutic agents that might normally be excluded by an intact BBB. Peripheral nerve injury and TRPV1-expressing primary afferent C-fibers cause opening of the blood-brain barrier Simon Beggs, Xue Jun Liu, Chun Kwan, Michael W Salter* Simon Beggs, Xue Jun Liu, Chun Kwan, Michael W Salter* © 2010 Beggs et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. MOLECULAR PAIN MOLECULAR PAIN Peripheral nerve injury and TRPV1-expressing primary afferent C-fibers cause opening of the blood-brain barrier Peripheral nerve injury and TRPV1-expressing primary afferent C-fibers cause opening of the blood-brain barrier Beggs et al. Beggs et al. Molecular Pain 2010, 6:74 http://www.molecularpain.com/content/6/1/74 MOLECULAR PAIN Peripheral nerve injury causes delayed opening of the blood-spinal cord barrier BSCB permeability is increased by chronic constriction injury (CCI) We assessed the integrity of the BSCB with intrave- nously administered Evans Blue dye. Evans Blue binds to albumin in the circulation producing a molecular complex of sufficient molecular size that it does not cross the intact BSCB or BBB. Thus, there is minimal accumulation of Evans Blue in the spinal cord, or brain, under basal conditions in naïve animals (Figure 1). To investigate whether BSCB permeability might be altered by CCI we administered Evans Blue at varying times after the nerve injury, induced by placing a poly- thene cuff around the nerve, and quantified the amount of Evans Blue in the dorsal spinal cord ipsilat- eral to the injury. We found that 6 hours after the CCI surgery, the level of Evans Blue accumulating was not different from that of the dorsal horn of naïve animals, or animals that we subjected to sham surgery (Figure 1A left). In contrast, 24 hours after surgery there was a significant increase in Evans Blue in the ipsilateral dor- sal horn of nerve-injured animals as compared with sham controls (Figure 1A left). Evans Blue accumula- tion in the dorsal horn of nerve-injured animals was also significantly increased when the dye was adminis- tered 3 days after surgery. However, when Evans Blue was administered 7 days after surgery the dye accumu- lation in the dorsal horn of nerve-injured animals was not different from that of sham controls or naïve animals. In peripheral tissues, vascular permeability is normally greater than in the CNS, although there is a vascular- tissue barrier that excludes, for example, large proteins from entering the tissue. It has long been known that peripheral vascular permeability in skin and other tis- sues can be markedly increased by antidromic dis- charges in primary sensory neurons, allowing large proteins to leak through capillaries thereby causing plasma extravasation [5-7]. This plasma extravasation, together with the vasodilation that is produced by sen- sory nerve stimulation, comprise neurogenic inflamma- tion which is mediated by the release of the peptides substance P and calcitonin gene-related peptide (CGRP) from peripheral terminals of peptidergic C-fibers [6,8,9]. In accordance with Dale’s Principle [10], discharge activity in peptidergic C-fibers also releases substance P and CGRP from the central terminals of primary affer- ents in the spinal cord dorsal horn [11,12]. Background cord - the blood-spinal cord barrier (BSCB), is a net- work of endothelial cells joined by tight junctions that line the blood vessels within the CNS [3,4]. The core ‘neurovascular unit’ comprises endothelial cells, peri- cytes and astrocytic endfeet embedded within their basal laminae. The space between the astrocytic endfeet, which make up the abluminal surface of CNS capillaries, and the endothelial cells/pericytes represents the inter- face between the blood and CNS. The BBB is highly The blood-brain barrier (BBB) is a highly specialized structure crucial for the maintenance of central nervous system (CNS) homeostasis [1,2]. The basis of the barrier in the brain, and the corresponding barrier in the spinal Beggs et al. Molecular Pain 2010, 6:74 http://www.molecularpain.com/content/6/1/74 Page 3 of 12 Beggs et al. Molecular Pain 2010, 6:74 http://www.molecularpain.com/content/6/1/74 Beggs et al. Molecular Pain 2010, 6:74 http://www.molecularpain.com/content/6/1/74 Results restrictive with only a subset of small molecular weight, diffusible molecules readily crossing from the blood into the CNS parenchyma. Thus, most substances are nor- mally precluded from entering the CNS by the BBB. However, in many CNS pathological states the BBB becomes disrupted, allowing entry of substances from blood into the CNS, and this disruption is considered a key step for disorders such as traumatic injury, stroke and neurodegeneration. Peripheral nerve injury causes delayed opening of the blood-spinal cord barrier BSCB permeability is increased by chronic constriction injury (CCI) Peripheral nerve injury causes delayed opening of the blood-spinal cord barrier BSCB permeability is increased by chronic constriction injury (CCI) Spared nerve injury (SNI) also causes increased BSCB permeability Peripheral nerve injury causes delayed opening of the blood-spinal cord barrier BSCB permeability is increased by chronic constriction injury (CCI) However, it has been found that vascular permeability in the dorsal horn is not increased by activating C-fibers, at least over the time course of peripheral neurogenic inflam- mation [13]. Therefore, it has been assumed that although activity in sensory nerves causes rapid increases in vascular permeability in peripheral tissues, this activity is not capable of causing vascular perme- ability to increase in the CNS. To independently assess the status of the BSCB we intravenously administered horse radishperoxidase (HRP) which, like Evans Blue bound to albumin, is not able to cross the normal intact BSCB. We found that HRP coats the lining of vessels but does not enter the parenchyma of the dorsal horn after intravenous injection in naïve animals (Figure 1B). However, when administered at 24 hours after CCI, HRP was readily visualized in the dorsal horn parenchyma. By quantify- ing HRP in the ipsilateral dorsal horn we found that the amount of HRP was significantly increased in the dorsal horn from nerve-injured animals as compared with that from control animals (Figure 1B). From the accumulation of HRP and of Evans Blue after nerve injury we conclude that the permeability of the BSCB increases after CCI. The increase in BSCB permeabil- ity is delayed by more than 6 hours and appears to peak about after 24 hours after the surgery. The increase in permeability is transient, with BSCB per- meability returning to control levels by 7 days after CCI surgery. Discharge activity in primary afferents, particularly that initiated by injury to sensory nerves, may have slowly developing and persistent consequences in the nervous system that can lead to chronic pain states [14]. Emerging evidence indicates that one such consequence of peripheral nerve injury (PNI) is the entry of mono- cytes [15] and T cells [16,17] from the circulation into the spinal dorsal horn. Because the accumulation of these normally circulating cells is apparent many hours or days after the nerve injury, we wondered whether the BSCB may be disrupted at these times, as has been sug- gested at longer times after spinal nerve transection [18]. Therefore, here we investigated whether PNI or activation of primary afferent C-fibers may cause increased vascular permeability in the CNS but over a time course beyond that of neurogenic inflammation. Preliminary results of portions of this work have been reported [19]. Beggs et al. Molecular Pain 2010, 6:74 http://www.molecularpain.com/content/6/1/74 Beggs et al. Peripheral nerve injury causes delayed opening of the blood-spinal cord barrier BSCB permeability is increased by chronic constriction injury (CCI) Molecular Pain 2010, 6:74 h // l l i / Page 4 of 12 http://www.molecularpain.com/content/6/1/74 Spared nerve injury (SNI) also causes increased BSCB permeability injury, we examined the effects of axotomy of the sciatic ner e in hich the common peroneal and tibial ner es Figure 1 Peripheral nerve injury (PNI) increases BSCB permeability. A: Increased permeability to Evans Blue of spinal cord from naïve rats, sham operated rats or rats with chronic constriction injury (CCI, left) or spared nerve injury (SNI, right). Inset: Standard curve of Evans blue and the representative reading of naïve rats or CCI rats. B: Increased permeability to horseradish peroxidase (HRP) following PNI. Left, representative spinal cord sections of HRP extravasation 24 hours after SNI, CCI or naïve rats. Right, histogram showing the integrated pixel density of HRP signaling. Data are presented as mean ± SEM; p < 0.05 compared to naïve; n = 4-6 per group. All measurements are from lumbar spinal cord ipsilateral to PNI stimulation. Figure 1 Peripheral nerve injury (PNI) increases BSCB permeability. A: Increased permeability to Evans Blue of spinal cord from naïve rats, sham operated rats or rats with chronic constriction injury (CCI, left) or spared nerve injury (SNI, right). Inset: Standard curve of Evans blue and the representative reading of naïve rats or CCI rats. B: Increased permeability to horseradish peroxidase (HRP) following PNI. Left, representative spinal cord sections of HRP extravasation 24 hours after SNI, CCI or naïve rats. Right, histogram showing the integrated pixel density of HRP signaling. Data are presented as mean ± SEM; p < 0.05 compared to naïve; n = 4-6 per group. All measurements are from lumbar spinal cord ipsilateral to PNI stimulation. Figure 1 Peripheral nerve injury (PNI) increases BSCB permeability. A: Increased permeability to Evans Blue of spinal cord from naïve rats, sham operated rats or rats with chronic constriction injury (CCI, left) or spared nerve injury (SNI, right). Inset: Standard curve of Evans blue and the representative reading of naïve rats or CCI rats. B: Increased permeability to horseradish peroxidase (HRP) following PNI. Left, representative spinal cord sections of HRP extravasation 24 hours after SNI, CCI or naïve rats. Right, histogram showing the integrated pixel density of HRP signaling. Data are presented as mean ± SEM; p < 0.05 compared to naïve; n = 4-6 per group. All measurements are from lumbar spinal cord ipsilateral to PNI stimulation. Spared nerve injury (SNI) also causes increased BSCB permeability injury, we examined the effects of axotomy of the sciatic nerve, in which the common peroneal and tibial nerves were transected. We found that 24 hours and 3 days after surgery Evans Blue accumulation in the dorsal horn was significantly increased in animals with SNI compared with To determine whether the increase in BSCB permeability was a unique consequence of CCI or whether increased permeability may be induced by other types of nerve Beggs et al. Molecular Pain 2010, 6:74 http://www.molecularpain.com/content/6/1/74 Page 5 of 12 Beggs et al. Molecular Pain 2010, 6:74 http://www.molecularpain.com/content/6/1/74 Figure 2 The effect of local lidocaine block and sequential nerve injury on CCI-induced increase in BSCB permeability. A: 2% of Lidocaine was applied to the sciatic nerve immediately following CCI, and Evans Blue extravasation in the ipsilateral lumbar spinal cord was measured 24 hours post CCI. B: A sequential CCI was performed on the right sciatic nerve after the initial CCI was performed in the left sciatic nerve 6 days previously. Evans blue extravasation was measured 7 days after initial CCI. All readings are from the left lumbar spinal cords (ipsilateral to the initial injury). Data are presented as mean ± SEM; p < 0.05 *p < 0.01 compared to naïve; n = 5 per group. that in animals with sham surgery or naïve control animals (Figure 1A right). There was no increase in Evans Blue accumulation 6 hours or 7 days after SNI. In addition, HRP was found to accumulate in the dorsal horn parench- yma when assessed at 24 hours after SNI (Figure 1B). Thus, we conclude that, like CCI, SNI causes a delayed and transient increase in BSCB permeability, and therefore that increased BSCB permeability may be a general conse- quence of injury to a peripheral nerve. Because the magni- tude and time course of the increase in Evans Blue and HRP accumulation were similar for CCI and SNI we used only one nerve injury model, CCI, and one approach to assess BSCB permeability, Evans Blue administration, for the remainder of the present study. CCI-induced increase in BSCB permeability is prevented by lidocaine To determine whether opening of the BSCB requires pro- pagation of action potentials from the site of nerve injury, we investigated the effect of the local anaesthetic lidocaine. Spared nerve injury (SNI) also causes increased BSCB permeability injury on CCI-induced increase in BSCB permeability. A: 2% of Lidocaine was applied to the sciatic nerve immediately following CCI, and Evans Blue extravasation in the ipsilateral lumbar spinal cord was measured 24 hours post CCI. B: A sequential CCI was performed on the right sciatic nerve after the initial CCI was performed in the left sciatic nerve 6 days previously. Evans blue extravasation was measured 7 days after initial CCI. All readings are from the left lumbar spinal cords (ipsilateral to the initial injury). Data are presented as mean ± SEM; p < 0.05 *p < 0.01 compared to naïve; n = 5 per group. Spared nerve injury (SNI) also causes increased BSCB permeability Lidocaine was applied directly to the nerves starting 30 min prior to injury to ensure that action potentials were blocked at the time of nerve transection. Gel foam soaked in lidocaine was placed around the nerves after transection and we administered Evans Blue 24 hours later. We found that administering lidocaine prevented the increase in Evans Blue accumulation after CCI (Figure 2A). From this finding we infer that action potential propagation in the transected nerves is required to cause the increase in BSCB opening after peripheral nerve injury. Recovery of BSCB integrity after CCI due to loss of signal to open The increase in BSCB permeability was transient with per- meability returning to control levels by 7 days after nerve injury. We reasoned that the recovery of BSCB integrity might be due to loss of a drive that sustains BSCB opening or that the BSCB might become resistant to the driving signals. To differentiate between these possibilities we made a second nerve injury, on the side contralateral to the first injury, 6 days after the first injury. We found that there was a significant increase in Evans Blue accumula- tion in the dorsal spinal cord 24 hrs after the second nerve injury (Figure 2B). Thus, the BSCB was not resistant to opening when a drive to open was presented on day 6. Therefore, we conclude that the recovery of BSCB perme- ability was due to a recovery of the action potential-depen- dent drive to open after the nerve injury. Figure 2 The effect of local lidocaine block and sequential nerve injury on CCI-induced increase in BSCB permeability. A: 2% of d l d h d l f ll Figure 2 The effect of local lidocaine block and sequential nerve injury on CCI-induced increase in BSCB permeability. A: 2% of Lidocaine was applied to the sciatic nerve immediately following CCI, and Evans Blue extravasation in the ipsilateral lumbar spinal cord was measured 24 hours post CCI. B: A sequential CCI was performed on the right sciatic nerve after the initial CCI was performed in the left sciatic nerve 6 days previously. Evans blue extravasation was measured 7 days after initial CCI. All readings are from the left lumbar spinal cords (ipsilateral to the initial injury). Data are presented as mean ± SEM; p < 0.05 p < 0.01 compared to naïve; n = 5 per group. Increase in BSCB permeability by C-fiber stimulation is blocked by lidocaine It is conceivable that short duration C-fiber stimulation initiates long-lasting discharge in primary afferents and to investigate this possibility we applied lidocaine to the sciatic nerve either before or immediately after the elec- trical stimulation. We found that applying lidocaine prior to the stimulation prevented the increase in Evans Blue accumulation 24 hrs later (Figure 3C). In contrast, applying lidocaine immediately after the electrical stimu- lation had no effect on the Evans Blue accumulation at 24 hrs. Under the conditions used, blockade of action potential propagation develops within 15-20 minutes and therefore we conclude the C-fiber discharge during the electrical stimulation, and possibly in the immediate few minutes thereafter, acts as a trigger to cause the delayed increase in BSCB permeability. Figure 3 C-fiber intensity stimulation increases BSCB permeability. A: Sciatic nerve stimulation with C-fiber intensity, but not A-fiber intensity increases permeability to Evans Blue in rat spinal cord 24 hours post stimulation. Data are presented as mean ± SEM; p < 0.01 compared to naïve, n = 4-8 per group. B: Representative spinal cord sections of HRP extravasation from naïve rats or rats 24 hours post C-fiber intensity stimulation of the sciatic nerve. C: C-fiber intensity stimulation-induced permeability is blocked by pre-treatment, but not post-treatment, with lidocaine. 2% lidocaine was applied to the sciatic nerve either 30 minutes before C-fiber stimulation (pre-block) or immediately after C-fiber stimulation (post-block). The duration of lidocaine treatment was 30 minutes in both cases. Data are presented as mean ± SEM; p < 0.01 compared to C-fiber stimulation, n = 4-8 per group. All the readings are from lumbar spinal cord ipsilateral to nerve stimulation. Capsaicin applied to the sciatic nerve causes increased BSCB permeability at 24 hrs Neurogenic plasma extravasation is mediated through activating primary afferent C-fibers that express TRPV1 and are hence activated by capsaicin [9,22]. To deter- mine whether activating this class of primary afferent might increase BSCB permeability we applied capsaicin to the sciatic nerve and measured Evans Blue in the dor- sal spinal cord 24 hrs later. We found that capsaicin, but not vehicle control, caused an increase in Evans Blue at 24 hrs (Figure 4A). In contrast applying capsaicin to the sciatic nerve led to peripheral plasma extravasation that was apparent within 45 min (Figure 4B) Thus, we con- clude that activating TRPV1-expressing C-fibers is suffi- cient to cause an increase in BSCB permeability. Figure 3 C fiber intensity stimulation increases BSCB Short-duration stimulation of C-fibers opens BSCB C-fiber but not A-fiber stimulation causes increase in BSCB permeability fibers. As electrical stimulation for only several minutes in duration can produce peripheral vasodilation and plasma extravasation [20], and can induce long-lasting pain hypersensitivity [21], we examined effects of short- duration stimulation on Evans Blue accumulation in the ipsilateral dorsal spinal cord. We found that electrical In order to determine which type of peripheral nerve fiber may drive opening of the BSCB, we made use of electrical stimulation of the intact sciatic nerve to differ- entially activate different classes of peripheral nerve Page 6 of 12 Beggs et al. Molecular Pain 2010, 6:74 http://www.molecularpain.com/content/6/1/74 stimulation that selectively activates A-fibers (1 mA; 10 Hz; 5 min) caused no change in Evans Blue accumula- tion (Figure 3A). By contrast, stimulation sufficient to recruit primary afferent C-fibers (10 mA; 10 Hz; 5 min) caused a delayed increase in Evans Blue accumulation: Evans Blue was not different from control 3 hrs after C- fiber stimulation (not illustrated), but was significantly increased 24 hrs after stimulation (Figure 3A). Electrical stimulation at C-fiber intensities also produced an increase in HRP in the ipsilateral dorsal spinal cord 24 hrs after the stimulation (Figure 3B). Thus, the short- duration electrical stimulation of C-fibers but not A- fibers was sufficient to cause a delayed increase in BSCB permeability. C-fiber stimulation and peripheral nerve injury cause delayed widespread opening of BSCB y In investigating the effects of electrical C-fiber stimula- tion we examined the contralateral as well as the ipsi- lateral lumbar dorsal spinal cord and found that the increase in Evans Blue accumulation 24 hrs after sti- mulation was produced in the contralateral, as well as the ipsilateral dorsal spinal cord (Figure 5A). Moreover the level of Evans Blue in the contralateral side was not different from on the side ipsilateral to the nerve stimulation. Increased Evans Blue accumulation in the contralateral dorsal cord was mimicked by applying capsaicin to the sciatic nerve (Figure 5A). The increases in Evans Blue accumulation in spinal cord was prevented by applying lidocaine to the sciatic nerve just prior to electrical stimulation (Figure 5A). Figure 3 C-fiber intensity stimulation increases BSCB permeability. A: Sciatic nerve stimulation with C-fiber intensity, but Figure 3 C-fiber intensity stimulation increases BSCB bilit A S i ti ti l ti ith C fib i t it b t Figure 3 C-fiber intensity stimulation increases BSCB permeability. A: Sciatic nerve stimulation with C-fiber intensity, but not A-fiber intensity increases permeability to Evans Blue in rat spinal cord 24 hours post stimulation. Data are presented as mean ± SEM; p < 0.01 compared to naïve, n = 4-8 per group. B: Representative spinal cord sections of HRP extravasation from naïve rats or rats 24 hours post C-fiber intensity stimulation of the sciatic nerve. C: C-fiber intensity stimulation-induced permeability is blocked by pre-treatment, but not post-treatment, with lidocaine. 2% lidocaine was applied to the sciatic nerve either 30 minutes before C-fiber stimulation (pre-block) or immediately after C-fiber stimulation (post-block). The duration of lidocaine treatment was 30 minutes in both cases. Data are presented as mean ± SEM; p < 0.01 compared to C-fiber stimulation, n = 4-8 per group. All the readings are from lumbar spinal cord ipsilateral to nerve stimulation. Figure 3 C-fiber intensity stimulation increases BSCB permeability. A: Sciatic nerve stimulation with C-fiber intensity, but not A-fiber intensity increases permeability to Evans Blue in rat spinal cord 24 hours post stimulation. Data are presented as mean ± SEM; p < 0.01 compared to naïve, n = 4-8 per group. B: Representative spinal cord sections of HRP extravasation from naïve rats or rats 24 hours post C-fiber intensity stimulation of the sciatic nerve. C: C-fiber intensity stimulation-induced permeability is blocked by pre-treatment, but not post-treatment, with lidocaine. C-fiber stimulation and peripheral nerve injury cause delayed widespread opening of BSCB 2% lidocaine was applied to the sciatic nerve either 30 minutes before C-fiber stimulation (pre-block) or immediately after C-fiber stimulation (post-block). The duration of lidocaine treatment was 30 minutes in both cases. Data are presented as mean ± SEM; p < 0.01 compared to C-fiber stimulation, n = 4-8 per group. All the readings are from lumbar spinal cord ipsilateral to nerve stimulation. e 3 C-fiber intensity stimulation increases BSCB Figure 3 C-fiber intensity stimulation increases BSCB permeability. A: Sciatic nerve stimulation with C-fiber intensity, but not A-fiber intensity increases permeability to Evans Blue in rat spinal cord 24 hours post stimulation. Data are presented as mean ± SEM; p < 0.01 compared to naïve, n = 4-8 per group. B: Representative spinal cord sections of HRP extravasation from naïve rats or rats 24 hours post C-fiber intensity stimulation of the sciatic nerve. C: C-fiber intensity stimulation-induced permeability is blocked by pre-treatment, but not post-treatment, with lidocaine. 2% lidocaine was applied to the sciatic nerve either 30 minutes before C-fiber stimulation (pre-block) or immediately after C-fiber stimulation (post-block). The duration of lidocaine treatment was 30 minutes in both cases. Data are presented as mean ± SEM; p < 0.01 compared to C-fiber stimulation, n = 4-8 per group. All the readings are from lumbar spinal cord ipsilateral to nerve stimulation. Beggs et al. Molecular Pain 2010, 6:74 http://www.molecularpain.com/content/6/1/74 Beggs et al. Molecular Pain 2010, 6:74 http://www.molecularpain.com/content/ Beggs et al. Molecular Pain 2010, 6:74 Page 7 of 12 http://www.molecularpain.com/content/6/1/74 Figure 4 Local application of capsaicin increases BSCB permeability. A: 1% Capsaicin or vehicle was applied onto the sciatic nerve and Evans Blue extravasation was measured 24 hours later. All readings are from lumbar spinal cord ipsilateral to capsaicin or vehicle. Data are presented as mean ± SEM; p < 0.01 compared to naïve, n = 5-9 per group. B: 1% Capsaicin was applied to the sciatic nerve and Evans Blue was injected immediately after capsaicin. Ipsilateral and contralateral paw images were captured 45 minutes after capsaicin treatment. Figure 4 Local application of capsaicin increases BSCB permeability. A: 1% Capsaicin or vehicle was applied onto the sciatic nerve and Evans Blue extravasation was measured 24 hours later. All readings are from lumbar spinal cord ipsilateral to capsaicin or vehicle. Data are presented as mean ± SEM; p < 0.01 compared to naïve, n = 5-9 per group. C-fiber stimulation and peripheral nerve injury cause delayed widespread opening of BSCB B: 1% Capsaicin was applied to the sciatic nerve and Evans Blue was injected immediately after capsaicin. Ipsilateral and contralateral paw images were captured 45 minutes after capsaicin treatment. Figure 4 Local application of capsaicin increases BSCB permeability. A: 1% Capsaicin or vehicle was applied onto the sciatic nerve and Evans Blue extravasation was measured 24 hours later. All readings are from lumbar spinal cord ipsilateral to capsaicin or vehicle. Data are presented as mean ± SEM; p < 0.01 compared to naïve, n = 5-9 per group. B: 1% Capsaicin was applied to the sciatic nerve and Evans Blue was injected immediately after capsaicin. Ipsilateral and contralateral paw images were captured 45 minutes after capsaicin treatment. post CCI (p < 0.05). Progressively more rostral brain areas showed lower levels of Evans Blue accumulation; occipital cortex p = 0.058; cerebellum and frontal cortex showed no significant increase. Thus, in addition to increasing BSCB permeability, PNI causes disruption of the BBB in some regions of the brain. Taking these findings together we conclude that the increase in BSCB permeability triggered by short-dura- tion electrical stimulation of C-fibers was not restricted to the ipisilateral spinal dorsal horn but was wide- spread in the spinal cord. PNI causes increase in BSCB and BBB permeability in thoracic and cervical spinal cord and in brain Discussion We therefore questioned whether PNI might also cause widespread opening of the blood-CNS barriers. We found that Evans Blue accumulation was significantly increased throughout the spinal cord 24 hrs after CCI and SNI (Figure 5B). Thus, like C-fiber stimulation, injury to a peripheral nerve causes extensive opening of the BSCB. In addition, we measured Evans Blue accu- mulation in brain regions following CCI. Significant increases were found in caudal brain regions; brain stem Evans Blue levels were significantly increased at 3 days Here we have discovered that peripheral nerve injury or electrical stimulation of C-fibers in the sciatic nerve pro- duce opening of the blood-spinal cord and blood-brain barriers. The increase in BSCB permeability is prevented by applying lidocaine to the nerve prior to the nerve injury or electrical stimulation, indicating that action potential discharge is required. By contrast, applying lidocaine directly after the electrical stimulation had no effect on the subsequent increase in BSCB permeability. Beggs et al. Molecular Pain 2010, 6:74 http://www.molecularpain.com/content/6/1/74 Beggs et al. Molecular Pain 2010, 6:74 Page 8 of 12 http://www.molecularpain.com/content/6/1/74 Increased BSCB permeability was also produced by permeability. Our findings thus reveal a previously Figure 5 Increased BSCB permeability extends throughout the spinal cord. A: left panel, representative spinal cord from naïve rats, rats 24 hours post C-fiber stimulation or rats with lidocaine block before C-fiber stimulation. Middle and right panels, C-fiber stimulation (middle) or local application of capsaicin onto sciatic nerve (right) increases contralateral spinal cord Evans Blue permeability 24 hours post treatment. B: Increased permeability to Evans Blue in the lumbar spinal cord contralateral to CCI (left panel) or SNI (middle) and in thoracic spinal cord (right). Upper picture: representative spinal cord and brain from naïve rats, or rats 24 hours post PNI. Data are presented as mean ± SEM; P < 0.05, p < 0.01, P < 0.001 compared to respective naïve groups, n = 6-9 per group. Figure 5 Increased BSCB permeability extends throughout the spinal cord. A: left panel, representative spinal cord from naïve rats, rats 24 hours post C-fiber stimulation or rats with lidocaine block before C-fiber stimulation. Middle and right panels, C-fiber stimulation (middle) or local application of capsaicin onto sciatic nerve (right) increases contralateral spinal cord Evans Blue permeability 24 hours post treatment. Discussion It was found that L4 nerve injury caused an endogenous albumin accumula- tion at time points of one to ten weeks after the transec- tion. The accumulation observed at those time points may reflect albumin that entered during the period of opening that we have discovered here. The recovery of BSCB integrity appears to be due to a gradual loss of the signals that are driving the opening, rather than to resistance of the BSCB to sustained signals to open, because we found here that the permeability increased upon a second nerve injury 7 days after the first. Thus, while the BSCB is only open for several days after nerve injury, repeated nerve injury may re-open the BSCB after it has recovered. Because the increase in BSCB permeability is delayed many hours after electrical C-fiber stimulation and the increase is not prevented by applying lidocaine to the nerve immediately after the stimulation, we infer that the activity evoked during the stimulation acts to trigger a cascade of events that culminates in the opening of the barrier. It is conceivable that the cascade may include transcription of critical genes and translation of the relevant gene products that act as mediators. In addition, or alternatively, to increased production of cer- tain gene products, key elements of the BBB might become reduced, and in preliminary experiments we have found a decrease in the level of aquaporin-4, a component of the BBB, preceding the increase in per- meability [19]. Given the widespread increase in vascular permeability in the CNS, a possible scenario may be that the cascade of events involves the production and release of a humoral mediator, or mediators, that act on the cellular and/or intercellular elements that maintain the intact barrier. Alternatively, there could be rostral spread of BSCB permeability from an initiation site in the lumbar dorsal horn or central release of diffusible mediator(s) that circulate within the cerebrospinal fluid. Or, the increased permeability might be caused by synaptically released mediators from neuronal pathways having widespread projections, such as those from brainstem or hypothalamic regions. Traumatic and ischemic injuries to the CNS are well- known to cause localized disruption of the BBB which is considered critical to the pathophysiology [1,33,34]. Discussion For example, fibronectin is known to cross the opened BBB [26] and this is known to stimulate P2X4R expression in microglia [27], and this increase in P2X4 is critical for mechanical hypersensitivity after PNI [28,29]. Also, matrix metalloproteinases (MMPs) have been implicated in opening the BBB [30], and MMPs in the dorsal horn are critical for pain hypersensitivity after PNI [31,32]. However, the PNI-induced increase in BBB permeability is not sufficient on its own to cause pain hypersensitivity because permeability is increased throughout the spinal cord whereas pain hypersensitivity is typically restricted to the region near the nerve injury. Our finding that the PNI-induced increase in BSCB permeability began within 24 hours and the permeability returning to the basal level by 7 days after PNI, is at earlier time points than a previous report using transec- tion of the L4 spinal nerve [18]. It was found that L4 nerve injury caused an endogenous albumin accumula- tion at time points of one to ten weeks after the transec- tion. The accumulation observed at those time points may reflect albumin that entered during the period of opening that we have discovered here. The recovery of BSCB integrity appears to be due to a gradual loss of the signals that are driving the opening, rather than to resistance of the BSCB to sustained signals to open, because we found here that the permeability increased upon a second nerve injury 7 days after the first. Thus, while the BSCB is only open for several days after nerve injury, repeated nerve injury may re-open the BSCB after it has recovered. Traumatic and ischemic injuries to the CNS are well- known to cause localized disruption of the BBB which is considered critical to the pathophysiology [1,33,34]. With the stimuli used presently – PNI, electrical C-fiber stimulation or capsaicin applied to the nerve – there is no direct injury to areas where the BSCB and BBB per- meability is increased. Thus, CNS injury cannot account for the increases in BSCB and BBB permeability we have activation. Plasma extravasation has been found to begin within tens of seconds of the start of C fiber stimulation [20,23]. By contrast, BSCB permeability has been found to not increase during or within minutes after C fiber stimulation [13] or with intravenous administration of capsaicin [24]. Discussion Thus, the onset of the effect of TRPV1- expressing afferents on vascular permeability in periph- eral tissues is dramatically different than that of the effect of these fibers on vascular permeability in the CNS. A second major difference between the effects of TRPV1-afferents on peripheral versus CNS vascular per- meability is the duration of the increase: peripheral plasma extravasation ends within minutes of terminating electrical C-fiber stimulation [23], whereas the increase in BSCB permeability persists for days after the stimula- tion. A third difference is the localization of the increase in vascular permeability: plasma extravasation in the periphery is highly localized to the territory innervated by the nerve that is stimulated [25] whereas the increase in permeability in the CNS is evoked far beyond the anatomical distribution of the central terminals, occur- ring throughout the spinal cord. These major differences imply that, beyond being initiated by TRPV1-expressing C-fibers, the mechanisms for the sensory neuron-evoked increase in vascular permeability in the CNS are highly divergent from those producing plasma extravasation in the periphery. The increase in BSCB permeability might contribute to the development of pain hypersensitivity after PNI. Increased permeability may facilitate the entry of circu- lating cells such as monocytes [15] and T cells [17] that home to the dorsal horn near the site of termination of the central endings of injured primary afferents. Increased BSCB permeability could also allow entry of soluble factors that are normally excluded but that could contribute to pain hypersensitivity in the dorsal horn. For example, fibronectin is known to cross the opened BBB [26] and this is known to stimulate P2X4R expression in microglia [27], and this increase in P2X4 is critical for mechanical hypersensitivity after PNI [28,29]. Also, matrix metalloproteinases (MMPs) have been implicated in opening the BBB [30], and MMPs in the dorsal horn are critical for pain hypersensitivity after PNI [31,32]. However, the PNI-induced increase in BBB permeability is not sufficient on its own to cause pain hypersensitivity because permeability is increased throughout the spinal cord whereas pain hypersensitivity is typically restricted to the region near the nerve injury. Our finding that the PNI-induced increase in BSCB permeability began within 24 hours and the permeability returning to the basal level by 7 days after PNI, is at earlier time points than a previous report using transec- tion of the L4 spinal nerve [18]. Discussion B: Increased permeability to Evans Blue in the lumbar spinal cord contralateral to CCI (left panel) or SNI (middle) and in thoracic spinal cord (right). Upper picture: representative spinal cord and brain from naïve rats or rats 24 hours post PNI Data are presented as mean ± SEM; P < 0 05 *p Figure 5 Increased BSCB permeability extends throughout the spinal cord. A: left panel, representative spinal cord from naïve rats, rats 24 hours post C-fiber stimulation or rats with lidocaine block before C-fiber stimulation. Middle and right panels, C-fiber stimulation (middle) or local application of capsaicin onto sciatic nerve (right) increases contralateral spinal cord Evans Blue permeability 24 hours post treatment. B: Increased permeability to Evans Blue in the lumbar spinal cord contralateral to CCI (left panel) or SNI (middle) and in thoracic spinal cord (right). Upper picture: representative spinal cord and brain from naïve rats, or rats 24 hours post PNI. Data are presented as mean ± SEM; P < 0.05, p < 0.01, P < 0.001 compared to respective naïve groups, n = 6-9 per group. permeability. Our findings thus reveal a previously unknown function of TRPV1-expressing C-fibers. permeability. Our findings thus reveal a previously unknown function of TRPV1-expressing C-fibers. Increased BSCB permeability was also produced by direct application of capsaicin to the sciatic nerve. Tak- ing our findings together, the most parsimonious expla- nation is that discharge activity in TRPV1-expressing C- fibers triggers a cascade of events which, after many hours, leads to an increase in BSCB and BBB This function of these afferents to cause opening of the BSCB was unanticipated as peripheral neurogenic plasma extravasation, which is also initiated by TRPV1- expressing C-fibers, develops rapidly upon electrical Beggs et al. Molecular Pain 2010, 6:74 http://www.molecularpain.com/content/6/1/74 Beggs et al. Molecular Pain 2010, 6:74 http://www.molecularpain.com/content/6/1/74 Beggs et al. Molecular Pain 2010, 6:74 http://www.molecularpain.com/content/6/1/74 Page 9 of 12 The increase in BSCB permeability might contribute to the development of pain hypersensitivity after PNI. Increased permeability may facilitate the entry of circu- lating cells such as monocytes [15] and T cells [17] that home to the dorsal horn near the site of termination of the central endings of injured primary afferents. Increased BSCB permeability could also allow entry of soluble factors that are normally excluded but that could contribute to pain hypersensitivity in the dorsal horn. Lidocaine block In summary, we have discovered that peripheral nerve injury and activation of TRPV1-expressing C-fibers causes extensive opening of the BSCB and BBB. The onset of the opening is delayed by many hours after the stimulus and persists for several days. The opening of the BSCB may contribute to the pathophysiology of neuropathic pain hypersensitivity by creating a permis- sive environment for recruitment and infiltration of circulating immune cells into the spinal cord parench- yma. Given the widespread effect on vascular perme- ability in the CNS it is possible that activating the sensory nerves causes the release of a circulating fac- tor, or factors, that causes breakdown of the barrier. Our findings have a number of potential clinical impli- cations for situations where there is activation of TPRV1-expressing C-fibers, such as trauma or other noxious peripheral stimuli. 0.1 ml of 2% lidocaine (Novocol Pharmaceutical, Canada) was applied topically to the sciatic nerve for 30 minutes before nerve injury or C-fiber stimulation. The efficacy of local lidocaine block was confirmed by lack of peripheral plasma extravasations 45 minutes after C-fiber stimulation. Electrical stimulation of sciatic nerve The sciatic nerve was isolated as described above and a bipolar stimulating hook electrode was used for electrical stimulation. The stimulation parameters for C-fiber strength stimulation were: 5 min train duration, 500 μsec stimulus pulse duration, 10 Hz, 10 mA. The stimulation parameters for A-fiber strength stimulation were: 5 min train duration, 150 μsec stimulus pulse duration, 10 Hz, 1 mA. Capsaicin stimulation Capsaicin (1% in Ethanol: Tween 80: saline 1:1:8, Sigma- Aldrich) or vehicle was applied to the sciatic nerve. Peripheral Nerve injury models p j y Spared nerve injury (SNI) was performed as described previously [36]. Briefly, rats were anaesthetized by iso- fluorane inhalation and the left sciatic nerve exposed under aseptic conditions. The distal trifurcation of the sciatic nerve was identified and the tibial and common peroneal branches ligated and cut, leaving the sural branch intact. The wound was sutured closed and the animals allowed to recover and returned to their hous- ing. Chronic constriction injury (CCI) was performed as described previously [37]. A polyethylene cuff (PE-60, 2 mm in length) was placed around the exposed left scia- tic nerve and the wound closed as above. Sham sur- geries, exposure of the sciatic nerve only, were also performed. To assess the effect of sequential nerve injury on the permeability of the BSCB, CCI was per- formed on the left sciatic nerve followed by a further CCI on the right nerve 7 days later. Discussion With the stimuli used presently – PNI, electrical C-fiber stimulation or capsaicin applied to the nerve – there is no direct injury to areas where the BSCB and BBB per- meability is increased. Thus, CNS injury cannot account for the increases in BSCB and BBB permeability we have found presently. Our findings open up the possibility that PNI and acti- vating TRPV1-expressing primary afferents may cause increased BSCB and BBB permeability in humans. If this is found to be the case, then there are several potential clinical implications of our findings. First, the BSCB and BBB would be much more permeable in situations of Page 10 of 12 Beggs et al. Molecular Pain 2010, 6:74 http://www.molecularpain.com/content/6/1/74 peripheral trauma, accidental or surgical, particularly when there is nerve damage. From this, one may expect that the pharmacokinetics of systemically administered drugs may be altered and the penetration into CNS might be enhanced. This enhanced drug penetration might increase the potency of agents where BBB perme- ability is limiting, but might also increase centrally- mediated side effects. A second potential clinical implica- tion is that our findings may point to development of approaches to purposefully open the BBB in order to facilitate entry of drugs that normally have limited access to central targets. While it is unlikely that direct nerve injury would be used therapeutically, it is conceivable that short-duration C-fiber stimulation, done under gen- eral anaesthesia as in the studies here in rats, might be tolerated. However, by far the best approach would be through ascertaining the mediator or mediators involved in the afferent-induced opening of the BBB and to then develop treatments based on mimicking, in a safe way, the mechanism. A third possibility is that widespread opening of the BBB caused by PNI or activation of TRPV1-expressing C-fibers might contribute to the so- called ‘sickness syndrome’ [35] that follows injury. A final, and much more speculative, possibility to consider pertains to non-traumatic, non-ischemic disorders where disruption of the BBB appears critical for the pathophy- siology, such as demyelinating, neuroimmune or neuro- degenerative disorders. Our findings raise the possibility that in some such diseases activity of TRPV1-expressing C-fibers may contribute to the BBB disruption. were approved by the Animal Care Committee of the Hospital for Sick Children. For all experiments male 250-300 g Sprague-Dawley (Charles River) were used. Evans Blue assay Blood spinal cord permeability was determined by Evans Blue extravasation into the spinal cord. Evan’s blue dye (2%, 4 ml/kg) was infused through the jugular vein of anaesthetized rats. After 45 minutes animals were per- fused with PBS. The spinal cord and supraspinal tissues were immediately dissected and dura mater removed. The lumbar spinal cord (L4-6), and middle thoracic cord were further dissected. Tissue was incubated in 600 μl of formamide (Sigma-Aldrich) at 60C for 72 h. Beggs et al. Molecular Pain 2010, 6:74 http://www.molecularpain.com/content/6/1/74 Beggs et al. Molecular Pain 2010, 6:74 http://www.molecularpain.com/content/6/1/74 Beggs et al. Molecular Pain 2010, 6:74 http://www.molecularpain.com/content/6/1/74 Beggs et al. Molecular Pain 2010, 6:74 http://www.molecularpain.com/content/6/1/74 Evan’s Blue concentration was then determind by spec- trophotometry at 620 nm. 9. Holzer P: Local effector functions of capsaicin-sensitive sensory nerve endings: involvement of tachykinins, calcitonin gene-related peptide and other neuropeptides. Neuroscience 1988, 24:739-768. 10. Dale HH: The chemical transmission of nerve impulses. Science 1934, 80:450. Acknowledgements d b d Supported by Canadian Institutes of Health Research (CIHR) grant MT 11219 to MWS as well as by the Krembil Foundation, the Ontario Neurotrauma Foundation and Jacob’s Ladder. CK was the recipient of a CIHR Fellowship. MWS is an International Research Scholar of the Howard Hughes Medical Institute and holds a Canada Research Chair (Tier I) in Neuroplasticity and Pain. 20. Gonzalez HL, Carmichael N, Dostrovsky JO, Charlton MP: Evaluation of the time course of plasma extravasation in the skin by digital image analysis. J Pain 2005, 6:681-688. 21. Hathway GJ, Vega-Avelaira D, Moss A, Ingram R, Fitzgerald M: Brief, low frequency stimulation of rat peripheral C-fibres evokes prolonged microglial-induced central sensitization in adults but not in neonates. Pain 2009, 144:110-118. Authors’ contributions SB participated in the design of the study, carried out experiments, analyzed data and wrote the paper. XJL carried out experiments, analyzed data and participated in writing of the paper. CK designed and carried out experiments, and analyzed data. MWS conceived of the study, participated i i d i d di i d h All h d d SB participated in the design of the study, carried out experiments, analyzed data and wrote the paper. XJL carried out experiments, analyzed data and participated in writing of the paper. CK designed and carried out experiments, and analyzed data. MWS conceived of the study, participated in its design and coordination, and wrote the paper. All authors read and approved the text. 22. Lynn B, Shakhanbeh J: Substance P content of the skin, neurogenic inflammation and numbers of C-fibres following capsaicin application to a cutaneous nerve in the rabbit. Neuroscience 1988, 24:769-775. 23. Carmichael NME, Dostrovsky JO, Charlton MP: Enhanced vascular permeability in rat skin induced by sensory nerve stimulation: evaluation of the time course and appropriate stimulation parameters. Neuroscience 2008, 153:832-841. in its design and coordination, and wrote the paper. All authors read and approved the text. Statistics 17. Costigan M, Moss A, Latremoliere A, Johnston C, Verma-Gandhu M, Herbert TA, Barrett L, Brenner GJ, Vardeh D, Woolf CJ, Fitzgerald M: T-cell infiltration and signaling in the adult dorsal spinal cord is a major contributor to neuropathic pain-like hypersensitivity. J Neurosci 2009, 29:14415-14422. Evan’s Blue concentration was assessed across all groups using one way ANOVA and Newman-Keuls post-hoc multiple comparisons. 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Following post- fixation and sucrose cryoprotection, spinal cord sections (50 μm) were cut on a microtome. Sections were incu- bated in cy3-labelled tyramide solution (Perkin Elmer) for 7 minutes to measure HRP activity. Resulting fluor- escence images were captured and integrated pixel den- sity measured using Volocity software. 12. Jancsó G: Pathobiological reactions of C-fibre primary sensory neurones to peripheral nerve injury. Exp Physiol 1992, 77:405-431. 12. Jancsó G: Pathobiological reactions of C fibre primary sensory neurones to peripheral nerve injury. Exp Physiol 1992, 77:405-431. 13. Koltzenburg M, Lewin G, McMahon S: Increase of blood flow in skin and spinal cord following activation of small diameter primary afferents. Brain Res 1990, 509:145-149. 13. Koltzenburg M, Lewin G, McMahon S: Increase of blood flow in skin and spinal cord following activation of small diameter primary afferents. Brain Res 1990, 509:145-149. 14. 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Gee MD, Lynn B, Cotsell B: The relationship between cutaneous C fibre type and antidromic vasodilatation in the rabbit and the rat. J Physiol (Lond) 1997, 503(Pt 1):31-44. Published: 2 November 2010 References 26. Nasu-Tada K, Koizumi S, Tsuda M, Kunifusa E, Inoue K: Possible involvement of increase in spinal fibronectin following peripheral nerve injury in upregulation of microglial P2X4, a key molecule for mechanical allodynia. Glia 2006, 53:769-775. 1. Huber JD, Egleton RD, Davis TP: Molecular physiology and pathophysiology of tight junctions in the blood-brain barrier. Trends Neurosci 2001, 24:719-725. 1. Huber JD, Egleton RD, Davis TP: Molecular physiology and pathophysiology of tight junctions in the blood-brain barrier. Trends Neurosci 2001, 24:719-725. 2. Persidsky Y, Ramirez SH, Haorah J, Kanmogne GD: Blood-brain barrier: structural components and function under physiologic and pathologic conditions. J Neuroimmune Pharmacol 2006, 1:223-236. 2. Persidsky Y, Ramirez SH, Haorah J, Kanmogne GD: Blood-brain barrier: structural components and function under physiologic and pathologic conditions. J Neuroimmune Pharmacol 2006, 1:223-236. 27. Tsuda M, Toyomitsu E, Komatsu T, Masuda T, Kunifusa E, Nasu-Tada K, Koizumi S, Yamamoto K, Ando J, Inoue K: Fibronectin/integrin system is involved in P2X(4) receptor upregulation in the spinal cord and neuropathic pain after nerve injury. Glia 2008, 56:579-585. 3. Abbott NJ, Rönnbäck L, Hansson E: Astrocyte-endothelial interactions at 3. Abbott NJ, Rönnbäck L, Hansson E: Astrocyte-endothelial interactions at the blood brain barrier Nat Rev Neurosci 2006 7 41 53 28. Tsuda M, Shigemoto-Mogami Y, Koizumi S, Mizokoshi A, Kohsaka S, Salter MW, Inoue K: P2X4 receptors induced in spinal microglia gate tactile allodynia after nerve injury. Nature 2003, 424:778-783. 4. Abbott NJ, Patabendige AAK, Dolman DEM, Yusof SR, Begley DJ: Structure and function of the blood-brain barrier. 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Methods Animals All animals were used in accordance with the guidelines of the Canadian Council on Animal Care. All protocols Page 11 of 12 Beggs et al. Molecular Pain 2010, 6:74 http://www.molecularpain.com/content/6/1/74 Beggs et al. Molecular Pain 2010, 6:74 http://www.molecularpain.com/content/6/1/74 31. Kawasaki Y, Xu Z, Wang X, Park JY, Zhuang Z, Tan P, Gao Y, Roy K, Corfas G, Lo EH, Ji R: Distinct roles of matrix metalloproteases in the early- and late-phase development of neuropathic pain. Nat Med 2008, 14:331-336. 32. Beggs S, Salter MW: Taking two cuts at pain. Nat Med 2008, 14:243-244. 33. Carvey PM, Hendey B, Monahan AJ: The blood-brain barrier in neurodegenerative disease: a rhetorical perspective. J Neurochem 2009, 111:291-314. 34. Weiss N, Miller F, Cazaubon S, Couraud P: The blood-brain barrier in brain homeostasis and neurological diseases. Biochim Biophys Acta 2009, 1788:842-857. 35. 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Carvey PM, Hendey B, Monahan AJ: The blood-brain barrier in neurodegenerative disease: a rhetorical perspective. J Neurochem 2009, 111:291-314. 34. Weiss N, Miller F, Cazaubon S, Couraud P: The blood-brain barrier in brain homeostasis and neurological diseases. Biochim Biophys Acta 2009, 1788:842-857. 36. Decosterd I, Woolf CJ: Spared nerve injury: an animal model of persistent peripheral neuropathic pain. Pain 2000, 87:149-158. 37. Pitcher GM, Ritchie J, Henry JL: Nerve constriction in the rat: model of neuropathic, surgical and central pain. Pain 1999, 83:37-46. doi:10.1186/1744-8069-6-74 Cite this article as: Beggs et al.: Peripheral nerve injury and TRPV1- expressing primary afferent C-fibers cause opening of the blood-brain barrier. Molecular Pain 2010 6:74. References Szolcsányi J: Antidromic vasodilatation and neurogenic inflammation. Agents Actions 1988, 23:4-11. 7. Szolcsányi J: Antidromic vasodilatation and neurogenic inflammation. Agents Actions 1988, 23:4-11. 8. 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https://openalex.org/W4280517393	https://www.frontiersin.org/articles/10.3389/fneur.2022.866261/pdf	English	null	Culture Effects on the Chinese Version Boston Naming Test Performance and the Normative Data in the Native Chinese-Speaking Elders in Mainland China	Frontiers in neurology	2,022	cc-by	6,906	BRIEF RESEARCH REPORT published: 13 May 2022 doi: 10.3389/fneur.2022.866261 BRIEF RESEARCH REPORT published: 13 May 2022 doi: 10.3389/fneur.2022.866261 Yan Li, Yuchen Qiao, Fen Wang, Cuibai Wei, Rui Wang, Hongmei Jin, Bingxin Xie, Jing You, Jianping Jia and Aihong Zhou* Innovation Center for Neurological Disorders and Department of Neurology, National Clinical Research Center for Geriatric Diseases, Xuanwu Hospital, Capital Medical University, Beijing, China Background: The Chinese version of Boston Naming Test (BNT-C) is administered in China widely. However, the neuropsychological parameter of BNT-C in native Chinese-speaking elders in mainland China has not been explored systematically. The aim of this study was to explore cultural inﬂuences on BNT-C performance and establish norms among native Chinese-speaking elders in Beijing. Edited by: Pedro Rosa-Neto, McGill University, Canada Reviewed by: Nilton Custodio, Peruvian Institute of Neurosciences (IPN), Peru Ding Ding, Fudan University, China Correspondence: Aihong Zhou zahxwh@163.com Edited by: Pedro Rosa-Neto, McGill University, Canada Reviewed by: Nilton Custodio, Peruvian Institute of Neurosciences (IPN), Peru Ding Ding, Fudan University, China Methods: A total of 161 native, Chinese-speaking, cognitively normal elders aged ≥55 years were enrolled from various communities in Beijing. The BNT-C was conducted on all the participants. The internal consistency, participants’ familiarity, and naming accuracy were analyzed and compared with data from Chinese areas outside the mainland and from American published previously. The inﬂuencing factors and stratiﬁed norms for BNT-C were established. Specialty section: This article was submitted to Dementia and Neurodegenerative Diseases, a section of the journal Frontiers in Neurology Results: The BNT-C showed good internal consistency (α = 0.738). Strong correlation between naming accuracy and object familiarity was found (r = 0.962, P < 0.001). Participants’ familiarity and correct naming rate for many items were notably different between the Chinese-speaking elders and English-speaking elders in America. The difference in some items’ correct naming rate also existed between Beijing, Taiwan, and Hongkong. Higher education was associated with higher scores, whereas age and gender had no effect on BNT-C performance. The recommended norms of total naming scores for elders with education ≤9 and >9 years were 16 and 23, respectively. Received: 31 January 2022 Accepted: 11 April 2022 Published: 13 May 2022 Keywords: Boston Naming Test, confrontation naming, cross-cultural, lexical familiarity, normative data, Chinese population, elders Culture Effects on the Chinese Version Boston Naming Test Performance and the Normative Data in the Native Chinese-Speaking Elders in Mainland China Yan Li, Yuchen Qiao, Fen Wang, Cuibai Wei, Rui Wang, Hongmei Jin, Bingxin Xie, Jing You, Jianping Jia and Aihong Zhou Yan Li, Yuchen Qiao, Fen Wang, Cuibai Wei, Rui Wang, Hongmei Jin, Bingxin Xie, Jing You, Jianping Jia and Aihong Zhou* Procedures and Scoring The BNT-C was administered to all the participants by trained raters as described by Cheung RW who, together with his colleagues, developed the BNT-C test (20). All 30 cards bearing the line-drawing objects or animals were presented to the elders in a ﬁxed order. Participants were instructed to name each object depicted on the cards. If the participant named one item correctly, one point was awarded and it was recorded among “scores of spontaneous naming (SN).” The examiner then proceeded to the next item. If the participant gave a wrong response or gave no response within 20 s, the participant’s response was recorded in detail and a standard semantic cue was provided (e.g., “it is a plant” for “tree”). A semantic cue was designed for each item as in the original version of BNT. If the participant gave the correct answer, one point was awarded and it was recorded among “scores after semantic cue (SC)” (20). In the original BNT, if the participants failed the semantic cue, then a phonemic cueing was supplied. Given that Chinese is a logographic language and the names of most objects consist of one sound, BNT-C adopted a multiple-choice recognition task including the target response, name of an object similar to the target in function (e.g., “cow” for “camel”), and name of an object similar to the target in appearance (e.g., “mountain” for “camel”). The participant’s response was recorded but no credit was given for choosing naming. The total score of BNT-C ranged from 0 to 30 (“SN” plus “SC”), the higher indicating the better naming ability. The Chinese version of BNT (BNT-C) was developed by Hongkong scholars by selecting 30 items from the original English version without item adjustment and replacement (20). The authors proved that BNT-C successfully distinguished naming impairment in Cantonese-speaking patients with a head injury from a normal control group. Since then, BNT-C has been widely used across China. Due to cultural diﬀerences, people’s familiarity with BNT items varies between the Chinese and Caucasian populations. For example, the igloo and harp are relatively unfamiliar to the Chinese, while the abacus, which is considered the most diﬃcult for Americans, is well- known to many Chinese. Consequently, the inﬂuential factors and norm data of the BNT-C may diﬀer markedly from those in the west. However, there has been no study exploring the familiarity of the BNT-C. Procedures and Scoring The per-item correct naming rate and striated norm of BNT-C in China mainland have not been evaluated and reported either. In this study, we administered BNT-C to 161 Chinese-speaking community elders in Beijing. The participants’ familiarity and naming performance for each BNT-C item were determined and compared with those from American (21) as well as other Chinese areas (Taiwan and Hongkong) published previously (5, 20). The correlations between naming accuracy and familiarity were further explored. The eﬀects of demographic variables (gender, age, and education) on naming performance were examined, and stratiﬁed norms were established considering signiﬁcant inﬂuential factors. Citation: Li Y, Qiao Y, Wang F, Wei C, Wang R, Jin H, Xie B, You J, Jia J and Zhou A (2022) Culture Effects on the Chinese Version Boston Naming Test Performance and the Normative Data in the Native Chinese-Speaking Elders in Mainland China. Front. Neurol. 13:866261. doi: 10.3389/fneur.2022.866261 Conclusion: The participants’ familiarity with BNT items differed between different cultures, which further affected the naming accuracy and total scores. The education stratiﬁed norms established here are helpful for the better application of BNT-C in mainland China. May 2022 \| Volume 13 \| Article 866261 Frontiers in Neurology \| www.frontiersin.org Chinese Elders BNT-C Performance Li et al. Li et al. Chinese Version of BNT The BNT-C used in this study consists of 30 items (Table 2) selected from the original 60 items without item adaption (20). The order of presentation followed the original sequence, and the design of the stimulus cards was identical to that of the original pictures (20). METHODS The Boston Naming Test (BNT) was compiled by Goodglass and Kaplan in 1983, which is composed of 60-line drawings of objects and animals, ranging from very familiar objects (trees and pencils) to unfamiliar objects (Sphinx and scaﬀolding) (1). By far, it is the most widely used confrontational naming test in the world and provides valuable diagnostic information for patients with aphasia and other cognitive- linguistic impairments from stroke (2), head injury (3), and neurodegenerative diseases such as frontotemporal dementia (4). This study was conducted from January 2018 to November 2019 at ﬁve communities in Beijing, China. The study protocol was approved by the Ethics Review Board of the Xuanwu Hospital, Beijing Capital Medical University. Written informed consent was obtained from all the participants. Subjects The participants were recruited from community volunteers in Beijing. The eligible samples for inclusion were (1) 55–85 years old, (2) native Chinese speakers, and (3) the Mini-Mental State Examination (MMSE) ≥24 (22). Neurologists interviewed all the subjects. Any individuals with a history of psychiatric or central nervous system diseases, hearing loss, learning disability, and any other condition that was likely to have an inﬂuence on performance in the BNT were excluded. The BNT was originally designed for English-speaking people in North America. Individuals’ familiarity with BNT items diﬀers among culture, populations, and countries, which likely aﬀect the naming performance and total scores (5). Therefore, it is important to explore the item familiarities and establish BNT norms according to local cultural and linguistic populations when used outside North America (5). So far, BNT has been adapted to many languages, including Danish (6), Spanish (7), Chinese (8), Dutch (9), Korean (10), French Canadian (11), Greek (12), Italian (13), Malay (14), and Swedish (15). Among the various versions of the BNT in diﬀerent languages, some of them adopted the original English items (6, 15, 16) and others made adjustments or replacements to some items to adapt to the local cultural background (12, 17, 18). Even in other English-speaking countries outside America, such as Australia (19), BNT also had to be adapted to local populations. Inﬂuential Factors and Stratiﬁed Norms for BNT-C Multiple linear regression analysis was used to explore the eﬀect of age, gender, and education on BNT-C performance (Table 3). No signiﬁcant correlations between age or gender and BNT-C scores were found. The analysis identiﬁed that education strongly correlated with BNT-C performance. Therefore, the subjects were subgrouped into the following four educational categories: (1) ≤6 years (n = 32); (2) 7–9 years (n = 40); (3) 10–12 years (n = 42), and (4) ≥13 years (n = 47). According to the post hoc analysis, there were no signiﬁcant diﬀerences between ≤6 years and 7–9 years of education and between 10–12 years and ≥13 years of education in scores for spontaneous naming (≤6 vs. 7–9 years: 20.29 ± 3.95 vs. 22.66 ± 3.31; 10–12 vs. ≥13 years: 25.00 ± 2.28 vs. 25.30 ± 2.37) and total scores after semantic cueing (≤6 vs. 7–9 years: 22.29 ± 3.58 vs. 23.94 ± 3.49; 10–12 vs. ≥13 years: 26.38 ± 1.83 vs. 26.65 ± 1.85). Accordingly, we subgrouped the participants into two education level groups (≤9 and >9 years) eventually. Internal Consistency of the BNT-C y The 30 items composing the BNT-C showed a high-internal reliability coeﬃcient (α = 0.738). Every item in the BNT-C was positively correlated with the total score and contributed positively to Cronbach’s α for the total score. Familiarity Rating The participants were instructed to rate each item of BNT- C for familiarity based on how usual or unusual the objects May 2022 \| Volume 13 \| Article 866261 Frontiers in Neurology \| www.frontiersin.org 2 Li et al. Li et al. Chinese Elders BNT-C Performance Per item correct naming rates for the 30 BNT-C items were recorded and compared with data from American normal elders (23) and other Chinese normal elders outside the mainland [Taiwan (5) and Hong Kong (20)]. The correct naming rate diﬀerence between populations >20% was considered signiﬁcant (5) (Table 2). Compared with the American residents, Beijing elders performed better in naming for compass, abacus, and protractor but performed worse in naming igloo, harp, dart, trellis, seahorse, cactus, and pyramid. The naming accuracy gap of igloo, harp, dart, protractor, compass, and abacus between the Chinese and the Americans was close to or >40%. Only 3.9% of the Beijing elders named igloo correctly compared with 98% of the American residents. In contrast, American residents got 55% and 46.7% correct naming rates for abacus and compass, respectively, lower than those of the Beijing residents (97.1 and 93.2%, respectively). Compared with the Hong Kong elders, the Beijing residents performed better in naming mushroom and protractor but worse in seahorse and dart. The Beijing residents named better in protractor and worse in seahorse, igloo, harp, and trellis than the Taiwan elders. Compared with the American elders, the Beijing, Taiwan, and Hong Kong elders were consistently good in naming compass and abacus but consistently worse in igloo, dart, and harp. All populations performed well in naming tree, pencil, scissors, ﬂowers, racquet, hanger, camel, saw, snail, funnel, escalator, wheelchair, with a correct rate of >90%. were in their experience, using a 5-point scale ranging from 1 (very unfamiliar) to 5 (very familiar). Familiarity was deﬁned as “the degree to which you come in contact with or think about the object.” RESULT In total, 161 cognitively normal elders, 74 men (46.0%) and 87 women (54.0%), were recruited for the current study. The average age was 71 years (range: 55–85 years). The average period of formal education was 11.4 years (range: 1–19 years). The average MMSE score was 28.03 (range: 25–31). Demographic data and MMSE scores stratiﬁed by education were presented in Table 1. In total, 161 cognitively normal elders, 74 men (46.0%) and 87 women (54.0%), were recruited for the current study. The average age was 71 years (range: 55–85 years). The average period of formal education was 11.4 years (range: 1–19 years). The average MMSE score was 28.03 (range: 25–31). Demographic data and MMSE scores stratiﬁed by education were presented in Table 1. Pearson correlation analysis was used to explore the correlation between correct naming and familiarity. The Pearson correlation coeﬃcient was 0.962 (P < 0.001), which indicated that naming accuracy was highly correlated with familiarity. Statistical Analysis Statistical analyses were performed with SPSS version 24.0 (SPSS Inc., Chicago, Ill., USA). Demographic and neuropsychological data were presented as mean ± SD or number and percentage. The mean data between two groups were analyzed using an independent-sample t-test or χ2 test (chi-square). The internal consistency of BNT-C was assessed by Cronbach’s alpha. Pearson’s correlation coeﬃcient was used to determine the correlation between naming accuracy and familiarity. A multiple linear regression analysis was used to explore the inﬂuence of age, gender, and education on BNT-C performance. For all tests, P < 0.05 was considered statistically signiﬁcant. Per Item Familiarity and Correct Naming Rate of BNT-C Participants’ familiarities for each BNT-C item were recorded and compared with those from a study that rated the 60 BNT pictures on familiarity in 30 elder native English speakers in America (21). However, the study adopted a scale ranging from one (not at all familiar) to seven (very familiar) instead of one to ﬁve. To facilitate comparison, we multiplied their results by ﬁve-sevenths. The average familiarity of Chinese in our study was 4.33, but it was 4.94 in the American residents. Our elders rated not familiar (<4) in seven items (igloo, harp, pyramid, seahorse, dart, cactus, and trellis) and rated familiar or very familiar in the remaining 23 items. American elders rated all 30 items as familiar or very familiar. Further analysis into items with a familiarity diﬀerence >0.5 showed that Americans were much more familiar with igloos than Chinese (4.98 vs. 1.95). They were also more familiar with harp (5 vs. 3.07), pyramid (4.98 vs. 3.35), cactus (5 vs. 3.79), dart (4.86 vs. 3.71), seahorse (4.81 vs. 3.67), and trellis (4.93 vs. 3.83). The familiarities with rhinoceros, harmonica, tongs, protractor, and tripod were also 0.5 points higher in the Americans than in the Chinese residents. Abacus was the only item that the Chinese residents were more familiar with than the Americans (4.80 vs. 4.55) (Table 2). The naming accuracy was signiﬁcantly higher in the high education group (>9 years) than in the lower education group (≤9 years) in seahorse (77.00 vs. 42.90%, P < 0.001), dart (68.90 vs. 47.60%, P = 0.031), rhinoceros (95.10 vs. 78.60%, P = 0.010), harp (54.10 vs. 26.20%, P = 0.005), pyramid (86.90 vs. 47.60%, P < 0.001), compass (100.00 vs. 83.30%, P = 0.004), tripod (90.20 vs. 52.40%, P < 0.001), tongs (91.80 vs. 71.40%, May 2022 \| Volume 13 \| Article 866261 Frontiers in Neurology \| www.frontiersin.org 3 Chinese Elders BNT-C Performance Li et al. TABLE 1 \| Demographic and neuropsychological data. Per Item Familiarity and Correct Naming Rate of BNT-C 23.36 ± 3.65, P < 0.001) than the lower education group (Table 1). We chose “mean-2∗SD” as the recommended cut-oﬀvalue. For the individuals with formal education >9 years, the appropriate cutoﬀwas 20 for spontaneous naming scores and 23 for total scores. For the individuals with formal education ≤9 years, the cutoﬀscores were 14 and 16, respectively. P = 0.006), and protractor (85.20 vs. 57.10%, P = 0.001) (Table 4). Consequently, the high education group achieved better spontaneous naming scores (25.14 ± 2.33 vs. 21.79 ± 3.75, P < 0.001) and total scores (26.47 ± 1.88 vs. 23.36 ± 3.65, P < 0.001) than the lower education group (Table 1). We chose “mean-2∗SD” as the recommended cut-oﬀvalue. For the individuals with formal education >9 years, the appropriate cutoﬀwas 20 for spontaneous naming scores and 23 for total scores. For the individuals with formal education ≤9 years, the cutoﬀscores were 14 and 16, respectively. DISCUSSION BNT was originally developed for the English-speaking populations in America. Because of its practicability and simplicity, many scholars have translated and adapted BNT for people with diﬀerent languages and cultural backgrounds. BNT-C was composed by selecting 30 items from the original English version and is widely used in China. However, the psychometrics of the BNT-C has been rarely reported on and evaluated in the native Chinese-speaking elderly in the mainland. This study explored the internal consistency, per item familiarity, and correct naming rate of the BNT-C for the ﬁrst time and generated striated norms for the Chinese-speaking elders in mainland China. We found good internal consistency in BNT-C, indicating that the items of this test reliably measure the same construct. Compared with the Americans, the Chinese elders were signiﬁcantly less familiar with many items in the BNT-C and achieved low naming accuracy and total scores. We identiﬁed a positive correlation between education and BNT-C performance and established norms according to diﬀerent education levels. A previous study indicated that picture familiarity facilitates the processing of BNT word representations (24). It is expected that people likely name objects correctly when they are familiar with them. Consistently, we found a strong positive correlation between naming accuracy and item familiarity in the BNT-C performance. This correlation further explained the diﬀerence in per item correct naming rate between the Chinese and American residents. The American residents performed better in naming igloo, cactus, harp, and pyramid which they were more familiar with than the Chinese. In contrast, they perfomed signiﬁcantly worse in naming abacus which they were least familiar with. The results further demonstrated how familiarity inﬂuenced naming accuracy. Per Item Familiarity and Correct Naming Rate of BNT-C Total Education (years) t P-value ≤9 >9 Number 161 72 89 / Male: female ratio 74:87 31:41 43:46 0.443 0.506 Age, years 71.00 ± 6.29 70.70 ± 6.69 71.19 ± 6.06 −0.414 0.680 Education, years 11.41 ± 3.54 7.74 ± 1.46 13.76 ± 2.22 15.245 <0.001 MMSE 28.03 ± 1.65 27.45 ± 1.90 28.43 ± 1.33 −3.058 0.003 BNT-C scores (SN) 23.83 ± 3.37 21.79 ± 3.75 25.14 ± 2.33 −5.488 <0.001 BNT-C scores (TS) 25.26 ± 3.10 23.36 ± 3.65 26.47 ± 1.88 −5.405 <0.001 BNT-C cut-off score (SN) 14 20 BNT-C cut-off score (TS) 16 23 MMSE, Mini-Mental State Examination; BNT-C, the Chinese version of the Boston Naming Test; SN, scores of spontaneous naming; TS, total scores (“scores of spontaneous naming” plus “scores after semantic cue”). TABLE 1 \| Demographic and neuropsychological data. life experiences, and education levels. However, familiarity data have been rarely evaluated and reported previously both in China and other countries. The current study found that the Chinese elders were not familiar with igloo, harp, pyramid, seahorse, dart, cactus, and trellis, which are not common in daily life or ordinary readings in China. Ferraro et al. rated the BNT pictures on familiarity in 30 elder native English speakers in America (21). As expected, the Chinese residents were less familiar with many items than the American residents. Most Chinese elders were very unfamiliar with igloo, which was very familiar to the Americans. In contrast, the Chinese residents were more familiar with abacus which was less familiar to the Americans. Many Chinese elders used the abacus for calculation in their early lives. The discrepancies in familiarities for BNT items truly reﬂected the cultural diﬀerence between China and America. Besides BNT- C, there were many adapted versions outside North America. Most modiﬁed versions replaced part test items according to local cultures (12, 17, 18). It seems appropriate to consider the possibility of replacing items that are not suitable for the Chinese individuals with more culturally representative items in the adapted Chinese BNT version. The development of new test items was outside the scope of this study, but future research should certainly address this consideration. P = 0.006), and protractor (85.20 vs. 57.10%, P = 0.001) (Table 4). Consequently, the high education group achieved better spontaneous naming scores (25.14 ± 2.33 vs. 21.79 ± 3.75, P < 0.001) and total scores (26.47 ± 1.88 vs. Object Familiarity and Naming Accuracy of BNT-C in Different Cultural Backgrounds Object Familiarity and Naming Accuracy of BNT-C in Different Cultural Backgrounds BNT consists of 60 items for participants to name, including common things such as beds and trees, as well as uncommon objects such as pyramids and sphynx. People vary in their familiarities with each item due to diﬀerent cultural backgrounds, BNT consists of 60 items for participants to name, including common things such as beds and trees, as well as uncommon objects such as pyramids and sphynx. People vary in their familiarities with each item due to diﬀerent cultural backgrounds, BNT-C was developed by the Hong Kong scholars and was used in Taiwan and the mainland widely. Compared with people from Hong Kong or Taiwan, Beijing elders performed better May 2022 \| Volume 13 \| Article 866261 Frontiers in Neurology \| www.frontiersin.org 4 Chinese Elders BNT-C Performance Li et al. TABLE 2 \| Per-item familiarity and correct naming rate of BNT-C in our study and other studies. Item Original item No. Item Familiarity Correct (%) No. Object Familiarity and Naming Accuracy of BNT-C in Different Cultural Backgrounds BJ USA (21) BJ HK (20) TW (5) USA (23) Number 161 30 161 77 264 60 Age range 55–85 56–86 55–85 23–79 60–92 40–78 Education, years 11.4 15.0 11.4 9.7 12.5 13.9 SN N/A N/A 23.8 (3.4) 24.9 (3.0) 24.7 (3.9) N/A TS N/A N/A 25.2 (3.1) 26.7 (2.8) N/A 54.5 1 2 Tree 4.84 5.00 100.0 100.0 100.0 100.0 2 3 Pencil 4.90 5.00 100.0 100.0 100.0 100.0 3 6 Scissors 4.92 5.00 100.0 100.0 100.0 98.3 4 8 Flowers 4.79 5.00 100.0 100.0 100.0 98.3 5 9 Saw 4.66 5.00 98.1 100.0 98.1 100.0 6 12 Broom 4.90 5.00 89.3 94.8 98.1 100.0 7 14 Mushroom 4.84 5.00 100.0 67.5 97.7 93.3 8 15 Hanger 4.91 5.00 99.0 100.0 99.2 100.0 9 16 Wheelchair 4.75 5.00 91.4 92.2 96.2 100.0 10 17 Camel 4.62 4.88 99.0 98.7 99.2 98.3 11 21 Racquet 4.76 5.00 100.0 97.4 98.1 100.0 12 22 Snail 4.60 4.95 98.1 90.9 96.2 100.0 13 24 Seahorse 3.67 4.81 63.1 90.9 82.6 91.7 14 25 Dart 3.71 4.86 61.4 83.1 73.9 98.3 15 30 Harmonica 4.24 5.00 80.6 83.1 84.5 86.7 16 31 Rhinoceros 4.13 4.95 88.4 81.8 93.6 83.0 17 33 Igloo 1.95 4.98 3.9 20.8 60.6 98.3 18 36 Cactus 3.79 5.00 71.8 85.7 86.4 100.0 19 37 Escalator 4.72 5.00 94.2 97.4 97.3 100.0 20 38 Harp 3.07 5.00 43.1 54.5 68.9 100.0 21 42 Stethoscope 4.67 5.00 95.2 83.1 87.9 96.7 22 43 Pyramid 3.35 4.98 70.9 83.1 79.5 95.0 23 46 Funnel 4.68 5.00 96.1 98.7 92.0 95.0 24 47 Accordion 4.57 4.95 96.1 77.9 84.8 91.7 25 50 Compass 4.63 4.91 93.2 90.9 92.0 46.7 26 52 Tripod 4.23 4.84 74.8 63.6 80.3 81.7 27 54 Tongs 4.20 4.95 83.5 81.8 96.6 81.7 28 57 Trellis 3.83 4.93 57.3 41.6 84.5 88.3 29 59 Protractor 4.16 4.81 73.8 33.8 44.7 35.0 30 60 Abacus 4.80 4.55 97.1 98.7 99.6 55.0 BNT-C, the Chinese version of the Boston Naming Test; “Original Item No.” refers to the original BNT (1). Values for education and BNT are means. N/A, Not available; SN, scores of TABLE 2 \| Per-item familiarity and correct naming rate of BNT-C in our study and other studies. BNT-C, the Chinese version of the Boston Naming Test; “Original Item No.” refers to the original BNT (1). Values for education and BNT are means. Object Familiarity and Naming Accuracy of BNT-C in Different Cultural Backgrounds Multicenter studies adopting the same inclusion criteria are required in future work for a more convincing comparison. Object Familiarity and Naming Accuracy of BNT-C in Different Cultural Backgrounds N/A, Not available; SN, scores of spontaneous naming; TS, total scores (“scores of spontaneous naming” plus “scores after semantic cue”); BJ, Beijing; HK, Hongkong; TW, Taiwan. TABLE 3 \| Results of multiple linear regression analysis of gender, age, and education on BNT-C score. Unstandardize B Coefﬁcients std. error Standardized coefﬁcients beta t P-value (Constant) 24.466 3.949 6.195 <0.001 Gender −1.043 0.587 −0.180 −1.778 0.079 Age −0.002 0.055 −0.004 −0.041 0.967 Education 1.854 0.632 0.310 2.936 0.004 BNT-C, the Chinese version of the Boston Naming Test. TABLE 3 \| Results of multiple linear regression analysis of gender, age, and education on BNT-C score. regression analysis of gender, age, and education on BNT-C scor May 2022 \| Volume 13 \| Article 866261 5 Frontiers in Neurology \| www.frontiersin.org Chinese Elders BNT-C Performance Li et al. TABLE 4 \| Percentage of correct BNT-C between two different education levels. Item Correct (%) χ2 P-value ≤9 years >9 years Tree 100.00 100.00 – – Pencil 100.00 100.00 – – Scissors 100.00 100.00 – – Flowers 100.00 100.00 – – Saw 95.20 100.00 2.96 0.164 Broom 88.10 90.20 0.00 0.992 Mushroom 100.00 100.00 – – Hanger 97.60 100.00 1.47 0.408 Wheelchair 95.20 88.50 0.69 0.406 Camel 100.00 98.40 0.70 1.000 Racquet 100.00 100.00 – – Snail 97.60 98.40 0.07 1.000 Seahorse 42.90 77.00 12.49 <0.001 Dart 47.60 68.90 4.68 0.031 Harmonica 76.20 83.60 0.87 0.35 Rhinoceros 78.60 95.10 6.59 0.010 Igloo 4.80 3.30 0.00 1.000 Cactus 64.30 77.00 2.00 0.157 Escalator 90.50 96.70 0.81 0.367 Harp 26.20 54.10 7.92 0.005 Stethoscope 92.90 96.70 0.19 0.667 Pyramid 47.60 86.90 18.58 <0.001 Funnel 92.90 98.40 0.81 0.367 Accordion 92.90 98.40 0.81 0.367 Compass 83.30 100.00 8.44 0.004 Tripod 52.40 90.20 18.82 <0.001 Tongs 71.40 91.80 7.49 0.006 Trellis 54.80 59.00 0.18 0.668 Protractor 57.10 85.20 10.16 0.001 Abacus 100.00 95.10 0.74 0.388 BNT-C, the Chinese version of the Boston Naming Test. BNT score of 55). Combining the familiarity diﬀerences and the correlation between familiarity and naming accuracy, we could infer that cultural background inﬂuences participants’ familiarities with BNT items, which aﬀects the naming accuracy further and impact the total scores ultimately. However, it should be noted that the per-item familiarity and correct naming rate of BNT-C in this study and other studies in HK, TW, and the USA may have limited comparability given the varied age range and educational levels. Frontiers in Neurology \| www.frontiersin.org Effects of Demographic Factors on the BNT-C Performance Demographic variables including age, gender, and education were repeatedly reported to impact the performance of cognition measures. Lower mean BNT scores with lower educational levels have been frequently found in the published works of literature (26–30). This study also demonstrated that education correlated signiﬁcantly with BNT-C scores. Our ﬁndings were consistent with other research. Cheung et al. (20) performed the BNT-C in 77 normal adults in Hong Kong and found a positive association between education and naming scores (r = 0.342, P < 0.01). Chen et al. (5) applied the same BNT-C to 264 native Chinese people with normal cognition aged >60 years in Taiwan. They also found years of education were positively correlated with BNT-C score (r = 0.376, P < 0.01). Our research further showed that high-educated participants made fewer errors than low- educated subjects on dart, harp, pyramid, tripod, and protractor. The results were supported by the study exploring the inﬂuence of schooling on the performance of aphasia examination, which indicated that confrontation naming demands a greater degree of semantic knowledge, which is proved with increasing years of formal education (31). This may be because these objects are not common in daily life but are acquired gradually in study and reading. The inﬂuence of gender and age on BNT performance remains controversial. Using the original 60-item BNT in normal elders in Middle Tennessee, Welch (30) found that age was signiﬁcantly involved in confrontational naming ability. Moreover, there was also a gender bias that men scored signiﬁcantly higher on 17 items than women. However, no gender eﬀect was found for the Turkish version of the BNT (32) and the adapted BNT version for the Portuguese speakers (33). The Korean version of the BNT scores was slightly aﬀected by age but rarely inﬂuenced by gender (10). For BNT-C, Cheung, who developed the BNT-C, found that gender and age have no eﬀects on naming performance in Hongkong Chinese population (20). Chen et al. (5) did not identify the eﬀect of either gender and age on BNT-C scores in the Taiwan residents. Consistent with previous research, we found no correlations between age or gender and BNT-C performance in this study. It likely indicated that gender and age aﬀect the performance less on this 30-item BNT-C version. in naming protractor but worse in seahorse, dart, igloo, and harp. Effects of Demographic Factors on the BNT-C Performance This was probably because the residents in Hong Kong and Taiwan were more inﬂuenced by western culture than the Chinese mainland residents. Compared with the Americans, the Chinese people including Beijing, Taiwan, and Hong Kong consistently performed better in naming compass and abacus but consistently worse in igloo, dart, and harp. This consistency reﬂected the long-term diﬀerences between the Chinese culture and the American culture. Hobson et al. (25) showed a reliable creation of an estimated 60-item BNT score from administrations of the 30-item BNT by multiplying the obtained score by two. By this method, the total BNT scores from our study and those from Hong Kong were multiplied by the two to allow comparisons across diﬀerent studies. The calculated total scores (“scores of spontaneous naming” plus “scores after semantic cuing”) for elders in Beijing (estimated BNT score of 50) were lower than that in Hong Kong (20) (estimated BNT score of 53) and America (23) (estimated This study has several limitations. First, our study mainly recruited the elders aged ≥55 years, so the data may not be generalized to the adults of all ages. Second, the major May 2022 \| Volume 13 \| Article 866261 6 Chinese Elders BNT-C Performance Li et al. subjects were educated and only few were illiterate. This might underestimate the education eﬀects on BNT-C performance. However, with the popularization of compulsory education, there were fewer illiterates now, and there will hardly be illiterates in the future. The results of this study will be suitable for future use. Third, the number of participants in each age-education cell was relatively small. Further research should expand the sample size. Fourth, the participants were recruited from communities in the urban areas of Beijing. In the future, urban and rural residents should be selected nationwide to make the results more representative. Another one that should be considered was that the American, Hong Kong, and Taiwan studies used for comparison were conducted 10 years or more before. The results may deviate from the real diﬀerences between the current population. However, the Chinese people rated less familiar and score on many items than the American decades later, indicating that cultural diﬀerences between China and America and their impact on BNT-C persist till now. accuracy for each BNT-C item between the Americans and native Chinese speakers, which was highly consistent with the cultural diﬀerences. REFERENCES without temporal lobe radionecrosis. Arch Neurol. (2000) 57:1347–52. doi: 10.1001/archneur.57.9.1347 without temporal lobe radionecrosis. Arch Neurol. (2000) 57:1347–52. doi: 10.1001/archneur.57.9.1347 1. 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ETHICS STATEMENT The studies involving human participants were reviewed and approved by Ethics Committee of Xuanwu Hospital, Capital Medical University. The patients/participants provided their written informed consent to participate in this study. DATA AVAILABILITY STATEMENT The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Effects of Demographic Factors on the BNT-C Performance The possibility of replacing items that are not suitable for the Chinese individuals with more culturally representative objects should be considered in future work. CONCLUSIONS AZ: conception, design, and revision of the article. YL, YQ, FW, and CW: data collection. YL: analysis and drafting of the article. RW, HJ, BX, and JY: data collection. JJ: revision of the article and approved the ﬁnal version. All authors contributed to the article and approved the submitted version. 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Arch Clin Neuropsychol. (2009) 24:343–54. doi: 10.1093/arclin/acp039 29. Frontiers in Neurology \| www.frontiersin.org May 2022 \| Volume 13 \| Article 866261 REFERENCES doi: 10.1002/gps.2533 Publisher’s Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their aﬃliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. 26. Piguet O, Millar JL, Bennett HP, Lye TC, Creasey H, Broe GA. Boston naming test:: normative data for older Australians. Brain Impairment. (2001) 2:131–9. doi: 10.1375/brim.2.2.131 Copyright © 2022 Li, Qiao, Wang, Wei, Wang, Jin, Xie, You, Jia and Zhou. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). 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https://openalex.org/W2556608150	https://europepmc.org/articles/pmc5104480?pdf=render	English	null	Regioselective Glucuronidation of Diosmetin and Chrysoeriol by the Interplay of Glucuronidation and Transport in UGT1A9-Overexpressing HeLa Cells	PloS one	2,016	cc-by	10,217	Editor: Alberto G Passi, University of Insubria, ITALY Published: November 10, 2016 Copyright: © 2016 Zeng et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. RESEARCH ARTICLE Data Availability Statement: All relevant data are within the paper. Data Availability Statement: All relevant data are within the paper. Funding: This research was supported by the joint fund set up by the National Natural Science Foundation of China (No. U1203204), the National Natural Science Foundation of China (No. 81460638, 81360671), Science and Technology Project of Guangzhou City (201509010004) and Guangdong Natural Science Foundation Province (2015AD030312012). Regioselective Glucuronidation of Diosmetin and Chrysoeriol by the Interplay of Glucuronidation and Transport in UGT1A9- Overexpressing HeLa Cells Xuejun Zeng1,2☯, Jian Shi2☯, Min Zhao2, Qingwei Chen1,2, Liping Wang2, Huangyu Jiang2, Feifei Luo2, Lijun Zhu2, Linlin Lu2, Xinchun Wang1, Zhongqiu Liu2 1 Department of Pharmacy, First Hospital Affiliated to Shihezi University, Shihezi, Xinjiang, 832002, China, 2 International Institute for Translational Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, 510006, China 1 Department of Pharmacy, First Hospital Affiliated to Shihezi University, Shihezi, Xinjiang, 832002, China, 2 International Institute for Translational Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, 510006, China a1111 ☯These authors contributed equally to this work. * liuzq@gzucm.edu.cn (ZL); cwjwxc@163.com (XW) OPEN ACCESS This study aimed to determine the reaction kinetics of the regioselective glucuronidation of diosmetin and chrysoeriol, two important methylated metabolites of luteolin, by human liver microsomes (HLMs) and uridine-50-diphosphate glucuronosyltransferase (UGTs) enzymes. This study also investigated the effects of breast cancer resistance protein (BCRP) on the efflux of diosmetin and chrysoeriol glucuronides in HeLa cells overexpressing UGT1A9 (HeLa—UGT1A9). After incubation with HLMs in the presence of UDP-glucuronic acid, diosmetin and chrysoeriol gained two glucuronides each, and the OH—in each B ring of diosmetin and chrysoeriol was the preferable site for glucuronidation. Screening assays with 12 human expressed UGT enzymes and chemical-inhibition assays demonstrated that glucuronide formation was almost exclusively catalyzed by UGT1A1, UGT1A6, and UGT1A9. Importantly, in HeLa—UGT1A9, Ko143 significantly inhibited the efflux of diosme- tin and chrysoeriol glucuronides and increased their intracellular levels in a dose-dependent manner. This observation suggested that BCRP-mediated excretion was the predominant pathway for diosmetin and chrysoeriol disposition. In conclusion, UGT1A1, UGT1A6, and UGT1A9 were the chief contributors to the regioselective glucuronidation of diosmetin and chrysoeriol in the liver. Moreover, cellular glucuronidation was significantly altered by inhibit- ing BCRP, revealing a notable interplay between glucuronidation and efflux transport. Dios- metin and chrysoeriol possibly have different effects on anti-cancer due to the difference of UGT isoforms in different cancer cells. Citation: Zeng X, Shi J, Zhao M, Chen Q, Wang L, Jiang H, et al. (2016) Regioselective Glucuronidation of Diosmetin and Chrysoeriol by the Interplay of Glucuronidation and Transport in UGT1A9-Overexpressing HeLa Cells. PLoS ONE 11(11): e0166239. doi:10.1371/journal. pone.0166239 Citation: Zeng X, Shi J, Zhao M, Chen Q, Wang L, Jiang H, et al. (2016) Regioselective Glucuronidation of Diosmetin and Chrysoeriol by the Interplay of Glucuronidation and Transport in UGT1A9-Overexpressing HeLa Cells. PLoS ONE 11(11): e0166239. doi:10.1371/journal. pone.0166239 Citation: Zeng X, Shi J, Zhao M, Chen Q, Wang L, Jiang H, et al. (2016) Regioselective Glucuronidation of Diosmetin and Chrysoeriol by the Interplay of Glucuronidation and Transport in UGT1A9-Overexpressing HeLa Cells. PLoS ONE 11(11): e0166239. doi:10.1371/journal. pone.0166239 Glucuronidation of Diosmetin and Chrysoeriol by the Interplay of Glucuronidation and Transport in UGT1A9-Overexpressing HeLa Cells. PLoS ONE 11(11): e0166239. doi:10.1371/journal. pone.0166239 Glucuronidation and Transportation of Diosmetin and Chrysoeriol pharmacological effects, including antioxidation, anti-inflammation, and anticarcinogenic activity [2,3]. A study reported that after luteolin’s oral administration, it undergoes methyla- tion as a major metabolic pathway. Isomers diosmetin (5,7,30-trihydroxy-40-methoxyflavone) and chrysoeriol (5,7,40-trihydroxy-30- methoxyflavone) are the two methylated metabolites [4] of luteolin. These isomers have been shown to exhibit common biological effects, such as oste- oporosis prevention [5]. Diosmetin is an active ingredient of some medications [6] and has been reported to display several biological properties, including anticancer effects [7,8] and antibacterial actions [9]-. Similar to diosmetin, chrysoeriol is mainly distributed in many plant products, such as parsley [10] and peanut hull [11]. Chrysoeriol could inhibit lipid peroxida- tion in low-density lipoprotein [11] and exhibit antioxidant activity and free radical scavenging ability [12]. The biological properties of flavone are well known to be severely limited by the compound’s low bioavailability resulting from extensive metabolism and excretion. However, studies on the mechanism and characteristics of the metabolism and excretion of diosmetin and chrysoeriol are few. Competing Interests: The authors have declared that no competing interests exist. Uridine-50-diphosphate glucuronosyltransferases (UGTs) are a superfamily of enzymes that catalyze the glucuronidation of many compounds [13]. Glucuronidation, a primary phase II conjugation reaction, is considered as a detoxification mechanism because the generated glu- curonides are highly polar and can be rapidly eliminated [14,15]. UGT isoforms involved in glucuronidation of phenolics and other relevant compounds discussed belong to UGT1A or UGT2B family [13], and these enzymes possess broad and overlapping substrate specificities. Our previous study demonstrated that diosmetin and chrysoeriol could be metabolized to their phase II metabolites by Ugts [16]. Thus, the UGT-catalyzed glucuronidation could play a key role in determining the bioavailability and clearance of diosmetin and chrysoeriol [13]. However, the characteristics of and difference between the glucuronidation of diosmetin and chrysoeriol by UGTs and the other major contributing enzymes have not been fully estab- lished. This knowledge is valuable in achieving better prediction of diosmetin and chrysoeriol disposition, which could be the main factors affecting the compounds’ bioavailability and bio- logical activities. Our findings could also add to the general understanding of the mechanisms of action of diosmetin and chrysoeriol in vivo. On the other hand, the high polarity of the glucuronides renders difficult their passive diffu- sion outward from the cell. As such, the glucuronides require efflux transporters to exit the cells. Introduction 81460638, 81360671), Science and Technology Project of Guangzhou City (201509010004) and Guangdong Natural Science Foundation Province (2015AD030312012). Luteolin (30,40,5,7-tetrahydroxyflavone), a typical catecholic flavonoid, is present in various dietary sources such as fruits, vegetables, wines, oils, tea etc [1]. It exhibits a wide range of PLOS ONE \| DOI:10.1371/journal.pone.0166239 November 10, 2016 1 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0166239 November 10, 2016 Cell Culture HeLa and HeLa—UGT1A9 cells were seeded into six-well plate at a density of 1.0 × 105 cells/well, and were grown using Dulbecco’s modified Eagle’smedium (DMEM) supplemented with 10% fetal bovine serum (FBS). After 3 ~ 4 days of seeding, the cells can be used for the glucuronidation experiments. The cellular glucuronidation of flavone is hence presumed to be affected by the activities of glucuronidation enzymes, relevant efflux transporters, and the interplay between UGT enzymes and efflux transporters [17]. Breast cancer resistance protein (BCRP), an important efflux pump in the cell membrane, is known to participate in the biliary and intestinal excretion of many flavone glucuronides [18–20]. Therefore, the interplay between BCRP and UGT enzymes could play a key role in the phase II disposition of flavone. Understanding the role of BCRP is important for assessing the contribution of BCRP in glucuronide disposition. Our group studied the effect of such interplay on luteolin disposition using established HeLa— UGT1A9 cells with UGT1A9 and BCRP to investigate the metabolism of luteolin and the excre- tion of its glucuronides [13]. Compared with Caco-2 and MDCK II cells, HeLa—UGT1A9 cells serve as a suitable model for the study of the interplay between transporters and UGT enzymes because of the functional expression of only one dominant UGT and one dominant efflux transporter in these cells [21,22]. Diosmetin and chrysoeriol are the main methylation metabo- lites of luteolin. Hence, the study of the effect of the interplay between transporters and UGT enzymes on diosmetin and chrysoeriol metabolism is important and useful for determining the mechanism and characteristics of the absorption and metabolism of luteolin in vivo. The present study aimed to determine and compare the reaction kinetics of the regioselec- tive glucuronidation of diosmetin and chrysoeriol by human liver microsomes (HLMs) and PLOS ONE \| DOI:10.1371/journal.pone.0166239 November 10, 2016 2 / 21 Glucuronidation and Transportation of Diosmetin and Chrysoeriol expressed UGT enzymes. The main UGT enzymes involved in such regioselective glucuroni- dation were identified and determined through incubation with a panel of 12 UGT enzymes and HLMs in the presence of specific UGT inhibitors. Kinetic parameters were derived by fit- ting and appropriate modeling of the data. UGT1A9 was one of important UGT enzymes in the liver involved in the metabolism of diosmetin and chrysoeriol as determined in this study. Hence, we used HeLa—UGT1A9 cells to explore how the interplay between UGT1A9 and BCRP affects diosmetin and chrysoeriol disposition. The results of our study would contribute to improving the understanding on the absorption and disposition of luteolin. Chemicals and Reagents Diosmetin (>98% purity) was purchased from Chengdu Must Pharmaceutical Co. Ltd. (Chengdu, China). Chrysoeriol was acquired from Shenzhen Zhenqiang Bio-Technology Co. Ltd. (Shenzhen, China). Tilianin (95% purity; internal standard; IS) was provided by the Department of Pharmacy, First Hospital Affiliated to Shihezi University. Diosmetin-7-O-glu- curonide (Dio-7-G), diosmetin-30-O-glucuronide (Dio-30-G), chrysoeriol-7-O-glucuronide (Chr-7-G) and chrysoeriol-40-O-glucuronide (Chr-40-G) were prepared and identified by our laboratory [16]. Carvacrol and troglitazone were purchased from Qiyun Bio-Technology Co. Ltd. (Guangzhou, China). Phenylbutazone, bilirubin, UDP glucuronic acid (UDPGA), ala- methicin, MgCl2, D-saccharic-1,4-lactone monohydrate, β-glucuronidase (Type HP-2 from Helix pomatia), Hanks’ Balanced Salts, HEPES (N-(2-Hydroxyethyl) piperazine-N’-(2-ethane- sulfonic acid)), MTT and Ko143 were purchased from Sigma—Aldrich Co. (St. Louis, MO, USA). Human expressed UGTs (Supersomes Enzymes), pooled HLMs (35 individuals), and human expressed UGT enzymes (UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, and 2B17) were purchased from BD Biosciences (Woburn, MA, USA). HeLa cells and HeLa cells stably transfected with UGT1A9 (HeLa—UGT1A9 cells) were provided by Dr. Ming Hu (Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, Texas, USA). HyClone fetal bovine serum and HyClone pen- icillin—streptomycin solution were obtained from Thermo Fisher Scientific (MA, USA). All other chemicals and solvents were of analytical grade or better. Glucuronidation and Transportation of Diosmetin and Chrysoeriol then allowed to stand for 30 min in a water bath. These reactions were terminated by adding 60 μL ice-cold acetonitrile containing the IS (25 nM tilianin). The samples were centrifuged at 13,000 rpm for 30 min, and the supernatant was analyzed by ultra high pressure liquid chroma- tography (UHPLC)–tandem mass spectrometry (MS/MS). All experiments were performed in triplicate. Kinetic Evaluation The enzyme kinetic parameters were obtained by fitting kinetic models to the experimental data using GraphPad Prism version 5.04 for Windows (GraphPad Software Inc., San Diego, CA, USA). The best model was selected based on visual inspection of the Eadie-Hofstee plots, the calculated r2 values, and corrected Akaike’s information criterion [26]. The HLMs or human expressed UGT enzymes were employed for kinetic studies at concentrations ranging from 0.0021 mg/mL to 0.0053 mg/mL. The rates of the glucuronidation were fitted to the fol- lowing equations: V ¼ Cmetabolite t Cprotein Where V (nmol/mg/min) is the rates of the glucuronidation, Cmetabolite (μM) and Cprotein (mg/mL) respectively are the concentration of the metabolite and the reaction protein, t (min) is the reaction time [2,22]. Where V (nmol/mg/min) is the rates of the glucuronidation, Cmetabolite (μM) and Cprotein (mg/mL) respectively are the concentration of the metabolite and the reaction protein, t (min) is the reaction time [2,22]. Model selection was based on visual inspection of Eadie—Hofstee plots [26] and it was reported in our previous study [25]. In brief, if the Eadie—Hofstee plot was linear, formation rates (V) of glucuronides at different substrate concentrations (C) were fitted to the standard Eq (1): V ¼ Vmax C Km þ C ð1Þ ð1Þ where Km is the Michaelis-Menten constant and Vmax is the maximum rate of glucuronidation The intrinsic clearance (CLint) was derived by Vmax/Km. When Eadie—Hofstee plots revealed substrate inhibition kinetics, the reaction rate (V) were fitted to Eq (2): V ¼ Vmax1 1 þ ðKm1=CÞ þ ðC=KsiÞ ð2Þ ð2Þ where C is the substrate concentration, V is the initial reaction rate, Vmax1 is the maximum enzyme velocity, Km1 is the substrate concentration required to achieve 50% of Vmax1, and Ksi is the substrate inhibition constant. The intrinsic clearance (CLint) was derived by Vmax1/Km1. Glucuronidation Assay All metabolism studies in vitro were approved by the First Hospital Affiliated to Shihezi Uni- versity Research Ethics Committee. The glucuronidation assay was performed as described in previous publications [23–25]. The stock solutions of diosmetin or chrysoeriol were prepared in methanol-dimethyl sulfoxide (DMSO) (1:1, v/v) and diluted with methanol—DMSO (1:1, v/v) to the desired concentrations immediately before use. The incubation volumes were 120 μL, and the total organic solvent content was 1%. The mixtures contained HLMs or expressed UGT enzymes, MgCl2 (0.88 mM), saccharolactone (4.4 mM), alamethicin (22 μg/mL), and diosmetin or chrysoeriol (at desired concentrations) in 50 mM potassium phosphate (pH 7.4). The reactions were initiated by adding UDPGA (3.5 mM) at 37˚C and 3 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0166239 November 10, 2016 MTT assay The cytotoxicity of diosmetin and chrysoeriol on A549 and HepG2 cells was measured using the MTT assay. Cells were seeded at a density of 3 × 103/well in 96-well plates and grown over- night, and then the medium was replaced with a fresh medium containing substrates (5 μM Ko143, 10 μM Ko143, 10 μM diosmetin or chrysoeriol contained or not contained 5 μM Ko143 or 10 μM Ko143). The cells were further incubated at 37˚C with 5% CO2 for 48 h. The culture medium was removed and replaced with 100 μL 0.5 mg/mL 3-(4, 5-dimethylthiozol- 2-yl)-3, 5-dipheryl tetrazolium bromide (MTT) solution and incubated at 37˚C for 4 h. The supernatants were discarded, and 150 μL of dimethyl sulfoxide was added to each well. The plates were shaken for 10 min at room temperature, and then, the stained formazan product was determined at 570 nm with a multimode plate reader. Assay with Human Expressed UGTs The method of this study as described in detail previously [25]. In this study, 12 human expressed UGTs (UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, and 2B17) were used for the glucuronidation of diosmetin and chrysoeriol. Three substrate concentra- tions (1.25, 2.5, and 10 μM) were employed in this experiment, and the incubation progress was performed as shown above. The incubation conditions were as follows: protein concentra- tion: 0.0053 mg/mL; temperature of water: 37˚C; shaker speed: 50 rpm; reaction time: 30 min. After incubation, all samples were pretreated as described above and analyzed by UHPLC— MS/MS. 4 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0166239 November 10, 2016 Glucuronidation and Transportation of Diosmetin and Chrysoeriol Glucuronides Excretion Experiments in the HeLa and HeLa—UGT1A9 Cell Model Glucuronides excretion experiments were performed as described in detail previously [17]. The HeLa cells were used to incubate diosmetin and chrysoeriol as the control. Brifely speak- ing, the cells were incubated at 37˚C with loading solution (2 mL). The loading solution were HBSS (9.801 g Hanks’ balanced salts, 0.372 g NaHCO3, 3.502 g glucose, 5.963 g HEPES and 1.164 g NaCl dissolved in 900 mL deionized water) which containing different diosmetin or chrysoeriol concentrations (1.25, 2.5, 5, 10, 20, and 40 μM with 0.1% DMSO) at the pre-set time, 500 μL of incubation medium from each well was collected, and the same volume of dios- metin or chrysoeriol solution was loaded in each well. Ko143, whenever applied, a chemical inhibitor of BCRP (Ki < 1 μM [30]), was used to determine whether the diosmetin or chry- soeriol glucuronides were the substrates of BCRP. Ko143 (5 or 10 μM) was diluted in a solution containing diosmetin or chrysoeriol (2.5 μM). The collected samples were mixed with 250 μL acetonitrile containing 25 nM tilianin and then analyzed by UHPLC—MS/MS after centrifu- gation at 13,000 rpm for 30 min. After the excretion experiment, the cells were washed and selected in 300 μL HBSS buffer, and then the cells were ultrasonicated. After centrifugation at 13,000 rpm for 30 min, the 200 μL supernatant was obtained and mixed with 100 μL acetoni- trile containing 25 nM tilianin. The mixture was then centrifuged at 13,000 rpm for 30 min and prepared for subsequent UHPLC—MS/MS analysis. The excretion rates, glucuronides intracellular, Glucuronides excretion, the clearance of efflux transporter (CL) and the fraction of the metabolized dose (fmet) experiments were calcu- lated as described in detail previously [17]. Chemical Inhibition Studies Bilirubin (UGT1A1 inhibitor)[12,27], phenylbutazone (UGT1A6 inhibitor)[25,28], or carva- crol (UGT1A9 inhibitor)[17,29] were used to determine glucuronidation activities of diosme- tin and chrysoeriol in the HLMs and expressed UGTs (UGT1A1, UGT1A6, and UGT1A9). The protein concentrations of the HLMs and expressed UGTs were 0.0021–0.0106 mg pro- tein/mL. All of the incubations were run at 37˚C for 30 min, and the way of pretreated and determined samples were as discribed above. UHPLC—MS/MS system Quantification of the analytes was carried out on an Agilent 1290 UHPLC system coupled to Agilent 6490 triple quadrupole MS/MS. The conditions were as follows: column, Acquity UPLC HSS T3, 1.8 μm, 2.1 mm × 100 mm (Waters, USA); mobile phase A, 100% aqueous 5 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0166239 November 10, 2016 Glucuronidation and Transportation of Diosmetin and Chrysoeriol buffer (0.1%, v/v formic acid, pH 2.5); mobile phase B, 100% acetonitrile; flow rate, 0.3 mL/ min; gradient for diosmetin and its metabolites, 0–1 min, 25–25% B, 1–3.5 min, 25–33% B, 3.5–5 min, 33–90% B, 5–6 min, 90–90% B, 6–7 min, 90–21% B; gradient for chrysoeriol and its metabolites, 0–5 min, 21–21% B, 5–7 min, 21–90% B, 7–8 min, 90–90% B, 8–9 min, 90–21% B; and injection volume, 2 μL. The mass spectrometric detection in this study was performed using an ESI source in posi- tive ionization modes, with capillary voltage set at 3.0 kV, sheath gas and desolvation tempera- tures set at 300 and 250˚C, respectively. The sheath gas and desolvation flows were 11 and 14 L/min, respectively. The nozzle voltage was set at 1.5 kV. Acquisition was performed in a mul- tiple-reaction monitoring (MRM) mode at m/z transitions of 447.41!285.20 for tilianin; 477.00!301.00 for Dio-7-G, Dio-30-G, Chr-7-G, and Chr-40-G; and 301.27!286.10 for dios- metin and chrysoeriol in the positive ESI mode. Collision energies were 20 V for tilianin, 24 V for diosmetin or chrysoeriol, and 32 V for diosmetin glucuronides or chrysoeriol glucuronides, respectively. Analysis of Diosmetin and Chrysoeriol and their Metabolites by UHPLC—MS/MS We already identified the metabolites of diosmetin and chrysoeriol in our previous study [16]. UHPLC—MS/MS analysis showed that two mono-glucuronides were formed in HLMs incu- bations with diosmetin and chrysoeriol in the presence of UDPGA. The retention times of diosmetin and its metabolites (Dio-7-G and Dio-30-G) were 5.47, 3.06, and 3.38 min, respec- tively (Fig 1a). The peak eluting times at 7.35, 5.54, and 6.21 min corresponded to those of chrysoeriol and its metabolites (Chr-7-G and Chr-40-G) (Fig 1b). Statistical Analysis One-way ANOVA with or without Tukey—Kramer multiple comparison (post hoc) tests were used to evaluate statistical difference. Data were expressed as mean ± standard deviation (S.D) (n = 3), and the level of significance was set at p < 0.05 (“” or “#”), p < 0.01 (“” or “##”), or p < 0.001 (“” or “###”). PLOS ONE \| DOI:10.1371/journal.pone.0166239 November 10, 2016 Kinetics of Diosmetin and Chrysoeriol Glucuronidation by HLMs Diosmetin and chrysoeriol can be metabolized by HLMs into two metabolites, respectively. Dio-7-G and Dio-3’-G were the metabolites of diosmetin, and the rate of formation Dio-30-G was much faster than that of Dio-7-G in HLMs within the tested concentration ranges. Hence, the preferred site for the catalysis of diosmetin glucuronidation was 30-OH rather than 7-OH. The Eadie—Hofstee plots (Fig 2a and 2b) were used as evidences to state that the glucuronida- tion of 7-OH glucuronidation exhibited substrate inhibition profiles (Fig 2A), whereas the glu- curonidation of 30-OH followed the classic Michaelis—Menten kinetics (Fig 2B). Chr-7-G and Chr-40-G were the metabolites of chrysoeriol, and the rate of formation of Chr-40-G was much faster than that of Chr-7-G in the HLMs within the tested concentration ranges. Therefore, the preferred site for the catalysis of chrysoeriol glucuronidation was 40-OH rather than 7-OH. The Eadie—Hofstee plots (Fig 2c and 2d) were used as evidences to state that the glucuronida- tion of 7-OH glucuronidation exhibited the classic Michaelis—Menten kinetics (Fig 2C), whereas 40-OH followed substrate inhibition profiles (Fig 2D). The kinetic parameters of the glucuronidation of diosmetin and chrysoeriol by HLMs are shown in Table 1. Dio-30-G in the HLMs exhibited a much lower Km value (0.19 μM for Dio- PLOS ONE \| DOI:10.1371/journal.pone.0166239 November 10, 2016 6 / 21 Glucuronidation and Transportation of Diosmetin and Chrysoeriol 30-G, 4.66 μM for Dio-7-G). The CLint of diosmetin in the HLMs through 30-OH was about 48 times that of the 7-OH pathway (6.25 mL/min/mg for Dio-30-G, 0.13 mL/min/mg for Dio- 7 G) O th th h d th HLM hibit d h l K f Ch 40 G th f Ch 7 G Fig 1. Analysis of diosmetin, chrysoeriol, and their metabolites by UHPLC—MS/MS. Fig 1a and 1b show the chromatogram of the incubation samples of diosmetin and chrysoeriol by HLMs, respectively. doi:10.1371/journal.pone.0166239.g001 Fig 1. Analysis of diosmetin, chrysoeriol, and their metabolites by UHPLC—MS/MS. Fig 1a and 1b show the chromatogram of the incubation samples of diosmetin and chrysoeriol by HLMs, respectively. doi:10.1371/journal.pone.0166239.g001 doi:10.1371/journal.pone.0166239.g001 doi:10.1371/journal.pone.0166239.g001 30-G, 4.66 μM for Dio-7-G). The CLint of diosmetin in the HLMs through 30-OH was about 48 times that of the 7-OH pathway (6.25 mL/min/mg for Dio-30-G, 0.13 mL/min/mg for Dio- 7-G). On the other hand, the HLMs exhibited a much lower Km of Chr-40-G than of Chr-7-G (0.15 μM for Chr-40-G, 3.83 μM for Chr-7-G). Kinetics of Diosmetin and Chrysoeriol Glucuronidation by HLMs As such, the CLint of chrysoeriol in the HLMs through 40-OH was about 32 times that of the 7-OH pathway (1.27 mL/min/mg for Chr-40-G, 0.04 mL/min/mg for Chr-7-G). Effects of Chemical Inhibitor on Diosmetin and Chrysoeriol Metabolism We determined the inhibitory effects of bilirubin (UGT1A1 inhibitor), phenylbutazone (UGT1A6 inhibitor), and carvacrol (UGT1A9 inhibitor) on the glucuronidation of diosmetin (Fig 4a1 and 4a2) and chrysoeriol (Fig 4b1 and 4b2) in the HLMs, UGT1A1, UGT1A6 and UGT1A9. As we can see in Fig 4, the phenylbutazone, calvacrol, calvacrol and bilirubin can significantly decrease the formation of Dio-7-G, Dio-3’-G, Chr-7-G and Chr-4’-G compared with control, respectively. And these chemical inhibitors can dose-dependently inhibited the formation of Dio-7-G, Dio-3’-G, Chr-7-G and Chr-4’-G, which further proved that UGT1A6, UGT1A9, UGT1A9 and UGT1A1 respectively were the main UGTs involved in the formation of Dio-7-G, Dio-3’-G, Chr-7-G and Chr-4’-G in HLMs. Main UGTs Responsible for the Glucuronidation of Diosmetin and Chrysoeriol In Vitro UGT2B family and UGT1A4 had no contribution to diosmetin or chrysoeriol UGT metabolism. Main UGTs Responsible for the Glucuronidation of Diosmetin and Chrysoeriol In Vitro We used UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15, UGT2B17 to explore which UGT isoform was the main iso- form in charge of diosmetin or chrysoeriol UGT metabolism (Fig 3). The results showed that the isoforms that produced the most rapid glucuronidation rates of Dio-7-G (0.21 ± 0.007 nmol/mg/min to 0.85 ± 0.03 nmol/mg/min), Dio-30-G (9.36 ± 0.23 nmol/mg/min to 12.38 ± 0.20 nmol/mg/min), Chr-7-G (0.44 ± 0.01 nmol/mg/min to 0.73 ± 0.01 nmol/mg/min), Chr-40-G (0.75 ± 0.09 nmol/mg/min to 2.37 ± 0.08 nmol/mg/min) were UGT1A6, UGT1A9, 7 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0166239 November 10, 2016 Glucuronidation and Transportation of Diosmetin and Chrysoeriol Fig 2. Kinetics of diosmetin and chrysoeriol glucuronidation by HLMs. The curves were estimated on the basis of fitted parameters generated using the Michaelis-Menten kinetics (B, C) or substrate inhibition (A, D) in HLMs. The Eadie—Hofstee plots are shown in right panal (a-d). Each data point corresponds to the average of three determinations with error bars representing S.D. doi:10.1371/journal.pone.0166239.g002 Fig 2. Kinetics of diosmetin and chrysoeriol glucuronidation by HLMs. The curves were estimated on the basis of fitted parameters generated using the Michaelis-Menten kinetics (B, C) or substrate inhibition (A, D) in HLMs. The Eadie—Hofstee plots are shown in right panal (a-d). Each data point corresponds to the average of three determinations with error bars representing S.D. doi:10.1371/journal.pone.0166239.g002 Fig 2. Kinetics of diosmetin and chrysoeriol glucuronidation by HLMs. The curves were estimated on the basis of fitted parameters generated using the Michaelis-Menten kinetics (B, C) or substrate inhibition (A, D) in HLMs. The Eadie—Hofstee plots are shown in right panal (a-d). Each data point corresponds to the average of three determinations with error bars representing S.D. doi:10.1371/journal.pone.0166239.g002 8 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0166239 November 10, 2016 Glucuronidation and Transportation of Diosmetin and Chrysoeriol Table 1. Kinetic parameters of diosmetin and chrysoeriol glucuronidation by HLMs. Km (μM) Vmax CLint nmol/mg/min (mL/min/mg) diosmetin Dio-7-G 4.66±0.72 0.61±0.05 0.13 Dio-30-G 0.19±0.02 1.16±0.03 6.25 chrysoeriol Chr-7-G 3.83±0.55 0.15±0.01 0.04 Chr-40-G 0.15±0.03 0.20±0.01 1.27 doi:10.1371/journal.pone.0166239.t001 Table 1. Kinetic parameters of diosmetin and chrysoeriol glucuronidation by HLMs. UGT1A9 and UGT1A10, respectively. UGT1A3, UGT1A9, UGT1A10 made a prominent con- tribution to forming Dio-7-G; UGT1A7 made a prominent contribution to forming Dio-30-G; UGT1A3 and UGT1A8 made a prominent contribution to the formation of Chr-7-G; UGT1A1 and UGT1A8 made a prominent contribution to the formation of Chr-40-G. Kinetics of Diosmetin and Chrysoeriol Glucuronidation by Human Expressed UGT enzymes According to above results, we used human expressed UGT enzymes to incubate different con- centration diosmetin and chrysoeriol and determined their glucuronidation rates. We used UGT1A6 and UGT1A9 to incubate diosmetin to further confirm if they were the main isoform of forming Dio-7-G and Dio-30-G, respectively; and we used UGT1A9 and UGT1A1 to incu- bate chrysoeriol to further confirm if they were the main isoform of forming Chr-7-G and Chr-40-G, respectively. Within the tested concentration ranges, The Eadie—Hofstee plots (Fig 5a and 5d) were used as evidences to state that both UGT1A6- and UGT1A1-mediated the for- mation of Dio-7-G (Fig 5A) and Chr-40-G (Fig 5D) exhibited substrate inhibition kinetic char- acteristics. And the Eadie—Hofstee plots (Fig 5b and 5c) were used as evidences to state that both UGT1A9-mediated formation of Dio-30-G (Fig 5B) and Chr-7-G (Fig 5C) exhibited clas- sic Michaelis-Menten kinetic characteristics. The kinetic parameters of the diosmetin and chrysoeriol glucuronidation catalyzed by human expressed UGT enzymes are shown in Table 2. These data accompanied the absence of large variation in Km value of the glucuronides except for Dio-7-G (3.02 μM for Dio-7-G in UGT1A6, 0.45 μM for Dio-30-G in UGT1A9, 0.26 μM for Chr-7-G in UGT1A9, and 0.28 μM for Chr-40-G in UGT1A1). The intrinsic clearance (CLint; Vmax/Km) of Dio-7-G, Dio-30-G, Chr-7-G, and Chr-40-G selectively in UGT1A6, UGT1A9, UGT1A9, and UGT1A1 were higher than that in HLMs. 9 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0166239 November 10, 2016 Glucuronidation and Transportation of Diosmetin and Chrysoeriol Fig 3. Glucuronidation of diosmetin and chrysoeriol by human expressed UGT isoforms. Experiments were conducted at concentrations 1.25, 2.5, and 10 μM. The concentration of each human expressed UGT isoform was 0.0053 mg/mL. The experiments were conducted at 37˚C for 30 min, and the amount of glucuronides formed were measured by UHPLC—MS/MS. The rates of glucuronidation were calculated as nmol/mg/min. Each column corresponds to the average of three determinations with error bars representing the S.D. doi:10.1371/journal.pone.0166239.g003 0166239 November 10 2016 10 / 21 Fig 3. Glucuronidation of diosmetin and chrysoeriol by human expressed UGT isoforms. Experiments were conducted at concentrations 1.25, 2.5, and 10 μM. The concentration of each human expressed UGT isoform was 0.0053 mg/mL. The experiments were conducted at 37˚C for 30 min, and the amount of glucuronides formed were measured by UHPLC—MS/MS. The rates of glucuronidation were calculated as nmol/mg/min. doi:10.1371/journal.pone.0166239.g003 Glucuronidation and Transportation of Diosmetin and Chrysoeriol Fig 4. Effects of chemical inhibitors on diosmetin and metabolism and UGT1A9- and UGT1A1-mediated chrysoeriol metabolism in HLMs. Fig 4 A shows the inhibitory effects of phenylbutazone on the 7-O-glucuronide of diosmetin in HLMs and UGT1A6. Fig 4 B displays the inhibitory effects of carvacrol on the 30-O-glucuronide of diosmetin in HLMs and UGT1A9. Fig 4 C presents the inhibitory effects of carvacrol on the 7-O-glucuronide of chrysoeriol in HLMs and UGT1A9. Fig 4 D shows the inhibitory effects of bilirubin on the 40-O-glucuronides of chrysoeriol in HLMs and UGT1A1. Each column corresponds to the average of three determinations with error bars representing the S.D. The “” symbol means a statistically significant difference compared with control at p < 0.05; “” means p < 0.01; “*” means p < 0.001. doi:10.1371/journal.pone.0166239.g004 Fig 4. Effects of chemical inhibitors on diosmetin and metabolism and UGT1A9- and UGT1A1-mediated chrysoeriol metabolism in HLMs. Fig 4 A shows the inhibitory effects of phenylbutazone on the 7-O-glucuronide of diosmetin in HLMs and UGT1A6. Fig 4 B displays the inhibitory effects of carvacrol on the 30-O-glucuronide of diosmetin in HLMs and UGT1A9. Fig 4 C presents the inhibitory effects of carvacrol on the 7-O-glucuronide of chrysoeriol in HLMs and UGT1A9. Fig 4 D shows the inhibitory effects of bilirubin on the 40-O-glucuronides of chrysoeriol in HLMs and UGT1A1. Each column corresponds to the average of three determinations with error bars representing the S.D. The “” symbol means a statistically significant difference compared with control at p < 0.05; “” means p < 0.01; “” means p < 0.001. Fig 4. Effects of chemical inhibitors on diosmetin and metabolism and UGT1A9- and UGT1A1-mediated chrysoeriol metabolism in HLMs. Fig 4 A shows the inhibitory effects of phenylbutazone on the 7-O-glucuronide of diosmetin in HLMs and UGT1A6. Fig 4 B displays the inhibitory effects of carvacrol on the 30-O-glucuronide of diosmetin in HLMs and UGT1A9. Fig 4 C presents the inhibitory effects of carvacrol on the 7-O-glucuronide of chrysoeriol in HLMs and UGT1A9. Fig 4 D shows the inhibitory effects of bilirubin on the 40-O-glucuronides of chrysoeriol in HLMs and UGT1A1. Each column corresponds to the average of three determinations with error bars representing the S.D. The “” symbol means a statistically significant difference compared with control at p < 0.05; “” means p < 0.01; “” means p < 0.001. doi:10.1371/journal.pone.0166239.g004 doi:10.1371/journal.pone.0166239.g004 Kinetics of Diosmetin and Chrysoeriol Glucuronidation by Human Expressed UGT enzymes Each column corresponds to the average of three determinations with error bars representing the S.D. doi:10.1371/journal.pone.0166239.g003 Fig 3. Glucuronidation of diosmetin and chrysoeriol by human expressed UGT isoforms. Experiments were conducted at concentrations 1.25, 2.5, and 10 μM. The concentration of each human expressed UGT isoform was 0.0053 mg/mL. The experiments were conducted at 37˚C for 30 min, and the amount of glucuronides formed were measured by UHPLC—MS/MS. The rates of glucuronidation were calculated as nmol/mg/min. Each column corresponds to the average of three determinations with error bars representing the S.D. doi:10 1371/journal pone 0166239 g003 Fig 3. Glucuronidation of diosmetin and chrysoeriol by human expressed UGT isoforms. Experiments were conducted at concentrations 1.25, 2.5, and 10 μM. The concentration of each human expressed UGT isoform was 0.0053 mg/mL. The experiments were conducted at 37˚C for 30 min, and the amount of glucuronides formed were measured by UHPLC—MS/MS. The rates of glucuronidation were calculated as nmol/mg/min. Each column corresponds to the average of three determinations with error bars representing the S.D. doi:10.1371/journal.pone.0166239.g003 10 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0166239 November 10, 2016 Effect of Ko143 on Glucuronides Excretion To estimate the effect of BCRP on the excretion of glucuronides in HeLa—UGT1A9 cells, the Ko143 was adopted (Fig 7). Due to at the excretion rate of diosmetin glucuronides or chrysoer- iol glucuronides reached a peak at 2.5 μM substrate concentration, the concentration of sub- strate were set at 2.5 μM. Co-incubation of 5 μM or 10 μM Ko143 with diosmetin or chrysoeriol led to the excretion rates of diosmetin or chrysoeriol glucuronides decreased and their intracellular level significantly increased. Ko143 (5 μM) inhibited Dio-7-G and Dio-30-G excretion by 19% and 16%, respectively; and Ko143 (10 μM) decreased the excretion by 40% and 29%, respectively (Fig 7a1 and 7a2). Moreover, 5 μM Ko143 inhibited Chr-7-G and Chr- 40-G excretion by 14% and 19%, respectively; and 10 μM Ko143 decreased both excretions by 43% (Fig 7a3 and 7a4). The effects of Ko143 on the total intracellular amount of glucuronides are displayed in Fig 7B. At 2.5 μM diosmetin, the total amount of intracellular Dio-7-G and Dio-30-G was increased by 1.1- and 1.3-fold, respectively, in the presence of 5 μM Ko143 and by 1.5- and 1.8-fold, respectively, in the presence of 10 μM Ko143 compared with the control (Fig 7b1). At 2.5 μM chrysoeriol, the total amount of intracellular Chr-7-G and Chr-40-G both increased by 1.3-fold in the presence of 5 μM Ko143 and by 1.7- and 1.8-fold, respectively, in the presence of 10 μM Ko143 compared with the control (Fig 7b2). Ko143 inhibited the CL of these four glucuronides (33% to 65%) (Fig 7C). The results of fmet (Fig 7D) indicated that the effect of Ko143 on diosmetin or chrysoeriol glucuronidation were small or insignificant. All the results indicated that the BCRP played a crucial role in excretion diosmetin glucuronides and chrysoeriol glucuronides. Glucuronidation and Transportation of Diosmetin and Chrysoeriol Table 2. Kinetic parameters of diosmetin and chrysoeriol glucuronidation by human expressed UGT enzymes. Km (μM) Vmax CLint nmol/mg/min mL/mg/min diosmetin Dio-7-G (UGT1A6) 3.02±0.59 0.92±0.06 0.30 Dio-30-G (UGT1A9) 0.45±0.05 4.27±0.13 9.52 chrysoeriol Chr-7-G (UGT1A9) 0.26±0.04 0.12±0.01 0.48 Chr-40-G (UGT1A1) 0.28±0.05 0.63±0.06 2.26 doi:10.1371/journal.pone.0166239.t002 ic parameters of diosmetin and chrysoeriol glucuronidation by human expressed UGT enzymes. 7-G and Chr-40-G reached a peak at 2.5 μM, (Fig 6a2). Intracellular glucuronide levels dose- dependently increased with diosmetin or chrysoeriol loading concentrations (Fig 6b1 and 6b2). CL (Fig 6c1 and 6c2) dose-dependently decreased with increasing diosmetin or chrysoer- iol concentration. And fmet (Fig 6d1 and 6d2) dose-dependently decreased with increasing diosmetin or chrysoeriol concentration except Chr-40-G. The fmet of Chr-40-G reached a peak at 2.5 μM chrysoeriol. 7-G and Chr-40-G reached a peak at 2.5 μM, (Fig 6a2). Intracellular glucuronide levels dose- dependently increased with diosmetin or chrysoeriol loading concentrations (Fig 6b1 and 6b2). CL (Fig 6c1 and 6c2) dose-dependently decreased with increasing diosmetin or chrysoer- iol concentration. And fmet (Fig 6d1 and 6d2) dose-dependently decreased with increasing diosmetin or chrysoeriol concentration except Chr-40-G. The fmet of Chr-40-G reached a peak at 2.5 μM chrysoeriol. Effects of Diosmetin and Chrysoeriol Concentrations on Glucuronide Excretion Six concentrations of diosmetin and chrysoeriol were chosen to explore the effects of diosme- tin and chrysoeriol concentrations on the excretion, intracellular amount, CL, and fmet in Hela and HeLa—UGT1A9 cells (Fig 6). Because it did not find the glucuronided metabolites of diosmetin or chrysoeriol in HeLa cells, we mainly determined the excretion, intracellular amount, CL, and fmet in HeLa—UGT1A9 cells. (Fig 6). The excretion rates of Dio-7-G reached a peak at 10 μM and Dio-30-G reached a peak at 2.5 μM (Fig 6a1). The excretion rates of Chr- 11 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0166239 November 10, 2016 Glucuronidation and Transportation of Diosmetin and Chrysoeriol Fig 5. Kinetics of diosmetin and chrysoeriol glucuronidation by human expressed UGT enzyme curves are estimated on the basis of fitted parameters generated using the substrate inhibition (A, D) or Michaelis-Menten kinetics (B, C) in UGT1A6, UGT1A1, UGT1A9, and UGT1A9, respectively. The Eadie Hofstee plots are shown in the right panal (a-d). Each data point corresponds to the average of three determinations with error bars representing the S.D. doi:10.1371/journal.pone.0166239.g005 0166239 November 10 2016 Fig 5. Kinetics of diosmetin and chrysoeriol glucuronidation by human expressed UGT enzymes. The curves are estimated on the basis of fitted parameters generated using the substrate inhibition (A, D) or Michaelis-Menten kinetics (B, C) in UGT1A6, UGT1A1, UGT1A9, and UGT1A9, respectively. The Eadie— Hofstee plots are shown in the right panal (a-d). Each data point corresponds to the average of three determinations with error bars representing the S.D. doi:10 1371/journal pone 0166239 g005 doi:10.1371/journal.pone.0166239.g005 PLOS ONE \| DOI:10.1371/journal.pone.0166239 November 10, 2016 12 / 21 Discussion Luteolin is widely found in our diet, such as carrots, peppers, celery, olive oil and peppermint [17]. It was reported that luteolin could be metabolized to diosmetin and chrysoeriol, which were the methylation metabolites. And the diosmetin and chrysoeriol could be metabolized in rats by Ugts [16]. Therefore, it was speculated that glucuronides of diosmetin and chrysoeriol would be formed in humans. The objective of this study was to the metabolism characteristic of diosmetin and chrysoeriol. The exposure of the glucuronides of diosmetin and chrysoeriol could influence the pharmacological action of luteolin. Hence, the metabolic elucidation of diosmetin and chrysoeriol is important to understand the pharmacokinetic characteristics and biological effects of luteolin in vivo. Diosmetin and chrysoeriol, which also distributed in many plant products, are pharmacologically valuable in many diseases, such as osteoporosis [4]. This study for the first time investigated the regioselectivity of the glucuronidation of diosmetin and chrysoeriol by kinetic profiling and identified the major enzymes responsible for the glucuroni- dation of the two compounds. In addition, this study also showed the effect of the BCRP— UGT1A9 interplay on diosmetin and chrysoeriol glucuronidation in vivo. The metabolic activities of the HLMs toward diosmetin and chrysoeriol were determined by kinetic profiling (Fig 2). These activities included the metabolic rates at various substrate concentrations. The CLint value, independent of substrate concentration, measures the cata- lytic efficiency of functional enzymes. The determination of regioselectivity was based on the CLint derived from kinetic profiling over a wide range of substrate concentrations. The eluci- dation of regioselectivity by kinetic determination was superior to that by rate determination at high substrate concentrations [31]. In addition, the use of CLint as indicator of UGT activity was more advantageous than the use of reaction rates because CLint is more relevant in predict- ing hepatic clearance in vivo compared with other kinetic parameters [32,33]. In HLMs, the CLint value of diosmetin through 30-OH was much higher than that of the 7-OH, and the CLint value of chrysoeriol through 40-OH was about 32 times than that of the 7-OH pathway. For another, the Km values of diosmetin and chrysoeriol glucuronides were lower than a series of 7-OH, 3’-OH or 4’-OH flavonoids, such as acacetin glucuronide (Km was 14.465 μM)[34] and genistein glucuronides (Km was 15.1 μM)[35] (Table 1). The effects of diosmetin and chrysoeriol on cell viability in A549 and HepG2 cells with untreated and treated Ko143 The “” (for Dio-7-G or Chr-7-G) or “#” (for Dio-30-G or Chr-40-G) symbol means a statistically significant difference between-group, at p < 0.05; “” or “##” means p < 0.01; “” or “###” means p < 0.001. doi:10.1371/journal.pone.0166239.g006 doi:10.1371/journal.pone.0166239.g006 The effects of diosmetin and chrysoeriol on cell viability in A549 and HepG2 cells with untreated and treated Ko143 We found that in A549 cells, 10 μM chrysoeriol with 5 μM or 10 μM Ko143 respectively decreased the A549 cell viability by 12% and 18% compared with the group that substrate was 10μM chrysoeriol, but the cytotoxicity of diosmetin had little change when treated Ko143. And in HepG2 cells, 10 μM diosmetin with 5 μM or 10 μM Ko143 respectively decreased the A549 cell viability by 12% and 13% compared with the group that substrate was 10 μM diosme- tin, but the cytotoxicity of chrysoeriol had little change when treated Ko143 (Fig 8). PLOS ONE \| DOI:10.1371/journal.pone.0166239 November 10, 2016 13 / 21 Fig 6. Effect of substrate concentrations on excretion rates (a), intracellular amounts (b), CL (c), an fmet (d) of Dio-7-G, Dio-30-G, Chr-7-G, and Chr-40-G. Three samples (500 μL) were obtained at 15, 30, an 60 min and replaced with fresh loading solution (500 μL) that contains diosmetin or chrysoeriol. The excreti rates of glucuronides were calculated from the slope of the amount-versus-time curves. The intracellular amounts of the glucuronides were determined at the end of the excretion experiments after the cells were Glucuronidation and Transportation of Diosmetin and Chrysoe / Glucuronidation and Transportation of Diosmetin and Chrysoeriol Fig 6. Effect of substrate concentrations on excretion rates (a), intracellular amounts (b), CL (c), and fmet (d) of Dio-7-G, Dio-30-G, Chr-7-G, and Chr-40-G. Three samples (500 μL) were obtained at 15, 30, and 60 min and replaced with fresh loading solution (500 μL) that contains diosmetin or chrysoeriol. The excretion rates of glucuronides were calculated from the slope of the amount-versus-time curves. The intracellular amounts of the glucuronides were determined at the end of the excretion experiments after the cells were PLOS ONE \| DOI:10.1371/journal.pone.0166239 November 10, 2016 14 / 21 Glucuronidation and Transportation of Diosmetin and Chrysoeriol washed twice with ice-cold HBSS. Each column corresponds to the average of three determinations with error bars representing the S.D. The “” (for Dio-7-G or Chr-7-G) or “#” (for Dio-30-G or Chr-40-G) symbol means a statistically significant difference between-group, at p < 0.05; “” or “##” means p < 0.01; “” or “###” means p < 0.001. washed twice with ice-cold HBSS. Each column corresponds to the average of three determinations with error bars representing the S.D. PLOS ONE \| DOI:10.1371/journal.pone.0166239 November 10, 2016 Discussion Three samples (500 μL) were obtained at 20, 40, and 60 min and replaced with fresh PLOS ONE \| DOI:10.1371/journal.pone.0166239 November 10, 2016 16 / 21 Glucuronidation and Transportation of Diosmetin and Chrysoeriol loading solution (500 μL) containing 2.5 μM diosmetin or chrysoeriol. Each data point (panel a) and each column (panel b-d) corresponds to the average of three determinations with error bars representing the S.D. The “” (for Dio-7-G or Chr-7-G) or “#” (for Dio-30-G or Chr-40-G) symbol means a statistically significant difference between-group at p < 0.05; “” or “##” means p < 0.01; “” or “###” means p < 0.001. doi 10 1371/journal pone 0166239 g007 loading solution (500 μL) containing 2.5 μM diosmetin or chrysoeriol. Each data point (panel a) and each column (panel b-d) corresponds to the average of three determinations with error bars representing the S.D. The “” (for Dio-7-G or Chr-7-G) or “#” (for Dio-30-G or Chr-40-G) symbol means a statistically significant difference between-group at p < 0.05; “” or “##” means p < 0.01; “” or “###” means p < 0.001. doi:10.1371/journal.pone.0166239.g007 1A10 was not further consideration. Therefore, UGT1A1, which was the second main isoform for Chr-40-G formation, and UGT1A6, UGT1A9 were used to the further inhibition assay, and these three isoforms are highly expressed in liver [37]. Bilirubin (UGT1A1 inhibitor), phenyl- butazone (UGT1A6 inhibitor) and carvacrol (UGT1A9 inhibitor) were used in this study. All the concentrations of diosmetin and chrysoeriol adopted in this study were based on the Km value of glucuronides. The results showed that bilirubin, phenylbutazone, and carvacrol inhib- ited the formation of the glucuronides in UGT enzymes and HLMs in a dose-dependent man- ner (Fig 4). Thus, the results suggested that UGT1A6, UGT1A9, UGT1A9, and UGT1A1 were the main expressed UGTs responsible for the formation of Dio-7-G, Dio-30-G, Chr-7-G, and Chr-40-G, respectively. The inhibition kinetics of these three inhibitors have been previously described to be substrate dependent, but the mechanism of inhibition of drug-metabolizing enzymes remains unclear [29,38]. Thus, the mechanism of inhibition of these inhibitors requires additional studies, and in vitro data on the inhibition should be interpreted with cau- tion. To further elucidate the characteristics of the glucuronidation metabolism of diosmetin and chrysoeriol, their rates of glucuronidation metabolism by UGT1A1, UGT1A6, and UGT1A9 were determined at different substrate concentrations (Fig 5). Discussion On the other hand, human expressed UGTs and chemical inhibition assays were used to determine the major UGTs involved in the glucuronidation metabolism of diosmetin and chrysoeriol. Among the 12 expressed UGTs, only the enzymes from the UGT1A family except UGT1A4 showed glucuronidation activity on diosmetin and chrysoeriol, and the UGT2B fam- ily offered no contribution to their UGT metabolism. According to the expressed UGT assay, the main UGT isoforms for Dio-7-G, Dio-30-G, Chr-7-G, and Chr-40-G formation were UGT1A6, UGT1A9, UGT1A9 and UGT1A10, respectively (Fig 3). Because the expression of individual UGTs at the mRNA level in the liver are different (UGT1A1 (7%), UGT1A6 (5%), UGT1A9 (6%))[36], and the UGT isoform showing the highest activity could not be the main contributor in HLMs. Therefore, the inhibition assay was further performed to determine the role of the UGT isoform in the metabolism of diosmetin and chrysoeriol. In addition, it was reported that UGT1A8 and 1A10, which were involved in the glucuronidation of diosmetin and chrysoeriol, were not present in the liver [37]. And the objective of this research was to study the hepatic clearance of diosmetin and chrysoeriol. Thus, the effect of UGT1A8 and PLOS ONE \| DOI:10.1371/journal.pone.0166239 November 10, 2016 15 / 21 Glucuronidation and Transportation of Diosmetin and Chrysoeriol Fig 7. Effect of BCRP-specific inhibitor Ko143 on the excretion rate of glucuronides (Dio-7-G, D G, Chr-7-G, and Chr-40-G) (a), total intracellular glucuronide amounts (Dio-7-G, Dio-30-G, Chr-7-G Chr-40-G) (b), CL (c), and fmet (d). Engineered HeLa cells stably overexpressing UGT1A9 grown on si plates (1 × 105 cells/well) were treated with 2.5 μM of diosmetin or chrysoeriol in the absence or presen Ko143 at 5 or 10 μM. Three samples (500 μL) were obtained at 20, 40, and 60 min and replaced with fre Fig 7. Effect of BCRP-specific inhibitor Ko143 on the excretion rate of glucuronides (Dio-7-G, Dio-30- G, Chr-7-G, and Chr-40-G) (a), total intracellular glucuronide amounts (Dio-7-G, Dio-30-G, Chr-7-G, and Chr-40-G) (b), CL (c), and fmet (d). Engineered HeLa cells stably overexpressing UGT1A9 grown on six-well plates (1 × 105 cells/well) were treated with 2.5 μM of diosmetin or chrysoeriol in the absence or presence of Ko143 at 5 or 10 μM. Discussion The glucuronide formation did not show substrate inhibition kinetics; hence, we speculated that the decrease in the excre- tion rates of the glucuronides at high diosmetin or chrysoeriol concentration was most likely caused by BCRP inhibition by diosmetin or chrysoeriol. Indeed, several flavonoids (e.g., aya- nin and retusin) have been reported to inhibit BCRP encoded by the ABCG2 gene with poten- cies only slightly lower than that of Ko143. Structural features found to contribute positively to BCRP inhibition included a hydroxyl group in position 5 and a double bond between position 2 and 3 [39], which were also found in the structures of diosmetin and chrysoeriol. Therefore, BCRP inhibition by diosmetin or chrysoeriol was believed to account for the decreased excre- tion rate of glucuronides in the HeLa—UGT1A9 cells observed at high diosmetin or chrysoer- iol concentrations. The role of BCRP in the excretion was studied using Ko143, a specific BCRP inhibitor that does not affect UGT activities as a general rule. Ko143 (5 μM or 10 μM) significantly inhibited the BCRP-mediated excretion of the glucuronides of diosmetin and chrysoeriol. Ko143 also significantly increased the accumulation of intracellular glucuronides at 2.5 μM (Fig 7). Impor- tantly, Ko143 inhibited glucuronide CL and fmet at 2.5 μM diosmetin or chrysoeriol, suggesting that BCRP-mediated excretion could be the predominant pathway for diosmetin and chry- soeriol disposition. According to the results of the effect of Ko143 on glucuronides excretion, we found that the total intracellular amount of glucuronides was increases after treated Ko143. But the effects of increased intracellular glucuronides in cells were not clear. Therefore, we used these A549 and HepG2 tumor cell lines to demonstrate the effects of diosmetin and chrysoeriol on cell viability with untreated and treated Ko143 inhibitor (Fig 8). Both A549 and HepG2 cells express BCRP [40,41]. A549 cells express UGT1A1, UGT1A3 and UGT2B7 [42], and HepG2 cells expresse UGT1A1, UGT1A6 and UGT1A9 [43]. According to the present results, we found that the increased total intracellular glucuronides could increase the cytotoxicity, and diosmetin and chrysoeriol had different effects on different cancer cells, possibly due to different isoform of UGTs in the different cancer cells. In conclusion, this study first demonstrated that the diosmetin and chrysoeriol could be metabolited into Dio-7-G, Dio-30-G, Chr-7-G and Chr-40-G by human in vitro. Discussion The results of compa- rable kinetic profiles of the expressed UGTs further explained the conclusion in chemical inhi- bition study (Table 2). In this study, UGT1A9 was the main expressed UGTs responsible for the formation of Dio- 30-G and Chr-7-G, and Dio-7-G and Chr-40-G also can be formed by UGT1A9. Therefore, the Fig 8. The effects of diosmetin and chrysoeriol on cell viability in A549 and HepG2 cells with untreated and treated Ko143. 5 μM Ko143, 10 μM Ko143, 10 μM diosmetin, 10 μM diosmetin with 5 μM Ko143 or 10 μM Ko143, 10μM chrysoeriol, 10μM chrysoeriol with 5 μM Ko143 or 10 μM Ko143 were used as substrates to determine the effects of diosmetin and chrysoeriol on cell viability in A549 and HepG2 cells with untreated and treated Ko143. Each column corresponds to the average of three determinations with error bars representing the S.D. The “” symbol means a statistically significant difference compared with control at p < 0.05; “” means p < 0.01; “” means p < 0.001). Fig 8. The effects of diosmetin and chrysoeriol on cell viability in A549 and HepG2 cells with untreated and treated Ko143. 5 μM Ko143, 10 μM Ko143, 10 μM diosmetin, 10 μM diosmetin with 5 μM Ko143 or 10 μM Ko143, 10μM chrysoeriol, 10μM chrysoeriol with 5 μM Ko143 or 10 μM Ko143 were used as substrates to determine the effects of diosmetin and chrysoeriol on cell viability in A549 and HepG2 cells with untreated and treated Ko143. Each column corresponds to the average of three determinations with error bars representing the S.D. The “” symbol means a statistically significant difference compared with control at p < 0.05; “” means p < 0.01; “*” means p < 0.001). doi:10.1371/journal.pone.0166239.g008 17 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0166239 November 10, 2016 Glucuronidation and Transportation of Diosmetin and Chrysoeriol HeLa—UGT1A9 cells, which were used to study how the interplay between UGT1A9 and BCRP affects drug disposition [21], were utilized to further elaborate diosmetin and chrysoer- iol metabolism in terms of glucuronide production. This work delineated the interplay between UGT1A9 and BCRP in diosmetin and chrysoeriol metabolism and disposition at the kinetic level. The excretion rates of Dio-7-G in HeLa—UGT1A9 peaked at 10 μM, and those of Dio-30-G, Chr-7-G, and Chr-40-G rapidly peaked at 2.5 μM (Fig 6). Discussion All the find- ings suggested that the oral bioavailability of luteolin, diosmetin and chrysoeriol would be greatly limited by first-pass glucuronidation in the liver. 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https://openalex.org/W2958275140	https://www.research-collection.ethz.ch/bitstream/20.500.11850/355889/3/document.pdf	English	null	Analysis of the equine “cumulome” reveals major metabolic aberrations after maturation in vitro	BMC genomics	2,019	cc-by	19,124	ETH Library Journal Article Author(s): Walter, Jasmin; Huwiler, Fabian; Fortes, Claudia; Grossmann, Jonas; Roschitzki, Bernd; Hu, Junmin; Naegeli, Hanspeter; Laczkó, Endre; Bleul, Ulrich Author(s): Walter, Jasmin; Huwiler, Fabian; Fortes, Claudia; Grossmann, Jonas; Roschitzki, Bernd; Hu, Junmin; Naegeli, Hanspeter; Laczkó, Endre; Bleul, Ulrich g Creative Commons Attribution 4.0 International Originally published in: Originally published in: BMC Genomics 20, https://doi.org/10.1186/s12864-019-5836-5 This page was generated automatically upon download from the ETH Zurich Research Collection. For more information, please consult the Terms of use. This page was generated automatically upon download from the ETH Zurich Research Collection. For more information, please consult the Terms of use. Walter et al. BMC Genomics (2019) 20:588 https://doi.org/10.1186/s12864-019-5836-5 Open Access Analysis of the equine “cumulome” reveals major metabolic aberrations after maturation in vitro Jasmin Walter1* , Fabian Huwiler1, Claudia Fortes2, Jonas Grossmann2, Bernd Roschitzki2, Junmin Hu2, Hanspeter Naegeli3, Endre Laczko2 and Ulrich Bleul1 * Correspondence: jwalter@vetclinics.uzh.ch * Correspondence: jwalter@vetclinics.uzh.ch 1Clinic of Reproductive Medicine, Vetsuisse Faculty, University of Zurich, 8057 Zurich, Switzerland Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Maturation of oocytes under in vitro conditions (IVM) results in impaired developmental competence compared to oocytes matured in vivo. As oocytes are closely coupled to their cumulus complex, elucidating aberrations in cumulus metabolism in vitro is important to bridge the gap towards more physiological maturation conditions. The aim of this study was to analyze the equine “cumulome” in a novel combination of proteomic (nano-HPLC MS/MS) and metabolomic (UPLC-nanoESI-MS) profiling of single cumulus complexes of metaphase II oocytes matured either in vivo (n = 8) or in vitro (n = 7). Results: A total of 1811 quantifiable proteins and 906 metabolic compounds were identified. The proteome contained 216 differentially expressed proteins (p ≤0.05; FC ≥2; 95 decreased and 121 increased in vitro), and the metabolome contained 108 metabolites with significantly different abundance (p ≤0.05; FC ≥2; 24 decreased and 84 increased in vitro). The in vitro “cumulome” was summarized in the following 10 metabolic groups (containing 78 proteins and 21 metabolites): (1) oxygen supply, (2) glucose metabolism, (3) fatty acid metabolism, (4) oxidative phosphorylation, (5) amino acid metabolism, (6) purine and pyrimidine metabolism, (7) steroid metabolism, (8) extracellular matrix, (9) complement cascade and (10) coagulation cascade. The KEGG pathway “complement and coagulation cascades” (ID4610; n = 21) was significantly overrepresented after in vitro maturation. The findings indicate that the in vitro condition especially affects central metabolism and extracellular matrix composition. Important candidates for the metabolic group oxygen supply were underrepresented after maturation in vitro. Additionally, a shift towards glycolysis was detected in glucose metabolism. Therefore, under in vitro conditions, cumulus cells seem to preferentially consume excess available glucose to meet their energy requirements. Proteins involved in biosynthetic processes for fatty acids, cholesterol, amino acids, and purines exhibited higher abundances after maturation in vitro. Conclusion: This study revealed the marked impact of maturation conditions on the “cumulome” of individual cumulus oocyte complexes. Under the studied in vitro milieu, cumulus cells seem to compensate for a lack of important substrates by shifting to aerobic glycolysis. These findings will help to adapt culture media towards more physiological conditions for oocyte maturation. Keywords: Oocyte, Cumulus, IVM, Proteomics, Metabolomics, Complement, Coagulation, Glucose, Oxygen, Purine © The Author(s). Abstract 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: jwalter@vetclinics.uzh.ch 1Clinic of Reproductive Medicine, Vetsuisse Faculty, University of Zurich, 8057 Zurich, Switzerland Full list of author information is available at the end of the article Background One major limitation for the proteomics approach is the large amount of COCs required for analysis [37, 38] as enrichment of proteins prior to ana- lysis is not possible. However, technical improvements for these techniques currently allow the analysis of small sample amounts [33, 39, 40]. Differences in the cumulus proteome through maternal ageing in humans [41], between cyclic and prepubertal whole porcine COCs [42, 43], and bovine cumulus cells and oocytes [44] were reported in studies using pooled CCs or COCs. How- ever, beyond some practical benefits, pooling samples has some drawbacks such as masking of outliers, dilu- tion of low abundance proteins and the loss of the possi- bility for the estimation of inter-individual variations within groups [45, 46]. These issues lead to the reduced applicability of pooled samples, especially for biomarker discovery [47]. Global protein expression profiling, with- out identification of altered protein spots, for human cumulus cells of single oocytes was performed in 2006 [48]. This previous study observed alterations in the protein expression profiles of cumulus cells under different stimulation protocols, as well as minor aberrations in fertilization outcomes using protein electrophoresis after metabolic labelling [48]. Only recently, intact- cellMALDI-TOF mass spectrometry (ICM-MS) in combination with top-down proteomics was investigated as tool for biomarker discovery in cumulus cells of single bovine oocytes [49]. Data on the equine cumulus cell proteome are not available in the literature. However, a characterization of the mare follicular fluid composition was performed during late follicular development using 2D-PAGE and mass spectrometry [50]. Similar metho- dology was used to characterize seasonal variation in equine follicular fluid [51]. Available clinical data reflect the gap between in vitro matured and in vivo matured oocytes with regard to the developmental competence of oocytes. Fundamental research to elucidate altered metabolism during IVM is necessary to bridge this gap. Currently, high-throughput “Omics” technologies provide the opportunity to obtain a more global view on complex biological processes in reproduction [13–16]. The cumulus complex (CC) makes intimate contact with its oocyte and is required to obtain the maturational competence of the oocyte [17–21]. After maturation and fertilization, the CC is not required for further development; thus, these cells can serve as a unique source to noninvasively investigate metabolism during oocyte maturation [22]. Background transcriptome between in vitro and in vivo matured COCs [31–34]. All these studies were performed in spe- cies other than the horse. An equine study on granulosa cells, which have a developmental origin similar to that of cumulus cells, observed age-related changes in their transcriptome [35]. Another transcriptomic study on equine granulosa and theca cells during dominant follicular development identified distinct expression profiles within these stages [36]. g Maturation of oocytes is the first step for in vitro pro- duction (IVP) of embryos across species. Oocyte matur- ation can occur under in vivo conditions where mature oocytes are collected from the donor for fertilization or under in vitro conditions. Usually, maturation in vitro is accompanied by decreased developmental competence among oocytes [1, 2]. The standard in vitro fertilization (IVF) protocol in human reproduction includes the ovar- ian stimulation of women with exogenous gonadotropins to mature oocytes in vivo [3]. Even though maturation and fertilization rates after in vitro maturation (IVM) are promising, IVM correlates with decreased implantation rates, increased miscarriage rates and increased live birth rates [3–5]. Therefore, candidates for IVM in human reproduction are mostly restricted to women at risk for ovarian hyperstimulation syndrome (OHSS) [3, 6]. For the equine species, direct comparisons of embryonic development after in vivo and in vitro are available. In one study in vitro matured oocytes were transferred into the oviduct for in vivo fertilization and further develop- ment. The results showed a highly decreased develop- mental capacity of the in vitro matured oocytes (9%) compared with that of the in vivo matured oocytes (82%) [1]. Blastocyst rates after intracytoplasmic sperm injection (ICSI) of in vitro matured oocytes achieved only up to 35%, which was distinctly lower than their in vivo matured counterparts (up to 70%) [2]. A special mystery in equine-assisted reproduction is the complete failure of classical IVF [7]. At present, this failure is reflected by only two foals born after classical IVF that originated from oocytes matured in vivo [8, 9]. All other equine IVP foals have been generated by ICSI [10, 11]. Whether the cause of this issue is located on the male or female side currently remains unclear [7, 12]. Studies focusing more closely on metabolism and the CC phenotype, e.g., using proteomics or metabolomics, are rare. Walter et al. BMC Genomics (2019) 20:588 Walter et al. BMC Genomics (2019) 20:588 Page 2 of 24 Page 2 of 24 Background Overrepresented KEGG pathways for the pro- teins overexpressed in in vitro matured cumulus were metabolic pathways (KEGG ID 01100), aminoacyl-tRNA biosynthesis (KEGG ID 00970), fatty acid metabolism (KEGG ID 01212) and fatty acid biosynthesis (KEGG ID 00061) (Fig. 2). [53]. Individual analysis of lipid metabolism by desorption electrospray ionization mass spectrometry (DESI-MS) revealed distinct lipid profiles for individual oocytes and embryos [54–56]. Simultaneous profiling of the cumulus cell proteome and metabolome on the single COC level is a technical challenge but provides the unique opportunity to reveal the metabolism as close as possible to the phenotype. This multi-omics (“cumulomics”) approach was chosen for the characterization of aberrations in equine CC metabolism during maturation in vitro. A highly sensi- tive method allowed the analysis on the single COC level, which provides the unique opportunity in the future to directly correlate the “cumulome” with the developmental competence of the corresponding oocyte. The goal is to bridge the gap between in vitro and in vivo maturation and improve the culture conditions for IVM. Equine oocytes also serve as an ideal model for trans- lational research towards clinical human-assisted re- productive technologies. Both species usually develop a single dominant follicle of large volume and have a similar follicular phase and interovulatory interval [37, 57, 58]. Additionally, the timing of ovulation seems to be similar, occurring 36–37 h after human chorionic gonadotrophin (hCG) administration [57]. Another bene- fit of equine CCs for the study of metabolism during maturation for translational research is the large amount of cumulus compared to that in other species. This abundance of cumulus allows for the collection of enough material from single CCs for analysis. Therefore, the results of this study may contribute to improving human IVM conditions, which could save a wide range of women from the exhausting process of ovarian stimu- lation in the future. Background Most of the available “Omics” studies on cumulus cells are transcrip- tomic analyses of pooled cumulus or cumulus oocyte complexes (COCs) that relate the gene expression profile to the developmental competence of the oocyte [23–30]. Other studies examined changes in the cumulus Data on the cumulus cell metabolome are even scarcer across species. Comparison of in vitro matured with in vivo matured COCs revealed altered cellular metabolism-related genes along with increased triglycerides in bovine cumulus cells matured in vitro [34]. Glycosidic residues showed significant quantitative and qualitative differences in equine and porcine COCs after in vitro and in vivo maturation [52]. In the horse, maternal obesity caused alterations in the lipid fingerprint of preovulatory follicles and oocytes Walter et al. BMC Genomics (2019) 20:588 Walter et al. BMC Genomics (2019) 20:588 Page 3 of 24 Page 3 of 24 FC ≥2; Table 1; Additional file 1: Table S1). Of these proteins, 95 were significantly underexpressed in vitro, and 86 of these proteins were linked to a unique ortho- logous human UniProt identitiy (ID; Fig. 1). In the in vitro group, 121 proteins (118 with unique orthologous human UniProt IDs) were significantly overexpressed (Fig. 2). Enrichment analysis of overrepresented Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways was performed in the STRING-DB version 10.5 [59]. The pathway “complement and coagulation cascades” (KEGG ID 4610) was significantly overrepresented in the underexpressed proteins in in vitro matured cumulus (Fig. 1). Overrepresented KEGG pathways for the pro- teins overexpressed in in vitro matured cumulus were metabolic pathways (KEGG ID 01100), aminoacyl-tRNA biosynthesis (KEGG ID 00970), fatty acid metabolism (KEGG ID 01212) and fatty acid biosynthesis (KEGG ID 00061) (Fig. 2). FC ≥2; Table 1; Additional file 1: Table S1). Of these proteins, 95 were significantly underexpressed in vitro, and 86 of these proteins were linked to a unique ortho- logous human UniProt identitiy (ID; Fig. 1). In the in vitro group, 121 proteins (118 with unique orthologous human UniProt IDs) were significantly overexpressed (Fig. 2). Enrichment analysis of overrepresented Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways was performed in the STRING-DB version 10.5 [59]. The pathway “complement and coagulation cascades” (KEGG ID 4610) was significantly overrepresented in the underexpressed proteins in in vitro matured cumulus (Fig. 1). Metabolome The quantifiable metabolome contained 906 metabolic compounds; of these compounds, 108 showed a signifi- cant difference (p < 0.05; FC > 2) in abundance between the two maturation groups. Compared to the in vivo matured cumulus, the in vitro matured cumulus ex- hibited 84 compounds with a higher abundance and 24 compounds with a lower abundance (Table 1; Additional file 2: Table S2). Putative metabolite IDs were found for 6 compounds with lower abundance and for 22 compounds with higher abundance after IVM. Essence of the altered “cumulome” in vitro For an integrative view on the proteomic and metabolo- mic results, compounds with significantly different abun- dances were summarized in the following 10 metabolic groups (Table 2, Fig. 3): oxygen supply (down in vitro: 5 proteins, 1 metabolite; up in vitro: 1 metabolite), glucose metabolism (down in vitro: 3 proteins; up in vitro: 1 pro- tein, 5 metabolites), fatty acid metabolism (down in vitro: 4 proteins; up in vitro: 5 proteins, 3 metabolites), oxidative phosphorylation (up in vitro: 1 protein, 1 metabolite), amino acid metabolism (down in vitro: 1 metabolite; up in vitro: 1 proteins; 2 metabolites), purine and pyrimidine metabolism (down in vitro: 1 protein; up in vitro: 7 Proteome A total of 1811 quantifiable equine proteins (NCBI-Ac- cessions) were identified in the 15 cumulus samples. For downstream analysis, the equine NCBI entries were blasted to human orthologous UniProt Accession, which yielded a total of 1714 unique entries. The proteome contained 216 differentially expressed proteins (p ≤0.05; Table 1 Summary of the proteomics and metabolomics results. The counts of proteins and metabolomic compounds that were quantifiable and the counts that showed different abundances (p < 0.05; fold change (FC) > 2) in the in vitro matured cumulus samples compared to those in the in vivo matured cumulus samples are presented Method Quantifiable Different abundance (p < 0.05; FC > 2) Down in vitro Up in vitro Proteomics (proteins) 1811 (1714)a 216 (204)a 95 (86)a 121 (118)a Metabolomics (compounds) 905 108 (28)b 24 (6)b 84 (22)b awith unique orthologous human UniProt ID bwith putative metabolite ID Table 1 Summary of the proteomics and metabolomics results. The counts of proteins and metabolomic compounds that were quantifiable and the counts that showed different abundances (p < 0.05; fold change (FC) > 2) in the in vitro matured cumulus samples compared to those in the in vivo matured cumulus samples are presented Method Quantifiable Different abundance (p < 0.05; FC > 2) Down in vitro Up in vitro Proteomics (proteins) 1811 (1714)a 216 (204)a 95 (86)a 121 (118)a Metabolomics (compounds) 905 108 (28)b 24 (6)b 84 (22)b awith unique orthologous human UniProt ID bwith putative metabolite ID Walter et al. BMC Genomics (2019) 20:588 Page 4 of 24 Fig. 1 Interaction network of proteins underexpressed in in vitro matured cumulus complexes (interaction confidence: high (> 0.7), database matches n = 82). Highly enriched KEGG pathway in the group of underrepresented proteins is the complement and coagulation cascade (red nodes, pathway ID 04610; n = 21; false discovery rate 1.3e−32). The proteins of the complement cascade are represented by the red nodes within the yellow circle (n = 8), whereas the proteins of the coagulation cascade are within the blue circle (n = 13) Fig. 1 Interaction network of proteins underexpressed in in vitro matured cumulus complexes (interaction confidence: high (> 0.7), database matches n = 82). Highly enriched KEGG pathway in the group of underrepresented proteins is the complement and coagulation cascade (red nodes, pathway ID 04610; n = 21; false discovery rate 1.3e−32). Proteome The proteins of the complement cascade are represented by the red nodes within the yellow circle (n = 8), whereas the proteins of the coagulation cascade are within the blue circle (n = 13) proteins, 3 metabolites), steroid metabolism (down in vitro: 2 metabolites; up in vitro: 1 protein), extracellular matrix (down in vitro: 8 proteins; up in vitro: 3 proteins, 1 metabolite), complement cascade (down in vitro: 14 proteins) and coagulation cascade (down in vitro: 17 pro- teins). These 91 manually selected and grouped com- pounds of the overall 232 metabolites and proteins with unique IDs and significant differences between the two maturation groups are listed in detail in Table 2 and Fig. 3. All significantly different proteins (Additional file 1: Table S1) and metabolic compounds (Additional file 2: Table S2) are listed in the article supplements. Discussion From a technical point of view, in this “cumulomics” study, a simultaneous profiling of the cumulus cells Walter et al. BMC Genomics (2019) 20:588 Page 5 of 24 Fig. 2 Interaction network of proteins overexpressed in in vitro matured cumulus complexes (interaction confidence: high (> 0.7), database matches n = 116). Enriched KEGG pathways in this group of proteins are metabolic pathways (red nodes, pathway ID 01100; n = 23; false discovery rate 3.6e−05), aminoacyl-tRNA biosynthesis (blue nodes, pathway ID 00970; n = 5; false discovery rate 0.0006), fatty acid metabolism (green nodes, pathway ID 01212; n = 5; false discovery rate 0.0007) and fatty acid biosynthesis (yellow nodes, pathway ID 00061, n = 2, false discovery rate 0.04) Fig. 2 Interaction network of proteins overexpressed in in vitro matured cumulus complexes (interaction confidence: high (> 0.7), database matches n = 116). Enriched KEGG pathways in this group of proteins are metabolic pathways (red nodes, pathway ID 01100; n = 23; false discovery rate 3.6e−05), aminoacyl-tRNA biosynthesis (blue nodes, pathway ID 00970; n = 5; false discovery rate 0.0006), fatty acid metabolism (green nodes, pathway ID 01212; n = 5; false discovery rate 0.0007) and fatty acid biosynthesis (yellow nodes, pathway ID 00061, n = 2, false discovery rate 0.04) conducted with DESI-MS [54–56]. For this study, a combination of bottom-up proteomics by nano-HPLC MS/MS and metabolome analysis by UPLC-nanoESI-MS in negative mode was used for the analysis of individual CCs. The detected “cumulome” included 1811 quanti- fiable proteins as well as 905 quantifiable metabolic compounds. Therefore, the analytical technique of the “cumulomics” approach proved to be a highly sensitive “holistic approach”, which can characterize metabolic alterations on the single COC level. proteome and metabolome from the very same samples was performed for the first time. This technical develop- ment opened the gate to unravel the metabolic pheno- type of cumulus cells under different maturation conditions. The analysis was performed on the level of single COCs, which holds the unique potential to di- rectly correlate the “cumulome” with the developmental competence of the corresponding oocyte. A novel com- bination linking methanol based metabolite extraction with filter-aided sample preparation (FASP) for proteo- mics was adapted to for the minute sample amount of single CCs [39]. Thus far, only studies focusing on single “Omics” technologies have been available. Discussion Recently, intact MALDI-TOF mass spectrometry was used to ana- lyse the proteome of individual bovine oocytes, cumulus cells and granulosa cells; a total of 439 shared peaks were detected, and identification of the peaks was per- formed using top-down proteomics on protein extracts of pooled samples [49]. An analysis of the lipid profile of single bovine oocytes and embryos was previously Twenty-eight metabolites with putative metabolite IDs and 204 proteins with unique UniProt IDs were signifi- cantly different between the two maturation groups (Table 1). These alterations affect a wide variety of meta- bolic pathways (Fig. 3). The experimental design used an available pool of slaughtered animals; thus, there was certain heterogeneity with regard to donor mares (e.g. breeds and ages) and follicles (Additional file 4). This reflects the typical situation when immature COCs are collected for assisted reproduction from client mares or Walter et al. Discussion BMC Genomics (2019) 20:588 Page 6 of 24 e 2 The essence of the altered “cumulome” after maturation in vitro olic Groups with their Proteins/ olites Abbreviation Figure Proteomics: Orthologous Human Accession Uniprot Proteomics: NCBI Accession Anova (Progenesis) Max fold change (Progenesis) Up/ Down in- vitro Metabolomics: HMDB ID Metabolomics: Compound (m/z) gen Supply (proteins: n = 5; metabolites n = 2) moglobin A HBA HBA_HUMAN NP_001078901.1 0.015 25.58 Down moglobin B HBB HBB_HUMAN NP_001157490.1;XP_001504241.1 0.007 52.65 Down uloplasmin CP CERU_HUMAN XP_001491539.1;XP_014587394.1;XP_014587022.1 < 0.001 140.94 Down nsferrin TF TRFE_HUMAN NP_001075415.2 0.003 10.25 Down mopexin HEMO HEMO_HUMAN XP_005612174.1 0.010 10.14 Down roxymethylbilane Hydroxymethylbilane HMDB01137 4.89_426.1165 m/z 0.001 16.86 Down ubin-Glucuronide Bilirubin-Gluc HMDB10332 6.37_741.2851 m/z 0.040 2.21 Up cose Metabolism (proteins n = 4; metabolites n = 5) tamine-fructose-6-phosphate notransferase 1 GFPT1 GFPT1_HUMAN XP_005599972.1 < 0.001 101.18 Down tamine-fructose-6-phosphate notransferase 2 GFPT2 GFPT2_HUMAN XP_014585826.1 0.024 2.80 Down dependend 6- osphofructokinase muscle type PFK-M PFKAM_HUMAN XP_005611133.1;XP_014596234.1 0.006 4.67 Up ute Carrier Family 2, facilitated cose transporter member 1 GLUT1 GTR1_HUMAN NP_001157443.1;XP_005607060.1 0.040 2.01 Down sphoenolpyruvate PEP HMDB00263 6.23_166.9749 m/z 0.018 157.36 Up actate / L-Lactate Lactate HMDB00190 6.41_111.0085 m/z 0.019 2.78 Up ose-Monophosphate Hexose-P not specified 6.41_259.0217 m/z 0.039 6.10 Up ate/Isocitrate Citrate HMDB00094 6.84_191.0191 m/z 0.024 4.04 Up hospho-d-glyceric acid 2PG HMDB03391 6.39_185.9927n 0.026 10.30 Up y Acid Metabolism (proteins n = 9; metabolites n = 3) olipoprotein A1 APOA1 APOA1_HUMAN XP_005611649.1 0.001 30.68 Down olipoprotein A2 APOA2 APOA2_HUMAN XP_001503846.1 0.003 48.44 Down olipoprotein H APOH APOH_HUMAN XP_001499728.1 < 0.001 30.74 Down ponectin ADIPO ADIPO_HUMAN XP_001499564.1 0.009 8.64 Down tyl-CoA carboxylase 1 ACACA ACACA_HUMAN XP_005597614.1;XP_001496980.1 < 0.001 6.07 Up y acid synthase FASN FAS_HUMAN XP_014591306.1 0.001 2.64 Up g-chain-fatty-acid-CoA ligase 3 ACSL ACSL3_HUMAN XP_001915556.1 0.013 4.14 Up y acid binding protein FABP FABPH_HUMAN NP_001157357.1;XP_005607059.1 0.001 46.96 Up l-CoA desaturase ACOD ACOD_HUMAN XP_001500414.1 < 0.001 7.97 Up e essence of the altered “cumulome” after maturation in vitro (Continued) oups with their Proteins/ Abbreviation Figure Proteomics: Orthologous Human Accession Uniprot Proteomics: NCBI Accession Anova (Progenesis) Max fold change (Progenesis) Up/ Down in- vitro Metabolomics: HMDB ID Metabolomics: Compound (m/z) oethanolamine O- Phosphoethanolamine HMDB00224 4.64_175.9943 m/z 0.002 2.76 Up rol (40:7) DAG (40:7) not specified 3.61_647.5261 m/z 0.024 11.18 Up olamine CDP-Ethanolamine HMDB01564 4.85_222.0249 m/z 0.021 2.75 Up hosporylation (proteins n = 1; metabolites n = 1) quinone oxidoreductase Mt-ND5 NU5M_HUMAN NP_007170.1 0.046 3.61 Up avin mononucleotide FMNH2 HMDB01142 7.19_439.0852 m/z < 0.001 4.37 Up d Metabolism (proteins n = 7; metabolites n = 4) erine aminotransferase PSAT1 SERC_HUMAN XP_001496412.1 0.022 9.40 Up -Serine SERINE HMDB00187; HMDB03406 4.98_104.0352 m/z 0.008 3.22 Down ydroxypyruvic acid PHPA HMDB01024 6.82_164.9577 m/z 0.000 19.00 Up oleucine L; Iso-L HMDB00687; HMDB00172 6.55_168.0429 m/z 0.003 7.84 Up Cystine HMDB00192 4.81_221.0093 m/z 0.008 2.67 Up NA ligase SARS SYSC_HUMAN XP_005610429.1 0.007 2.17 Up RNA ligase YARS SYYC_HUMAN XP_014592779.1 0.015 2.06 Up yl t-RNA ligase NARS SYNC_HUMAN XP_001488269.1 0.016 3.19 Up t-RNA ligase TARS SYTC_HUMAN XP_001498225.2;XP_001491149.3 0.005 2.11 Up e t-RNA ligase MARS SYMC_HUMAN XP_001488941.1;XP_005611399.1 0.039 2.12 Up NA ligase AARS SYAC_HUMAN XP_001501062.3 0.013 34.61 Down midin Metabolism (proteins n = 8; metabolites n = 3) osphate hokinase PRPS1 PRPS1_HUMAN XP_001491425.1;XP_001489096.2;XP_001496502.1 0.011 2.09 Up bosylformylglycinamidine PFAS PUR4_HUMAN XP_001918417.1 0.006 14.09 Up al purine biosynthesis PURH PUR_9_HUMAN XP_005610644.2 0.007 2.01 Up monophosphate nase 2 IMDH2 IMDH2_HUMAN XP_001494600.4;XP_005600741.2 0.004 3.17 Up onophosphate Synthase GMPS GUAA_HUMAN XP_014587031.1 0.003 2.23 Up ted RNA polymerases I, II, unit RPABC1 POLR2 RPAB1_HUMAN XP_001496313.1;XP_014596768.1 0.026 3.93 Up osyl)-triphosphatase ENPP4 ENPP4_HUMAN XP_001502639.1 0.005 5.49 Down Walter et al. Discussion BMC Genomics (2019) 20:588 Page 7 of 24 Walter et al. BMC Genomics (2019) 20:588 Page 7 of 24 Table 2 The essence of the altered “cumulome” after maturation in vitro (Continued) Metabolic Groups with their Proteins/ Metabolites Abbreviation Figure Proteomics: Orthologous Human Accession Uniprot Proteomics: NCBI Accession Anova (Progenesis) Max fold change (Progenesis) Up/ Down in- vitro Metabolomics: HMDB ID Metabolomics: Compound (m/z) O-Phosphoethanolamine O- Phosphoethanolamine HMDB00224 4.64_175.9943 m/z 0.002 2.76 Up Diacyglycerol (40:7) DAG (40:7) not specified 3.61_647.5261 m/z 0.024 11.18 Up CDP-Ethanolamine CDP-Ethanolamine HMDB01564 4.85_222.0249 m/z 0.021 2.75 Up 4 Oxidative Phosporylation (proteins n = 1; metabolites n = 1) NADH-ubiquinone oxidoreductase chain 5 Mt-ND5 NU5M_HUMAN NP_007170.1 0.046 3.61 Up reduced flavin mononucleotide FMNH2 HMDB01142 7.19_439.0852 m/z < 0.001 4.37 Up 5 Amino Acid Metabolism (proteins n = 7; metabolites n = 4) phosphoserine aminotransferase PSAT1 SERC_HUMAN XP_001496412.1 0.022 9.40 Up L-Serine; D-Serine SERINE HMDB00187; HMDB03406 4.98_104.0352 m/z 0.008 3.22 Down Phosphohydroxypyruvic acid PHPA HMDB01024 6.82_164.9577 m/z 0.000 19.00 Up Leucine/Isoleucine L; Iso-L HMDB00687; HMDB00172 6.55_168.0429 m/z 0.003 7.84 Up L-Cystine Cystine HMDB00192 4.81_221.0093 m/z 0.008 2.67 Up Serine t-RNA ligase SARS SYSC_HUMAN XP_005610429.1 0.007 2.17 Up Tyrosine t-RNA ligase YARS SYYC_HUMAN XP_014592779.1 0.015 2.06 Up Asparaginyl t-RNA ligase NARS SYNC_HUMAN XP_001488269.1 0.016 3.19 Up Threonine t-RNA ligase TARS SYTC_HUMAN XP_001498225.2;XP_001491149.3 0.005 2.11 Up Methionine t-RNA ligase MARS SYMC_HUMAN XP_001488941.1;XP_005611399.1 0.039 2.12 Up Alanine t-RNA ligase AARS SYAC_HUMAN XP_001501062.3 0.013 34.61 Down 6 Purin & Pyrimidin Metabolism (proteins n = 8; metabolites n = 3) Ribose-phosphate pyrophosphokinase PRPS1 PRPS1_HUMAN XP_001491425.1;XP_001489096.2;XP_001496502.1 0.011 2.09 Up Phosphoribosylformylglycinamidine synthase PFAS PUR4_HUMAN XP_001918417.1 0.006 14.09 Up Bifunctional purine biosynthesis protein PURH PUR_9_HUMAN XP_005610644.2 0.007 2.01 Up Inosine-5′-monophosphate dehydrogenase 2 IMDH2 IMDH2_HUMAN XP_001494600.4;XP_005600741.2 0.004 3.17 Up Guanine Monophosphate Synthase GMPS GUAA_HUMAN XP_014587031.1 0.003 2.23 Up DNA-directed RNA polymerases I, II, and III subunit RPABC1 POLR2 RPAB1_HUMAN XP_001496313.1;XP_014596768.1 0.026 3.93 Up Bis(5′-adenosyl)-triphosphatase ENPP4 ENPP4_HUMAN XP_001502639.1 0.005 5.49 Down Walter et al. Discussion BMC Genomics (2019) 20:588 Page 8 of 24 e 2 The essence of the altered “cumulome” after maturation in vitro (Continued) olic Groups with their Proteins/ olites Abbreviation Figure Proteomics: Orthologous Human Accession Uniprot Proteomics: NCBI Accession Anova (Progenesis) Max fold change (Progenesis) Up/ Down in- vitro Metabolomics: HMDB ID Metabolomics: Compound (m/z) oxanthine HypoX HMDB00157 4.75_172.9976 m/z 0.014 2.62 Up nosine Guano HMDB00133 4.63_318.0790 m/z 0.033 4.29 Up xycytidine monophosphate dCMP HMDB01202 6.89_343.9941 m/z 0.025 2.25 Up ine 5′-monophosphate synthase UMPS UMPS_HUMAN XP_001500089.4 0.009 3.17 Up oid Metabolism (proteins n = 2; metabolites n = 2) hosphomevalonate arboxylase MVD MVD1_HUMAN XP_001488083.1;XP_014593934.1 0.007 6.21 Up wth Regulation by Estrogen in st Cancer Gene 1 GREB GREB1_HUMAN XP_014586792.1 0.002 4.26 Up anyl Pyrophosphate Geranyl-PP HMDB01285 4.89_314.0589n 0.002 3.04 Down one Sulfate Estrone-S HMDB01425 6.98_385.0719 m/z < 0.001 2.94 Down cellular Matrix/Proteoglycans (proteins n = 11; metabolites n = 1) onectin VN VTNC_HUMAN XP_001504173.3 0.001 19.83 Down onectin FN FINC_HUMAN XP_001489154.4 < 0.001 152.88 Down r-alpha-trypsin inhibitor heavy n H1 ITIH1 ITIH1_HUMAN XP_001492576.1;XP_014587106.1 < 0.001 76.91 Down r-alpha-trypsin inhibitor heavy n H2 ITIH2 ITIH2_HUMAN XP_001916967.1 < 0.001 45.40 Down r-alpha-trypsin inhibitor heavy n H3 ITIH3 ITIH3_HUMAN XP_014587105.1 < 0.001 14.74 Down r-alpha-trypsin inhibitor heavy n H4 ITIH4 ITIH4_HUMAN XP_005600608.1;XP_014587103.1 0.011 26.91 Down ement-membrane specific aran sulfate proteoglycan core ein HSPG2 PGBM_HUMAN XP_014592730.1 0.014 3.17 Down ermal Growth Factor Receptor EGFR EGFR_HUMAN XP_005609210.2 0.009 2.24 Up troglycan DAG1 DAG1_HUMAN XP_001497663.1;XP_005600719.1;XP_005600720.1;XP_005600721.1 0.011 2.35 Up ne Protease HTRA1 HTRA1 HTRA1_HUMAN XP_005602601.1 0.013 3.92 Up junction alpha 1 protein; nexin-43 GJA1 CXA1_HUMAN NP_001296155.1;XP_014584258.1;XP_014584259.1 0.008 2.69 Down ndroitin-4-sulphate CHOND4 HMDB00652 4.75_496.0430 m/z 0.012 3.61 Up plement Cascade (proteins n = 14) ma protease C1 inhibitor IC1 IC1_HUMAN; SERPING1 XP_001498388.1 < 0.001 56.92 Down mplement 4 (A/B) C4 CO4B_HUMAN XP_001492943.1 0.007 7.78 Down Walter et al. Discussion BMC Genomics (2019) 20:588 Page 8 of 24 Table 2 The essence of the altered “cumulome” after maturation in vitro (Continued) Metabolic Groups with their Proteins/ Metabolites Abbreviation Figure Proteomics: Orthologous Human Accession Uniprot Proteomics: NCBI Accession Anova (Progenesis) Max fold change (Progenesis) Up/ Down in- vitro Metabolomics: HMDB ID Metabolomics: Compound (m/z) Hypoxanthine HypoX HMDB00157 4.75_172.9976 m/z 0.014 2.62 Up Guanosine Guano HMDB00133 4.63_318.0790 m/z 0.033 4.29 Up Deoxycytidine monophosphate dCMP HMDB01202 6.89_343.9941 m/z 0.025 2.25 Up Uridine 5′-monophosphate synthase UMPS UMPS_HUMAN XP_001500089.4 0.009 3.17 Up 7 Steroid Metabolism (proteins n = 2; metabolites n = 2) Diphosphomevalonate decarboxylase MVD MVD1_HUMAN XP_001488083.1;XP_014593934.1 0.007 6.21 Up Growth Regulation by Estrogen in Breast Cancer Gene 1 GREB GREB1_HUMAN XP_014586792.1 0.002 4.26 Up Geranyl Pyrophosphate Geranyl-PP HMDB01285 4.89_314.0589n 0.002 3.04 Down Estrone Sulfate Estrone-S HMDB01425 6.98_385.0719 m/z < 0.001 2.94 Down 8 Extracellular Matrix/Proteoglycans (proteins n = 11; metabolites n = 1) Vitronectin VN VTNC_HUMAN XP_001504173.3 0.001 19.83 Down Fibronectin FN FINC_HUMAN XP_001489154.4 < 0.001 152.88 Down Inter-alpha-trypsin inhibitor heavy chain H1 ITIH1 ITIH1_HUMAN XP_001492576.1;XP_014587106.1 < 0.001 76.91 Down Inter-alpha-trypsin inhibitor heavy chain H2 ITIH2 ITIH2_HUMAN XP_001916967.1 < 0.001 45.40 Down Inter-alpha-trypsin inhibitor heavy chain H3 ITIH3 ITIH3_HUMAN XP_014587105.1 < 0.001 14.74 Down Inter-alpha-trypsin inhibitor heavy chain H4 ITIH4 ITIH4_HUMAN XP_005600608.1;XP_014587103.1 0.011 26.91 Down Basement-membrane specific heparan sulfate proteoglycan core protein HSPG2 PGBM_HUMAN XP_014592730.1 0.014 3.17 Down Epidermal Growth Factor Receptor EGFR EGFR_HUMAN XP_005609210.2 0.009 2.24 Up Dystroglycan DAG1 DAG1_HUMAN XP_001497663.1;XP_005600719.1;XP_005600720.1;XP_005600721.1 0.011 2.35 Up Serine Protease HTRA1 HTRA1 HTRA1_HUMAN XP_005602601.1 0.013 3.92 Up Gap junction alpha 1 protein; Connexin-43 GJA1 CXA1_HUMAN NP_001296155.1;XP_014584258.1;XP_014584259.1 0.008 2.69 Down Chondroitin-4-sulphate CHOND4 HMDB00652 4.75_496.0430 m/z 0.012 3.61 Up 9 Complement Cascade (proteins n = 14) Plasma protease C1 inhibitor IC1 IC1_HUMAN; SERPING1 XP_001498388.1 < 0.001 56.92 Down Complement 4 (A/B) C4 CO4B_HUMAN XP_001492943.1 0.007 7.78 Down Page 9 of 24 Page 9 of 24 Walter et al. Discussion BMC Genomics (2019) 20:588 e 2 The essence of the altered “cumulome” after maturation in vitro (Continued) bolic Groups with their Proteins/ bolites Abbreviation Figure Proteomics: Orthologous Human Accession Uniprot Proteomics: NCBI Accession Anova (Progenesis) Max fold change (Progenesis) Up/ Down in- vitro Metabolomics: HMDB ID Metabolomics: Compound (m/z) b-binding protein alpha chain C4BP C4BPA_HUMAN XP_014594908.1 0.001 48.62 Down b-binding protein alpha chain C4BP C4BPA_HUMAN XP_001492582.2;XP_005609671.1;XP_005609672.1;XP_005609676.1;XP_ 005609677.1;XP_005609678.1;XP_014594955.1;XP_014594956.1;XP _014594957.1;XP_014594958.1 < 0.001 73.54 Down = Complement C3 C3 CO3_HUMAN XP_014596564.1 0.005 9274.92 Down = Complement C3 C3 CO3_HUMAN XP_001915589.1 0.001 748.41 Down mplement C5 C5 CO5_HUMAN XP_014591698.1;zz\|FGCZCont0049_P01031\|CO5_HUMAN 0.044 5.66 Down mplement C7 C7 CO7_HUMAN XP_005604353.1 0.004 33.92 Down mplement factor I CFI CFAI_HUMAN XP_014593434.1;XP_014593435.1;XP_014593436.1 < 0.001 16.51 Down mplement factor B CFB CFAB_HUMAN XP_001492602.1 0.008 33.85 Down mplement factor H X1 CFH CFAH_HUMAN XP_001491754.3 0.019 70.12 Down mplement factor H X5 CFH CFAH_HUMAN XP_005608148.2 0.002 12.74 Down usterin CLUS CLUS_HUMAN NP_001075413.1 0.049 3.44 Down ronectin VN VTNC_HUMAN XP_001504173.3 0.001 19.83 Down oagulation Cascade (proteins n = 17) rinogen alpha chain FGA FIBA_HUMAN XP_005607860.1 0.001 43.25 Down rinogen beta chain FGB FIBB_HUMAN XP_003364583.1 < 0.001 55.46 Down rinogen gamma chain FGG FIBG_HUMAN XP_001914833.2 < 0.001 36.00 Down inogen-1 KNG KNG1_HUMAN XP_001499389.1;XP_005601930.1 0.008 14.66 Down othrombin F2 THRB_HUMAN XP_001490892.3 0.001 6.66 Down parin Cofactor 2 HEP2 HEP2_HUMAN; SERPIND1 XP_003365492.1 0.003 46.20 Down sma serine protease inhibitor IPSP IPSP_HUMAN; SERPINA5 XP_001496026.2 0.031 44.21 Down tithrombin 3 ANT3 ANT3_HUMAN; SERPINC1 XP_014594947.1;zz\|FGCZCont0237_P01008\|ANT3_HUMAN 0.001 8.46 Down sma protease C1 inhibitor IC1 IC1_HUMAN; SERPING1 XP_001498388.1 <0.001 55.27 Down pha-2-antiplasmin A2AP A2AP_HUMAN; SERPINF2 XP_001504386.3;XP_005597710.1 0.005 10.84 Down pha-2-macroglobulin A2M A2MG_HUMAN XP_014596179.1 0.015 49.65 Down pha-2-macroglobulin A2M A2MG_HUMAN XP_014596181.1;XP_014596182.1 0.014 40.92 Down pha-2-macroglobulin A2M A2MG_HUMAN XP_001499173.2 0.000 15.00 Down sminogen PLG PLMN_HUMAN XP_001500552.3 0.027 16.17 Down Page 10 of 24 Walter et al. Discussion Donor age was already shown to influence the equine granulosa cells transcriptome [35] and human cumulus proteome [41]. Gene expression pro- files in granulosa cells vary throughout folliculoge- nesis, for the bovine species this was illustrated on the GranulosaIMAGE interactive web interface [60]. For the in vitro matured group it is not possible to specify exactly the developmental phase of the donor follicles, which can also impact the results. The picture of altered metabolism after IVM is of course influenced by maturation conditions. Differences in media composition, gas concentrations or cell hand- ling would obviously affect the altered “cumulome”. from slaughterhouse animals for research purposes. Therefore, factors as age and follicular size cannot fully be ruled out as confounding factors. Mean mare age was higher in the in vitro group, still not significantly different. Donor age was already shown to influence the equine granulosa cells transcriptome [35] and human cumulus proteome [41]. Gene expression pro- files in granulosa cells vary throughout folliculoge- nesis, for the bovine species this was illustrated on the GranulosaIMAGE interactive web interface [60]. For the in vitro matured group it is not possible to specify exactly the developmental phase of the donor follicles, which can also impact the results. The picture of altered metabolism after IVM is of course influenced by maturation conditions. Differences in media composition, gas concentrations or cell hand- ling would obviously affect the altered “cumulome”. Discussion BMC Genomics (2019) 20:588 Table 2 The essence of the altered “cumulome” after maturation in vitro (Continued) Metabolic Groups with their Proteins/ Metabolites Abbreviation Figure Proteomics: Orthologous Human Accession Uniprot Proteomics: NCBI Accession Anova (Progenesis) Max fold change (Progenesis) Up/ Down in- vitro Metabolomics: HMDB ID Metabolomics: Compound (m/z) Alpha-1-antiproteinase 2 precursor A1AT A1AT_HUMAN NP_001108005.1 0.001 35.00 Down Alpha-1-antiproteinase 2 precursor A1AT A1AT_HUMAN XP_005605481.1 0.001 33.45 Down Alpha-1-antiproteinase 2 precursor A1AT A1AT_HUMAN XP_001495905.2 0.001 27.21 Down Table 2 The essence of the altered “cumulome” after maturation in vitro (Continued) Metabolic Groups with their Proteins/ Metabolites Abbreviation Figure Proteomics: Orthologous Human Accession Uniprot Proteomics: NCBI Accession Anova (Progenesis) Max fold change (Progenesis) Up/ Down in- vitro Metabolomics: HMDB ID Metabolomics: Compound (m/z) Alpha-1-antiproteinase 2 precursor A1AT A1AT_HUMAN NP_001108005.1 0.001 35.00 Down Alpha-1-antiproteinase 2 precursor A1AT A1AT_HUMAN XP_005605481.1 0.001 33.45 Down Alpha-1-antiproteinase 2 precursor A1AT A1AT_HUMAN XP_001495905.2 0.001 27.21 Down Page 11 of 24 Walter et al. BMC Genomics (2019) 20:588 Fig. 3 The essence of the altered “cumulome” after maturation in vitro. Schematic view on aberrant metabolism in cumulus cells after maturation in vitro compared to maturation in vivo. Mapped are 91 compounds with significantly different abundance in in vitro matured cumulus (78 proteins and 21 metabolites, full names are listed in Table 2). Relevant related proteins, metabolites or pathways not detected in this study were imputed (dashed lines). Items with higher abundance after maturation in vitro are coloured in purple, items with lower abundance in green. Circles surround proteins and hexagons metabolites Fig. 3 The essence of the altered “cumulome” after maturation in vitro. Schematic view on aberrant metabolism in cumulus cells after maturation in vitro compared to maturation in vivo. Mapped are 91 compounds with significantly different abundance in in vitro matured cumulus (78 proteins and 21 metabolites, full names are listed in Table 2). Relevant related proteins, metabolites or pathways not detected in this study were imputed (dashed lines). Items with higher abundance after maturation in vitro are coloured in purple, items with lower abundance in green. Circles surround proteins and hexagons metabolites Some of these potential effects are discussed in relevant chapters in the discussion. from slaughterhouse animals for research purposes. Therefore, factors as age and follicular size cannot fully be ruled out as confounding factors. Mean mare age was higher in the in vitro group, still not significantly different. Glucose metabolism Glucose metabolism plays a central role in the altered compounds under in vitro conditions. First, glucose transporter member 1 (GLUT1) was significantly un- derrepresented in in vitro matured cumulus. GLUT1 was the highest expressed glucose transporter at the mRNA and protein levels in mouse cumulus cells where insulin stimulation resulted in glucose uptake [70]. Bovine cumulus responded to hypoxia during IVM through upregulation of GLUT1 gene expression (Slc2a1), and HIF1α seems to play a role in mediating this response [64]. For equine cumulus cells, the ex- pression of the GLUT1 gene (Slc2a1) was previously documented, with significantly higher abundance in expanded CC than in compact cumulus cells [71]. These studies corroborate the observed GLUT1 under- representation after maturation under the hyperoxic (20%) in vitro condition in this study. Another hint for the potential oxygen binding/trans- porting role of Hb in the cumulus oophorus is an analyte that was also significantly underrepresented in the in vitro matured group and seems to match hydroxy- methylbilane (HMB), a precursor of heme biosynthesis. The transcripts of enzymes involved in heme biosyn- thesis were previously detected in COCs including the enzyme directly related to HMB synthesis (Hmbs; hydroxymethylbilane synthase) [63]. The in vitro underrepresented proteins ceruloplasmin and transferrin work together in the transport of Fe2+, which is required for heme synthesis. The underrepre- sentation of these proteins contribute to the observed underrepresentation of HBA, HBB and HMB. The simultaneous underrepresentation of proteins and meta- bolites involved in in vitro matured COCs strengthens the hypothesis regarding an oxygen binding role of hemoglobin within the COC, which seems to be defi- cient in in vitro matured cumulus cells. Nevertheless, heme is a constituent of hemoglobin and is required for cytochromes, cholesterol biosynthesis (see below) and hydrogen peroxide degradation (heme containing monofunctional catalases). Therefore, other functions of this molecule in this setting are also possible. The importance of glucose metabolism for the matur- ation of COCs is well described in the literature. Oocytes possess only a limited capacity to metabolise glucose; they depend on cumulus cells for the valorisation of glucose [72]. The glycolytic compounds upregulated in in vitro matured cumulus cells are phosphoenolpyruvate (PEP) and lactate (Table 2, Fig. 3). The higher abundance of these potential metabolites indicates an increased glycolytic rate compared to that in in vivo conditions. Oxygen supply An interesting group of proteins underexpressed in in vitro matured cumulus is related to oxygen supply; first and foremost among these proteins is hemoglobin (Hb) A and B. The expression of Hb in non-thyroid cells was illustrated through recent advances in Hb research [61]. Recent studies documented hemoglobin B (HBB) expres- sion in murine and human cumulus cells and exhibited underrepresentation of HBB after IVM [49, 62–64]. The function of HBB in these cells remains unknown. HBB can ensure the necessary oxygen supply for the oocyte in the reduced oxygen environment of the maturing follicle [61, 65]. Under in vivo conditions, the estimated oxygen concentrations in the follicle were approximately 1–5% Page 12 of 24 Page 12 of 24 Walter et al. BMC Genomics (2019) 20:588 Walter et al. BMC Genomics (2019) 20:588 The addition of ferrohemoglobin (FE2+) to the IVM medium improved blastocyst rates [63]. Moreover, the in vitro condition used for the present study included a high oxygen environment (20% oxygen), which seems to result in the underrepresentation of these proteins compared to that in COCs matured in vivo under low oxygen conditions. [66], which is tremendously lower than the common 20% under IVM conditions [67]. Hypoxia induces the expres- sion of HBB through hypoxia inducible factor 1 (HIF-1) [61, 68]. Higher levels of hypoxia inducible factor 1 α (HIF1α) protein were detected in cumulus cells exposed to low oxygen compared to those exposed to 20% oxygen during IVM [64]. Therefore, the high oxygen concen- tration under IVM conditions seems to be responsible for the underrepresentation of hemoglobin A (HBA) and HBB under in vitro conditions in this study. Hemopexin, a scavenger enzyme for Hb, was also underrepresented in the in vitro matured cumulus. When heme is released through degradation of Hb, it is transferred to hemopexin. Therefore, hemopexin acts as a detoxifying agent for free heme with its pro-inflammatory and pro-oxidant effects. Glucose metabolism The switch from TCM199-based ma- turation medium (with a more physiological glucose con- centration) to DMEM/F12-based maturation medium for equine oocytes resulted in significantly better cleavage and blastocyst rates, without alteration of the maturation rates [75]. Therefore, the high glucose concentration during IVM seems to be beneficial for the developmental com- petence of equine oocytes. Nevertheless, a substantial discrepancy for the “cumulome” between in vivo and in vitro matured COCs was observed. In summary, energy generation in vivo does not seem to heavily depend on glycolysis unlike that in vitro. glycosylation of proteins [72, 73]. The role of HBP can explain the general observation of better expanded CCs after maturation in vivo compared to that in vitro. For the interpretation of these alterations in glucose metab- olism, the available glucose under the two experimental conditions must be taken in account. The concentration of glucose in the natural surroundings of COCs, the follicular fluid, is usually in the range of plasma levels. Compared to other species, equine follicular fluid con- tains slightly higher glucose concentrations, ranging from 4.7 mmol/l in small follicles, with a drop to 3.3 mmol/l in larger follicles [74]. DMEM/F12, used for IVM in this study, contains an almost 4-fold higher (17.5 mmol/l) glucose concentration compared to that in follicular fluid. The switch from TCM199-based ma- turation medium (with a more physiological glucose con- centration) to DMEM/F12-based maturation medium for equine oocytes resulted in significantly better cleavage and blastocyst rates, without alteration of the maturation rates [75]. Therefore, the high glucose concentration during IVM seems to be beneficial for the developmental com- petence of equine oocytes. Nevertheless, a substantial discrepancy for the “cumulome” between in vivo and in vitro matured COCs was observed. In summary, energy generation in vivo does not seem to heavily depend on glycolysis unlike that in vitro. inhibition of ß-oxidation resulted in increased glucose consumption. This finding are reflected in the altered equine “cumulome” of in vitro matured COCs where increased protein amounts for fatty acid synthesis and glycolysis were major findings in the altered metabol- ism after IVM. Interestingly, very similar results were observed in a bovine study where an increase in the lipid content of in vitro matured cumulus cells was accompanied by upregulation of genes related to gly- colysis and fatty acid synthesis, whereas ß-oxidation was decreased after maturation in vitro [34]. Glucose metabolism Adipo- nectin (ADIPO) was also upregulated in in vitro ma- tured cumulus cells. This regulator prevents energy deficits in cells, which was present in in vitro matured COCs as indicated by a lower ATP/ADP ratio compared to that in in vivo matured COCs [34]. In addition to the altered lipid related proteome, meta- bolic compounds that can be related to metabolites in fatty acid metabolism and membrane lipids were upregulated after maturation in vitro. One analyte can be attributed to DAG (40:7), a diglyceride that can be a precursor for tri- glycerides, products of membrane lipid degradation or serve as second messengers (https://pubchem.ncbi.nlm.nih. gov). Two other potential compounds upregulated in vitro, O- phosphoethanolamine and CDP-ethanolamine, belong to the glycerophospholipid metabolism pathway (KEGG pathway 00564). These metabolites are precursors for phos- pholipids or products of phospholipid breakdown (https:// pubchem.ncbi.nlm.nih.gov). Metabolomic analysis of bo- vine IVM COCs and denuded oocytes after IVM revealed that cumulus cells modulate the lipid profile. Triacylgly- cerols and phospholipids were higher in COCs than in de- nuded oocytes [80]. Phospholipid metabolism compounds accumulated in bovine IVM medium during maturation, which makes production and secretion by COCs into the medium likely [81]. In summary, the presented equine results revealed major aberrations in fatty acid metabolism after maturation in vitro on both levels of the analysed “cumulome”. The synthesis of fatty acids in cumulus cells seems to compensate for the insufficient supply of fatty acids in COCs in vitro. Fatty acid metabolism Under the experimental condition of this study, fatty acid synthesis seems to be upregulated after maturation in vitro. Three main enzymes involved in fatty acid synthesis (ACSL; ACACA; FASN) were overrepresented in in vitro matured cumulus. This overrepresentation is most likely the result of an insufficient supply of fatty acid in the maturation medium compared to that in the follicular environment in vivo. The only source of fatty acids in the medium was the supplemented serum, which contains less essential linoleic acid than is contained in the DMEM/F12-based medium used. In potential support of this hypothesis, three compo- nents of lipoproteins (APOA1 and 2; APOH) were underrepresented after IVM. These proteins are in- volved in lipid transport and storage and play an im- portant role in providing lipids for the COCs. APOA1 was identified as crucial component of mare follicular fluid throughout the reproductive seasons [51]. Lipid metabolism is an important player in COC metabol- ism and oocyte developmental competence [76]. The special role of cumulus cells in supporting oocyte de- velopmental competence through fatty acid synthesis and oxidation was described in a bovine study [77]. Meiotic resumption was compromised by inhibition of ß-oxidation in mouse [78, 79] and cow oocytes [77]. There was a close link to glucose consumption as Glucose metabolism This theory is supported by overexpression of ATP dependent 6-phosphofructokinase muscle type (PFK-M), the main rate-limiting enzyme in glycolysis, after matu- ration in vitro. PFK-M is regulated by ATP concen- trations; a high ATP/ADP ratio downregulates the glycolytic rate. Therefore, the overrepresentation after IVM seems to be the result of an energy (ATP) deficit or increased ATP consumption. For the equine species, reduced glucose metabolism was detected for expanded COCs. These COCs also exhibited higher maturational competence (50% versus 21.7%). These expanded COCs showed significantly lower glucose consumption along with decreased pyruvate and lactate production [71]. Heme catabolism results in the production of bilirubin, a yellow compound that seems to be responsible for the yellow colour of follicular fluid in humans [69]. One analyte overrepresented in in vitro matured cumulus (6.37_741.2851 m/z) can be attributed to the conjugated form of bilirubin, bilirubin glucuronide. The higher abundance can be a result of increased heme catabolism, therefore contributing to the underrepresentation of Hb in IVM cumulus in this study. In summary, the underrepresentation of all these proteins and metabolites related to oxygen supply in in vitro matured cumulus cells play an important role in the further developmental potential of COCs. This underrepresentation may be one reason for the impaired developmental competence of in vitro matured COCs. Alternatively, the rate-limiting enzyme of the hexosa- mine biosynthetic pathway (HBP) is glutamine-fructose- 6-phosphate aminotransferase 1 and 2 (GFPT1 and 2), which was significantly underexpressed in in vitro ma- tured cumulus cells. HBP participates in the production of hyaluronic acid and products for O-linked Walter et al. BMC Genomics (2019) 20:588 Page 13 of 24 Page 13 of 24 glycosylation of proteins [72, 73]. The role of HBP can explain the general observation of better expanded CCs after maturation in vivo compared to that in vitro. For the interpretation of these alterations in glucose metab- olism, the available glucose under the two experimental conditions must be taken in account. The concentration of glucose in the natural surroundings of COCs, the follicular fluid, is usually in the range of plasma levels. Compared to other species, equine follicular fluid con- tains slightly higher glucose concentrations, ranging from 4.7 mmol/l in small follicles, with a drop to 3.3 mmol/l in larger follicles [74]. DMEM/F12, used for IVM in this study, contains an almost 4-fold higher (17.5 mmol/l) glucose concentration compared to that in follicular fluid. Steroid metabolism Diphosphomevalonate decarboxylase (MVD), an enzyme catalysing the last reaction in the mevalonate pathway, was overrepresented in in vitro matured cumulus. The mevalonate pathway converts mevalonate in sterol iso- prenoids (cholesterol) or non-sterol isoprenoids (e.g., heme-A or ubiquinone) [83]. MVD is responsible for the synthesis of isopentenyl diphosphate (IPP). IPP is con- densed with dimethylallyl diphosphate to form geranyl diphosphate (geranyl-PP), a metabolite attributed to a metabolomic compound that was underrepresented in vitro [84]. MVD shows the highest expression in the liver for cholesterol production [84], but its expression was also documented in mouse ovarian follicles in the antral stage [85]. Cumulus cells provide cholesterol to their oocytes, which is transferred through gap junc- tions. Synthesis of cholesterol in cumulus cells is stimu- lated by oocyte secreted factors [86]. Cholesterol is the precursor for steroidogenesis in mammalian cumulus cells during maturation [87, 88]. Another metabolite MS signal underrepresented after maturation in vitro was assigned to the steroid hormone oestrone sulfate (oestrone-s). Estrone-s is biologically inactive, but through sulfotransferases, it can be con- verted into biologically active unconjugated estrone. Therefore, estrone-s can serve as reservoir for estrone. Expression of local steroid sulfatases was documented in human cumulus cells [89]. The results of this study revealed a deficit in potential metabolites in mevalonate/ steroid metabolism with upregulation of a key enzyme of the mevalonate pathway in in vitro matured cumulus cells. This finding can be interpreted as the struggle of IVM COCs to produce sufficient steroids in compensa- tion of the missing steroidogenic follicular surrounding. Oxidative phosphorylation Oxidative phosphorylation occurs at the mitochon- drial membrane and produces energy (ATP) using the electrons generated from glucose in the TCA cycle. A central part in the mitochondrial respiratory chain is NADH-ubiquinone oxidoreductase chain 5 (Mt-ND5), which was overrepresented after maturation in vitro. This enzyme transfers electrons from NADH out of the TCA cycle to the respiratory chain. The inter- mediate electron acceptor for the enzyme is FMN, which is reduced to FMNH2 in the electron transport chain and was also overrepresented in in vitro Page 14 of 24 Page 14 of 24 Walter et al. BMC Genomics (2019) 20:588 Page 14 of 24 This issue would result in increased energy requirements for nucleotide production compared to those in in vivo conditions, which explains the shift in glucose metabo- lism towards the PPP (PRPP production) and glycolysis (ATP production). matured cumulus cells. The overrepresentation of these enzymes in oxidative phosphorylation might be the consequence of an energy deficit in COCs during maturation in vitro. Purine and pyrimidine metabolism In in vitro matured cumulus, metabolomic compounds (3) and proteins (7) that can be associated with purine and pyrimidine metabolism were significantly overrepre- sented. The first important protein linking the pentose phosphate pathway (PPP) with purine and pyrimidine biosynthesis is ribose-phosphate pyrophosphokinase (PRPS1), which was overrepresented after maturation in vitro. PRPS1 produces the metabolite phosphoribosyl pyrophosphate (PRPP) from the substrate ribose-5- phosphate from the PPP. PRPP is used for purine and pyrimidine biosynthesis as well as purine salvage path- ways. Purine and pyrimidine metabolism in cumulus cells plays an important role in the orchestration of meiotic resumption. The increase in enzymes involved in purine biosynthesis in the cumulus of equine in vitro matured cumulus cells can be a result of decreased purine salvage compared to that in in vivo matured cu- mulus cells. Energy requirements (ATP) for the purine salvage pathways are distinctly lower (5x) as for de novo synthesis [82]. However, the rate of salvage pathway depends on PRPP concentration; therefore, it can be hypothesized that in vitro maturated cumulus cells suffer from a lack of PRPP. Upregulation of enzymes in de novo synthesis tries to compensate for this lack of PRPP. Hemoglobin A (HBA) is a non-steroid isoprenoid product of the mevalonate pathway, which was also un- derrepresented after maturation in vitro (see the section oxygen supply). Steroids can stimulate porphyrin (heme) biosynthesis [90] and increase Fe incorporation [91]. Therefore, the lack of steroids in in vitro matured cumu- lus cells can also result in suppressed heme biosynthesis. Amino acid metabolism The altered metabolome after maturation in vitro also indicates potential aberrations in the amino acid dis- tribution. Phosphohydroxypyruvate (PHPA) and phos- phoserine aminotransferase (PSAT1), which catalyse the conversion to phosphoserine in the serine biosyn- thesis pathway, were overrepresented after maturation in vitro. This result can be explained by the under- representation of a compound attributed to serine after IVM. Serine is not an essential amino acid; thus, the potential lack of this compound is most likely the result of a higher demand e.g., for nucleotide synthe- sis (see the section Purine and Pyrimidine Metabol- ism). The overrepresentation of compounds in serine biosynthesis support this hypothesis for an increased serine demand in vitro. Opposed to serine, a meta- bolic compound that can be attributed to leucine/iso- leucine was overrepresented after maturation in vitro. These aberrations in the equine “cumulome” after IVM raise the question of whether higher concentra- tions of serine in maturation media contribute to more physiological culture conditions for COC maturation. Extracellular matrix/proteoglycans Extracellular matrix (ECM) composition plays an impor- tant role in the fertilization of the oocyte. The ECM expands during the maturation process, and only success- fully expanded ECM around cumulus allows correct adhe- sion and oviductal pick-up of the COC, influencing sperm Walter et al. BMC Genomics (2019) 20:588 Page 15 of 24 Page 15 of 24 capacitation in human sperm [106]. FN content in hu- man follicular fluid seemed to be a marker for oocyte quality, maturity and fertilization capability [107, 108]. Recently, for mice COCs, the FN-integrin pathway was shown to play an important role in cumulus expansion during ovulation [109]. In human cumulus samples, FN in younger women was higher than that in older women [110]. Both findings illustrate that FN expression in cumulus cells is positively associated with the oocyte developmental potential. A novel specific splice variant of bovine FN was observed in cumulus cells, which raised the hypothesis of a special function of this variant in cumulus cells [111]. In vitro matured equine oocytes also possess a reduced capacity for further development, and reduced expression of FN was observed in this study. These data contribute to the available literature and indicate a role of FN as a cumulus marker for the developmental competence of corresponding oocytes. Additionally, a role in the mediation of equine sperm-oocyte contact seems likely but needs further investigation. motility and adhesion as well as fertilization [92–95]. Therefore, it is not surprising that a substantial portion of alterations in the “cumulome” after IVM is associated with ECM composition. p Through the gonadotrophin surge, cumulus cells are stimulated to form a hyaluronan-rich ECM responsible for the expansion of the CC [96]. However, inter-alpha- trypsin-inhibitor heavy chains 1–4 (ITIH1–4) were un- derrepresented in in vitro matured cumulus. This group of proteins is responsible for retaining hyaluronic acid (HA) in the cumulus matrix for expansion [97, 98]. A putative upregulated compound in in vitro matured CCs was chondroitin-4-sulfate (CHOND4). For mice, the binding of HA during maturation in the cumulus oophorus was hypothesized to be enabled by the release of chondroitin sulfate into the culture medium (in vitro) or follicular fluid (in vivo). This exchange of chondroitin sulfate with HA leads to stabilization of the cumulus ECM through a covalent interaction of HA and ITIH [99]. Extracellular matrix/proteoglycans These results are in accordance with the presented equine “cumulome” data where ITIHs were underrepre- sented after IVM and CHOND4 was overrepresented. That is, during IVM, CHOND4 might be not sufficiently released into the culture medium and replaced by HA. Mouse in vivo matured COCs were more resistant to shear stress than in vitro matured COCs, which supports this hypothesis [99]. g Interestingly, serine protease HTRA1 was significantly overrepresented in the in vitro matured cumulus. This protease has a variety of targets but especially degrades FN in the ECM. ECM remodelling by HTRA1 affects a variety of pathobiological conditions such as osteoarth- ritis, cancer, and Alzheimer’s disease [112]. In human cumulus cells, HTRA1 expression was significantly higher than that in granulosa cells [113], which can be attributed to a special role of this protein in cumulus cell ECM production. The upregulation of this protein in in vitro matured cumulus might be responsible for the un- derrepresentation of FN after maturation in vitro. Another protein underrepresented after IVM in this context was heparan sulfate proteoglycan 2 (HSPG2, Perlecan). HSPG2 is a core protein attached to three glycosaminoglycan chains (heparan sulfate or chondro- itin sulfate). HSPG2 is a main component of basement membranes [100], but its expression in cumulus cells of germinal vesicle stage oocytes from women [101] and granulosa cells from cows [60] was previously docu- mented. Heparan sulfate proteoglycan expression peaked in rat preovulatory granulosa cells where the core proteins, such as HSPG2, remaining constant through- out the cycle. These proteoglycans can bind and activate antithrombin III (also underrepresented in in vitro matured cumulus); thus, they also possess a role in the control of proteolysis and fibrin formation [102] (see chapter coagulation cascade). p Another protein that was underrepresented in in vitro matured cumulus and plays an important role in the ability of oocytes to achieve their full developmental competence is gap junction alpha 1 protein (GJA1; Connexin-43 (CX43)). Oocytes share close bidirectional communication with their surrounding oocytes. The exchange of small molecules and cellular coupling via gap junctions between cumulus cells and their surround- ing oocyte is especially important [114]. The crucial role of CX43-mediated cumulus oocyte communication for the meiotic maturation of oocytes was documented for bovine [115] as well as human oocytes [116]. The pre- sence of open gap junctions in granulosa cells is neces- sary for the maintenance of the oocyte in meiotic arrest. Extracellular matrix/proteoglycans Granulosa cells deliver cGMP through gap junctions to the oocyte; this process maintains high levels of cAMP in the oocyte high as well as meiotic arrest [117]. Diffe- rences in gap-junctional coupling of equine oocytes were found with regard to breeding season; 90% coupling was detected in breeding season versus 55% interrupted communication in the non-breeding season [118]. Vitronectin (VN), which was underrepresented after IVM, is a glycoprotein found in ECM that promotes cell adhesion. VN plays a role in the cytolytic complement pathway through the regulation of membrane attack complex (MAC) formation [103] (Fig. 3). VN in bovine cumulus ECM showed a negative effect on sperm moti- lity [104] as well as a dose dependent effect on oocyte- sperm interactions [105]. Another important protein in ECM that was underrepresented after IVM is fibronectin (FN), another adhesive glycoprotein. FN is secreted by cumulus cells during maturation [93], and induced Page 16 of 24 Walter et al. BMC Genomics (2019) 20:588 Page 16 of 24 Walter et al. BMC Genomics (2019) 20:588 Walter et al. BMC Genomics (2019) 20:588 Meiotic resumption of equine oocytes seemed to be associated with a decline in CX43 protein in cumulus cells, with significantly higher levels in in vitro than in in vivo matured cumulus cells [119]. Nonetheless, available equine gene expression data for GJA1 (CX43) in equine cumulus cells showed no variation between in vivo and in vitro maturated cumulus cells. Therefore, altered protein expression seems to be a result of post-tran- scriptional regulation [120]. For bovine oocytes, the ex- pression level of CX43 in cumulus cells significantly correlated with the developmental competence of the cor- responding oocyte [114]. Whether this correlation is also valid for the equine species needs further investigations. downregulated in the in vitro matured cumulus. In total, 11 unique proteins are associated with the complement cascade (Table 1, Fig. 3). The complement system is part of the innate immune system and includes 30 proteins overall. In this study, proteins of the classical and alternative pathway of complement activation are represented in the group of proteins with significantly lower expression. In addition to the complement factors C3, C4, C5 and C7 and the positive regulator CFB, nega- tive regulators of the complement cascade (IC1, C4BP, CFI, CFH, CLUS, VN) belonged to the underexpressed proteins in the in vitro matured cumulus cells (Fig. 3). Extracellular matrix/proteoglycans p ( g ) An active complement system in the follicular fluid is relevant for ovulation in vivo [127–129]. Previous studies on the follicular fluid of mares revealed the pre- sence of complement proteins in follicular fluid [50] with seasonal variation [51]. Several complement pro- teins seem to be shed within cell secreted vesicles into the follicular fluid of mares [130]. Confirmation of gene expression for several complement proteins in human granulosa cells supports the hypothesis that these cells are able to actively secrete complement factors [131]. For component C3, available studies indicate a role in the developmental competence of the oocyte [132–134]. The reduced presence of 11 proteins of the complement cascade supports the hypothesis that the lack of com- plement proteins is responsible for the reduced develop- mental competence of equine oocytes matured in vitro. Moreover, complement component 3 plays a role in sperm-oocyte-interactions [135, 136]. Transferring this context to the equine species, the lack of complement factors in equine CCs after IVM contribute to the in- ability of equine sperm to fertilize COCs. Further ex- periments are necessary to clarify the role of the complement system in the maturation process and the mediation of equine sperm-oocyte interactions. Thus far, the discussed alterations with regard to ECM composition were mostly compounds identified as under- represented after maturation in vitro. Some candidates showed an opposite trend with overrepresentation after IVM. One of these candidates, dystroglycan (DAG1), is an ECM glycoprotein that is poorly characterized in follicular cells compared to some of the previously discussed mo- lecules. In porcine granulosa cells, DAG1 showed a de- crease in concentration with increasing follicle size [121]. g Overrepresented epidermal growth factor receptor (EGFR) is the transmitter of extracellular events in the cells. EGFR kinase activity is responsible for gap junction closure in response to LH, which results in meiotic resumption [117]. The functionality of EGFR seems to be one key factor for the oocyte to acquire developmen- tal competence [122]. The addition of epidermal growth factor (EGF) to the maturation medium of equine oocytes significantly increases maturation rates [123]. For bovine oocytes, EGFR expression in cumulus cells seems to be a marker for oocyte developmental compe- tence [23]. All these data indicate a positive effect of EGFR activity on oocyte developmental competence. Extracellular matrix/proteoglycans The overexpression of EGFR receptor in the in vitro ma- tured samples can be a result of the underrepresentation of a metabolic compound assigned to oestrone-s (under- represented in in vitro matured cumulus, see the chapter steroid metabolism). In breast cancer, oestrogen was responsible for maintaining low levels of EGFR expres- sion [124]. Another explanation for this upregulation delivers the addition of 50 mg/ml EGF to the maturation medium. EGF showed a positive effect on maturation in many species and is routinely added to equine ma- turation media [125]. In hepatic epithelial cells, EGF increased EGFR mRNA 3–5-fold [126]. Coagulation Cascade In in vitro matured cumulus, a wide variety of factors par- ticipating in the coagulation cascade were significantly un- derrepresented (n = 13; Table 2; Fig. 3). Three fibrinogen chains (A,B,G), which are converted into fibrin in the coagulation process by thrombin (F2a), belong to this protein group. Thrombin is derived through enzymatic cleavage of prothrombin (F2), which was also under- represented in in vitro matured cumulus. Additionally, the following group of thrombin inhibitors with anticoagulant activity was underrepresented: heparin cofactor 2 (HEP2), plasma serine protease inhibitor (IPSP), antithrombin 3 (ANT3), and plasma protease C1 inhibitor (IC1). More- over, proteins involved in fibrinolysis were underrepre- sented. Plasmin (PLM) is the enzyme responsible for fibrinolysis; the precursor of PLM, plasminogen (PLG), as well as the following proteins with inhibitory effects on Complement Cascade The most dominant proteome result is the massive over- representation of the complement and coagulation cascades (pathway ID 04610; String Enrichment Analysis n = 21 proteins; complement cascade n = 8 proteins; false discovery rate 1.3e−32) in the group of proteins Page 17 of 24 Page 17 of 24 Page 17 of 24 Walter et al. BMC Genomics (2019) 20:588 Walter et al. BMC Genomics (2019) 20:588 Walter et al. BMC Genomics (2019) 20:588 biosynthesis in cumulus cells. VN, FN, complement C3, A2M and antithrombin III are potential players in equine sperm-oocyte interaction and attraction and were underrepresented after maturation in vitro. These candidates deserve more attention to improve equine IVF success in the future. PLM were underrepresented in the in vitro matured group: alpha-2-antiplasmin (A2AP); alpha-2-macroglobulin (A2M) and alpha-1-antiproteinase 2 precursor (A1AT). In sum- mary, the in vitro matured cumulus appears to lack fibrino- gen as well as the two main progenitors for coagulation (F2) and fibrinolysis (PLG). The shortage of these factors seems to be the result of massive consumption during COC collection or IVM. In support of this hypothesis, inhibitory factors for coagulation (IPSP, IC1, ANT3, and HEP2) as well as inhibitory factors for fibrinolysis (A1AT, A2AP, IC1, and A2M) were also underrepresented in the in vitro matured cumulus. Overall, the presented alterations in the “cumulome” after IVM point towards the future direction for the development of more physiological IVM conditions. Fine adjustment of media composition needs to focus on fatty acids, amino acids and purines. This adjustment will contribute to overcome the need for supraphysiological oxygen and glucose concentrations, which seem to aid the COC only by compensating for actual shortcomings in media. The coagulation system is a dominant part of equine follicular fluid [50, 51]. F2 and PLG were detected mainly during spring anovulatory season. Fluctuation in follicular coagulation factors throughout the reproductive season de- livers explanations for different incidences for hemorrhagic anovulatory follicles [51]. Fibrinogen concentrations in equine follicular fluid were measured 40% higher than those in plasma [137]. Therefore, there must be local production/secretion of fibrinogen from follicular cells. Analysis of gene expression from human granulosa and cumulus cells of preovulatory follicles revealed the selective expression of fibrinogen in granulosa cells [138]. Coagula- tion system proteins that were upregulated in follicular fluid from women who underwent IVF successfully included fibrinogen, kininogen-1, prothrombin and coagu- lation factor XII [139]. In vivo COC collection Oestrous was induced by injection of 1 ml Cloprostenol (Estrumate®, MSD Animal Health GmbH, Luzern, Switzerland) in mares (n = 7) owned by the University of Zurich that were designated for slaughter for non- reproductive reasons (Additional file 4: Table S4). Mares were regularly checked by transrectal ultrasonography; when a follicle over 35 mm along with uterine oedema was detected, ovulation was induced by injection of 2500 I.U. hCG (Chorulon®, MSD Animal Health GmbH, Luzern, Switzerland). Slaughter was scheduled 30 h after injection, and ovaries were excised from the carcasses to harvest COCs from the dominant follicle by follicular scraping. As expected, all recovered COCs had a nicely expanded CC. Each COC was washed four times in 100 μl phosphate buffered saline solution containing bovine serum albumin (PBS-BSA). In the last step, oocytes were denuded using The Stripper® (Cooper Surgical Fertility &Genomic Solutions, Malov, Denmark), and 3 μl of the CC was collected for analysis. Denuded oocytes were scanned under an inverted microscope for extrusion of polar bodies. All COCs used in the analysis (n = 8) were successfully matured, with extrusion of the first polar body. Animal testing authorisation for collection of in vivo matured oocytes was permitted by the Zurich cantonal veterinary office (authorisation number 153/13). Complement Cascade Regarding the role of the coagula- tion system on oocyte maturation, available data are rare. The analysis of bovine cumulus cell gene expression after IVM revealed a significant enrichment of genes (n = 5) involved in the complement and coagulation pathway [32]. Through upregulation of the transcriptional machinery for these proteins, the cells try to compensate for the increased consumption of these proteins during IVM. Beyond the more general role of the coagulation system, maturation-specific functions in the fertilization process are possible. Antithrombin III (Serpin C1) plays a role as chemoattractant for sperm [140]. A derivate of A2M stimulates spermatozoa-zona pellucida binding in the human cumulus matrix [141]. Therefore, the study revealed specific candidates that hold the potential to improve the fertilization of equine COCs. Untargeted metabolomics analysis Metabolites of single CCs were profiled using nano high- performance liquid chromatography mass spectrometry (UPLC–nanoESI-MS) in negative mode similar to that suggested by Paglia et al. [142]. In brief, cells were lysed and extracted in 100 μl MeOH/H2O (9:1 v:v) using 4 freeze/thaw cycles (1 min liquid nitrogen; 5 min high in- tensity focused ultrasound (HIFU)). After centrifugation (15 min at 13000 rpm, at 4 °C), the supernatant with lipids and metabolites was collected and stored at −20 °C. The pellet was collected and immediately processed for pro- teomics analyses (see below). Prior to analysis, the extracts were dried under a stream of nitrogen and reconstituted in 20 μl water, further diluted with 80 μl 50 mM ammo- nium acetate in acetonitrile/MeOH (90:9 v:v) adjusted with ammonium hydroxide to pH 9. Metabolites were se- parated on nanoAcquity UPLC (Waters) equipped with a BEH-Amide capillary column (200 μm × 150 mm, 1.7 μm particle size, Waters) by applying a gradient of 0.5 mM ammonium acetate in water adjusted with ammonium hydroxide to pH 9 (A) and 0.5 mM ammonium acetate in acetonitrile adjusted with ammonium hydroxide to pH 9 (B) from 90% B to 50% B. The injection volume was 1 μl. The UPLC was coupled to a Synapt HDMS G2 mass spectrometer (Waters) by a nanoESI source. MS data were acquired using negative polarization and all ion fragmen- tation (MSE) over a mass range of 50 to 1200 m/z at a resolution of 22,000 (MS and MSMS). All solvents used were of quality HPLC grade (Chromasolv, Sigma-Aldrich). Metabolite data sets were evaluated with Progenesis QI software (Nonlinear Dynamics, A Waters Company), which aligns the ion intensity maps based on a reference data set, followed by peak picking on an aggregated ion intensity map. Detected ions were identified based on accurate mass, and detected adduct patterns and isotope patterns by comparison with entries in the Human Meta- bolome Database (HMDB). A mass accuracy tolerance of 0.025 Da was set for the searches. Fragmentation patterns were considered for the identifications of metabolites. Quality controls were run on pooled samples and Analysis of all biological samples (n = 15) was performed in one analytical run in random order using reverse- phaseLC-MS/MS on an Orbitrap Fusion mass spectrom- eter (Thermo Scientific) coupled to a nano HPCL system (EASY-nLC 1000, Thermo Scientific) in data dependent acquisition (DDA) mode. In vitro COC collection In summary, according to these key findings, metabolism under in vitro conditions seems to focus on fuelling cells with energy via aerobic glycolysis as important candidates involved in oxygen supply and glucose metabolism were altered. This alteration can be the result of the culture system used with high levels of oxygen and glucose. With the help of these compounds, a lack of other important substrates (purines, cholesterol/steroids, lipids, and amino acids) for the COC might be overcome by increased Oocytes for IVM (n = 7) were collected from mares (n = 5) slaughtered at local abattoirs. The animals were out of oestrous and slaughtered for non-reproductive reasons (Additional file 4: Table S4). Ovaries were excised from the carcasses, and COCs from non-dominant follicles were recovered by scraping. Only oocytes with compact cumulus oophorus were selected for the IVM process. All cells in close connection to the oocyte were defined as Page 18 of 24 Walter et al. BMC Genomics (2019) 20:588 reference compound mixtures to determine technical accuracy and stability. cumulus oophorus. IVM was performed for 30 h in 30 μl droplets of Advanced DMEM/F12 based maturation medium (Advanced DMEM/F-12, Thermo Fisher Scien- tific, with 2.5 mM Glutamax, 2.2 g/l NaHCO3, 1 ml/100 ml foetal bovine serum, 50 ng/ml EGF, 10 IU/ml PMSG and 5 IU/ml hCG). The maturation rate was 62%. After the IVM period, COCs were treated as described for the in vivo matured group with washing and denudation in PBS-BSA. Only cumulus samples from successfully matured oocytes were used for this study. All cumulus samples were snap frozen and stored in liquid nitrogen until preparation for “cumulomics” analysis. cumulus oophorus. IVM was performed for 30 h in 30 μl droplets of Advanced DMEM/F12 based maturation medium (Advanced DMEM/F-12, Thermo Fisher Scien- tific, with 2.5 mM Glutamax, 2.2 g/l NaHCO3, 1 ml/100 ml foetal bovine serum, 50 ng/ml EGF, 10 IU/ml PMSG and 5 IU/ml hCG). The maturation rate was 62%. After the IVM period, COCs were treated as described for the in vivo matured group with washing and denudation in PBS-BSA. Only cumulus samples from successfully matured oocytes were used for this study. All cumulus samples were snap frozen and stored in liquid nitrogen until preparation for “cumulomics” analysis. Proteomics analysis After initial methanol extraction for metabolomics ana- lysis, the pellet was immediately prepared for proteomic analysis. Therefore, a sonoreactor-based cell lysis protocol (SR) [143] was combined with FASP (adapted from [39]). The SR-FASP protocol was specifically developed for the analysis of the proteome for single COCs [40]. The pellet was dissolved in 30 μl SDS lysis buffer (4% SDS, 100 mM Tris/HCL pH 8.2, 0.1 M DTT–dithiothreitol) and incu- bated at 95 °C for 5 min. In the next step, samples were treated with HIFU for 10 min with amplitude of 65% in cycle 0.5 (Sonoreactor UTR200; Hielscher Ultrasonics GmbH). After cell lysis, protein concentration was estimated with a Qubit® Protein Assay Kit (Life Technolo- gies). A total of 10 μg of proteins were used for the adapted FASP protocol [39]. Proteins were diluted in 200 μl UT buffer (Urea 8 M in 100 nM Tris/HCL, pH 8.2) and loaded on a Microcon-30 kDa Centrifugal Filter Unit with Ultracel-30 membrane (Merck Millipore). The unit was centrifuged at 14,000 g for 25 min at room temperature. A wash with 200 μl UT buffer followed by centrifugation at 14,000 g for 25 min was performed. Re- duced proteins were alkylated with 100 μl iodoacetamide 0.05 M in UT buffer during an incubation of 5 min, followed by three washing steps with 199 μl UT and two steps with 100 μl NaCl 0.5 M. Protein digestion on the fil- ter unit was performed overnight in a wet chamber at room temperature using 120 μl 0.05 M triethylammonium bicarbonate buffer (pH 8.5) with trypsin (Promega) in a ra- tio of 1:50 (w/w). After elution at 14,000 g, the peptide so- lution was acidified using trifluoroacetic acid (TFA) to a final concentration of 0.5%. Peptides were desalted using Finisterre solid phase extraction C18 columns (Teknok- roma), dried in a vacuum concentration and resolubilized in LC-MS solution (3% acetonitrile, 0.1% formic acid). Untargeted metabolomics analysis A homemade frit-column (75 μm × 150 mm) packed with reverse phase material (ReproSil-Pur 120, C18-AQ, 1.9 μm (Dr. Maisch HPLC GmbH)) was coupled to the MS with a fused-silica spray emitter (20 μm × 8 cm, tip: 10 ± 1 μm; New Objective). A defined amount of 500 ng peptides per sample was loaded to the column and analyzed by LC-MS/MS. For channel A, the solvent composition was 0.1% formic acid in water, and for channel B, it was 0.1% formic acid in acetonitrile. Elution of peptides was performed using a flow rate of 300 nl/min with a gradient of 1 to 35% acetonitrile over Page 19 of 24 Page 19 of 24 Walter et al. BMC Genomics (2019) 20:588 Walter et al. BMC Genomics (2019) 20:588 120 min, followed by a cleaning period for 10 min with 98% acetonitrile. Full-scan mass spectra (300–1500 m/z) were acquired with a resolution of 60,000 at 200 m/z after accumulation to a target value of 5e5. Look mass correc- tion (371,1010 and 445,12,003 m/z) was used for internal calibration, and the maximum cycle time between pre- cursor masses was set to 3 s. Data dependent MS/MS were recorded in a linear ion trap using quadrupole isola- tion in a window of 0.7 Da. Selected ions were fragmented with 30% fragmentation energy. The ion trap was run in rapid scan mode with 1e2 as the target value and a ma- ximum injection time of 35 ms. Precursor ions with charge state from + 2 to + 6 and a signal intensity of at least 1e4 were selected for fragmentation. For 25 s, a dynamic exclusion list was applied with activation of maximum parallelizing ion injections. As reference, a pool containing 5 μl of each cumulus sample was analysed in the same analytical run and used as reference for aligning in data analysis. discovery rate for quantifiable proteins with at least two peptides was estimated at 0.2% using the target-decoy strategy [144]. For protein quantification, the average of the normalised abundance from the most intense 3 peptide ions of each protein group were calculated indi- vidually for each sample [146]. This generates the norma- lised quantitative protein abundance. Statistical testing was performed on hyperbolic arcsine transformed values using ANOVA. Differentially expressed proteins were defined with a fold change > 2 along with p ≤0.05. Untargeted metabolomics analysis Enrich- ment analysis of overexpressed proteins in KEGG path- ways was performed online using STRING-Database (http://string-db.org) [147]. All mass spectrometry pro- teomics data were handled using the local laboratory in- formation management system [148] and all relevant data have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identi- fier PXD011086 [149]. Integrated analysis of proteomic and metabolomic results was performed using KEGG Mapper v3.1 (release date October 1st 2017; http://www.genome.jp/kegg/map- per.html). Label-free quantification was conducted with Progen- esis QI for Proteomics Software (Nonlinear Dynamics, A Waters Company). The reference for automatic aligning was the raw-file of the sample pool. For peak picking, the high sensitivity option was chosen, and only peptides with charge 2, 3 and 4 were used for analysis. The top five mass spectra were exported for a database search using charge deconvolution and deisotoping options with a minimum fragment count of 200 peaks per MS/ MS [144]. Spectra were searched against the NCBI data base for horses (NCBI Taxonomy ID 9796, release date 20,170,523). For functional downstream analysis of pro- teins, the database was blasted to human homologous proteins from the canonical UniProt database (Tax ID: 9606, 20,161,209: file: fgcz_9606_reviewed_cnl_conta- minantNoHumanCont_20,161,209.fasta). The database was concatenated with reversed sequence information for estimation of the false discovery rate [144]. The search was run on Mascot Server v.2.4.3. (Matrix Science), with a tolerance of 10 ppm for precursor ion mass and 0.5 Da for fragment ion tolerance. Enzyme spe- cificity was restricted to trypsin with an allowed maximum of 2 missed cleavage sites. As fixed modification, only carbamidomethylation of cysteine was specified, and for variable modifications, deamidation of glutamine and asparagine as well as protein n-terminus acetylation were selected. Protein probabilities by the protein prophet algo- rithm [145] were analysed in Scaffold v4.1.1 (Proteome Software Inc.). Proteins containing similar peptides that could not be discriminated by the MS/MS analysis were grouped according to the principles of parsimony. To link the MS1 features in Progenesis QI for proteomics with peptide and protein information, we uploaded a Scaffold spectrum report filtered for false discovery rates on the peptide (5%) and protein (10%) levels. The overall false Additional files Additional file 1: Table S1. All 216 differentially expressed proteins (p ≤0.05; FC ≥2); 204 with unique UniProt IDs. Of these proteins, 95 were significantly underexpressed in the in vitro matured group, and 86 of these proteins were linked to a unique orthologous human UniProt ID (Fig. 1). In the in vitro group, 121 proteins (118 with unique orthologous human UniProt IDs) were significantly overexpressed. (XLSX 140 kb) Additional file 2: Table S2. All 108 metabolic compounds with a significant different (p < 0.05; FC > 2) abundance between the two maturation groups. Here, 84 compounds showed a higher abundance in the in vitro group and 24 compounds a lower abundance compared to those in the in vivo matured group. For 28 compounds, putative metabolite IDs were found (6 with lower abundance and 22 with higher abundance after in vitro maturation). (XLSX 84 kb) Additional file 3: Table S3. All 905 measured metabolomic compounds (Progenesis QI Output Measurements). (XLSX 481 kb) Additional file 3: Table S3. All 905 measured metabolomic compounds (Progenesis QI Output Measurements). (XLSX 481 kb) Additional file 4: Table S4. Additional information on donor mares and follicles for the in vivo and in vitro matured groups. Mann-Whitney test indicated no significant difference for mare age (p = 0.2) and a significant difference for follicle size (p = 0.04) between the two maturation groups. (XLSX 10 kb) (XLSX 10 kb) (XLSX 10 kb) 2PG: 2-phospho-d-glyceric acid; A1AT: Alpha-1-antiproteinase 2 precursor (A1AT); A2AP: Alpha-2-antiplasmin; A2M: Alpha-2-macroglobulin; AARS: Alanine t-RNA ligase; ACACA: Acetyl-CoA carboxylase 1; ACOD: Acyl- CoA desaturase; ACSL: Long-chain-fatty-acid-CoA ligase 3; ADIPO: Adiponectin; ANT3: Antithrombin 3; APOA1 and 2: Apolipoprotein A1 and 2; APOH: Apolipoprotein H; C3: Complement C3; C4: Complement 4 A/B (C4); C4BP: C4b-binding protein alpha chain; C5: Complement C5; C7: Complement C7; CC: Cumulus complex; CFB: Complement factor B; CFH: Complement factor H X1; CFI: Complement factor I; CHOND4: Chondroitin-4-sulfate; CLUS: Clusterin; COC: Cumulus oocyte complex; CP: Ceruloplasmin; DAG1: Dystroglycan; dCMP: Deoxycytidine monophosphate; DESI: Desorption electrospray ionization mass spectrometry; DTT: Dithiothreitol; EGFR: Epidermal Growth Factor Receptor; ENPP4: Bis(5′- adenosyl)-triphosphatase; F2: Prothrombin; FABP: Fatty acid binding protein; Abbreviations 2PG 2 h h 2PG: 2-phospho-d-glyceric acid; A1AT: Alpha-1-antiproteinase 2 precursor (A1AT); A2AP: Alpha-2-antiplasmin; A2M: Alpha-2-macroglobulin; AARS: Alanine t-RNA ligase; ACACA: Acetyl-CoA carboxylase 1; ACOD: Acyl- CoA desaturase; ACSL: Long-chain-fatty-acid-CoA ligase 3; ADIPO: Adiponectin; ANT3: Antithrombin 3; APOA1 and 2: Apolipoprotein A1 and 2; APOH: Apolipoprotein H; C3: Complement C3; C4: Complement 4 A/B (C4); C4BP: C4b-binding protein alpha chain; C5: Complement C5; C7: Complement C7; CC: Cumulus complex; CFB: Complement factor B; CFH: Complement factor H X1; CFI: Complement factor I; CHOND4: Chondroitin-4-sulfate; CLUS: Clusterin; COC: Cumulus oocyte complex; CP: Ceruloplasmin; DAG1: Dystroglycan; dCMP: Deoxycytidine monophosphate; DESI: Desorption electrospray ionization mass spectrometry; DTT: Dithiothreitol; EGFR: Epidermal Growth Factor Receptor; ENPP4: Bis(5′- adenosyl)-triphosphatase; F2: Prothrombin; FABP: Fatty acid binding protein; 2PG: 2-phospho-d-glyceric acid; A1AT: Alpha-1-antiproteinase 2 precursor (A1AT); A2AP: Alpha-2-antiplasmin; A2M: Alpha-2-macroglobulin; AARS: Alanine t-RNA ligase; ACACA: Acetyl-CoA carboxylase 1; ACOD: Acyl- CoA desaturase; ACSL: Long-chain-fatty-acid-CoA ligase 3; ADIPO: Adiponectin; ANT3: Antithrombin 3; APOA1 and 2: Apolipoprotein A1 and 2; APOH: Apolipoprotein H; C3: Complement C3; C4: Complement 4 A/B (C4); C4BP: C4b-binding protein alpha chain; C5: Complement C5; C7: Complement C7; CC: Cumulus complex; CFB: Complement factor B; CFH: Complement factor H X1; CFI: Complement factor I; CHOND4: Chondroitin-4-sulfate; CLUS: Clusterin; COC: Cumulus oocyte complex; CP: Ceruloplasmin; DAG1: Dystroglycan; dCMP: Deoxycytidine monophosphate; DESI: Desorption electrospray ionization mass spectrometry; DTT: Dithiothreitol; EGFR: Epidermal Growth Factor Receptor; ENPP4: Bis(5′- adenosyl)-triphosphatase; F2: Prothrombin; FABP: Fatty acid binding protein; Page 20 of 24 Walter et al. BMC Genomics (2019) 20:588 Walter et al. BMC Genomics (2019) 20:588 Zurich, 8057 Zurich, Switzerland. 3Institute of Pharmacology and Toxicology, Vetsuisse Faculty, University of Zurich, 8057 Zurich, Switzerland. Abbreviations 2PG 2 h h FASN: Fatty acid synthase; FASP: Filter-aided sample preparation; FDR: False discovery rate; FGA: Fibrinogen alpha chain; FGB: Fibrinogen beta chain; FGG: Fibrinogen gamma chain; FMNH2: Reduced flavin mononucleotide (FMNH2); FN: Fibronectin; Geranyl-PP: Geranyl Pyrophosphate; GFPT1&2: Glutamine-fructose-6-phosphate aminotransferase 1&2; GJA1: Gap junction alpha 1 protein; GLUT1: Solute Carrier Family 2, facilitated glucose transporter member 1; GMPS: Guanine Monophosphate Synthase; Guano: Guanosine; HBA: Hemoglobin A; HBB: Hemoglobin B; hcG: Human chorionic gonadotrophin; HEMO: Hemopexin; HEP2: Heparin Cofactor 2; Hexose-P: Hexose-Monophosphate; HMB: Hydroxymethylbilane; HMDB: Human metabolome database; HSPG2: Basement membrane-specific heparan sulfate proteoglycan core protein; HTRA1: Serine Protease HTRA1; HypoX: Hypoxanthine; I.U.: International units; IC1: Plasma protease C1 inhibitor; IC1: Plasma protease C1 inhibitor (IC1); ICSI: Intracytoplasmic sperm injection; ID: Identities; IMDH2: Inosine-5′-monophosphate dehydrogenase 2; IPSP: Plasma serine protease inhibitor; ITIH1–4: Inter-alpha-trypsin-inhibitor heavy chain H1–4; IVF: In vitro fertilization; IVM: In vitro maturation; IVP: In vitro production; KEGG: Kyoto encyclopedia of genes and genomes; KNG: Kininogen-1; MAC: Membrane attack complex; Mt-ND5: NADH-ubiquinone oxidoreductase chain 5; MVD: Diphosphomevalonate decarboxylase (MVD);; Oestrone-S: Oestrone sulfate (Estrone-S); PANT: Pantetheine; PEP: Phosphoenolpyruvate; PFAS: Phosphoribosylformylglycinamidine synthase; PFK-M: ATP dependent 6-phosphofructokinase muscle type; PHPA: Phosphohydroxypyruvic acid (PHPA); PLG: Plasminogen; POLR2: DNA- directed RNA polymerases I, II, and III subunit RPABC1; PRPS1: Ribose-phosphate pyrophosphokinase; PURH: Bifunctional purine biosynthesis protein; SERC: Phosphoserine aminotransferase; TF: Transferrin; TFA: Trifluoroacetic acid; UMPS: Uridine 5′-monophosphate synthase; VN: Vitronectin Zurich, 8057 Zurich, Switzerland. 3Institute of Pharmacology and Toxicology, Vetsuisse Faculty, University of Zurich, 8057 Zurich, Switzerland. Zurich, 8057 Zurich, Switzerland. 3Institute of Pharmacology and Toxicology, Vetsuisse Faculty, University of Zurich, 8057 Zurich, Switzerland. Funding Th “ 13. Kosteria I, Anagnostopoulos AK, Kanaka-Gantenbein C, Chrousos GP, Tsangaris GT. The use of proteomics in assisted reproduction. In Vivo. 2017;31:267–83. The “cumulomics” project was funded by the “Forschungskredit” of the University of Zurich (“Clinomics-Project”; Grant FK-13-062), which supports young researchers (JW) of the University of Zurich. 14. Seli E, Robert C, Sirard M-A. OMICS in assisted reproduction: possibilities and pitfalls. Mol hum Reprod, vol. 16; 2010. p. 513–30. Available from: http:// www.ncbi.nlm.nih.gov/pubmed/20538894. 2010 ed Acknowledgements 7. Leemans B, Gadella BM, Stout TAE, De Schauwer C, Nelis H, Hoogewijs M, et al. Why doesn't conventional IVF work in the horse? The equine oviduct as a microenvironment for capacitation/fertilization. Reproduction. 2016;152:R233–45. 7. Leemans B, Gadella BM, Stout TAE, De Schauwer C, Nelis H, Hoogewijs M, et al. Why doesn't conventional IVF work in the horse? The equine oviduct as a microenvironment for capacitation/fertilization. Reproduction. 2016;152:R233–45. The authors gratefully acknowledge the help of Benita Pineroli in the IVF laboratory and thank Aldona Pieńkowska-Schelling for providing her microscopy expertise. 8. Palmer E, Bezard J, Magistrini M, Duchamp G. In-vitro fertilization in th horse - a retrospective study. J Reprod Fertil. 1991;44:375–84. 8. Palmer E, Bezard J, Magistrini M, Duchamp G. In-vitro fertilization horse - a retrospective study. J Reprod Fertil. 1991;44:375–84. Received: 2 October 2018 Accepted: 23 May 2019 Received: 2 October 2018 Accepted: 23 May 2019 References S TJ 1. Scott TJ, Carnevale EM, Maclellan LJ, Scoggin CF, Squires EL. Embryo development rates after transfer of oocytes matured in vivo, in vitro, or within oviducts of mares. Theriogenology. 2001;55:705–15 Available from: http://www.sciencedirect.com/science/article/pii/S0093691X01004381. 2. Foss R, Ortis H, Hinrichs K. Effect of potential oocyte transport protocols on blastocyst rates after intracytoplasmic sperm injection in the horse. Equine Vet J Suppl. 2013;45:39–43. 3. Practice Committees of the American Society for Reproductive Medicine and the Society for Assisted Reproductive Technology. In vitro maturation: a committee opinion. Fertil Steril. 2013;99:663–6. 4. Buckett WM, Chian R-C, Dean NL, Sylvestre C, Holzer HEG, Tan SL. Pregnancy loss in pregnancies conceived after in vitro oocyte maturation, conventional in vitro fertilization, and intracytoplasmic sperm injection. Fertil Steril. 2008;90:546–50 Available from: http://www.ncbi.nlm.nih.gov/pubmed/ 17904128. 2007 ed. 5. Child TJ, Phillips SJ, Abdul-Jalil AK, Gulekli B, Tan SL. A comparison of in vitro maturation and in vitro fertilization for women with polycystic ovaries. Obstet Gynecol. 2002;100:665–70 2002nd ed. PHPA: Phosphohydroxypyruvic acid (PHPA); PLG: Plasminogen; POLR2: DNA- directed RNA polymerases I, II, and III subunit RPABC1; PRPS1: Ribose-phosphate pyrophosphokinase; PURH: Bifunctional purine biosynthesis protein; 6. Walls ML, Hunter T, Ryan JP, Keelan JA, Nathan E, Hart RJ. In vitro maturation as an alternative to standard in vitro fertilization for patients diagnosed with polycystic ovaries: a comparative analysis of fresh, frozen and cumulative cycle outcomes. Hum Reprod. 2015;30:88–96. 6. Walls ML, Hunter T, Ryan JP, Keelan JA, Nathan E, Hart RJ. In vitro maturation as an alternative to standard in vitro fertilization for patients diagnosed with polycystic ovaries: a comparative analysis of fresh, frozen and cumulative cycle outcomes. Hum Reprod. 2015;30:88–96. SERC: Phosphoserine aminotransferase; TF: Transferrin; TFA: Trifluoroacetic acid; UMPS: Uridine 5′-monophosphate synthase; VN: Vitronectin SERC: Phosphoserine aminotransferase; TF: Transferrin; TFA: Trifluoroacetic acid; UMPS: Uridine 5′-monophosphate synthase; VN: Vitronectin Authors’ contributions JW: concept of the study, experimental design, analysis and interpretation of data and writing the manuscript; FH: clinical part of the study, involvement in IVM laboratory part of the study; CF: experimental design, proteome analysis; JG: experimental design, proteomic data analysis and interpretation; BR: concept of the study, experimental design, proteome analysis and interpretation of data; JH: metabolome analysis; HN: knowledge transfer, critical discussion on experimental design and data; EL: experimental design, metabolome analysis, analysis and critical discussion of data; UB: concept of the study, experimental design, critical discussion of the data. 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https://openalex.org/W4309131653	https://zenodo.org/record/7159278/files/2022%20IEEE%20escience%20-%20compsys%20-%20final.pdf	English	null	Towards a Dynamic Composability Approach for using Heterogeneous Systems in Remote Sensing	arXiv (Cornell University)	2,022	cc-by	820	Towards a Dynamic Composability Approach for Using Heterogeneous Systems in Remote Sensing Ilkay Altintas1, Ismael Perez1, Dmitry Mishin1, Adrien Trouillaud2, Christopher Irving1, John Graham1, Mahidhar Tatineni1, Thomas DeFanti1, Shawn Strande1, Larry Smarr1, Michael L. Norman1 1 University of California, San Diego 2 Admiralty, https://admiralty.io/ Towards a Dynamic Composability Approach for Using Heterogeneous Systems in Remote Sensing Ilkay Altintas1, Ismael Perez1, Dmitry Mishin1, Adrien Trouillaud2, Christopher Irving1, John Graham1, Mahidhar Tatineni1, Thomas DeFanti1, Shawn Strande1, Larry Smarr1, Michael L. Norman1 1 University of California, San Diego 2 Admiralty, https://admiralty.io/ Ismael Perez (i3perez@sdsc.edu) Ismael Perez (i3perez@sdsc.edu) Ismael Perez (i3perez@sdsc.edu) Expanse Partitions Expanse Partitions Two partitions exist in Expanse • Slurm • Kubernetes Dynamic provision via cm-scale (Bright Computing) User Web Portal (ACCESS) Ismael Perez (i3perez@sdsc.edu) Ismael Perez (i3perez@sdsc.edu) utilus – National Research Platform Nautilus – National Research Platform Image source: https://pacificresearchplatform.org/nautilus/ Image source: https://pacificresearchplatform.org/nautilus/ Federation Layer Federation Layer Admiralty Federation Topologies • Topologies • Central control plane • Cloud Bursting • Decentralized • Connections form a directed graph • Cluster A is a source B == Cluster B is a target A • Bilateral connections • Target themselves p g Central control plane Cloud Bursting Decentralized onnections form a irected graph Cluster A is a source B == Cluster B is a target A Bilateral connections Target themselves • Connections form a directed graph • Cluster A is a source B == Cluster B is a target A • Bilateral connections • Target themselves Motivation for Wildfire Modeling Case Study re Modeling Case ●Wildland fires have destroyed 4% of California in 2020, the worst fire season in history. 2019 Maria Fire Source:https://abcnews.go.com/US/california-wildfires-live-updates-maria-fire-explodes- 8000/story?id=66686573 2019 Maria Fire Source:https://abcnews.go.com/US/california-wildfires-live-updates-maria-fire-explodes- 8000/story?id=66686573 2019 Maria Fire y ●Major destruction by fires is a worldwide trend, and fire behavior is changing due to a combination of natural and anthropogenic factors, and frequency of catastrophic fires is increasing ●Major destruction by fires is a worldwide trend, and fire behavior is changing due to a combination of natural and anthropogenic factors, and frequency of catastrophic fires is increasing Source:https://abcnews.go.com/US/california-wildfires-live-updates-maria-fire-explodes- 8000/story?id=66686573 Ismael Perez (i3perez@sdsc.edu) Ismael Perez (i3perez@sdsc.edu) Fire Modeling Workflow Fire Modeling Workflow • Input Data • Fuel • Weather (sensors or forecast) • Camera Imagery • Heterogenous Compute • Edge • AI • HPC nput Data Fuel Weather (sensors or forecast) Camera Imagery eterogenous Compute Edge AI Smoke Detector App running at the Edge • Collaborative effort between NSF NEON, NSF Sage, NSF Konza Prairie Biological Station to prescribed a burn and deploy a MDP with Sage Nodes • Mobile Deployment Unit (in red) • If you want to explore the data we collected from the controlled burn event, please visit: https://doi.org/10.57719/S54W2 D EON, NSF Sage, NSF Prairie Biological n to prescribed a burn eploy a MDP with Sage s e Deployment Unit (in red) want to explore the data lected from the olled burn event, please //doi.org/10.57719/S54W2 NSF al a burn h Sage it (in red) the data please • If you want to explore the data we collected from the controlled burn event, please visit: https://doi.org/10.57719/S54W2 D Implementation of Smoke and Fire Modeling on Composable Systems Three workflows • Smoke – Sage Edge App • Fire simulator • AI Training Both the fire simulator and training workflows are can be run on Expanse or Nautilus through the federation layer • Both the fire simulator and training workflows are can be run on Expanse or Nautilus through the federation layer Map between Heterogenous Workflows to Composable Systems • Other projects • Burnpro3D: control fire planning platform) • JupyterHub: Sage Edge App Development • Neurokube: AI Neuroscience imaging Conclusion Conclusion • Federation architecture to enable a composable infrastructure to ensure seamless integration of heterogenous compute and storage using Kubernetes ecosystem • We can target heterogenous workflows to b accomplish our fire modeling goals The authors would like to thank the WIFIRE and WorDS teams for their collaboration and support of the case study. Expanse , PRP, CHASE-CI and SAGE are supported through the NSF grants 1928224, 1541349, 1730158 and 1935984, respectively. The wildfire modeling case study was supported by NSF grants 1331615 and 2040676. Resources Expanse: https://www.sdsc.edu/services/hpc/expanse/ National Research Platform: https://pacificresearchplatform.org/ WIFIRE: https://wifire.ucsd.edu/ NVIDIA Bright Cluster manager: https://www.nvidia.com/en-us/data-center/bright- cluster-manager/ Sage: https://sagecontinuum.org/ Kubernetes: https://kubernetes.io/ Resources Extra Slides Extra Slides Admiralty Scheduling Diagram y g g JupyterHub - federation layer ●Testbed using Kind ●Three Kubernetes clusters ○Two k8s ○One k3s ●End goal is to develop Edge Applications to run on the Sage Platform and still being App and Cloud native ●Testbed using Kind ●Three Kubernetes clusters ○Two k8s ○One k3s ●End goal is to develop Edge Applications to run on the Sage Platform and still being App and Cloud native Testbed using Kind Three Kubernetes clusters ○Two k8s ○One k3s End goal is to develop Edge Applications to run on the Sage Platform and still being App and Cloud native ●End goal is to develop Edge Applications to run on the Sage
https://openalex.org/W4317648898	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/859ED9251490495CB23D34B89036CA3F/S1743921322001260a.pdf/div-class-title-stellar-activity-effects-on-the-atmospheric-escape-of-hot-jupiters-div.pdf	English	null	Stellar activity effects on the atmospheric escape of hot Jupiters	Proceedings of the International Astronomical Union	2,020	cc-by	3,555	Stellar activity eﬀects on the atmospheric escape of hot Jupiters Hiroto Mitani1 , Riouhei Nakatani2, and Naoki Yoshida1,3,4 1Department of Physics, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033 Hiroto Mitani1 , Riouhei Nakatani2, and Naoki Yoshida1,3,4 1Department of Physics, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033 Hiroto Mitani1 , Riouhei Nakatani2, and Naoki Yoshida1,3,4 1Department of Physics, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033 email: hiroto.mitani@phys.s.u-tokyo.ac.jp email: hiroto.mitani@phys.s.u-tokyo.ac.jp 4Research Center for the Early Universe, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033 4Research Center for the Early Universe, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033 Abstract. Transit observations have revealed the existence of atmospheric escape in several hot Jupiters. High energy photons from the host star heat the upper atmosphere and drive the hydrodynamic escape. The escaping atmosphere can interact with the stellar wind from the host star. We run radiation hydrodynamics simulations with non-equilibrium chemistry to investigate the wind eﬀects on the escape and the transit signature. Our simulations follow the planetary outﬂow driven by the photoionization heating and the wind interaction in a dynamically coupled, self-consistent manner. We show that the planetary mass-loss rate is almost independent of the wind strength, which however aﬀects the Ly-α transit depth considerably. But the Hα transit depth is almost independent of the wind strength because it is largely caused by the lower hot layer. We argue that observations of both lines can solve the degeneracy between the EUV ﬂux from the host and the wind strength. Keywords. hydrodynamics – methods: numerical – planets and satellites: atmospheres – planets and satellites: physical evolution The Predictive Power of Computational Astrophysics as a Discovery Tool Proceedings IAU Symposium No. 362, 2023 D. Bisikalo, D. Wiebe & C. Boily, eds. doi:10.1017/S1743921322001260 The Predictive Power of Computational Astrophysics as a Discovery Tool Proceedings IAU Symposium No. 362, 2023 D. Bisikalo, D. Wiebe & C. Boily, eds. doi:10.1017/S1743921322001260 © The Author(s), 2023. Published by Cambridge University Press on behalf of International Astronomical Union. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. https://doi.org/10.1017/S1743921322001260 Published online by Cambridge University Press https://doi.org/10.1017/S1743921322001260 Published online by Cambridge University Press 1. Introduction For close-in exoplanets, the extreme irradiation from the host star can drive the atmo- spheric escape. Such escape process can be important in planetary evolution and can even shape the statistical properties of observed close-in exoplanets (sub-Jovian desert; Szab´o and Kiss (2011), sub-Neptune desert; Fulton et al. (2017)). Transit observations have revealed the extended atmosphere for close-in exoplanets (Vidal-Madjar et al. 2003; Ehrenreich et al. 2015). Extreme-Ultraviolet (EUV> 13.6 eV) photons ionize the hydrogen atoms and heat the atmospheric gas through thermalization of the photoelectrons. The photoionization heat- ing contributes to atmospheric escape. Radiation hydrodynamics simulations identiﬁed the important physical processes and allowed detailed studies of the atmospheric struc- ture (Murray-Clay et al. 2009). Interaction with the stellar wind is also investigated in several studies (Bisikalo et al. 2013, 2018; Cherenkov et al. 2018; Vidotto and Cleary 2020; Carolan et al. 2021). Often the Ly-α transit depth is considered because of its large absorption. Ly-α photons from the host star can be easily absorbed by the inter- stellar medium between the star-plane system and the earth, which makes it diﬃcult 159 Wind eﬀects on the atmosphere of hot Jupiters to observe directly. Recent observations by ground-based telescopes use other lines (e.g. Helium triplet line, Hα line) which are more useful to detect the extended atmosphere. to observe directly. Recent observations by ground-based telescopes use other lines (e.g. Helium triplet line, Hα line) which are more useful to detect the extended atmosphere. Recent observations have also detected hot Jupiters around young active stars. Vigorous activities of such young stars can cause a strong inﬂuence on the planetary atmosphere, especially on the close-in planets. Strong winds can conﬁne the upper atmo- sphere and reduce both the mass loss and the transit depth. The wind eﬀect is expected to be important particularly in young systems with strong activities. Numerical simulations so far that have used to investigate the wind conﬁnement and Ly-α and Hα transits do not treat photoionization heating and the launched outﬂow in a self-consistent manner. To study the wind eﬀect on the planetary atmosphere, self- consistent radiation hydrodynamics simulations are necessary to follow the launching of the outﬂow because Hα absorption may be signiﬁcant in the lower atmospheric layers. We run simulations with varying the strength of the stellar wind and calculate the Ly-α and Hα transit depths. 1. Introduction We discuss the possibility that the strong stellar activity changes the absorption signatures. 2. Methods We ﬁrst introduce our simulations. We use the hydrodynamics simulation code PLUTO (Mignone et al. 2007) with the EUV radiation transfer module (Nakatani et al. 2018). Detailed implementation is described in (Nakatani et al. 2018; Mitani et al. 2021). Our simulations solve the following 2D axisymmetric hydrodynamic equations and non-equilibrium chemistry: ∂ρ ∂t + ∇· ρ⃗v = 0 (2.1) ∂ρvR ∂t + ∇· (ρvR⃗v) = −∂P ∂R −ρ∂Ψ ∂R (2.2) ∂ρvz ∂t + ∇· (ρvz⃗v) = −∂P ∂z −ρ∂Ψ ∂z (2.3) ∂ρE ∂t + ∇· (ρH⃗v) = −ρ⃗v · ∇Ψ + ρ(Γ −Λ) (2.4) ∂nHyi ∂t + ∇· (nHyi⃗v) = nHRi (2.5) ∂ρ ∂t + ∇· ρ⃗v = 0 (2.1) ∂ρvR ∂t + ∇· (ρvR⃗v) = −∂P ∂R −ρ∂Ψ ∂R (2.2) ∂ρvz ∂t + ∇· (ρvz⃗v) = −∂P ∂z −ρ∂Ψ ∂z (2.3) ∂ρE ∂t + ∇· (ρH⃗v) = −ρ⃗v · ∇Ψ + ρ(Γ −Λ) (2.4) ∂nHyi ∂t + ∇· (nHyi⃗v) = nHRi (2.5) (2.1) (2.5) where ρ, ⃗v, P are density, velocity, pressure of the gas. The potential Ψ includes contri- butions of the star and the planet and also incorporates the centrifugal force due to the orbital motion. We also follow the non-equilibrium chemistry including photoionization of the hydrogen atoms which can be important in the hydrogen absorption signatures. yi = ni/nH and Ri represent the abundance and the reaction rate, respectively. The incorporated chemical species are H, H+, H2, e−. where ρ, ⃗v, P are density, velocity, pressure of the gas. The potential Ψ includes contri- butions of the star and the planet and also incorporates the centrifugal force due to the orbital motion. We also follow the non-equilibrium chemistry including photoionization of the hydrogen atoms which can be important in the hydrogen absorption signatures. yi = ni/nH and Ri represent the abundance and the reaction rate, respectively. The incorporated chemical species are H, H+, H2, e−. The heating and cooling rates are denoted as Γ, Λ, respectively. We calculate the EUV photoionization heating rate by ray-tracing: Fν = Φν 4πa2 exp[−σνNHI] (2.6) Fν = Φν 4πa2 exp[−σνNHI] (2.6) Γph = 1 ρnHI ∞ ν0 dν σνh(ν −ν0)Fν (2.7) (2.6) Γph = 1 ρnHI ∞ ν0 dν σνh(ν −ν0)Fν (2.7) (2.7) where σν is the absorption cross section as a function of ν (Osterbrock and Ferland 2006) and NHI is the column density of hydrogen atoms and hν0 = 13.6 eV. 2. Methods We implement Ly-α cooling and hydrogen recombination cooling (Spitzer 1978; Anninos et al. 1997). https://doi.org/10.1017/S1743921322001260 Published online by Cambridge University Press 160 H. Mitani, R. Nakatani & N. Yoshida Table 1. Model parameters in the ﬁducial run. Stellar parameters Stellar Mass M∗ 1 M⊙ Stellar Radius R∗ 1 R⊙ Stellar EUV photon emission rate Φν 1.4 × 1038 s−1 Stellar wind velocity 540 km/s Stellar wind temperature 2 × 106 K Stellar wind density 2.5 × 103 g/cm3 Planetary parameters Planet Mass Mp 0.3 MJ Planet Radius Rp 1 RJ Semi-major axis a 0.045 AU Figure 1. The atmospheric structure of our simulations. The EUV photons and the wind from the host star are injected from the left side of the ﬁgure. The stellar mass loss rates are ˙M∗= 1 ˙M⊙(left), 10 ˙M⊙(middle), 100 ˙M⊙(right). In each ﬁgure, the upper panel shows the density and lower panel shows the temperature. 160 H. Mitani, R. Nakatani & N. Yoshida Table 1. Model parameters in the ﬁducial run. Table 1. Model parameters in the ﬁducial run. Stellar parameters Stellar Mass M∗ 1 M⊙ Stellar Radius R∗ 1 R⊙ Stellar EUV photon emission rate Φν 1.4 × 1038 s−1 Stellar wind velocity 540 km/s Stellar wind temperature 2 × 106 K Stellar wind density 2.5 × 103 g/cm3 Planetary parameters Planet Mass Mp 0.3 MJ Planet Radius Rp 1 RJ Semi-major axis a 0.045 AU Figure 1. The atmospheric structure of our simulations. The EUV photons and the wind from the host star are injected from the left side of the ﬁgure. The stellar mass loss rates are ˙M∗= 1 ˙M⊙(left), 10 ˙M⊙(middle), 100 ˙M⊙(right). In each ﬁgure, the upper panel shows the density and lower panel shows the temperature. In our simulations, Ly-α cooling is a major radiative cooling process, and the adiabatic cooling dominates the overall cooling processes. In our simulations, Ly-α cooling is a major radiative cooling process, and the adiabatic cooling dominates the overall cooling processes. g g p Table 1 shows the stellar and planetary parameters of our ﬁducial simulation. We also run simulations with various stellar wind strength by varying the stellar wind density. https://doi.org/10.1017/S1743921322001260 Published online by Cambridge University Press 3. Results and Implications Figure 1 shows the atmospheric structure of our simulations. The strong wind con- ﬁnes the outﬂow. The wind-outﬂow structure is shaped by the balance between the ram pressure of the wind and the thermal pressure of the outﬂow. The balanced point can be estimated as kBρp(r) Tp(r)/μmH = ρ∗(r) v2 ∗(r) (3.1) (3.1) where ρp(r), Tp(r) are the density and temperature of the planetary atmosphere at the contact point, μ is the mean molecular weight, mH is the hydrogen atomic mass, and ρ∗(r), v∗(r) are the density and velocity of the wind. The balanced point depends on both the velocity and density of the wind because the ram pressure determines the structure, implying that the mass-loss rate of the star does not uniquely determine the structure of the atmosphere and the observational signatures. Table 2 shows that the mass-loss rates of the planet are almost independent of the wind strength and that the value is approximately 1010 g/s because the wind can suppress the outﬂow only when it strongly aﬀects the atmosphere around the launching point. This is achieved for ˙M∗> 1000 ˙M⊙. https://doi.org/10.1017/S1743921322001260 Published online by Cambridge University Press Wind eﬀects on the atmosphere of hot Jupiters Wind eﬀects on the atmosphere of hot Jupiters 161 Wind eﬀects on the atmosphere of hot Jupiters 161 Table 2. Planetary Mass-loss rates with diﬀerent stellar winds. Stellar Wind strength Mass-loss rate (g/s) 1 ˙ M⊙ 2.9 × 1010 10 ˙ M⊙ 2.2 × 1010 100 ˙ M⊙ 2.3 × 1010 Figure 2. left panel: Ly-α transit depth at mid-transit. The shaded region is the line center region where the local interstellar medium can absorb. right panel: Hα transit depth. Figure 2. left panel: Ly-α transit depth at mid-transit. The shaded region is the line center region where the local interstellar medium can absorb. right panel: Hα transit depth. Figure 2 shows the Ly-α and Hα transit of our outputs. The peak of the Ly-α transit is blue-shifted due to the wind. This is consistent with the previous studies (McCann et al. 2019). To calculate Hα absorption, we assume the n = 2 level population using the 2p, 2s population of Christie et al. https://doi.org/10.1017/S1743921322001260 Published online by Cambridge University Press 3. Results and Implications (2013): n2 n1 = n2p + n2s n1s ≃10−9 5R∗ a 2 e16.9−(10.2 eV/kBTLyα,∗) + 1.627 × 10−8 T 104 K 0.045 e11.84−118400 K/T (3.2) × 8.633 log(T/T0) −γ where TLyα,∗∼7000 K is the excitation temperature for the solar Lyman-α, T0 = 1.02 K and γ = 0.57721 . . . is the Euler-Mascheroni constant. In our simulations, the strong wind can reduce the Ly-α transit depth while the Hα signature is almost independent of the strength. The Hα absorption by the atmosphere is signiﬁcant in lower hot region because the n = 2 level population is larger there (n2/n1 ∼10−9). where TLyα,∗∼7000 K is the excitation temperature for the solar Lyman-α, T0 = 1.02 K and γ = 0.57721 . . . is the Euler-Mascheroni constant. In our simulations, the strong wind can reduce the Ly-α transit depth while the Hα signature is almost independent of the strength. The Hα absorption by the atmosphere is signiﬁcant in lower hot region because the n = 2 level population is larger there (n2/n1 ∼10−9). The transit signatures due to the extended atmosphere are also dependent on the EUV ﬂux from the host star. The Ly-α transit depth depends on the EUV ﬂux and the wind strength, whereas the Hα transit signature does not sensitively depend on the wind strength unless the wind is extremely strong with ˙M∗> 1000 ˙M⊙. Our simulations are 2D axisymmetric and neglect the tail contribution to the transit signatures. The tail contribution should be signiﬁcant in Ly-α blue-wing but is likely unimportant in Hα absorption because the lower hot atmospheric layer matters. The diﬀerence between the wind eﬀects on Ly-α and Hα would be essentially the same even if we consider the tail contribution to the signature. We argue that observations of both signatures can solve the degeneracy of the stellar EUV luminosity and the wind properties in close-in gas giants. The stellar activities have a signiﬁcant impact on the planetary atmosphere and the observational signatures (Zhilkin et al. 2020). The rate of the ﬂare activities is well https://doi.org/10.1017/S1743921322001260 Published online by Cambridge University Press 162 H. Mitani, R. Nakatani & N. Yoshida known for the Sun (Maehara et al. 2017). Strong ﬂares are accompanied by coronal mass ejections (CMEs) in many cases, and thus we can estimate the rate of the strong mass-loss ˙M∗> 10 ˙M⊙due to the activities. 3. Results and Implications known for the Sun (Maehara et al. 2017). Strong ﬂares are accompanied by coronal mass ejections (CMEs) in many cases, and thus we can estimate the rate of the strong mass-loss ˙M∗> 10 ˙M⊙due to the activities. ∞ 1032 erg f(Eﬂare) dEﬂare ∼1 −100 year−1 (3.3) (3.3) For solar type stars, the possibility that the strong CME changes the observational Ly-α is expected to be small. For solar type stars, the possibility that the strong CME changes the observational Ly-α is expected to be small. For young stars, stellar activities should be stronger than that of the sun. Also the rate becomes higher. In the case of a very young host star (< 50 Myr), the probability becomes an order of magnitude larger (Feinstein et al. 2020). We note that, in many cases, the age of the host star in the observed exoplanets is older than 50 Myr and the eﬀect can be small. Interestingly, the activities are also stronger (Maehara et al. 2014) for cooler stars. The spectral type dependence of the stellar activity is also investigated. The ﬂare frequency in M dwarfs is a few orders of magnitude larger than in G-type stars. The strong CME frequency becomes larger in late-type stars, and the CME happens almost always in every transit around M dwarfs. References Anninos, P., Zhang, Y., Abel, T., & Norman, M. L. 1997, Cosmological hydrodynamics with multi-species chemistry and nonequilibrium ionization and cooling. New Astron., 2(3), 209–224. 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A. & Cleary, A. 2020, Stellar wind eﬀects on the atmospheres of close-in giants: a possible reduction in escape instead of increased erosion. MNRAS, 494(2), 2417–2428. Zhilkin, A. G., Bisikalo, D. V., & Kaygorodov, P. V. 2020, Coronal Mass Ejection Eﬀect on Envelopes of Hot Jupiters. Astronomy Reports, 64(2), 159–167. https://doi.org/10.1017/S1743921322001260 Published online by Cambridge University Press Discussion Hanawa: I am wondering whether your simulation resolved the bow shock and contact discontinuity. Hanawa: I am wondering whether your simulation resolved the bow shock and contact discontinuity. Hiroto: We talked about the force balance which describes the shock front. Our simulations resolve the shock front and contact discontinuity. Hiroto: We talked about the force balance which describes the shock front. Our simulations resolve the shock front and contact discontinuity. Bisikalo: Do you take into account orbital motion of planets? Because I cannot see the inﬂuence of the Coriolis force. Bisikalo: Do you take into account orbital motion of planets? Because I cannot see the inﬂuence of the Coriolis force. Hiroto: Our simulations are 2D axisymmetric and we consider the centrifugal force but neglect the Coriolis force. The Ly-α transit signature can be aﬀected by the 3D eﬀect because of the tail contribution. That can be important. Hα transit absorption can be independent of the existence of the tail because the absorption is signiﬁcant around a relatively lower region in which the gas temperature is high. The diﬀerence of the stellar wind strength dependence between Ly-α and H α may be qualitatively similar to the 3D. Rony: In the case of Jupiter, the strong magnetic ﬁeld plays a role of having magnetic (ram) pressure, setting the size of the planetary interaction with the solar wind, will you eventually do MHD simulations as well? And what is your boundary condition at the planet: you just give a radial subsonic (or supersonic) outﬂow? Hiroto: Magnetic pressure can indeed be important. I would run MHD simulations eventually but do not have an immediate plan currently. In our simulations, the EUV- driven outﬂow is excited from the hydrostatic, stratiﬁed gas, and the outﬂow base is well above the planet boundary. We use the boundary condition where the gas is hydrostatic across the boundary. https://doi.org/10.1017/S1743921322001260 Published online by Cambridge University Press
W2969027600.txt	https://periodicos.utfpr.edu.br/rbect/article/download/7371/pdf	pt		Entre crenças e aparências: compreensões sobre ciência por licenciandos em ciências da natureza	Revista brasileira de ensino de ciência e tecnologia	2,019	cc-by	8,152	https://periodicos.utfpr.edu.br/rbect Entre crenças e aparências: compreensões sobre ciência por licenciandos em ciências da natureza RESUMO Eril Medeiros da Fonseca erilmf@gmail.com 0000-0001-9529-2357 Universidade Federal do Pampa, Dom Pedrito, Rio Grande do Sul, Brasil. Leandro Duso dusoleandro@gmail.com 0000-0002-2605-0829 Universidade Federal do Pampa, Dom Pedrito, Rio Grande do Sul, Brasil. O presente artigo investigou e discutiu as concepções de licenciandos de um curso de Ciências da Natureza em relação à Natureza da Ciência e o perfil do cientista no fazer científico, no âmbito de um componente curricular de experimentação no ensino de ciências. Possui caráter qualitativo, em que os dados foram coletados por meio da aplicação de um questionário e pela construção de um diário de aula, que contribuiu na compreensão do discurso dos sujeitos da pesquisa. Através da Análise de Conteúdo sistematizaram-se os resultados em duas categorias, das quais: visão deformada sobre a natureza da Ciência e imagem reducionista do cientista. Os resultados apontaram uma compreensão acrítica sobre a Ciência, pautada por uma visão salvacionista, reducionista e utilitarista do trabalho científico, indicando a necessidade de se discutir e repensar a abordagem de tais questões e o modo como são abordadas na formação de professores. PALAVRAS-CHAVE: Natureza da ciência. Fazer científico. Ensino de ciências. Página \| 73 R. bras. Ens. Ci. Tecnol., Ponta Grossa, v. 12, n. 2, p. 73-93, mai./ago. 2019. INTRODUÇÃO A configuração da Ciência e sua produção é consequência de uma tendência positivista, relacionada a uma racionalidade científica, um modelo totalitário, que não considerava as ideias de sendo comum, pautada pela quantificação do conhecimento, culminando na redução da complexidade do mundo, prevalecendo à ordem e a previsibilidade. A Ciência acadêmica, privilegiando a ilustração, verificação e memorização de um corpo de conhecimentos perfeitamente estabelecido, não controverso, apresenta a Ciência como um processo objetivo, isento de valores, que conduz a verdades absolutas, inquestionáveis, através da observação rigorosa de regularidades nos fenômenos e do estabelecimento de generalizações. Embora a Ciência tenha ganhado rigor e autonomia, acarretou no desinteresse do conhecimento, na proletarização, divisão dos centros de investigação e no domínio do desenvolvimento científico por poucos (SANTOS, 2006). Santos (2006) menciona que a produção de conhecimento pela Ciência deve ser intensificada pelo diálogo entre saber científico e senso comum, sabendo que a Ciência não é um conhecimento restrito apenas ao espaço acadêmico, mas faz parte de uma instância maior, social, que pelos meios de comunicação torna-se mais ampla, influenciando decisões éticas, políticas e econômicas. Delizoicov, Angotti e Pernambuco (2011, p. 23) definem Ciência como “um conteúdo cultural relevante para viver, compreender e atuar no mundo contemporâneo”, engendrados com métodos e atividades comprometidas com um trabalho coletivo entre professores e alunos, tanto na academia/escola como na sociedade, permitindo tratar o conhecimento de forma ampla, não dicotomizando teoria e prática. Na opinião de Cachapuz et al. (2000), o grande objetivo da aprendizagem em Ciência deverá consistir na familiarização do aluno com as características do trabalho científico, ajudando-o a compreender os seus percursos, bem como as suas múltiplas facetas, colocando-o numa situação de cidadão ativo, apto a decidir em situações pluridisciplinares, nas quais a Ciência é uma entre as várias vozes da sociedade. Freire Jr. (2003) está convicto de que o desenvolvimento de uma imagem mais realista e multifacetada da Ciência poderá contribuir para a melhoria da relação entre a Ciência e a sociedade, representando o melhor antídoto contra tendências irracionalistas contemporâneas. Harres (2003) menciona que embora os currículos de Ciência variem na direção de diferentes contextos, buscando atender a demandas emergentes, a visão historicamente marcada sobre a Ciência é refletida por uma distinção entre epistemologia e a própria natureza da Ciência (NdC). As visões sobre os currículos de Ciência comumente empregadas referem-se a uma tradição empirista, que consideram o caráter teórico e observacional da Ciência de forma fragmentada, onde o conhecimento é visto de modo fechado, apartado da ação do mundo (HARRES, 2003). Essas visões refletem na ação docente sobre o que ensinar e como ensinar. Página \| 74 Nesta mesma perspectiva, Gil-Perez et al. (2001), descrevem sete visões deformadas dos professores em relação ao trabalho científico, destacamos R. bras. Ens. Ci. Tecnol., Ponta Grossa, v. 12, n. 2, p. 73-93, mai./ago. 2019. algumas que julgamos serem relevantes no saber-fazer científico, das quais: visão rígida; visão aproblemática e ahistórica e visão individualista e elitista da Ciência. A visão rígida apresenta o método científico como um conjunto de etapas seguido de forma mecânica e exata, evitando qualquer tipo de criatividade e/ou ambiguidade. A visão aproblemática e ahistórica caracteriza-se por uma perspectiva fechada e sem relações históricas da produção científica e epistemológica. A compreensão do conhecimento dá-se de modo pontual, desconsiderando as referências anteriores que o produziram. Na visão individualista e eletista os conhecimentos científicos são descritos como obras acabadas de “gênios” que concluíram seus resultados de modo particular, sem inter-relações a outros sujeitos ou hipóteses. Do mesmo modo, tal conhecimento é reservado a uma mínima parcela, eminentemente masculina e na maioria das vezes acessível a poucos. Estas visões deformadas da Ciência não são de estranhar, pois a maior parte dos professores raramente teve contato com a atividade científica ou oportunidade de refletir e de aprender sobre o funcionamento da Ciência. Em vários países, os cursos de formação inicial de professores não valorizam nem proporcionam os conhecimentos necessários ao ensino da NdC, pois centram-se na aquisição dos produtos intelectuais da Ciência e ignoram o processo de produção de conhecimento (GALLAGHER, 1991). Diante do exposto Chassot (2003a; 2003b) menciona que há uma predominância da presença masculina na área da Ciência, na qual tem sua origem europeia, de modo que a própria História da Ciência está intensamente ligada a História da Humanidade e não pode ser vista sem as diferentes conexões existentes entre ambas. Diante do exposto, acreditamos que a Ciência e a própria produção científica caracterizam-se como uma atividade humana, com interdependências entre sujeitos e contextos, portanto, não neutra, com intencionalidades e influenciada por fatos, fenômenos e relações sócio-históricas. Logo, os professores tendem a sub-valorizar a NdC na sua prática de ensino (LAKIN; WELLINGTON, 1994), limitando-se a transpor os modelos de trabalho dos seus formadores, marcados pelo domínio da dimensão pedagógica e pela desvalorização ou inexistência de reflexão epistemológica consciente (CACHAPUZ et al., 2000; PRAIA; CACHAPUZ, 1998). Nesta perspectiva, o presente artigo destina-se a analisar e discutir as concepções de licenciandos de um curso de Ciências da Natureza sobre a NdC. CONCEPÇÕES DE CIÊNCIA E SUAS IMPLICAÇÕES NO ENSINO DE CIÊNCIAS Página \| 75 Fonseca (2002, p. 11) define Ciência como “[...] o saber produzido através do raciocínio lógico associado à experimentação prática. Caracteriza-se por um conjunto de modelos de observação, identificação, descrição, investigação experimental e explanação teórica de fenômenos”. Considera-se, portanto, que a Ciência é mais uma das formas de compreender o mundo e as transformações que nele ocorrem, gerando conhecimentos que tornem os sujeitos aptos a interagir com tais transformações, intervindo em sua realidade. R. bras. Ens. Ci. Tecnol., Ponta Grossa, v. 12, n. 2, p. 73-93, mai./ago. 2019. Por definição Ciência é um conjunto de conhecimentos socialmente adquiridos ou produzidos e historicamente acumulados, constituídos de uma objetividade que permite sua transmissão, estruturada com métodos, teorias e linguagens próprias, visando compreender e orientar a natureza e as atividades humanas (FERREIRA, 1986). Borges (2007) na obra Em debate: cientificidade e Educação em Ciências, sistematiza algumas visões sobre a Ciência, consideradas ao longo do tempo, por diversos teóricos, descritas a seguir. Lakatos descreve uma visão tradicional da Ciência, pautada pelo método científico empirista-indutivista, a partir de observações e formulação de teorias, possui um critério único, atemporal, não histórico e universal. Segundo Lakatos e Musgrave (1979, p.217) dentro de um programa de pesquisa deve-se “[...] infalivelmente planejar suas teorias auxiliares na esteira dos fatos, sem antecipar outros”. Para Popper a Ciência evolui através de refutações, pois nunca se pode provar que algo é totalmente verdadeiro, demonstrando que algumas ideias são falsas. A partir disso obtém-se uma nova teoria e se aperfeiçoa outra (BORGES, 2007). A visão contextualista de Kuhn considera que o conhecimento depende do que é importante ou do que é valorizado por alguém ou comunidade, é conservado, resistindo a mudanças. Na visão do “anarquismo metodológico” de Feyerabend não existe uma regra metodológica rígida de pesquisa, pois toda regra de pesquisa já foi violada, sendo necessária para o progresso (BORGES, 2007). Bachelard, na visão dialética, vê o conhecimento científico como racionalista e realista, pois toda observação/experimentação antecede uma teoria, rompendo o diálogo entre conhecimento científico e senso comum, desconfiando de resultados imediatos (BORGES, 2007). Bachelard (1996) também considera dentre outros obstáculos à superação da natureza verbal, nela uma única imagem ou palavra constitui a explicação para um fenômeno da natureza, através de metáforas. Segundo o autor a atribuição de imagens para explicar determinados fatos e/ou fenômenos podem suprimir a necessidade de explicação, deixando tudo muito simples, seria a criação de esquemas gerais, que reprimem maiores aprofundamentos, prejudicando a razão, impedindo a visão abstrata e a evolução do pensamento. A visão externalista da Ciência, abordada também por Bunge, considera as influências externas dadas ao conhecimento. Nessa visão as temáticas da produção científica não atendem apenas aos interesses de uma comunidade científica, mas dialogam com fatores sociopolíticos, econômicos e culturais. Página \| 76 Além disso, frequentemente, a mídia transmite uma imagem distorcida da Ciência e ideias estereotipadas acerca dos cientistas e da sua atividade, com um impacto considerável nas concepções e na confiança do público acerca dos empreendimentos científicos e tecnológicos (NELKIN, 1995). Segundo Nelkin (1995) a investigação científica é muitas vezes apresentada aos estudantes e a sociedade, como uma atividade esotérica, misteriosa e extremamente complexa, cuja compreensão e prática só está ao alcance de um número restrito de iluminados que vivem completamente isolados e absorvidos pelo seu trabalho. R. bras. Ens. Ci. Tecnol., Ponta Grossa, v. 12, n. 2, p. 73-93, mai./ago. 2019. Assim, de acordo com Harres (1999, p. 198), “as concepções sobre a NdC inadequadas dos estudantes mais comuns encontradas incluem, entre outros aspectos”, os seguintes:  a consideração do conhecimento científico como absoluto;  a ideia de que o principal objetivo dos cientistas é descobrir leis naturais e verdades;  lacunas para entender o papel da criatividade na produção do conhecimento;  lacunas para entender o papel das teorias e sua relação com a pesquisa;  incompreensão da relação entre experiências, modelos e teorias. Consequentemente, as visões postas ao Ensino de Ciências refletem algumas concepções descritas acima, que foram hegemônicas, ditando conceitos e regras pré-estabelecidas de compreender a definição de Ciência e quem a produz. O processo de aprendizagem era vinculado a aspectos apenas técnicos, colocando o estudante como sujeito passivo, desvinculado de valores pessoais e profissionais. Na década de 60 e 70, no Ensino de Ciências, buscava-se pensar de forma lógica e crítica, vivenciando o método científico com discussões de pesquisas realizadas no laboratório. Ao invés de se considerar o produto, passava-se a considerar o processo (KRASILCHIK, 1987). Assim, constituiu-se duas visões em relação à Ciência: uma defendia a ideia de que a mesma ajudaria na resolução de problemas cotidianos, e a outra visava à formação acadêmica, que previa a formação de cientistas. Esta última prevaleceu de maneira formal, baseada em definições, deduções e equações, trabalhando com experimentos em que os resultados são pré-estabelecidos (KONDER, 1998). Esta concepção é reafirmada pelo desenvolvimento industrial, admitindo a Ciência e a tecnologia como aspectos essenciais na economia brasileira. Levando a criação de unidades escolares em áreas como Física, Química e Geologia, preparando indivíduos para ensinar tais áreas. A Biologia surge posteriormente por trabalhar com conceitos mais complexos (KONDER, 1998). A preocupação em constituir áreas específicas do saber estritamente para a profissionalização, contribuiu para a fragmentação no Ensino de Ciências, afetando o currículo das instituições de ensino. Na década de 80, visava-se, no Ensino de Ciências, analisar implicações sociais no desenvolvimento científico e tecnológico. A tendência era trabalhar com resolução de problemas, através de jogos e simulações, realizando articulações com Centros de Ciências e universidades (KRASILCHIK, 1987). Página \| 77 Diante do exposto, percebeu-se a necessidade de um ambiente que se oponha a essa concepção tecnocrática de ensino, demandando uma discussão de cunho democrático. Desta forma, o Ensino de Ciências caracteriza-se como uma possibilidade de desenvolver um conhecimento que vá além de conceitos e ideias, da cultura científica, de modo que os estudantes percebam os fenômenos da natureza e com isso estejam aptos a elaborar hipóteses, concepções e organizar ideias, preparando-os para agirem fora do ambiente escolar (CARVALHO, 2011). R. bras. Ens. Ci. Tecnol., Ponta Grossa, v. 12, n. 2, p. 73-93, mai./ago. 2019. O Ensino de Ciências é definido segundo Chassot (2011), como uma linguagem para possibilitar uma leitura do mundo. Esta leitura possibilita uma apropriação de conceitos científicos, ampliando a dimensão de conhecimentos essenciais para a formação de uma postura em relação ao ambiente em que se vive. Segundo Marco (1999, p. 95) formar cidadãos cientificamente cultos não significa dotá-los de uma linguagem científica, e sim ensinar a desmistificar e decodificar as crenças aderidas a Ciência e aos cientistas, abstrair de sua aparente neutralidade, entrar nas questões epistemológicas e nas terríveis desigualdades ocasionadas pelo mal uso da Ciência e suas condições sócio-políticas. Com isso, a apropriação de conhecimentos científicos deixaria de atender a necessidades relacionadas somente aos interesses políticos, econômicos, profissionais e/ou pessoais, para desenvolver a compreensão concreta de conceitos que componham e auxiliem no entendimento da conjuntura da realidade local, desencadeando assim um processo que contemple a cultura vivida. Harres (2003) afirma que algumas características em relação ao Ensino de Ciências evidenciam uma concepção deturpada que se criou sobre a mesma, pautada por um vocabulário técnico, com memorização de fatos e domínio de fórmulas, em uma perspectiva individual e não-social. O autor menciona que as consequências dessa visão para o ensino são bastante evidentes, materializadas na transmissão de conhecimento de forma acabada e não dinâmica, aplicação do método científico de forma acrítica e desconsideração do caráter evolutivo, especulativo e humano do conhecimento científico. Auler e Delizoicov (2006) buscando perceber as compreensões de professores e futuros professores de Ciências sobre interações entre a Ciência, a Tecnologia e a Sociedade, descrevem a passividade diante do desenvolvimento científico, necessitando a superação da perspectiva salvacionista atribuída a Ciência. Em relação aos cientistas, mencionam que, de modo geral, são considerados pessoas objetivas, imparciais, possuidoras de verdade e por vezes loucas lutando pelo bem da humanidade. Krasilchik (1987) aponta que as Ciências têm sido ensinadas como uma coleção de fatos e descrições de fenômenos, sem relações com mecanismos históricos. A percepção apática e fechada de Ciência culmina com as limitações das aulas de Ciências, o que faz com que o componente curricular do mesmo se torne irrelevante e sem significado. Portanto, mesmo com a diversidade de diferenças sociais, culturais, econômicas e regionais que uma instituição de ensino abarca, origina-se, cada vez mais, um distanciamento entre o que se ensina, o que se vive e o que se faz, limitando o rendimento do ensino. METODOLOGIA E CONTEXTO DA PESQUISA Página \| 78 Os sujeitos da pesquisa foram vinte e dois (22) licenciandos do terceiro semestre do componente curricular de Práticas pedagógicas: experimentação no Ensino de Ciências do curso de licenciatura em Ciências da Natureza, da Universidade Federal do Pampa – Campus Dom Pedrito/RS. R. bras. Ens. Ci. Tecnol., Ponta Grossa, v. 12, n. 2, p. 73-93, mai./ago. 2019. O curso visa formar profissionais que compreendam a realidade social, na qual se insere a escola em que atua, estimulando a curiosidade científica com seus educandos, de modo que reflita eticamente perante a sociedade e natureza. Possui caráter interdisciplinar e seus acadêmicos estão aptos a atuarem nos anos finais do Ensino Fundamental e Ensino Médio (UNIPAMPA, 2015). O componente curricular Práticas pedagógicas: experimentação no Ensino de Ciências visa discutir os modelos atuais de Ensino de Ciências: transmissão/recepção; redescoberta e mudança conceitual; aprendizagem significativa e novas possibilidades no ensino e aprendizagem em Ciências. Como objetivo, pretende: Compreender a abordagem epistemológica da história da experimentação no Ensino de Ciências, com ênfase na análise do valor pedagógico e da contribuição no processo e aprendizagem do Ensino de Ciências Naturais na perspectiva do Ensino Básico (UNIPAMPA, 2015). Optamos por trabalhar com a concepção da NdC e a visão que os licenciandos têm dos cientistas, por ser uma questão anterior à discussão sobre experimentação que se propõe o componente curricular, já que a visão sobre Ciência que cada um possui, baliza as práticas e concepções em relação ao Ensino de Ciências e a própria experimentação. A coleta dos dados baseou-se na aplicação de um questionário, no início do componente, com questões abertas, que versavam sobre a compreensão da NdC e o perfil físico e social de um cientista. As questões buscavam compreender a concepção dos licenciandos em relação à Ciência, quem é considerado um cientista e que atividades o mesmo desenvolve. Durante as aulas foram realizadas anotações em um diário de aula (ZABALZA, 2004) das falas dos licenciandos que teve como objetivo verificar a mudança do discurso em relação às concepções de Ciência e fazer científico. Algumas considerações sobre o que havia sido discutido eram anotadas no diário após cada aula, bem como possíveis avanços ou limitações relacionadas ao entendimento de cada licenciando. A turma era composta por vinte e oito (28) estudantes, porém no decorrer do componente houve desistência de seis (06), portanto, restaram vinte e dois (22) licenciandos que participaram integralmente do componente. Vale ressaltar que a participação na pesquisa teve o consentimento de todos os envolvidos. Uma vez organizados os dados coletados, passamos a tratá-los por meio da Análise de Conteúdo (BARDIN, 2009), que consiste em três etapas. Na primeira, Pré-análise, fase de sistematização das ideias para criação de um plano de análise, ocorreu a leitura atenta dos questionários e registros feitos no diário de aula. Na segunda etapa, Exploração do material, realizou-se a codificação, enumerando as falas dos licenciandos para a criação de possíveis categorias. Já na terceira etapa, Tratamento dos resultados obtidos e interpretação, foi feito a síntese dos resultados, confrontando-os com as dimensões teóricas. Página \| 79 Para organizar o conjunto dos fragmentos referentes às falas dos licenciandos foi adotada a sistemática de grafá-las com a letra “L”. Estes foram numerados de 1 a 22 sem que a numeração guarde qualquer relação com o respondente. Tais fragmentos aparecem grafados em itálico e entre aspas no texto. R. bras. Ens. Ci. Tecnol., Ponta Grossa, v. 12, n. 2, p. 73-93, mai./ago. 2019. DISCUTINDO OS RESULTADOS A utilização dos fragmentos das falas dos licenciandos possibilitou aprofundar a discussão de alguns elementos, que foram sistematizados nas seguintes categorias: visão deformada sobre a NdC e imagem reducionista do cientista. É importante mencionar que as respostas dos licenciandos foram mantidas na íntegra, pois não foram feitas correções gramaticais ou mudanças em relação à língua portuguesa. Visão deformada sobre a NdC Neste item discutimos as visões dos licenciandos em relação à NdC e ao fazer científico, modos e meios de produção. As compreensões envolvem uma variedade de atribuições sobre Ciência que em um dado momento integram as concepções sobre o Ensino de Ciências. Entendemos que tais visões são influenciadas por diferentes segmentos sociais (meios de comunicação, vivência familiar, ambiente escolar, tipo de formação inicial) que contribuem para reforçar o entendimento que cada um possui sobre Ciência e o seu fazer. A partir das respostas dos questionários dadas pelos licenciandos percebeuse que alguns acadêmicos descreveram a Ciência de forma naturalista, relacionando com o estudo dos fenômenos naturais, seres vivos e fatos cotidianos, demonstrando uma relação entre Ciência e questões ambientais, exemplificadas pelas respostas do L9 e L2, respectivamente: “estudo dos fenômenos naturais, físicos e químicos de extrema importância para o esclarecimento dos fatos do cotidiano” e “estudo de todos os seres vivos no meio ambiente”. Essas visões aproximam-se do chamado naturalismo ontológico, mencionado por Silveira (2014), pois a natureza é tudo que existe a respeito do que compõem a realidade e a Ciência estaria diretamente relacionada a questões naturais, por estudar esses fenômenos. Segundo Kosminsky e Giordan (2002) essa visão é corroborada pelos veículos de comunicação e divulgação científica, pois desempenham uma contribuição determinante para moldar as visões de Ciências, exercendo uma influência devido à difusão pelas estruturas sociais. Percebemos no discurso do L9 uma mudança em sua concepção naturalista para uma visão mais sistêmica, “Ciência é todo conhecimento adquirido através do estudo ou da prática, de qualquer área do conhecimento”. O mesmo percebemos na resposta do L2: “Engloba todo conhecimento adquirido, estudos e nos explica os acontecimentos”. Esse naturalismo também foi identificado através da descrição do L3, porém identificamos alguns elementos que se aproximam das ideias de Popper quando menciona que a Ciência é o “conhecimento que é adquirido através de estudos e práticas, baseados em reflexões e ideias que podem ser abandonadas, renovadas ou criadas novas”. Página \| 80 Percebemos o movimento dinâmico, não estanque, que o discente agrega a Ciência, que se harmoniza com as proposições de Popper considerando a substituição do método científico tradicional pelo método científico hipotético- R. bras. Ens. Ci. Tecnol., Ponta Grossa, v. 12, n. 2, p. 73-93, mai./ago. 2019. dedutivo. Nessa perspectiva hipóteses são elaboradas e reformuladas, possibilitando um aperfeiçoamento das teorias e o avanço do conhecimento (BORGES, 2007). O L2 e L4 também mencionaram que as atividades realizadas pelos cientistas são vinculadas a realização de experimentos e práticas observacionais, a saber: “Experimentos, pesquisas e descobertas, atividade de pesquisa e práticas” (L2). “Experimentação, práticas sobre algo científico e observações”. “Experimentação, observação” (L4). Nessa visão a Ciência associada à experimentação cresce por sucessivas conjecturas e refutações. Uma teoria científica é refutada por experimentos que podem a contradizer (BUENO, 2015), demonstrando a construção histórica atribuída ao fazer científico e a ideia de admitir a produção científica pelo seu próprio caráter humano. Na reinterpretação de Lakatos (1979) ao falseacionismo de Popper, as teorias não são refutadas por experimentos, mas através de outras teorias, a partir de programas de pesquisa, o que difere das afirmações de L27 sobre Ciência: “Conhecimento adquirido através de experimentos, estudos e hipóteses”. Lakatos propõe que o progresso científico é encaminhado, pelo que denomina de programas de pesquisa, que seriam diretrizes metodológicas correspondentes a construção e modificação de teorias. A partir dos programas as teorias desenvolvem-se e sobrevivem a possíveis refutações, e a competição entre elas é uma característica do processo do desenvolvimento científico, que são resguardadas/garantidas em cinturões protetores (LABURÚ; ARRUDA; NARDI, 1998). Nesse sentido, “as teorias não são elementos isolados, mas pertencentes a um determinado programa” (LABURÚ; ARRUDA; NARDI, 1998, p. 4), e qualquer fenômeno que destoa das ideias defendidas tornam-se um desafio a ser explicado dentro de um programa. Através dos programas de pesquisa as teorias tendem a evoluírem e permanecerem, enquanto outras incapazes de ofertar soluções satisfatórias a determinados problemas investigados acabam desaparecendo (BORGES, 2007). Já na fala de L20, percebemos uma visão mais conteudista relacionada ao Ensino de Ciências, a saber: “Tem como objetivo passar as matérias de Biologia que estuda a vida, Química que estuda as formas e o porquê delas, e a Física que estuda o que seria movimentos”. “É a matéria que abrange vários temas que ocorrem no nosso dia a dia, com conteúdos que abrangem os conteúdos de Física, Química e Biologia”. Essa visão provavelmente teve influência de uma ideia clássica de Ciência, herdada de um paradigma cartesiano, pautado no determinismo, racionalismo e mecanicismo (LAURINO; PINTO; NOVELLO, 2013). Nessa concepção, segundo Laurino, Pinto e Novello (2013, p. 2) o cientista é aquele que descobre algo que já está posto, objetivo, sem considerar sua subjetividade. Essa visão influencia também as concepções de práticas educacionais, pois pressupõem representações científicas já construídas, que acabam apenas sendo transmitidas como verdades aos estudantes. Página \| 81 R. bras. Ens. Ci. Tecnol., Ponta Grossa, v. 12, n. 2, p. 73-93, mai./ago. 2019. Nesse sentido, aceitar/acreditar em uma única verdade independente das ações humanas é negar o potencial criativo e investigativo do estudante, e para tal, negar a responsabilidade de cada um sobre o mundo. Por isso, os objetivos do Ensino de Ciências deveriam ser de educar cientificamente a população para que tenham consciência dos problemas do mundo e de sua possibilidade de atuação sobre os mesmos, de sua capacidade de modificar situações, incluso as amplamente aceitas. O L15 e L22, respectivamente, atribuíram uma visão salvacionista ao papel do cientista: “Desenvolvem pesquisas, procuram descobertas na área de saúde, procuram cura, através de remédios e vacinas para alimentação, pesquisam melhorias que não afetem tanto no ser humano, no meio ambiente, sempre à procura de mantê-lo habitável e sustentável”. “Pesquisa de grande e profunda importância, sobre doenças, remédios, sobre o meio em que vivemos”. Percebemos o papel redentor associado à Ciência, em que as atividades realizadas são relacionadas à busca de soluções a problemáticas como saúde pública e equilíbrio ambiental, atribuindo uma posição neutra no fazer científico, e de certo modo desconsiderando influências e intencionalidades políticas, sociais e econômicas. Delizoicov e Auler (2011, p. 251) consideram que na produção científica devem-se levar em conta alguns parâmetros como “investimento financeiro para a pesquisa científico-tecnológica e interesses estratégicos, das nações e governos, ao planejarem e promoverem seus projetos de pesquisa e desenvolvimento (P&D)”. Assim, os problemas científicos assumem uma dimensão não apenas temporal, mas espaço-temporal, pois demonstram uma intencionalidade através de sua localização e formulação (DELIZOICOV; AULER, 2011), pressupostos que contribuem para compreender o caráter não-neutro da gênese da Ciência e do próprio sistema científico-tecnológico. A compreensão de uma Ciência neutra ainda permanece fortemente presente em vários âmbitos da sociedade, em instituições como a academia e laboratórios de pesquisa e, conforme detectado por Gil Perez et al. (2001), além dos meios de comunicação, também parece estar presente em alguns setores da Universidade, na sociedade em geral e na Educação em Ciências. Para Oliveira (2008), assumir a neutralidade da ciência, isolando-a da esfera valorativa, significa uma Ciência fora do alcance de questionamentos em termos de valores sociais, dimensão esta impregnada na cultura ocidental. Auler (2002) identifica e analisa quatro dimensões interdependentes sobre a não neutralidade da Ciência e Tecnologia, sendo elas: 1) O direcionamento dado à atividade científico tecnológica (processo) resulta de decisões políticas; 2) A apropriação do conhecimento científico-tecnológico (produto) também resulta de decisões políticas; 3) O conhecimento científico produzido (produto) não é resultado apenas dos tradicionais fatores epistêmicos: lógica + experiência e 4) O aparato ou produto científico-tecnológico incorpora, materializa interesses, desejos de sociedade ou de grupos sociais hegemônicos. Página \| 82 Diante disso, as atividades de pesquisa deveriam ser direcionadas pela formulação dos problemas significativos para um determinado grupo e cuja solução é buscada pelos conhecimentos e práticas por ele compartilhados R. bras. Ens. Ci. Tecnol., Ponta Grossa, v. 12, n. 2, p. 73-93, mai./ago. 2019. (DELIZOICOV, 2004). Deste modo, a caracterização da não-neutralidade, deve ser direcionada aos problemas a serem investigados pela área de Ensino de Ciências, no Brasil. Dentre as várias possibilidades de se enfrentar o desafio dessa busca de sintonia, não se pode deixar de mencionar a contribuição que Freire (2016) dá através da proposição da investigação temática. Através dela, segundo argumenta esse educador, a equipe de pesquisadores pode identificar, em um processo dialógico e participativo, as temáticas significativas que são próprias de uma unidade epocal (FREIRE, 2016), ou seja, de características historicamente situadas, e que são manifestações locais de contradições sociais mais amplas, conforme também argumenta Santos (1977). Nesse sentido, convém mencionar, assim como assinala Carvalho (2012), que para fazer, falar, ler e escrever Ciência é necessário combinar de muitas maneiras o discurso verbal, as expressões matemáticas e representações gráficas. Nesse cenário, as linguagens falada e escrita são sistemas simbólicos que são utilizados para elaborar, descrever e apresentar processos e construir argumentos científicos. Segundo a autora, tais habilidades e competências deveriam ser suscitadas no ensino de Ciências desde os primeiros anos do Ensino Fundamental, de modo a desenvolver uma linguagem comum, do cotidiano a uma linguagem científica, utilizando de ferramentas científicas. Além do caráter não neutro identificamos também a visão utilitarista vinculada a Ciência e Tecnologia, remetendo a ideia de que essas instâncias trarão a cura a doenças e “descobrirão” medicamentos, tendo como princípio balizador a preservação da vida, atribuída como um “bem comum” a todos. Essas visões são descritas por L14 “descobertas de medicamentos para cura de doenças”. O mesmo percebemos do L16, quando menciona: “criação de medicamentos para proteger a vida”, e ajudar pessoas, no caso de tecnologias, nanotecnologias, medicamentos”. O entendimento de L19 relaciona-se ao desenvolvimento científicotecnológico, pois engloba vários temas, com objetivos diferenciados, como estudo da natureza e produção de remédios, assim menciona que Ciência “é o que estuda o porquê das coisas, buscando sempre uma real explicação dos acontecimentos pensando sempre na evolução”. O L25 também considera: “Estudo dos fenômenos naturais e tecnológicos, graças a ela não vivemos na época da pedra”. Percebemos o caráter utilitarista que os acadêmicos atribuem a Ciência, já que uma característica marcante na fala dos mesmos é a finalidade que o desenvolvimento científico possui: servir para. Nesta perspectiva, Bachelard (1996) caracteriza um aspecto marcante do pensamento pré-científico, a ideia de que todo fenômeno, necessariamente, precisa ter uma utilidade para ser validado, pois se não ganhar uma característica particular parece que não foi explicado. Esses aspectos constituem o obstáculo a ser superado do conhecimento unitário e pragmático. Página \| 83 Essa concepção, comumente aceita, transmite uma imagem deformada, descontextualizada e ingênua sobre o desenvolvimento científico. Os cientistas passam a ser considerados como seres “acima do bem e do mal”, fechados em torres de marfim, que proporcionam soluções a problemáticas de cada R. bras. Ens. Ci. Tecnol., Ponta Grossa, v. 12, n. 2, p. 73-93, mai./ago. 2019. sociedade/tempo (GIL-PEREZ et al., 2001). Nesse pensamento a Ciência não deveria servir “para”, mas partir de um “desejo”, um “querer”, já que para Bachelard (1996) é considerada como uma linguagem própria, específica, com conceitos próprios. Essa visão utilitarista, segundo Auler e Delizoicov (2006) é bastante difundida, já que se tem a ideia de que em algum momento, do presente ou futuro, Ciência e Tecnologia irão “resolver” os problemas existentes, propiciando um bem-estar social. Tanto Ciência como Tecnologia são associadas como redentoras, ignorando-se as relações sociais em que ambas estão inseridas. Porém, é relevante mencionar que na medida em que a Ciência penetrou na indústria, esta “foi profundamente industrializada” (JAPIASSU, 1988, p. 146). Em sua análise, Japiassu considera que isto não quer dizer que os fins meramente utilitários predominem na orientação da Ciência, mas que as normas intelectuais e éticas dos cientistas sofrem os efeitos de “novos imperativos”, passando, cada vez mais, a depender das decisões e dos financiamentos externos ao mundo científico. Assim, “as escolhas dos cientistas, que a princípio eram ‘livres’, tiveram que se dobrar às opções estranhas e ao interesse imanente à Ciência” (JAPIASSU, 1988, p. 146). Santos e Mortimer (2002) criticam a cientificidade atribuída a produção do conhecimento científico, pois é visto como um conhecimento superior a outras formas de saber, e que por si só todos os problemas são solucionados. Já Gonçalves (2015, p. 21) cita o determinismo científico na perspectiva de assegurar a Ciência e também a Tecnologia os benefícios e resoluções de problemas da humanidade. Nessa visão o conhecimento científico é sempre verdadeiro, superior e inquestionável. Sobre a descrição das atividades realizadas por um cientista, L22 relaciona à transmissão do conhecimento: “Desenvolve atividade de pesquisa onde depois de chegar a conclusões de seus experimentos transmite aos seus alunos”. Esta descrição se aproxima da discussão de Educação Bancária, conceituada por Freire (2016) como uma forma de controle na relação educador-educando, em que os conhecimentos são repassados do primeiro para o segundo, de modo que o educando é considerado sujeito passivo, sem consciência. Nessa concepção não há condições de criatividade e autonomia, pois para o autor “ensinar não é transferir conhecimento, mas criar as possibilidades para a sua produção ou a sua construção” (FREIRE, 2013, p. 24). Imagem reducionista do cientista Neste item discutimos as visões dos licenciandos em relação à imagem atribuída a um cientista, tanto física como social. As compreensões dizem respeito a uma imagem eurocêntrica que traduz a ideia de um indivíduo que está apartado da sociedade em geral ou é superior a ela, tendo um estereótipo bem definido, talvez influenciado pela formação escolar, reforçada pelos livros didáticos e mídia social. Página \| 84 Alguns acadêmicos agregaram a visão social de cientista vinculada a uma perspectiva individualista, como um indivíduo sério e “antissocial”, a exemplo: “Muito sério, de pouco assunto, meio antissocial” (L3); “Estuda em laboratórios o R. bras. Ens. Ci. Tecnol., Ponta Grossa, v. 12, n. 2, p. 73-93, mai./ago. 2019. benefício que o experimento trás para a sociedade” (L5); “Trabalha diretamente em laboratórios” (L15); “Antissocial, excluído, deveria interagir com a sociedade” (L7). É importante que a sociedade tenha clareza de que a Ciência é, acima de tudo, uma produção social, realizada por seres humanos, que reflete os interesses e ideologias de determinadas classes sociais, e que, embora internamente estruturada, apresenta divergências. Cachapuz et al. (2011) refletindo sobre o estudo e produção da Ciência, mencionam que os fundamentos e métodos, seu crescimento e contexto de descoberta não se constituem uma construção racional isolada, pois faz parte de uma teia de relações, que muitas vezes é ocultada, mas que convém aprofundálas em uma Educação em Ciências. Da mesma forma em que essas concepções distorcidas incidem sobre o ensino praticado e consequentemente na aprendizagem dos estudantes, que seguem uma orientação epistemologicamente pré-concebida. Os licenciandos ainda associaram a imagem do cientista a uma pessoa antissocial e excluída, que praticamente “vive” em um laboratório. Destacamos o caráter mutável que tanto o fazer científico como o cientista possuem, por este último ser humano, passível a erros, acertos e correlacionado a uma série de fatores que não apenas a produção científica. Favorável a essa discussão Bueno (2015, p. 39) discorre que “não existiu um método único e universal para chegar a todos os conhecimentos”, pois a maior parte dos estudos científicos foram feitos em equipe, as discussões, os debates coletivos, as posições e contraposições foram justamente o que contribuíram para a evolução do conhecimento. O autor menciona que os programas ou as tradições de investigação desenvolveram-se com os mesmos pressupostos de qualquer atividade humana. Porém, ainda destaca que as comunidades cientificas foram sempre muito conservadoras, pois aqueles que, de certa forma, afastaram-se da Ciência oficial não foram facilmente aceitos (BUENO, 2015). Diante disso, percebemos que historicamente a Ciência construiu uma imagem de cientista que a tempos é aceita, descrita pelo gênero masculino, magro e que usa óculos, e esse perfil parece sustentar-se até hoje. Em relação aos aspectos físicos os licenciandos descreveram essa visão clássica de cientista: “Usa óculos, jaleco branco é um pouco doido” (L2); “Masculino, alto, magro, sério e de óculos” (L3). Segundo Chassot (2003a) a quase ausência de mulheres na História da Ciência, ainda nas primeiras décadas do século XX, estava culturalmente definida como uma carreira imprópria para a mulher, da mesma maneira que, ainda na segunda metade do século XX, se dizia quais eram as profissões de homens e quais as de mulheres. Página \| 85 O número de mulheres que se dedicam às Ciências, em termos globais, é ainda menor que o de homens, mesmo que se possa dizer que nas décadas que nos são mais próximas tem havido uma presença significativa das mulheres nas mais diferentes áreas da Ciência, mesmo naquelas que antes pareciam domínio quase exclusivo dos homens (CHASSOT, 2003a; 2003b). Parece que usualmente não se valorizam significativamente as contribuições femininas. R. bras. Ens. Ci. Tecnol., Ponta Grossa, v. 12, n. 2, p. 73-93, mai./ago. 2019. Fourez (1995, p. 127) esclarece que: quando se fala de Ciência, pode-se distinguir dois tipos de atitudes. Uma delas chamada de idealista, caracterizada pela aceitação de normas universais e eternas, que determinam de que modo é e como deve ser o real. Uma outra, denominada histórica, vê as configurações assumidas pela Ciência como resultado de uma evolução, que não obedece necessariamente a leis eternas. Assim, se nos afiliarmos como “históricos” podemos entender que essa concepção de uma Ciência masculina ocorre, como resultado de uma História, humanamente construída, logo falível, e, dessa forma, estaremos sendo agentes desta construção e temos possibilidades de fazer modificações. Ou, se for ao contrário, caso dermos razão àqueles “idealistas” que assumem o conservadorismo de um modelo pronto e imutável, estaremos contribuindo para reforçar preconceitos milenares (CHASSOT, 2003b). Diante do exposto, as visões que são postas e repassadas em relação ao cientista e as atividades realizadas por este acabam sendo deformadas e empobrecidas, contribuindo para uma imagem pública da Ciência e do cientista como alheia e inatingível, quando não se torna recusável, fazendo também com que o interesse dos jovens seja diminuído drasticamente (CACHAPUZ et al., 2011). Por isso Cachapuz et al. (2011) argumentam sobre a necessária aposta em uma educação científica para a formação de cidadãos, em detrimento da orientada preparação de futuros cientistas, mesmo com grandes resistências de alguns professores, que defendem que a sociedade necessita de cientistas e tecnólogos, que devem ser selecionados desde cedo. Vale ressaltar que o discurso de L3 e L22 (respectivamente) em relação ao perfil de um cientista modificou-se ao longo do componente, a saber: “qualquer pessoa”; “uma pessoa com vida social, como outra qualquer”. Provavelmente as compreensões destes licenciandos alteraram-se devido as leituras e aprofundamentos teórico-práticos em que foram trabalhos no componente. ALGUMAS CONSIDERAÇÕES Este estudo investigou as compreensões de licenciandos em Ciências da Natureza sobre a NdC e o fazer científico, tanto no aspecto social como físico, no âmbito de um componente curricular sobre experimentação no Ensino de Ciências. Intencionamos discutir essas questões tendo em vista o direcionamento da prática docente, que é influenciado pelas visões atribuídas a concepção de Ciência e da atividade científica. Quanto à primeira categoria identificamos algumas visões deformadas do trabalho científico: naturalista, salvacionista e utilitarista. Tais compreensões podem ter sido atribuídas pela própria formação escolar e humana dos licenciandos que pode não ter possibilitado o papel da Ciência diante do contexto social e tecnológico. Página \| 86 A partir da segunda categoria percebemos o caráter reducionista atribuído ao perfil do cientista, pois prevaleceram as definições de uma pessoa do gênero R. bras. Ens. Ci. Tecnol., Ponta Grossa, v. 12, n. 2, p. 73-93, mai./ago. 2019. masculino, que usa óculos e antissocial. Estes discursos descrevem uma visão unitária da Ciência e da produção científica. É provável que tais visões também direcionem o trabalho desenvolvido pelos licenciandos futuramente em sala de aula e no modo como inserem o papel da experimentação, já que possivelmente os procedimentos adotados estarão assinalados por suas concepções sejam elas acrítica, salvacionista, reducionista ou utilitarista. Nesse sentido, a extensão das visões deformadas sobre Ciência é resultado de uma ausência, quase absoluta, de uma reflexão epistemológica e também de aceitação acrítica de um ensino pautado por transmissão de conhecimento, sendo necessário uma reflexão crítica sobre tais questões, no intuito de se apropriar com certa facilidade da atividade científica, o que perpassa pensar os objetivos do ensino, enquanto promoção da (re)construção do conhecimento (CACHAPUZ et al., 2011). Assim, no contexto do componente curricular que também trabalhou essas questões acreditamos que o mesmo possibilitou refletir sobre a concepção da NdC e do papel do cientista, muitos licenciandos despertaram um olhar mais crítico sobre as atribuições da Ciência, as influências que a mesma possui e como este entendimento é transposto a prática educativa. Pode-se dizer que embora a imagem de um cientista seja fortemente marcada por um perfil eurocêntrico vislumbra-se a inserção de outros sujeitos ao fazer científico, inclusive à possibilidade de serem os próprios licenciandos. No caso da construção do conhecimento científico, pode-se pensar que a priori não há um roteiro de onde se quer chegar, nem um caminho definitivo para realizar. Nas aulas de Ciências, o professor deve conhecer o produto e o processo que pretende discutir com seus estudantes, e assim, auxiliar nas situações problemas. Aprender Ciências não é só aprender conceitos. É mais, a aprendizagem conceitual depende da estrutura das concepções, da forma de argumentar, das estratégias de resolução de problemas, da coerência de raciocínio, da utilização das interpretações, e do alcance das intervenções. Assim sendo, apontamos para a importância da discussão sobre as concepções de Ciências, da NdC e do fazer cientifico para o campo de formação de professores, tanto em caráter inicial como continuado, tendo em vista a relevância de se pensar a discussão sobre aspectos da História e Filosofia da Ciência, desmistificando crenças e aparências à tempos aceitas, para promover definições coerentes com a produção do conhecimento científico. Página \| 87 R. bras. Ens. Ci. Tecnol., Ponta Grossa, v. 12, n. 2, p. 73-93, mai./ago. 2019. The beliefs and appearances: understanding on science by licensing in natural sciences ABSTRACT The present article investigated and discussed the conceptions of graduates of a course in Natural Sciences in relation to the Nature of Science and the profile of the scientist in the scientific making, within the framework of a curricular component of experimentation in science teaching. It is a qualitative research, in which the data were collected through the application of a questionnaire and the construction of a class diary, which contributed to the understanding of the discourse of the research subjects. Through Content Analysis the results were systematized into two categories, of which: deformed vision about the nature of Science and the reductionist image of the scientist. The results pointed to an uncritical understanding of science, guided by a salvationist, reductionist and utilitarian view of scientific work, indicating the need to discuss and rethink the approach to such questions and how they are approached in teacher training. KEYWORDS: Nature of science. Make scientific. Science teaching. Página \| 88 R. bras. Ens. Ci. Tecnol., Ponta Grossa, v. 12, n. 2, p. 73-93, mai./ago. 2019. REFERÊNCIAS AULER, D. Interações entre Ciência-Tecnologia-Sociedade no Contexto da Formação de Professores de Ciências. Tese de doutorado em Educação Científica e Tecnológica – Universidade Federal de Santa Catarina, Florianópolis, 2002. ______.; DELIZOICOV, D. Ciência-Tecnologia-Sociedade: relações estabelecidas por professores de ciências. Revista Electrónica de Enseñanza de Las Ciencias, v. 5, n. 2, 2006. Disponível em: <http://reec.uvigo.es/volumenes/volumen5/ART8_Vol5_N2.pdf>. Acesso em: 05 nov. 2017. BACHELARD, G. 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Diários de aula: um instrumento de pesquisa e desenvolvimento profissional. Porto Alegre: Artmed. 2004. Página \| 92 R. bras. Ens. Ci. Tecnol., Ponta Grossa, v. 12, n. 2, p. 73-93, mai./ago. 2019. Recebido: 2017-11-20 Aprovado: 2019-02-27 DOI: 10.3895/rbect.v12n2.7371 Como citar: FONSECA, E. M.; DUSO, L. Entre crenças e aparências: compreensões sobre ciência por licenciandos em ciências da natureza.. Revista Brasileira de Ensino de Ciência e Tecnologia, v. 12, n. 2, 2019. Disponível em: <https://periodicos.utfpr.edu.br/rbect/article/view/7371>. Acesso em: xxx. Correspondência: Eril Medeiros da Fonseca - erilmf@gmail.com Direito autoral: Este artigo está licenciado sob os termos da Licença Creative Commons-Atribuição 4.0 Internacional. Página \| 93 R. bras. Ens. Ci. Tecnol., Ponta Grossa, v. 12, n. 2, p. 73-93, mai./ago. 2019.
https://openalex.org/W2784892229	https://europepmc.org/articles/pmc5794197?pdf=render	English	null	The fungal myosin I is essential for Fusarium toxisome formation	PLOS pathogens	2,018	cc-by	14,457	RESEARCH ARTICLE Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Guangfei Tang1☯, Yun Chen1☯, Jin-Rong Xu2, H. Corby Kistler3, Zhonghua Ma1,4* 1 Institute of Biotechnology, Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Zhejiang University, Hangzhou, China, 2 Department of Botany and Plant Pathology, Purdue University, West Lafayette, Indiana, United States of America, 3 Department of Plant Pathology, University of Minnesota, St. Paul, Minnesota, United States of America, 4 State Key Laboratory of Rice Biology, Zhejiang University, Hangzhou, China ☯These authors contributed equally to this work. * zhma@zju.edu.cn a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Abstract Myosin-I molecular motors are proposed to function as linkers between membranes and the actin cytoskeleton in several cellular processes, but their role in the biosynthesis of fungal secondary metabolites remain elusive. Here, we found that the myosin I of Fusarium grami- nearum (FgMyo1), the causal agent of Fusarium head blight, plays critical roles in mycotoxin biosynthesis. Inhibition of myosin I by the small molecule phenamacril leads to marked reduction in deoxynivalenol (DON) biosynthesis. FgMyo1 also governs translation of the DON biosynthetic enzyme Tri1 by interacting with the ribosome-associated protein FgAsc1. Disruption of the ATPase activity of FgMyo1 either by the mutation E420K, down-regulation of FgMyo1 expression or deletion of FgAsc1 results in reduced Tri1 translation. The DON biosynthetic enzymes Tri1 and Tri4 are mainly localized to subcellular structures known as toxisomes in response to mycotoxin induction and the FgMyo1-interacting protein, actin, participates in toxisome formation. The actin polymerization disruptor latrunculin A inhibits toxisome assembly. Consistent with this observation, deletion of the actin-associated pro- teins FgPrk1 and FgEnd3 also results in reduced toxisome formation. Unexpectedly, the FgMyo1-actin cytoskeleton is not involved in biosynthesis of another secondary metabolite tested. Taken together, this study uncovers a novel function of myosin I in regulating myco- toxin biosynthesis in filamentous fungi. The fungal myosin I is essential for Fusarium toxisome formation Guangfei Tang1☯, Yun Chen1☯, Jin-Rong Xu2, H. Corby Kistler3, Zhonghua Ma1,4* OPEN ACCESS Citation: Tang G, Chen Y, Xu J-R, Kistler HC, Ma Z (2018) The fungal myosin I is essential for Fusarium toxisome formation. PLoS Pathog 14(1): e1006827. https://doi.org/10.1371/journal. ppat.1006827 Editor: Alex Andrianopoulos, University of Melbourne, AUSTRALIA Editor: Alex Andrianopoulos, University of Melbourne, AUSTRALIA Editor: Alex Andrianopoulos, University of Melbourne, AUSTRALIA Received: August 15, 2017 Accepted: December 18, 2017 Published: January 22, 2018 Copyright: © 2018 Tang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Received: August 15, 2017 Accepted: December 18, 2017 Published: January 22, 2018 Copyright: © 2018 Tang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Introduction Fusarium head blight (FHB) caused predominately by Fusarium graminearum is an economi- cally devastating disease of small grain cereal crops [1]. This disease not only reduces yield and seed quality but also poses a great risk to human and animal health owing to its ability to con- taminate grains with mycotoxins. The common mycotoxins associated with F. graminearum are deoxynivalenol (DON), nivalenol (NIV) and zearalenone (ZEA) [2]. Among them, DON is the most frequently detected mycotoxin in cereal grains throughout the world [3]. DON can inhibit protein synthesis by binding to the ribosome, and cause emetic effects, anorexia and immune dysregulation as well as growth, reproductive and teratogenic effects in mammals [4]. To minimize human and animal exposure to DON, regulatory organizations have established maximum permissible levels for DON in cereals and their products in many countries [5, 6]. However, DON contamination has become a challenging social issue because of the increased frequency and severity of FHB epidemics [7, 8]. Competing interests: The authors have declared that no competing interests exist. DON contamination is closely linked to the severity of FHB disease in the field. The best way to prevent DON contamination would be to manage FHB in the field during crop cultiva- tion. Currently, application of chemical fungicides is still a major approach against F. grami- nearum infection due to the lack of highly resistant wheat cultivars. However, application of several commercialized fungicides at sub-lethal concentrations could trigger DON biosynthe- sis [3, 9–11]. Recently, a novel cyanoacrylate fungicide phenamacril (JS399-19) has been mar- keted for FHB management and sale of phenamacril in China was approximately $40 million in 2016–2017. Interestingly, this small molecule compound (S1 Fig) exhibits highly specific antifungal activity against mycelial growth of a few Fusarium species including F. grami- nearum, F. asiaticum, F. verticillioides and F. oxysporum but not other fungal pathogens [12]. It shows excellent efficacy in controlling FHB in field trials [12, 13]. Combining inferences from genetic and biochemical results, we recently discovered that this compound acts on a novel tar- get, the class I myosin (FgMyo1) in F. graminearum, which is homologous to Myo3p and Myo5p in Saccharomyces cerevisiae [12]. FgMyo1 is essential for F. graminearum growth. At the beginning of this study, we found that phenamacril not only suppressed the mycelial growth of F. graminearum, but also significantly inhibited DON production. Functions of FgMyo1 in mycotoxin biosynthesis Dabeinong Funds for Discipline Development and Talent Training in Zhejiang University to ZM. Further support was by award 2014-67013-21561 from the Agriculture and Food Research Initiative of the National Institute of Food and Agriculture, United States Department of Agriculture to HCK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. was involved in toxisome formation but not for the biosynthesis of another secondary metabolite tested. Taken together, these results indicate for the first time that myosin I plays critical roles in mycotoxin biosynthesis. was involved in toxisome formation but not for the biosynthesis of another secondary metabolite tested. Taken together, these results indicate for the first time that myosin I plays critical roles in mycotoxin biosynthesis. Author summary The mycotoxin deoxynivalenol (DON) is the most frequently detected secondary metabo- lite produced by Fusarium graminearum and other Fusarium spp. To date, relatively few studies have addressed how mycotoxin biosynthesis occurs in fungal cells. Here we found that myosin I governs translation of DON biosynthetic enzyme Tri1 via interacting with the ribosome-associated protein FgAsc1. Moreover, the key DON biosynthetic enzymes Tri1 and Tri4 are mainly localized to the toxisomes derived from endoplasmic reticulum under toxin inducing conditions. We further found that the FgMyo1-actin cytoskeleton Funding: This research was supported by the National Natural Science Fund for Distinguished Young Scholar (31525020) to ZM, National Science Foundation (31672064) to YC, Natural Science Foundation of Zhejiang Province for Distinguished Young Scholar (LR17C140001) to YC, China Agriculture Research System (CARS-3- 1-15) to ZM, the Fundamental Research Funds for the Central Universities (2017FZA6014) to ZM, and 1 / 23 PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1006827 January 22, 2018 PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1006827 January 22, 2018 Functions of FgMyo1 in mycotoxin biosynthesis the isoprenoid intermediate farnesyl pyrophosphate to DON involves 15 genes encoding the biosynthetic enzymes, a DON transporter and regulatory proteins, which are located on differ- ent chromosomes: the 25 kb Tri5 cluster containing 12 genes on chromosome 2, the Tri1-Tri16 locus with two genes on chromosome 1 and the single gene locus for Tri101 on chromosome 3 [19–21]. Recent studies suggested that there is a cellular compartmentalization of biosynthetic enzymes for DON biosynthesis in F. graminearum [22]. Hydroxymethylglutaryl (HMG) CoA reductase (Hmr1) is a key enzyme in the mevalonate pathway for generating farnesyl pyro- phosphate and indispensable for DON production. Fluorescent labeled Hmr1-GFP localized to the reticulate peripheral and perinuclear endoplasmic reticulum (ER) in toxin non-inducing conditions, while the ER was remodeled to form spherical and ovoid structures in the tricho- thecene biosynthesis inducing (TBI) conditions [16, 22]. In addition, the enzymes trichodiene oxygenase (Tri4) and calonectrin oxygenase (Tri1) catalyzing the early and late steps in the DON biosynthetic pathway were co-localized and showed the same localization patterns as Hmr1 in TBI medium [22, 23]. These novel cellular structures containing DON biosynthesis enzymes were named "Fusarium toxisomes" (“toxisomes” in shorter form in this study) [16, 22, 23]. However, the molecular mechanism of toxisome formation remains elusive. The object of this study was to uncover the underlying mechanism of a myosin I inhibitor in regulating DON biosynthesis. Our results showed that myosin I plays critical roles in the translation of a Tri enzyme and in toxisome formation in F. graminearum. The importance of myosin I in the development of the mycotoxin biosynthetic machinery in F. graminearum may apply to other toxigenic pathogens. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1006827 January 22, 2018 Introduction These prelimi- nary results suggested that the myosin I might also be involved in the secondary metabolism. Class I myosins are widely expressed, single headed and membrane-associated members of the myosin superfamily that participate in regulating membrane dynamics and structure in nearly all eukaryotic cells [14, 15]. However, the underlying function of myosin I in mycotoxin bio- synthesis was totally unknown. Enzymes for secondary metabolite synthesis may be compartmentalized at conserved sub- cellular sites in fungi, potentially channeling precursors, sequestering intermediates and prod- ucts from the rest of the cell, thus promoting the efficiency of biosynthesis pathways [16]. In Penicillium chrysogenum, the major facilitator-type secondary transporter PenM promotes translocation of isopenicillin N from the cytosol to the peroxisomal lumen where it could be further metabolized to penicillin [17]. In Aspergillus, aflatoxin biosynthetic enzymes flow from peroxisomes to the motile vesicles termed aflatoxisomes in which aflatoxin biosynthesis takes place [18]. In F. graminearum, and other Fusarium spp, the biosynthetic pathway leading from PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1006827 January 22, 2018 2 / 23 Fig 1. Phenamacril disrupted toxisome formation and subsequently inhibited DON production. (A) Tri1-GFP localized to the spherical structures (termed as toxisomes) in hyphae grown in TBI but not in PDB or MM. Images were taken after each strain was incubated at 28 ˚C for 48 h. Bar = 10 μm. DIC indicates differential interference contrast. (B) After growth in TBI for 48 h, hyphae of ΔTri1::Tri1-GFP were stained with the ER-tracker red and examined for GFP and ER tracker signals. Bar = 10 μm. (C) After growth in TBI for 48 h, hyphae of PH-1::Tri1-GFP+H1-RFP were examined for the co- localization of H1-RFP and Tri1-GFP. Bar = 10 μm. (D) Hyphae of ΔTri1::Tri1-GFP were treated with 0.5 μg/ml phenamacril, or 1.4 μg/ml carbendazim for 24 h in TBI before examination for GFP signals. The solvent DMSO was used as a control. Bar = 10 μm. (E) Western blots of proteins isolated from the same set of samples used in 1D were detected with the anti-GFP or anti-GAPDH antibody. (F) DON production was assayed for the wild-type PH-1 growth in TBI supplemented with 0.5 μg/ml phenamacril or 1.4 μg/ml carbendazim. The solvent DMSO was used as a control. Values on the bars followed by the same letter are not significantly different according to a Fisher’s least significant difference (LSD) test at P = 0.05. (G) Phenamacril inhibited toxisome formation Functions of FgMyo1 in mycotoxin biosynthesis Functions of FgMyo1 in mycotoxin biosynthesis Fig 1. Phenamacril disrupted toxisome formation and subsequently inhibited DON production. (A) Tri1-GFP localized to the spherical structures (termed as toxisomes) in hyphae grown in TBI but not in PDB or MM. Images were taken after each strain was incubated at 28 ˚C for 48 h. Bar = 10 μm. DIC indicates differential interference contrast. (B) After growth in TBI for 48 h, hyphae of ΔTri1::Tri1-GFP were stained with the ER-tracker red and examined for GFP and ER tracker signals. Bar = 10 μm. (C) After growth in TBI for 48 h, hyphae of PH-1::Tri1-GFP+H1-RFP were examined for the co- localization of H1-RFP and Tri1-GFP. Bar = 10 μm. (D) Hyphae of ΔTri1::Tri1-GFP were treated with 0.5 μg/ml phenamacril, or 1.4 μg/ml carbendazim for 24 h in TBI before examination for GFP signals. The solvent DMSO was used as a control. Bar = 10 μm. The trichothecene biosynthetic enzymes are localized at toxisomes under toxin inducing conditions TRI1 encodes calonectrin oxygenase that catalyzes calonectrin to 7, 8-dihydroxycalonetrin, which is a late step of DON biosynthesis in F. graminearum [24]. To characterize expression patterns and the sub-cellular localization of Tri1 protein under various conditions, the TRI1 open reading frame tagged with GFP (green fluorescent protein) was introduced into a ΔTri1 F. graminearum PH-1 background, and the complemented strain expressing the Tri1-GFP (ΔTri1::Tri1-GFP) was used in the following study. In the toxin non-induction minimal (MM) or potato dextrose broth (PDB) media, Tri1-GFP displayed faint signals and was mainly asso- ciated with cell endomembrane (Fig 1A, left and middle panels). Tri1-GFP was highly induced and localized at the spherical structures (toxisomes) after 48 hours of incubation in the tricho- thecene biosynthesis induction (TBI) medium (Fig 1A right panel; S2 Fig) and in planta (Fig 1G, left panel). In addition, ER (endoplasmic reticulum)-tracker red staining indicated that Tri1-GFP was mainly localized to the ER in TBI cultures (Fig 1B), which is consistent with a previous finding that the toxisomes were identified as reorganization of the endoplasmic retic- ulum [22]. To determine whether the spherical structures were associated with the nucleus, we visualized nuclei by tagging the histone1 protein encoded by the FGSG_10800 locus with red fluorescent protein (RFP), which was designated as H1-RFP in the PH-1::Tri1-GFP strain. The H1-RFP/Tri1-GFP dual labeled strain was grown in TBI for 48 h, and localization of H1-RFP with Tri1-GFP was examined. As shown in Fig 1C, Tri1-GFP surrounded the H1-RFP labelled nuclei when the strain was cultured in the TBI medium. Moreover, the trichodiene oxygenase (Tri4) catalyzing the early step of DON biosynthesis had the same localization pattern as Tri1 (S3 Fig). Taken together, several lines of evidence suggested that trichothecene biosynthetic enzymes were clustered and localized to toxisomes derived from ER under the toxin inducing conditions. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1006827 January 22, 2018 3 / 23 (E) Western blots of proteins isolated from the same set of samples used in 1D were detected with the anti-GFP or anti-GAPDH antibody. (F) DON production was assayed for the wild-type PH-1 growth in TBI supplemented with 0.5 μg/ml phenamacril or 1.4 μg/ml carbendazim. The solvent DMSO was used as a control. Values on the bars followed by the same letter are not significantly different according to a Fisher’s least significant difference (LSD) test at P = 0.05. (G) Phenamacril inhibited toxisome formation PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1006827 January 22, 2018 4 / 23 Functions of FgMyo1 in mycotoxin biosynthesis in hyphae of ΔTri1::Tri1-GFP inoculated on wheat leaf. (H) Efficiencies of phenamacril (375 g/ha) and carbendazim (750 g/ha) in controlling Fusarium head blight (FHB) and DON contamination in the field trials. Values on the bars for disease incidence or DON production followed by the same letter are not significantly different according to a Fisher’s least significant difference (LSD) test at P = 0.05. https://doi.org/10.1371/journal.ppat.1006827.g001 https://doi.org/10.1371/journal.ppat.1006827.g001 https://doi.org/10.1371/journal.ppat.1006827.g001 The myosin I inhibitor restrains toxisome formation and mycotoxin biosynthesis Since the toxisomes are important for DON biosynthesis, a compound disrupting the toxisome formation may be very well effective against DON biosynthesis. To test this hypothesis, we established a "toxisome formation inhibitor screening" assay to quickly screen active com- pounds for their ability to restrict toxisome formation (see Material and methods). Briefly, the reporter strain expressing Tri1-GFP was grown in 24-wells plates supplemented with TBI medium. After 24 h incubation, individual compounds were added to wells. After incubation for another 24 h, the fluorescent intensity in each well was scanned with the plate-reader for the first round of screening. The wells with low or no fluorescent signals were further observed by microscopy. A total of 131 compounds including 11 commercial fungicides were tested for their activity against toxisome formation. Phenamacril was found to be the most efficient com- pound to inhibit toxisome formation and DON production (S4B and S4C Fig). The Tri1-GFP fluorescent signals were reduced dramatically and no typical toxisomes were observed in the mycelia treated with 0.5 μg/ml (approximately EC90 against mycelial growth) phenamacril for 6 h (S5B Fig) or 24 h (Fig 1D) in comparison with those in the non-treatment control. In addi- tion, the beta-tubulin inhibitor, carbendazim, did not inhibit toxisome formation (Fig 1D). As shown in Fig 1E, the translation levels of Tri1-GFP were further verified by immunoblot assay using an anti-GFP antibody. Consistent with the microscopic observation, the intensity of the Tri1-GFP band from the strain treated with carbendazim increased more than 2-fold as com- pared with the non-treated control. In contrast, a faint immunoblot band was detected in the same strain treated with phenamacril (Fig 1E). Correspondingly, DON in the mycelia treated with phenamacril was below the level detectable by LC-MS (liquid chromatography-mass spectrometer) (Fig 1F). Furthermore, we tested the efficiency of phenamacril against DON production in planta and in the field. As shown in Fig 1G, phenamacril also clearly inhibited toxisome formation in hyphae of F. graminearum inoculated on wheat leaf. In the field trials, this antifungal compound was very effective against FHB and DON production in comparison with the control chemical carbendazim (Fig 1H). The class I myosin (named FgMyo1) of F. graminearum has been identified as the target of phenamacril [12]. Taken together, these results strongly indicated that the myosin I inhibitor phenamacril was able to inhibit DON biosynthesis in F. graminearum. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1006827 January 22, 2018 Functions of FgMyo1 in mycotoxin biosynthesis Fig 2. FgMyo1 is required for toxin biosynthesis. (A) Localization of FgMyo1-RFP in hyphae of PH-1::FgMyo1-RFP growth in toxin non- inducing media PDB and MM at 28˚C for 48 h. Bar = 10 μm. (B) FgMyo1-RFP was co-localized with Tri1-GFP under the toxin inducing condition. Bar = 10 μm. (C) The interaction of FgMyo1 with Tri1 was confirmed by co-immunoprecipitation (Co-IP) analysis. Total proteins (input) extracted from the strain bearing FgMyo1-3×Flag and Tri1-GFP constructs or a single construct (FgMyo1-3×Flag or Tri1-GFP) were subjected to SDS-PAGE, and immunoblots were incubated with anti-Flag and anti-GFP antibodies, as indicated (Input panel). Each protein sample was pulled down using anti-Flag agarose and further detected with anti-GFP antibody (Flag pull-down panel). The protein samples were also incubated with the anti-GAPDH antibody as a reference. (D) The interaction of FgMyo1 with Tri1 was confirmed by bimolecular fluorescence complementation (BiFC) analysis. The constructs of pFgTri1-YFPN and pFgMyo1-YFPC were co-transformed into PH-1 to Fig 2. FgMyo1 is required for toxin biosynthesis. (A) Localization of FgMyo1-RFP in hyphae of PH-1::FgMyo1-RFP growth in toxin non- inducing media PDB and MM at 28˚C for 48 h. Bar = 10 μm. (B) FgMyo1-RFP was co-localized with Tri1-GFP under the toxin inducing condition. Bar = 10 μm. (C) The interaction of FgMyo1 with Tri1 was confirmed by co-immunoprecipitation (Co-IP) analysis. Total proteins (input) extracted from the strain bearing FgMyo1-3×Flag and Tri1-GFP constructs or a single construct (FgMyo1-3×Flag or Tri1-GFP) were subjected to SDS-PAGE, and immunoblots were incubated with anti-Flag and anti-GFP antibodies, as indicated (Input panel). Each protein sample was pulled down using anti-Flag agarose and further detected with anti-GFP antibody (Flag pull-down panel). The protein samples were also incubated with the anti-GAPDH antibody as a reference. (D) The interaction of FgMyo1 with Tri1 was confirmed by bimolecular fluorescence complementation (BiFC) analysis. The constructs of pFgTri1-YFPN and pFgMyo1-YFPC were co-transformed into PH-1 to Fig 2. FgMyo1 is required for toxin biosynthesis. (A) Localization of FgMyo1-RFP in hyphae of PH-1::FgMyo1-RFP growth in toxin non- d d d f h ( ) l l d h d h d Fig 2. FgMyo1 is required for toxin biosynthesis. (A) Localization of FgMyo1-RFP in hyphae of PH-1::FgMyo1-RFP growth in toxin non- inducing media PDB and MM at 28˚C for 48 h. Bar = 10 μm. (B) FgMyo1-RFP was co-localized with Tri1-GFP under the toxin inducing condition. Bar = 10 μm. The myosin I is essential for toxisome formation Given that the myosin I inhibitor significantly reduces DON biosynthesis, myosin I may be critical for toxisome formation. In order to verify this, we tagged FgMyo1 with RFP to deter- mine its subcellular localization. In toxin non-induction media MM and PDB, FgMyo1-RFP protein was detected as diffuse fluorescent signal in the cytoplasm, mainly localized at hyphal tips (Fig 2A). However, in the TBI medium, most FgMyo1-RFP fluorescence accumulated in subapical spherical structures (Fig 2A, right panel). To determine whether myosin I was local- ized to the toxisomes, a strain labeled with FgMyo1-RFP and Tri1-GFP was constructed and cultured in TBI. As indicated in Fig 2B, both proteins were mainly co-localized at the toxi- somes. Additionally, Co-IP and BiFC (Bimolecular Fluorescence Complementation) assays showed that FgMyo1 interacted with Tri1 in toxin inducing condition (Fig 2C and 2D). PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1006827 January 22, 2018 5 / 23 Functions of FgMyo1 in mycotoxin biosynthesis generate the strain FgTri1-YFPN+FgMyo1-YFPC. The strains bearing a single construct (FgMyo1-YFPC or FgTri1-YFPN) were used as negative controls. The YFP signals in hyphae of each strain grown in the TBI medium were examined under a confocal microscope. Bar = 10 μm. (E) The sensitivity of FgMyo1 derived mutants towards phenamacril. The wild-type PH-1, FgMyo1 silencing mutant FgMyo1-S2, inducible mutant Pzear-FgMYO1, and the point mutation strain FgMyo1E420K were incubated on PDA supplemented with 0.3 μg/ml phenamacril (left panel). For the inducible mutant, PDA was also added with (+) or without (-) the inducer 30 μg/ml β-estradiol. Mycelial growth inhibition of each strain by phenamacril was quantified (right panel). Values on the bars followed by the same letter are not significantly different according to a Fisher’s least significant difference (LSD) test at P = 0.05. (F) The toxisome formation patterns in FgMyo1 derived mutants. Each strain was grown in TBI, and images were taken after incubation for 48 h (left-upper panel). The accumulation of Tri1-GFP protein in each strain was determined by western blot assay with the anti-GFP antibody. The protein samples were also incubated with the anti-GAPDH antibody as a reference (left-lower panel). The intensities of GFP signals in each strain were also quantified. Values on the bars followed by the same letter are not significantly different according to a Fisher’s least significant difference (LSD) test at P = 0.05 (right panel). (G) The DON production of FgMyo1 derived mutants. DON was extracted from mycelia of each strain grown in TBI for 7 days. Values on the bars followed by the same letter are not significantly different according to a Fisher’s least significant difference (LSD) test at P = 0.05. https://doi.org/10.1371/journal.ppat.1006827.g002 https://doi.org/10.1371/journal.ppat.1006827.g002 https://doi.org/10.1371/journal.ppat.1006827.g002 Affinity capture mass spectrometry (ACMS) was then used to identify interacting proteins upon toxin-induction conditions using the dual tagged protein ZZ-Tri1-Flag as the bait. In the ACMS assay, FgMyo1 was captured by Tri1 (S1 Table). Furthermore, ten of the 30 Tri1-inter- acting proteins were described previously [22] as components of the toxisome, including the three cytochrome P-450 enzymes Tri1, Tri4 and Tri11 as well as HMG-CoA reductase. These results indicated that FgMyo1 interacts with Tri1 and thus has the potential for involvement in toxisome formation. To verify the role of FgMyo1 in toxisome formation, we used a knock-down approach because FgMYO1 is an essential gene in F. graminearum [12]. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1006827 January 22, 2018 (C) The interaction of FgMyo1 with Tri1 was confirmed by co-immunoprecipitation (Co-IP) analysis. Total proteins (input) extracted from the strain bearing FgMyo1-3×Flag and Tri1-GFP constructs or a single construct (FgMyo1-3×Flag or Tri1-GFP) were subjected to SDS-PAGE, and immunoblots were incubated with anti-Flag and anti-GFP antibodies, as indicated (Input panel). Each protein sample was pulled down using anti-Flag agarose and further detected with anti-GFP antibody (Flag pull-down panel). The protein samples were also incubated with the anti-GAPDH antibody as a reference. (D) The interaction of FgMyo1 with Tri1 was confirmed by bimolecular fluorescence complementation (BiFC) analysis. The constructs of pFgTri1-YFPN and pFgMyo1-YFPC were co-transformed into PH-1 to PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1006827 January 22, 2018 6 / 23 The myosin I is indispensable for translation of Tri1 To gain an insight into the function of FgMyo1 in toxisome formation, we further conducted an ACMS assay using the dual tagged protein ZZ-FgMyo1-Flag as the bait. In the ACMS assay, the ribosome-associated protein Asc1 (hereafter named FgAsc1,) was captured by FgMyo1. Unexpectedly, FgAsc1 was also pulled down by Tri1 (S1 Table). In addition, the interaction of FgMyo1 and FgAsc1 was confirmed by Co-IP assay (Fig 3A, left panel), while the directly interaction between these two proteins was not verified by BiFC. Given that the translation level of Tri1-GFP protein was inhibited dramatically by phenamacril (Fig 1D), and Asc1 is a conserved ribosomal protein and is required for efficient protein translation [30,31], we inferred that FgMyo1 might regulate Tri1 translation via interacting with Asc1. To test this hypothesis, we first examined co-localization of FgMyo1-GFP and FgAsc1 tagged with RFP. In toxin non-inducing conditions, FgAsc1-RFP was detected as diffuse fluorescent signal in the cytoplasm (Fig 3B). However, in the TBI medium, most FgAsc1-RFP accumulated in spherical structures and co-localized with FgMyo1 (Fig 3A, right panel) and also with Tri1 at the peri- nuclear positions (Fig 3B, lower panel). Since Asc1 is ribosome-associated protein, to further visualize localization of ribosomes, FgRpL25 (an essential component of 60S subunit of ribo- some [32, 33]) was tagged with mCherry under the control of its own promoter, and trans- formed into the wild type. Confocal microscopic examination showed that most FgRpL25- mCherry accumulated at the perinuclear positions in the toxin inducing conditions (S7 Fig, bottom panel). In contrast, FgRpL25- mCherry was mainly localized in the cytoplasm in the toxin non-inducing conditions (S7 Fig, upper panel). These results indicated that FgMyo1 interacts with the ribosome protein FgAsc1 in toxin inducing conditions. To further understand the role of FgAsc1 in Tri1 translation, we constructed a deletion mutant of FgAsc1. As expected, the mutant exhibited dramatically reduced hyphal growth (Fig 3C, upper panel). The translation level of Tri1-GFP in this mutant was decreased markedly in comparison with that in the wild type (Fig 3D, right panel), and subsequently, toxisome forma- tion and DON production was not detected in this mutant cultured in TBI medium (Fig 3D, left panel; Fig 3E). Functions of FgMyo1 in mycotoxin biosynthesis attenuated in virulence toward flowering wheat heads (S6 Fig). These results confirmed that FgMyo1 plays an important role in toxisome formation. The myosin I is indispensable for translation of Tri1 It is very interesting that the translation level of FK506-binding protein Fg_Fkbp54 (FGSG_01059) was not altered in ΔFgAsc1 as compared to that in the wild type (Fig 3F, right panel), suggesting that FgAsc1 controls the translation of some proteins (at least Tri1) but not all proteins, which is agreement with a role for Asc1 in regulating the translation of specific mRNAs in S. cerevisiae [31, 34]. Taken together, these results indicated that FgMyo1 was indispensable for translation of Tri1 protein by interacting with the ribosome protein FgAsc1. First, we took the advantage of the RNA interfering (RNAi) pathway to induce FgMYO1 silencing with hairpin RNA (hpRNA), which has been proven to be efficient in knockdown of mRNA expression for target genes in F. graminearum [25]. The recombinant plasmid pSilent-FgMYO1, designed for gener- ating the hpRNA of an FgMYO1 fragment (540 bp), was introduced into the wild-type PH-1. Predicting that transformants with reduced expression of FgMYO1 may grow poorly on the medium supplemented with phenamacril, we screened for transformants with increased sensi- tivity towards this compound and then verified the FgMYO1 expression level by reverse tran- scription-PCR. Among the 20 transformants tested, four showed increased sensitivity to phenamacril, and the expression levels of FgMYO1 were decreased 65%-90% in these silencing transformants in comparison with the wild type. The FgMYO1-S2 transformant, having the lowest FgMYO1 expression (10% of the parent strain PH-1), was selected for further character- ization. It had normal growth rate on PDA but failed to grow on PDA supplemented with phe- namacril at 0.3 μg/ml (approximately EC50 against mycelial growth) (Fig 2E). As expected, the toxisome formation indicated by Tri1-GFP was significantly impaired and only faint fluores- cent signals were observed in the mycelia of FgMYO1-S2 harboring Tri1-GFP (Fig 2F). Next, we replaced the native promoter of FgMYO1 with the zearalenone (ZEA)-inducible promoter (Pzear) [26] to generate a transformant that conditionally expressed FgMYO1. The resulting transformant (termed as Pzear-FgMYO1) without ZEA induction was unable to grow on PDA supplemented with 0.3 μg/ml phenamacril (Fig 2E). Consistently, this strain formed very faint toxisomes in TBI without the inducer as compared to the wild type (Fig 2F upper panel). The defects in mycelial growth and toxisome formation of Pzear-FgMYO1 were partially recovered by adding the inducer β-estradiol (Fig 2E and 2F, upper panel). In addition, translation levels of Tri1-GFP protein in above strains quantified by the western blotting assay were consistent with fluorescent signals (Fig 2F, lower panel). All of the above mutants, whether constructed by silencing or conditional expression, revealed significantly reduced DON production in TBI (Fig 2G). Since DON is a critical virulence factor and plays a significant role in the spread of pathogen within host tissues [27–29], it follows that each of these strains was severely PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1006827 January 22, 2018 7 / 23 PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1006827 January 22, 2018 The myosin I-actin cytoskeleton participates in toxisome formation In a previous study, we found that the ATPase activity of FgMyo1 is dependent on actin. FgMyo1E420K bearing a mutation at the actin interacting domain of FgMyo1, which caused the actin-activated ATPase activity of FgMyo1E420K was reduced to 5% as that of the wild-type FgMyo1 [12]. Correspondingly, toxisome formation in this strain was markedly decreased in comparison with that of the wild type (Fig 2E, upper panel). Moreover, we found that compo- nents of the actin cytoskeleton were enriched in the ACMS with Tri1 and FgMyo1 as the bait (S1 Table), suggesting that actin cytoskeleton may be associated with toxisome formation in F. graminearum. To address this possibility, we further constructed a strain bearing actin-RFP and Tri1-GFP. Then, the interaction between actin-RFP and Tri1-GFP was further verified by PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1006827 January 22, 2018 8 / 23 Functions of FgMyo1 in mycotoxin biosynthesis localization (left panel) and translation level (right panel) of the FK506-binding protein Fkbp54 tagged with GFP in the wild type and in the ΔFgAsc1 mutant. Bar = 10 μm. https://doi.org/10.1371/journal.ppat.1006827.g003 localization (left panel) and translation level (right panel) of the FK506-binding protein Fkbp54 tagged with GFP in the wild type and in the ΔFgAsc1 mutant. Bar = 10 μm. https://doi.org/10.1371/journal.ppat.1006827.g003 https://doi.org/10.1371/journal.ppat.1006827.g003 https://doi.org/10.1371/journal.ppat.1006827.g003 Co-IP assay (Fig 4A). In S. cerevisiae, the myosin I interacts with the actin and is required for polarization of the actin cytoskeleton [35]. Consistent with what is known in S. cerevisiae, actin was also associated with FgMyo1 in the ACMS assay using FgMyo1 as the bait (S1 Table). In addition, the interaction of FgMyo1-GFP and actin-RFP was further confirmed by the Co-IP assay (Fig 4B). Since actin is essential for F. graminearum growth, we were unable to obtain a knockout mutant of the ACTIN gene. Thus, to further investigate the function of actin in DON biosyn- thesis, the actin polymerization inhibitor latrunculin A was used to mimic impaired function Fig 4. The actin cytoskeleton is involved in toxisome formation. (A) The interaction of Actin-RFP and Tri1-GFP was verified by the Co-IP assay. Total proteins (input) extracted from the strain bearing Actin-RFP and Tri1-GFP constructs or a single construct (Actin-RFP or Tri1-GFP) were subjected to SDS-PAGE, and immunoblots were incubated with anti-GFP and anti-RFP antibodies, as indicated (Input panel). Each protein sample was pulled down using anti-GFP agarose and further detected with anti-RFP antibody (GFP pull-down panel). The protein samples were also incubated with the anti- GAPDH antibody as a reference. (B) Co-IP analysis for verification of the interaction between FgMyo1-GFP and Actin-RFP. Total proteins (input) extracted from the strain bearing Actin-RFP and FgMyo1-GFP constructs or a single construct (Actin-RFP or FgMyo1-GFP) were subjected to SDS-PAGE, and immunoblots were incubated with anti-Flag and anti-GFP antibodies, as indicated (Input panel). Each protein sample was pulled down using anti-GFP agarose and further detected with anti-RFP antibody (GFP pull-down panel). The protein samples were also incubated with the anti- GAPDH antibody as a reference. (C) The actin polymerization inhibitor latrunculin A inhibited toxisome formation. After growth in TBI for 24 h, ΔTri1:: Tri1-GFP was treated with 0.1 μg/ml latrunculin A for another 24 h before examination (left panel). The solvent DMSO was used as a control. Bar = 10 μm. Functions of FgMyo1 in mycotoxin biosynthesis Fig 3. FgMyo1 regulates translation of Tri1 via interacting with the ribosomal protein FgAsc1. (A) The interaction of FgMyo1-GFP and Asc1-RFP was verified by the Co-IP assay (left panel). FgMyo1-GFP was co-localized with FgAsc1-RFP under the toxin inducing conditions. Bar = 10 μm. (B) Localization of FgAsc1-RFP in hyphae of PH-1::FgAsc1-RFP+Tri1-GFP grown in toxin non-inducing medium PDB (upper panel) or toxin inducing medium TBI (lower panel) for 48 h. Bar = 10 μm. (C) ΔFgAsc1 exhibited dramatically reduced hyphal growth on PDA. (D) Toxisome formation was not detected in ΔFgAsc1 grown in TBI medium (left panel). Bar = 10 μm. The accumulation of Tri1-GFP protein in ΔFgAsc1 was determined by a western blot assay with the anti-GFP antibody (right panel). (E) DON production was under a detectable level in ΔFgAsc1. Values on the bars followed by the same letter are not significantly different according to a Fisher’s least significant difference (LSD) test at P = 0.05. (F) Comparisons in \| htt //d i /10 1371/j l t 1006827 J 22 2018 9 / 23 Fig 3. FgMyo1 regulates translation of Tri1 via interacting with the ribosomal protein FgAsc1. (A) The interaction of FgMyo1-GFP and Asc1-RFP was verified by the Co-IP assay (left panel). FgMyo1-GFP was co-localized with FgAsc1-RFP under the toxin inducing conditions. Bar = 10 μm. (B) Localization of FgAsc1-RFP in hyphae of PH-1::FgAsc1-RFP+Tri1-GFP grown in toxin non-inducing medium PDB (upper panel) or toxin inducing medium TBI (lower panel) for 48 h. Bar = 10 μm. (C) ΔFgAsc1 exhibited dramatically reduced hyphal growth on PDA. (D) Toxisome formation was not detected in ΔFgAsc1 grown in TBI medium (left panel). Bar = 10 μm. The accumulation of Tri1-GFP protein in ΔFgAsc1 was determined by a western blot assay with the anti-GFP antibody (right panel). (E) DON production was under a detectable level in ΔFgAsc1. Values on the bars followed by the same letter are not significantly different according to a Fisher’s least significant difference (LSD) test at P = 0.05. (F) Comparisons in PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1006827 January 22, 2018 9 / 23 Functions of FgMyo1 in mycotoxin biosynthesis Fig 5. Deletion of the actin associated protein genes FgPrk1 or FgEnd3 hinders toxisome formation. (A) Toxisome formation in ΔFgPrk1 and ΔFgEnd3 (left panel). The images were taken after each strain bearing Tri1-GFP was incubated in TBI for 48 h. Bar = 10 μm. The accumulation of Tri1-GFP protein in each strain was determined by using a western blot assay with the anti-GFP antibody (right panel). The protein samples were also incubated with the anti-GAPDH antibody as a reference. (B) Production of DON in ΔFgPrk1 and ΔFgEnd3 after each strain was cultured in TBI for 7 days. (C) The sensitivity of ΔFgPrk1, ΔFgEnd3 and their complementation strains (ΔFgPrk1-C and ΔFgEnd3-C) towards phenamacril and carbendazim. Each strain was cultured on PDA supplemented with 0.3 μg/ml phenamacril or carbendazim (left panel). Mycelial growth inhibition of each strain by phenamacril or carbendazim was quantified (right panel). Values on the bars for each fungicide treatment followed by different letters are significantly different according to a Fisher’s least significant difference (LSD) test at P = 0.05. https //doi org/10 1371/jo rnal ppat 1006827 g005 Fig 5. Deletion of the actin associated protein genes FgPrk1 or FgEnd3 hinders toxisome formation. (A) Toxisome formation in ΔFgPrk1 and ΔFgEnd3 (left panel). The images were taken after each strain bearing Tri1-GFP was incubated in TBI for 48 h. Bar = 10 μm. The accumulation of Tri1-GFP protein in each strain was determined by using a western blot assay with the anti-GFP antibody (right panel). The protein samples were also incubated with the anti-GAPDH antibody as a reference. (B) Production of DON in ΔFgPrk1 and ΔFgEnd3 after each strain was cultured in TBI for 7 days. (C) The sensitivity of ΔFgPrk1, ΔFgEnd3 and their complementation strains (ΔFgPrk1-C and ΔFgEnd3-C) towards phenamacril and carbendazim. Each strain was cultured on PDA supplemented with 0.3 μg/ml phenamacril or carbendazim (left panel). Mycelial growth inhibition of each strain by phenamacril or carbendazim was quantified (right panel). Values on the bars for each fungicide treatment followed by different letters are significantly different according to a Fisher’s least significant difference (LSD) test at P = 0.05. htt //d i /10 1371/j l t 1006827 005 Fig 5. Deletion of the actin associated protein genes FgPrk1 or FgEnd3 hinders toxisome formation. (A) Toxisome formation in ΔFgPrk1 and ΔFgEnd3 (left panel). The accumulation of Tri1-GFP protein was further verified by western blotting assay using the anti-GFP antibody (right panel). The protein samples were also incubated with the anti-GAPDH antibody as a reference. (D) DON was extracted from mycelia of PH-1 grown in TBI supplemented with 0.1 μg/ml latrunculin A. The solvent DMSO was used as a control. Values on the bars followed by different letters are significantly different according to a Fisher’s least significant difference (LSD) test at P = 0.05. Fig 4. The actin cytoskeleton is involved in toxisome formation. (A) The interaction of Actin-RFP and Tri1-GFP was verified by the Co-IP assay. Total proteins (input) extracted from the strain bearing Actin-RFP and Tri1-GFP constructs or a single construct (Actin-RFP or Tri1-GFP) were subjected to SDS-PAGE, and immunoblots were incubated with anti-GFP and anti-RFP antibodies, as indicated (Input panel). Each protein sample was pulled down using anti-GFP agarose and further detected with anti-RFP antibody (GFP pull-down panel). The protein samples were also incubated with the anti- GAPDH antibody as a reference. (B) Co-IP analysis for verification of the interaction between FgMyo1-GFP and Actin-RFP. Total proteins (input) extracted from the strain bearing Actin-RFP and FgMyo1-GFP constructs or a single construct (Actin-RFP or FgMyo1-GFP) were subjected to SDS-PAGE, and immunoblots were incubated with anti-Flag and anti-GFP antibodies, as indicated (Input panel). Each protein sample was pulled down using anti-GFP agarose and further detected with anti-RFP antibody (GFP pull-down panel). The protein samples were also incubated with the anti- GAPDH antibody as a reference. (C) The actin polymerization inhibitor latrunculin A inhibited toxisome formation. After growth in TBI for 24 h, ΔTri1:: Tri1-GFP was treated with 0.1 μg/ml latrunculin A for another 24 h before examination (left panel). The solvent DMSO was used as a control. Bar = 10 μm. The accumulation of Tri1-GFP protein was further verified by western blotting assay using the anti-GFP antibody (right panel). The protein samples were also incubated with the anti-GAPDH antibody as a reference. (D) DON was extracted from mycelia of PH-1 grown in TBI supplemented with 0.1 μg/ml latrunculin A. The solvent DMSO was used as a control. Values on the bars followed by different letters are significantly different according to a Fisher’s least significant difference (LSD) test at P = 0.05. https://doi.org/10.1371/journal.ppat.1006827.g004 https://doi.org/10.1371/journal.ppat.1006827.g004 10 / 23 PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1006827 January 22, 2018 PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1006827 January 22, 2018 Functions of FgMyo1 in mycotoxin biosynthesis Fig 6. The myosin I-actin cytoskeleton is not associated with aurofusarin biosynthesis. (A) Comparisons of red pigment (aurofusarin) biosynthesis among the wild type and various mutants constructed in this study. Images were taken after each strain was grown on PDA or in liquid PDB. The myosin I inhibitor phenamacril and the actin polymerization inhibitor latrunculin A did not inhibit aurofusarin biosynthesis. (B) Co-localization analysis for Tri1-GFP or the peroxisome indicator FgPex3-GFP with the aurofusarin biosynthetic enzyme AurJ-RFP. A strain dual-labeled with either AurJ-RFP and Tri1-GFP or AurJ-RFP and FgPex3-GFP was grown in TBI for 48 h before observation. Bar = 10 μm. (C) Phenamacril and latrunculin A did not affect cellular localization of AurJ-RFP. Fig 6. The myosin I-actin cytoskeleton is not associated with aurofusarin biosynthesis. (A) Comparisons of red pigment (aurofusarin) biosynthesis among the wild type and various mutants constructed in this study. Images were taken after each strain was grown on PDA or in liquid PDB. The myosin I inhibitor phenamacril and the actin polymerization inhibitor latrunculin A did not inhibit aurofusarin biosynthesis. (B) Co-localization analysis for Tri1-GFP or the peroxisome indicator FgPex3-GFP with the aurofusarin biosynthetic enzyme AurJ-RFP. A strain dual-labeled with either AurJ-RFP and Tri1-GFP or AurJ-RFP and FgPex3-GFP was grown in TBI for 48 h before observation. Bar = 10 μm. (C) Phenamacril and latrunculin A did not affect cellular localization of AurJ RFP https://doi.org/10.1371/journal.ppat.1006827.g006 https://doi.org/10.1371/journal.ppat.1006827.g006 Furthermore, these mutants produced significantly less DON as compared with the wild type (Fig 5B) and both mutants showed increased sensitivity to the myosin I inhibitor phenamacril (Fig 5C). Taken together, these results strongly indicated that the myosin I-actin cytoskeleton is essential for the toxisome formation in F. graminearum. The images were taken after each strain bearing Tri1-GFP was incubated in TBI for 48 h. Bar = 10 μm. The accumulation of Tri1-GFP protein in each strain was determined by using a western blot assay with the anti-GFP antibody (right panel). The protein samples were also incubated with the anti-GAPDH antibody as a reference. (B) Production of DON in ΔFgPrk1 and ΔFgEnd3 after each strain was cultured in TBI for 7 days. (C) The sensitivity of ΔFgPrk1, ΔFgEnd3 and their complementation strains (ΔFgPrk1-C and ΔFgEnd3-C) towards phenamacril and carbendazim. Each strain was cultured on PDA supplemented with 0.3 μg/ml phenamacril or carbendazim (left panel). Mycelial growth inhibition of each strain by phenamacril or carbendazim was quantified (right panel). Values on the bars for each fungicide treatment followed by different letters are significantly different according to a Fisher’s least significant difference (LSD) test at P = 0.05. https://doi.org/10.1371/journal.ppat.1006827.g005 of the actin cables. After treatment with latrunculin A at 0.1 μg/ml (approximately EC90 against mycelial growth of F. graminearum), the typical toxisome structures could not be observed, and Tri1-GFP was detected as diffuse fluorescent signal in the cytoplasm (Fig 4C, left panel). In addition, Tri1-GFP was noticeably decreased in the western blot assay upon latrunculin A treatment (Fig 4C, right panel). Subsequently, latruncunlin A showed strong inhibition of DON production (Fig 4D). These results indicated that the actin cytoskeleton is involved in toxisome formation in F. graminearum. In S. cerevisiae, Prk1 and End3 are involved in the organization of the actin cytoskeleton [36, 37]. To better understand the roles of the myosin I-actin cytoskeleton in toxisome forma- tion, we therefore were interested in constructing deletion mutants of their orthologs FgPrk1 (FGSG_05586) and FgEnd3 (FGSG_09721). Toxisome formation in mycelia of these two gene deletion mutants harboring the tagged Tri1-GFP was examined. The Tri1-GFP signals decreased noticeably in both ΔFgPrk1 and ΔFgEnd3 mutants (Fig 5A, left panel). In addition, western blot assays confirmed the amount of Tri1-GFP protein in these mutants was consider- ably lower than that of the wild type under the toxin inducing condition (Fig 5A, right panel). PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1006827 January 22, 2018 11 / 23 PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1006827 January 22, 2018 Discussion Trichothecenes are synthesized from acetyl-CoA as the basic precursor though the isoprenoid intermediate farnesyl pyrophosphate (FPP) and ultimately the trichothecene biosynthesis pathway [38]. The enzymes Tri1 and Tri4 are delivered to the specific cellular compartment known as the toxisome under the DON induction condition (Fig 1A, S2 Fig). This process is largely dependent on various environmental factors or stimuli, including nitrogen and carbon sources [10, 11, 39], amines [40], pH [41], light [42], and reactive oxygen species (ROS) [3]. Accumulating evidence indicates that some fungicides also stimulate DON biosynthesis. Milus and Parsons reported that propiconazole and tebuconazole treatments could result in a 50% increase in DON contamination in field trials [9]. Application of fluquinconazole or azoxystro- bin reduced disease incidence on wheat spikes but led to a significant increase in DON pro- duction by F. culmorum or F. graminearum in the harvested grains [10]. The fungicides epoxyconazole and propiconazole could also stimulate DON production in vitro and in wheat grains [11]. Therefore, the effects for disease management by application of fungicides may not be consistent with the impacts on mycotoxin biosynthesis. In this study, we tested the effect of 131 antifungal compounds on DON biosynthesis and found that phenamacril showed significant inhibition against DON biosynthesis. In agreement with previous studies, other fungicides including the carbendazim and azoles at sub-lethal concentrations could stimulate DON biosynthesis. Therefore, the chemical fungicides for FHB management should be care- fully considered to avoid stimulating mycotoxin biosynthesis. y g y y In eukaryotic cells, myosins participate in a wide variety of cellular processes, including cytokinesis, organellar transport, cell polarization, transcriptional regulation, intracellular transport, and signal transduction [43, 44]. They bind to the filamentous actin or other bind- ing partners, and produce physical forces by hydrolyzing ATP, therefore converting chemical energy into mechanical force [12–14, 44, 45]. The conserved head domain is accompanied by a broad diversity of N-terminal or C-terminal domains that bind to different molecular cargos, providing the functional specificity of myosin proteins [46]. A total of 31 defined myosin clas- ses have been identified in eukaryotes based on genomic surveys and phylogenetic analyses [15,46]. Three myosins: an essential class II myosin FgMyo2 (FGSG_08719), a class V myosin FgMyo2B (FGSG_07469), and the essential class I myosin FgMyo1 (FGSG_01410) are recog- nized in F. graminearum [47]. FgMyo2 is specifically localized to the delimiting septum of phialides and conidia, and required for septation [48]. Functions of FgMyo1 in mycotoxin biosynthesis These results indicate that aurofusarin might be synthesized in peroxisomes. In addition, the cellular localization and fluorescent intensity of AurJ-RFP was not discernibly affected by treatment with phenamacril or latrunculin A (Fig 6C). In summary, the myosinI-actin cyto- skeleton is not involved in aurofusarin pigmentation in F. graminearum. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1006827 January 22, 2018 The myosin I-actin cytoskeleton is not involved in pigmentation To test whether or not the myosin I-actin cytoskeleton is also necessary for biosynthesis of other secondary metabolites (SM), we examined aurofusarin biosynthesis because aurofusarin is a red polyketide pigment and easily visualized. As shown in Fig 6A, the FgMyo1 point muta- tion (FgMyo1E420K) and FgMYO1 knockdown mutants had similar red pigmentation in com- parisons with the wild type PH-1, as well as ΔTri1 and ΔTri4 mutants after incubation for 3 days on PDA or 5 days in liquid PDB. Consistent with these observations, phenamacril and latrunculin A did not inhibit aurofusarin biosynthesis in the wild type (Fig 6A). As controls, deletion mutants of aurofusarin biosynthesis genes AurJ and AurF did not produce the red pigment (Fig 6A). These results suggest that the myosinI-actin cytoskeleton is dispensable for aurofusarin pigmentation. To further confirm this finding, the aurofusarin biosynthesis gene AurJ was tagged with RFP and transformed into the wild type bearing Tri1-GFP or the peroxi- somal structural protein FgPex3-GFP. As indicated in Fig 6B (left panel), AurJ-RFP was mainly located in the cytoplasm and presented in a punctuate pattern that was different from the Tri1-GFP localization. However, AurJ-RFP was clearly co-localized with FgPex3-GFP. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1006827 January 22, 2018 12 / 23 Functions of FgMyo1 in mycotoxin biosynthesis that, results of the current study further confirmed that Tri1 and Tri4 are often localized in the perinuclear ER under the toxin inducing condition (Fig 1B), and that the ER was remodeled from thin reticulate ER (S8 Fig) in the toxin non-inducing conditions to thickened ER in the TBI conditions (Fig 1A). In addition, the ER remodeling is further supported by accumulation of the perinuclear ribosomes under the TBI conditions (S7 Fig) since ribosomes are often attached the rough ER. The toxisome structures were predicted to confer multiple beneficial biological functions including clustering of DON biosynthetic enzymes, promoting the efficiency of DON biosyn- thesis, as well as serving as a self-protection system against the self-toxicity of the Tri products and reaction intermediates [9, 22]. To date, four proteins including Tri1, Tri4, Tri14 and Hmr1 were validated to be localized to toxisomes [16, 22, 23]. However, the molecular mechanism for the ER remodeling to toxisome remains unknown. In eukaryotic cells, structures and functions of ER are dynamically changed by various intercellular and extracellular stimuli. For example, the ER network of Arabidopsis undergoes extensive remodeling, which is critically depended on a myosin-actin cytoskeleton system [50]. The plant specific myosin XI provides the force to propel ER streaming and the dynamic rearrangement of the ER network depends on the pro- pelling action of myosin-XI over actin coupled with a SYP73-mediated bridging [51]. Since F. graminearum doesn’t contain a myosin XI homologous protein, we infer that the FgMyo1-actin cytoskeleton may be involved in the ER remodeling for toxisome formation in F. graminearum. This inference is supported by multiple lines of evidence. First, FgMyo1 is comprised of the motor domain that binds to and interacts with actin [12, 18], an isoleucine and glutamine (IQ) motif, and a C-terminal tail. The tail domain contains a pleckstrin homology (PH) motif that is known to bind the anionic phospholipids in cellular membranes (S9 Fig) [52, 53]. The presence of a lipid-binding domain in the tail and an actin binding region in the motor domain equips the myosin I for cellular roles that link membranes to the actin cytoskeleton [54]. Second, dys- function of FgMyo1 and actin by inhibitors disrupts the toxisome formation (Figs 1D and 4C). PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1006827 January 22, 2018 Discussion In addition, the expression levels of TRI5 and TRI6 were obviously higher in the FgMyo2B heterokaryotic disruption mutant than those in the wild type [16,49]. These studies indicated that FgMyo2 and FgMyo2B may not be involved in mycotoxin biosynthesis directly. In the current study, we found that FgMyo1 is necessary for toxisome formation. Moreover, we further proved that FgMyo1 was not essential for the biosynthesis of the polyketide secondary metabolite, aurofusarin. These data suggest that the myosin I, but not other myosin motors, participates in DON biosynthesis in F. graminearum. The cellular compartmentalization (toxisome) for DON biosynthesis in F. graminearum was first described though the dynamic localization of fluorescent labeled Tri1 and Tri4 [23]. More recently, the toxisome was further identified as reorganization of the endoplasmic reticu- lum with pronounced expansion at perinuclear-and peripheral positions [22]. Consistent with PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1006827 January 22, 2018 13 / 23 Functions of FgMyo1 in mycotoxin biosynthesis that of the wild-type FgMyo1) [12]. This finding is similar to a previous report that the Myo1 mutants of Aspergillus nidulans with no more than 1% of the actin-activated ATPase activity of wild-type Myo1 in vitro and no detectable in vitro motility activity can support fungal cell growth, albeit with a delay in germination time and a reduction in hyphal elongation [61]. Therefore, the myosin I mediated endocytosis process is not connected with the toxisome for- mation in F. graminearum. The myosin-actin system also involves in the movement of organelles within cells, includ- ing the organelles for secondary metabolites organization. For instance, the short transporta- tion of melanosomes for the skin pigment melanin biosynthesis at the peripheral region of the mammalian cell is largely dependent on the Rab27a, melanophilin, myosinV-actin filament complex [62]. In Fusarium spp. Tri12 is suggested to play a role in export of trichothecene mycotoxins, which forms vacuoles and vesicles during the mycotoxin inducing condition [20, 21]. A previous study suggested that Tri12 interacted with toxisomes and may transfer the trichothecenes from toxisomes into the vesicles and vacuoles for further export [23]. The motility of vesicles containing Tri12 was reversibly inhibited by latrunculin A, indicating that movement was dependent upon the filamentous actin [21, 23]. The motor proteins are needed for the cellular motility of Tri12 by mechanical driving force on the filamentous actin. There are three major super-families of motor proteins: kinesins, dyneins, and myosins. The first two act as motors on microtubule filaments, while myosins function on actin [63]. Thus, it would be interesting to further study the functions of myosins in the transport of toxins that may accumulate in Tri12-linked vacuoles and vesicles in F. graminearum and in other toxigenic fungi. Taken together, our data support a model in which FgMyo1 is essential for toxisome for- mation under the DON induction conditions in F. graminearum by interacting with FgAsc1 indirectly for regulating the Tri protein biosynthesis and by directly participating in the endo- plasmic reticulum (ER) remodeling via the myosin-actin cytoskeleton system. In addition, the small molecule phenamacril is able to suppress the toxisome formation by inhibiting the ATPase activity of FgMyo1 (Fig 7). Fungal strains and growth assays The F. graminearum wild-type strain PH-1 (NRRL 31084) was used as a parental strain. The wild-type strain and transformants generated in this study were grown on potato dextrose agar (PDA) and minimal medium (MM) for hyphal examination. The carboxymethyl cellulose (CMC) liquid medium was used for conidiation assays [64]. For toxisome observation and trichothecene production analysis, each strain was grown in liquid trichothecene biosynthesis inducing (TBI) medium [38] at 28 ˚C in a shaker (150 rpm) in the dark. Each experiment was repeated three times. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1006827 January 22, 2018 Third, knockdown expression of FgMyo1 or the deletion of actin cytoskeleton organization related genes FgPrk1 and FgEnd3 resulted in a defect in toxisome formation and a reduction in DON production (Figs 2F, 5A and 5B). Finally, the point mutation FgMyo1E420K allowing only 5% of the wild-type ATPase activity also affected toxisome formation (Fig 2F), which is in agreement with the interpretation that the hydrolysis of ATP in FgMyo1 coverts the chemical energy into mechanical force and might provide the physical forces for ER remodeling. In addition to providing the force for membrane dynamics, the myosin I motors have also been suggested to function as anchors or tethers between membranes and other proteins. In opossum kidney epithelial cells, Myo1b was found to tether amino acid transporters to the api- cal plasma membrane, thereby facilitating neutral amino acid transport across the membrane [55]. Similarly, Myo1a is important for the retention and localization of sucrose isomaltase in the intestinal brush border membrane [56]. Furthermore, the spatial association of nuclear myosin I with the ribosome protein S6 plays an important role in the export of small ribosomal subunits through the nuclear pores [57]. In current study, we found that FgMyo1 interacts with the ribosome-associated protein Asc1, thereby facilitating translation of toxin biosynthe- sis enzymes, and further contributing to toxisome formation in the toxin inducing conditions. In eukaryotic cells, the myosin-actin system also plays important roles in endocytosis [58– 60]. Consistent with that, deletion mutants of actin cytoskeleton organizing gene orthologs, Prk1 and End3 resulted in the defects in both endocytosis and toxisome formation in F. grami- nearum. However, the mutants of two conserved endocytic components (Apm4 and Abp1) still formed typical toxisomes in TBI (S10C Fig). Importantly, the FgMyo1E420K mutant that exhibits the defect in toxisome formation (Fig 2F) retains the capability of endocytosis (S10A Fig), while the actin-activated ATPase activity of FgMyo1E420K is very low (circa 5% as PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1006827 January 22, 2018 14 / 23 Strain construction The strains ΔFgPrk1, ΔFgEnd3, ΔFgTri1, ΔFgTri4, ΔFgAsc1, ΔFgAurJ and ΔFgAurF were con- structed using the protocol described previously [65]. Briefly, the open reading frame (ORF) of each gene was replaced with hygromycin resistance cassette (HPH) and subsequent deletion mutants were identified by PCR assays with relevant primers (S2 Table). For complementa- tion, each ORF fused with a tag and geneticin resistance gene was introduced into correspond- ing mutant, and transformants were selected with geneticin. To construct FgMyo1 silenced mutants, a 540 bp fragment was amplified and inserted forward and reverse into the pSilent-1 plasmid, and the recombination hairpin RNA silencing plasmid was introduced into PH-1 as previous described [25]. To replace the FgMYO1 promoter with Pzear, the HPH and Pzear PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1006827 January 22, 2018 15 / 23 Functions of FgMyo1 in mycotoxin biosynthesis Fig 7. A proposed model showing the role of FgMyo1 in toxisome formation. Trichothecene biosynthesis enzymes (Tri proteins) are produced at a low level under toxin noninducing conditions. In toxin inducing conditions, FgMyo1 directly participates in remodeling the endoplasmic reticulum (ER) via the myosin-actin cytoskeleton to form the spherical and ovoid structures termed “Fusarium toxisomes.” In addition, FgMyo1 interacts with FgAsc1 indirectly to enhance the translation of Tri proteins. Phenamacril is able to suppress toxisome formation by inhibiting the ATPase activity of FgMyo1, and subsequently reduces the biosynthesis of DON in Fusarium graminearum. Fig 7. A proposed model showing the role of FgMyo1 in toxisome formation. Trichothecene biosynthesis enzymes (Tri proteins) are produced at a low level under toxin noninducing conditions. In toxin inducing conditions, FgMyo1 directly participates in remodeling the endoplasmic reticulum (ER) via the myosin-actin cytoskeleton to form the spherical and ovoid structures termed “Fusarium toxisomes.” In addition, FgMyo1 interacts with FgAsc1 indirectly to enhance the translation of Tri proteins. Phenamacril is able to suppress toxisome formation by inhibiting the ATPase activity of FgMyo1, and subsequently reduces the biosynthesis of DON in Fusarium graminearum. https://doi.org/10.1371/journal.ppat.1006827.g007 https://doi.org/10.1371/journal.ppat.1006827.g007 fragments were amplified respectively and fused by overlap PCR. Subsequently, the “HPH-Pzear” fragment was further fused with the 50 and 30 flanking regions of the FgMYO1 gene. The resulting fusion fragment was purified and transformed into PH-1. To induce the Pzear replacement, the inducer β-estradiol at 30 μM was added to the medium during the regeneration and mutant selection processes [66]. Strain construction To construct the FgTri1-GFP fusion cassette, the FgTri1 fragment containing the native promoter and ORF (without stop codon) was amplified with primers A15 + A16 (S1 Table). The resulting PCR products were co-transformed with Xho1-digested pYF11 into XK1-25. The alkali-cation yeast transformation kit (MP Biomedicals, Solon, USA) was used to generate the recombined FgTri1-GFP fusion vector. Subsequently, the FgTri1-GFP fusion vector was recovered from the yeast transformant by using the yeast plasmid extract kit (Solarbio, Beijing, China) and then transferred into E.coli strain DH5α for amplification. Using the same strategy, other GFP or RFP fusion cassettes were also constructed. Each recombination plasmid was transformed into PH-1 or the corresponding mutant for generating fluorescent label strains. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1006827 January 22, 2018 Microscopic examinations The fluorescent intensity and localization of tagged proteins were observed with a Zeiss LSM780 confocal microscopy (Gottingen, Niedersachsen, Germany). For observation of toxi- some formation patterns in PH-1 and derived mutants, each strain labeled with FgTri1-GFP was cultured in TBI for 48 h before examination. All samples were mounted on glass slides and sealed with cover glasses. The following parameter sets of the confocal microscopy were used: Plan-Neofluar 40x/1.30 Oil DIC M27 objective; laser: at 488 nm at 50% power for green fluorescence; dimension of X = 70.78 μm, Y = 70.78 μm; pinhole: 90 μm; digital gain: 1.00. To observe toxisomes in planta, fresh mycelial plugs of the fluorescent reporter strain were inocu- lated on the leaves of wheat seedlings of a susceptible cultivar Jimai 22. After incubation at 25˚C and 100% RH (relative humidity) for 5 days, the infected leaves were taken for toxisome examination observed under Plan-Neofluar 20x/0.50 M27 objective. j The following filter sets were used for other fluorescent or dye staining: the laser excitation wavelength was set at 405 nm for DAPI (blue fluorescence), at 561 nm for FM4-64 or RFP/ mCherry (red fluorescence), at 514 nm for YFP (yellow fluorescence). The endoplasmic reticu- lum (ER) was stained with ER-Tracker Red (Beyotime technology Co., Ltd), and laser was set at 587 nm for red fluorescence. The intensity of fluorescence was acquired using the Zeiss ZEN 2010 software. Bimolecular Fluorescence Complementation (BiFC) assays For BiFC assays, the final plasmid constructs of pYFPN-FgTri1 and pFgMyo1-YFPC were ver- ified by sequencing and then co-transformed into the protoplasts of PH-1 in pairs. Transfor- mants resistant to both hygromycin and neomycin were isolated and confirmed by PCR. The recombination plasmid pYFPN-FgTri1 or pFgMyo1-YFPC was transformed into PH-1, and resulted transformants were used as negative controls. YFP signals in the mycelia grown in TBI for 48 h were examined under a Zeiss LSM780 confocal microscope (Gottingen, Nieder- sachsen, Germany). Functions of FgMyo1 in mycotoxin biosynthesis 104 conidia/mL was added into a 24-well plate (2.0 mL/well). After 24 h static incubation at 28 ˚C, each tested compound was added into a well and the plate was incubated for another 48 h. Then, the fluorescent intensity in each well was scanned with the Varioskan Flash Multi- mode Reader (Thermo Scientific, MA, USA) for first round screening. The wells with lower or no fluorescent signals compared with that of the control treatment (the same volume of solvent dimethyl sulfoxide, DMSO) were further observed by a confocal microscopy. A total of 131 antifungal compounds including 11 commercialized fungicides were tested for the activity against toxisome formation. For each compound, there were three-well replicates, and the experiment was repeated three times. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1006827 January 22, 2018 Screening for toxisome formation inhibitors The strain expressing the FgTri1-GFP in the ΔTri1 background was used as the fluorescent reporter strain for anti-toxisome formation screening. The TBI medium supplemented with PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1006827 January 22, 2018 16 / 23 Functions of FgMyo1 in mycotoxin biosynthesis Affinity capture-mass spectrometry analysis The bait protein FgMyo1 was dual labeled with ZZ tag and 3×Flag at its N-terminus and C-ter- minus, respectively. The resulting fusion cassette was transferred into PH-1. The resulting transformant (PH-1::ZZ-FgMyo1-3×Flag) was used for protein extraction as previous described previously [65] and the affinity capture was conducted by the following procedures. After protein extraction, supernatant (25 ml) was transferred into a sterilized tube. The first run affinity capture was conducted using rabbit IgG agarose beads (Haoran Biotech Co., Shanghai, China), which was immuno-interacted with the ZZ tag. A total of 500 μl IgG agarose beads were added into the above supernatant to capture ZZ-FgMyo1-3×Flag interacting pro- teins, following the manufacturer’s instructions (General Electric Company, GA, USA). Then, the washed beads were subjected for the second run capture with anti-Flag agarose beads according to the manufacturer’s instructions (Abmart, NJ, USA). The final ZZ-FgMyo1- 3×Flag interacting proteins captured by the anti-Flag agarose beads were eluted with TBS sup- plemented with 10% SDS. In addition, the ZZ-FgTri1-3×Flag was constructed and the interact- ing proteins were captured using the same strategy. The captured proteins were digested with trypsin and further analyzed by mass spectrometry using a previous published protocol [69]. Enrichment for proteins assigned to particular functional categories (FunCat) was calculated as described previously [20, 28]. Analysis of mycotoxin production To quantify the mycotoxin production, each strain was grown in TBI medium or inoculated on wheat kernels. DON was extracted, and then purified, and quantified using the LC-MS/MS system as described previously [5, 68]. Co-immunoprecipitation (Co-IP) assays The GFP, RFP, 3× Flag, or mCherry-fusion constructs were verified by DNA sequencing and transformed in pairs into PH-1. Transformants expressing pairs of fusion constructs were con- firmed by western blot analysis. In addition, the transformants expressing a single fusion con- struct were used as references. For Co-IP assays, total proteins were extracted and incubated with the anti-GFP (ChromoTek, Martinsried, Germany) or anti-Flag (Abmart, Shanghai, China) agarose as described above. Proteins eluted from agarose were analyzed by western blot detection with a polyclonal anti-Flag A9044 (Sigma, St. Louis, MO), or an anit-GFP antibody (Abcam, Cambridge, UK). The protein samples were also detected with monoclonal anti- GAPDH antibody EM1101 (Hangzhou Huaan Biotechnology Co., Ltd.) as a reference. Each experiment was repeated twice. Western blotting hybridization The protein isolation was performed as described previously [67]. The resulting proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Immobilon-P transfer membrane (Millipore, Billerica, MA, USA). The poly- clonal anti-Flag A9044 (Sigma, St. Louis, MO) and monoclonal anti-GFP ab32146 (Abcam, Cambridge, UK) antibodies were used at a 1:5000 to 1:10 000 dilution for immunoblot analy- ses. The samples were also detected with monoclonal anti-GAPDH antibody EM1101 (Hang- zhou HuaAn Biotechnology Co., Ltd.) as a reference. The intensity of immunoblot bands were quantified using the ImageQuantTL software. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1006827 January 22, 2018 17 / 23 PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1006827 January 22, 2018 (A) Schematic structures of the FgMyo1 protein in F. graminearum. (B) Alignment of the FgMyo1 pleckstrin homology (PH) domain (residues 814–837 aa) with its orthologs of Homo sapiens. Red resi- dues indicate the conserved basic residues that are important for membrane binding in PH domain. Accession numbers for the proteins listed are indicated. (TIF) S10 Fig. The myosin I mediated endocytosis process is not connected with the toxisome formation in F. graminearum. (A) Time-course of FM4-64 internalization via the endocytic pathway in the wild type, FgMyo1E420K, ΔFgPrk1 and ΔFgEnd3. Living cells grown in PDB were stained with 8 mM FM4-64. Bar = 10 mm. (B) Hyphal growth patterns of endocytosis S3 Fig. Tri4-RFP was co-localized with Tri1-GFP at toxisomes in hyphae of PH-1:: Tri1-GFP+Tri4-RFP grown in the toxin inducing medium TBI. DIC indicates differential interference contrast. Bar = 10 μm. (TIF) S4 Fig. Inhibition of antifungal compounds against toxisome formation. (A) The inhibition of each compound (at 0.5 μg/ml) against mycelial growth of F. graminearum on PDA. The sol- vent DMSO was used as a control. (B) Toxisome formation in the mycelia of ΔTri1::Tri1-GFP treated with each antifungal compound. After the strain was cultured in TBI for 24 h, each fun- gicide was added into TBI at the final concentration at 0.5 μg/ml. Subsequently, the strain was incubated for another 24 h before observation. The DMSO is the solvent control. (C) Produc- tion of DON in each treatment. DON was extracted from mycelia of each strain cultured in TBI for 7 days. Values on the bars followed by different letters are significantly different according to a Fisher’s least significant difference (LSD) test at P = 0.05. (TIF) S5 Fig. Examination for toxisome formation in hyphae of ΔTri1::Tri1-GFP treated with 0.5 μg/ml phenamacril for different times. (A) Toxisome formation patterns in ΔTri1:: Tri1-GFP grown in TBI for the times as indicated in the figure. Bar = 10 μm. (B) Phenamacril abolished the toxisome formation in ΔTri1::Tri1-GFP. After ΔTri1::Tri1-GFP was grown in TBI for 24 h, the culture was then treated with phenamacril for the additional time (from 6 to 48 h) as indicated in the figure. Bar = 10. (TIF) S1 Fig. Chemical structure of the novel antifungl compound phenamacril. (TIF) S1 Fig. Chemical structure of the novel antifungl compound phenamacril. (TIF) S2 Fig. Time course analysis of accumulation and localization of Tri1-GFP in the toxin inducing conditions. (A) Examination for toxisome formation in time. The images were taken after the strain ΔTri1::Tri1-GFP was incubated in TBI at the corresponding time indi- cated in the figure. Bar = 10 μm. (B) The abundance of Tri1-GFP protein at the corresponding time was determined by the western blot assay with the anti-GFP antibody. The protein sam- ples were also incubated with the anti-GAPDH antibody as a reference. (C) Time course analy- sis of production of DON by ΔTri1::Tri1-GFP in TBI. (TIF) 18 / 23 PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1006827 January 22, 2018 PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1006827 January 22, 2018 Functions of FgMyo1 in mycotoxin biosynthesis S3 Fig. Tri4-RFP was co-localized with Tri1-GFP at toxisomes in hyphae of PH-1:: Tri1-GFP+Tri4-RFP grown in the toxin inducing medium TBI. DIC indicates differential interference contrast. Bar = 10 μm. (TIF) S4 Fig. Inhibition of antifungal compounds against toxisome formation. (A) The inhibition of each compound (at 0.5 μg/ml) against mycelial growth of F. graminearum on PDA. The sol- vent DMSO was used as a control. (B) Toxisome formation in the mycelia of ΔTri1::Tri1-GFP treated with each antifungal compound. After the strain was cultured in TBI for 24 h, each fun- gicide was added into TBI at the final concentration at 0.5 μg/ml. Subsequently, the strain was incubated for another 24 h before observation. The DMSO is the solvent control. (C) Produc- tion of DON in each treatment. DON was extracted from mycelia of each strain cultured in TBI for 7 days. Values on the bars followed by different letters are significantly different according to a Fisher’s least significant difference (LSD) test at P = 0.05. (TIF) S5 Fig. Examination for toxisome formation in hyphae of ΔTri1::Tri1-GFP treated with 0.5 μg/ml phenamacril for different times. (A) Toxisome formation patterns in ΔTri1:: Tri1-GFP grown in TBI for the times as indicated in the figure. Bar = 10 μm. (B) Phenamacril abolished the toxisome formation in ΔTri1::Tri1-GFP. After ΔTri1::Tri1-GFP was grown in TBI for 24 h, the culture was then treated with phenamacril for the additional time (from 6 to 48 h) as indicated in the figure. Bar = 10. (TIF) S6 Fig. FgMyo1 derived mutants and the actin associated protein gene deletion mutants ΔFgPrk1 and ΔFgEnd3 attenuated virulence on flowering wheat heads. Infected wheat heads were examined 15 days after inoculation with conidial suspension of each strain. The inoculation sites were indicated as black dots. (TIF) S7 Fig. Comparisons in localization of the ribosomal 60S subunit protein L25 (FgRpL25 tagged with mCherry) in toxin non-inducing (upper panel) and toxin inducing conditions (lower panel). The strain was also stained with a nucleus tracker DAPI (40, 6-diamidino- 2-phenylindole). Bar = 10 μm. (TIF) S8 Fig. Thin reticulate ER patterns in mycelia of F. graminearum grown in the non-toxin inducing medium. The mycelia of PH-1 grown in PDB for 48 h were used for staining with the ER-tracker Red. Bar = 10 μm. (TIF) S9 Fig. Schematic structures of FgMyo1 and the pleckstrin homology motif. S2 Table. A list of primers used in this study. (DOCX) S2 Table. A list of primers used in this study. (DOCX) Project administration: Zhonghua Ma. Resources: Jin-Rong Xu, H. Corby Kistler, Zhonghua Ma. Resources: Jin-Rong Xu, H. Corby Kistler, Zhonghua Ma. Supervision: Yun Chen, Zhonghua Ma. Validation: Guangfei Tang, Yun Chen, Zhonghua Ma. Visualization: Yun Chen, Zhonghua Ma. Visualization: Yun Chen, Zhonghua Ma. Writing – original draft: Yun Chen, Zhonghua Ma. Writing – review & editing: Jin-Rong Xu, H. Corby Kistler, Zhonghua Ma. S1 Table. Identification of Tri1 and FgMyo1-interacting proteins by the affinity capture- mass spectrometry assay. (DOCX) S1 Table. Identification of Tri1 and FgMyo1-interacting proteins by the affinity capture- mass spectrometry assay. (DOCX) S6 Fig. FgMyo1 derived mutants and the actin associated protein gene deletion mutants ΔFgPrk1 and ΔFgEnd3 attenuated virulence on flowering wheat heads. Infected wheat heads were examined 15 days after inoculation with conidial suspension of each strain. The inoculation sites were indicated as black dots. (TIF) S6 Fig. FgMyo1 derived mutants and the actin associated protein gene deletion mutants ΔFgPrk1 and ΔFgEnd3 attenuated virulence on flowering wheat heads. Infected wheat heads were examined 15 days after inoculation with conidial suspension of each strain. The inoculation sites were indicated as black dots. (TIF) S7 Fig. Comparisons in localization of the ribosomal 60S subunit protein L25 (FgRpL25 tagged with mCherry) in toxin non-inducing (upper panel) and toxin inducing conditions (lower panel). The strain was also stained with a nucleus tracker DAPI (40, 6-diamidino- 2-phenylindole). Bar = 10 μm. (TIF) S7 Fig. Comparisons in localization of the ribosomal 60S subunit protein L25 (FgRpL25 tagged with mCherry) in toxin non-inducing (upper panel) and toxin inducing conditions (lower panel). The strain was also stained with a nucleus tracker DAPI (40, 6-diamidino- 2-phenylindole). Bar = 10 μm. (TIF) S8 Fig. Thin reticulate ER patterns in mycelia of F. graminearum grown in the non-toxin inducing medium. The mycelia of PH-1 grown in PDB for 48 h were used for staining with the ER-tracker Red. Bar = 10 μm. (TIF) S9 Fig. Schematic structures of FgMyo1 and the pleckstrin homology motif. (A) Schematic structures of the FgMyo1 protein in F. graminearum. (B) Alignment of the FgMyo1 pleckstrin homology (PH) domain (residues 814–837 aa) with its orthologs of Homo sapiens. Red resi- dues indicate the conserved basic residues that are important for membrane binding in PH domain. Accession numbers for the proteins listed are indicated. (TIF) S10 Fig. The myosin I mediated endocytosis process is not connected with the toxisome formation in F. graminearum. (A) Time-course of FM4-64 internalization via the endocytic pathway in the wild type, FgMyo1E420K, ΔFgPrk1 and ΔFgEnd3. Living cells grown in PDB were stained with 8 mM FM4-64. Bar = 10 mm. (B) Hyphal growth patterns of endocytosis PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1006827 January 22, 2018 19 / 23 PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1006827 January 22, 2018 Author Contributions Data curation: Guangfei Tang, Yun Chen. Formal analysis: Guangfei Tang, Yun Chen, Jin-Rong Xu, H. Corby Kistler, Zhonghua Ma. Funding acquisition: Yun Chen, H. Corby Kistler, Zhonghua Ma. Investigation: Guangfei Tang, Yun Chen. Methodology: Guangfei Tang, Yun Chen, Zhonghua Ma. Methodology: Guangfei Tang, Yun Chen, Zhonghua Ma. Project administration: Zhonghua Ma. Functions of FgMyo1 in mycotoxin biosynthesis mutant ΔFgAPM4 and ΔFgAbp1 on PDA. (C) Toxisome formation of ΔFgAPM4 and ΔFgAbp1 grown in TBI medium. (D) The DON production of ΔFgAPM4, ΔFgAbp1 and their complemented strains. The DON was extracted from mycelia of each strain grown in TBI for 7 days. Values on the bars followed by the same letter are not significantly different according to a Fisher’s least significant difference (LSD) test at P = 0.05. (TIF) mutant ΔFgAPM4 and ΔFgAbp1 on PDA. (C) Toxisome formation of ΔFgAPM4 and ΔFgAbp1 grown in TBI medium. (D) The DON production of ΔFgAPM4, ΔFgAbp1 and their complemented strains. The DON was extracted from mycelia of each strain grown in TBI for 7 days. Values on the bars followed by the same letter are not significantly different according to a Fisher’s least significant difference (LSD) test at P = 0.05. (TIF) Functions of FgMyo1 in mycotoxin biosynthesis 8. Liu YP, Lu Y, Wang LY, Chang FQ, Yang LX. Occurrence of deoxynivalenol in wheat, Hebei Province, China. 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https://openalex.org/W3204618264	https://www.hal.inserm.fr/inserm-03465016/document	English	null	Reasons for using cannabidiol: a cross-sectional study of French cannabidiol users	Journal of cannabis research	2,021	cc-by	3,288	To cite this version: Davide Fortin, Vincent Di Beo, Sophie Massin, Yann Bisiou, Patrizia Carrieri, et al.. Reasons for using cannabidiol: a cross-sectional study of French cannabidiol users. Journal of Cannabis Research, 2021, 3 (1), pp.46. 10.1186/s42238-021-00102-z. inserm-03465016 Reasons for using cannabidiol: a cross-sectional study of French cannabidiol users Davide Fortin, Vincent Di Beo, Sophie Massin, Yann Bisiou, Patrizi Tangui Barré © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Abstract Background: Cannabidiol and cannabidiol-based products are proliferating in many countries. This recent and rapid diffusion prompts investigating the reasons for its use. Methods: We analyzed data from an online survey among cannabidiol users in the French general population (n = 1166) selected for their interest in such products. We described the reported reasons for using cannabidiol. We performed logistic regressions to identify the correlates of declaring well-being and other specific reasons for using cannabidiol. We also provided descriptive data regarding the cannabidiol patterns of use. Results: Well-being was the most cited primary reason for use (27% of the sample). Declaring well-being as a primary reason for using cannabidiol was inversely associated with cigarette smoking, cannabis use, and employment. Among cannabidiol users reporting well-being as their primary reason for use, stress and sleep improvements were the most- cited specific reasons. In the whole study sample, the most common modes of use were smoking cannabidiol-rich cannabis (61%) and ingesting cannabidiol oil sublingually (19%). Conclusions: In a sample of cannabidiol users from France, well-being was the most-cited primary reason for use, and smoking was the first route of administration. Further research is needed to clarify to what extent expected effects are scientifically sound and to understand country-related specificities regarding patterns of use. Keywords: Cannabidiol, France, Well-being, Cannabis, Stress (Moltke & Hindocha, 2021). Using an online question- naire, they found that self-perceived anxiety, sleep prob- lems, stress, and general health and well-being were the top 4 reasons for CBD use. Moreover, female users were less likely to declare general health and well-being as a reason for CBD use, and sublingual administration was by far the most-cited route of CBD administration. HAL Id: inserm-03465016 https://inserm.hal.science/inserm-03465016v1 Submitted on 3 Dec 2021 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Fortin et al. J Cannabis Res (2021) 3:46 https://doi.org/10.1186/s42238-021-00102-z Journal of Cannabis Research Open Access Resultsh The sample size included 1166 CBD users. Almost all lived in France (98%). The sample was predominantly male (70%), with a median age of 36 (interquartile range 28–44) years (Table 1). The four most-cited pri- mary reasons to use CBD were “for my well-being” (27%), “to heal my disease or reduce associated symp- toms” (25%), “to reduce the use of tobacco or other sub- stances” (12%), and “because I had difficulties obtaining regular (i.e., illicit) cannabis” (9%). Using a design quite similar to that of Moltke et al., we conducted an anonymous online survey among CBD users mostly living in France. The protocol fol- lowed the guidelines of the Declaration of Helsinki, and the INSERM Ethics Committee provided ethical approval (approval #20-677 dated April 23, 2020). An online link to the survey was distributed via media outlets specializing in cannabis-based products, CBD user groups on Facebook, and a community of people with chronic health conditions. Therefore, partici- pants were enrolled from the general population. Data were collected using a Google survey form between 23 April 2020 and 30 March 2021. In the survey, “CBD” included all legal CBD-rich products, including CBD- rich cannabis. Certain questions regarded patterns of use and collected, among other information, time since first CBD use, number of days CBD was used in the previous 30 days, time of day when CBD was used, CBD purchase locations in the previous 30 days, and principal type of CBD product used over the same period. Another question asked about the respondents’ primary reason for using CBD in the previous 30 days (only one answer possible) with several options regard- ing well-being, psychoactive substance use reduction, curiosity, and socialization. People who answered “for my well-being” were asked to check from a setlist the effects on their well-being which they expected to obtain from using CBD (multiple responses possible) including improved sleep, reduced anxiety/depression, increased concentration, headache relief, diminished stress, fewer cutaneous problems, increased energy, and reduced pain or inflammation. No questions related to the general health status of the participants or psychiatric disorders were asked. In the sub-population which answered well-being (n = 311), the effects of CBD most expected by respond- ents were diminished stress (63% of that group), fol- lowed by improved sleep (60%), reduced anxiety/ depression (43%), reduced pain or inflammation (41%), increased concentration (16%), and headache relief (16%). Resultsh Over half the study sample (56%) had first used CBD more than a year prior to the survey, and half (50%) had used it at least 20 of the previous 30 days (Table 2). The most common purchase locations were on the Internet (66%) and in specialized shops (20%). The most common modes of use were smoking CBD- rich cannabis (61%) and ingesting CBD oil sublingually (19%). In the multivariable analysis performed on the whole sample (Table 1), in terms of tobacco cigarette smoking patterns and the likelihood of reporting well-being as the primary reason to use CBD, smokers were 27% less likely to report it (adjusted odds ratio (aOR) 95% confidence interval (CI): 0.73 [0.55–0.97], p = 0.031) than non- smokers, while e-cigarette users were 97% more likely to report it (1.97 [1.2–3.24], p = 0.007). Moreover, individu- als who smoked illegal cannabis in the previous 30 days (0.73 [0.56–0.95], p = 0.021) and those who were actively employed (0.73 [0.54–0.99], p = 0.043) were less likely to report well-being as the primary reason. Neither gender nor age was associated with this outcome. The study group comprised respondents who reported using CBD in the previous 30 days and who answered the survey question about their primary reason for using CBD. Statistics related to patterns of CBD use for the whole sample were described. We per- formed logistic regressions to identify the correlates of CBD use, both for well-being (whole sample) as the primary reason and for the specific expected effects listed above. Only variables with a liberal p-value < 0.20 (Wald test) in the univariable analyses were con- sidered eligible for the multivariable models. The final multivariable models were built using a backward pro- cedure, and the likelihood ratio test (p < .05) was used to define the variables to be maintained in the final models. The Stata/SE 14.2 software (StataCorp LP, College Station, TX) was used for all analyses. Among those who reported well-being as their primary reason to use CBD, improved sleep was more frequently reported by individuals with lower self-reported income levels and those who were overweight, while individu- als who did not use illegal cannabis and those with lower self-reported income levels were more likely to report reduced anxiety/depression as a specific expected effect of using CBD. Introduction Cannabidiol (CBD)-based products are proliferating in countries with different legislation regarding the use of cannabis-based products (Walker et al., 2020). Con- cerns about the safety of CBD-based products have been raised (Lachenmeier et al., 2019). Given the high risk of misleading information and subsequent confusion about CBD’s legal status and its effects, there is a need to eluci- date motivations and patterns of use among CBD users. Recently, in the UK, Moltke et al. investigated the reasons for CBD use and patterns of use among 387 CBD users By using data from an online survey among CBD users, we aimed to identify sociodemographic and behavioral correlates of using CBD for general well-being and more specific purposes in French users, as well as present asso- ciated patterns of use. By doing so, we aimed at replicat- ing Moltke et al.’s investigations and potentially highlight country-specific differences. *Correspondence: tangui.barre@inserm.fr 2 Aix Marseille Univ, Inserm, IRD, SESSTIM, Sciences Economiques & Sociales de la Santé & Traitement de l’Information Médicale, ISSPAM, Marseille, France Full list of author information is available at the end of the article Page 2 of 5 Page 2 of 5 Fortin et al. J Cannabis Res (2021) 3:46 Page 2 of 5 Fortin et al. J Cannabis Res (2021) 3:46 Fortin et al. J Cannabis Res Resultsh Alcohol abstinence, illegal cannabis use, and a higher educational level (> secondary school diploma) were all associated with the desire for increased concentration. Finally, younger people and those who did not smoke tobacco cigarettes were more likely to use CBD for headache relief (Table 1). No characteris- tic was associated with the following specific reasons to use CBD: diminished stress, fewer cutaneous problems, increased energy, and pain or inflammation relief. Page 3 of 5 Fortin et al. J Cannabis Res (2021) 3:46 Fortin et al. J Cannabis Res Table 1 Sociodemographic and behavioral characteristics of cannabidiol users (n = 1166) and logistic regressions of cannabidiol users’ expected effects as outcomes in those who declared “well-being” to be the primary reason for using cannabidiol (n = 311) a Income level was self-reported as subjectively assessed as compared to an “average level” estimated by the participants b Body mass index was calculated as the body weight (in kg) divided by the squared height (in m). Resultsh A body mass index over 25 kg/m2 denotes overweight or obesity Variable Whole sample (n = 1166), N (%) Users declaring well-being as the primary reason for cannabidiol use (n = 311), N (%) Improved sleep (n = 306) Reduced anxiety/ depression (n = 306) Increased concentration (n = 290) Headache relief (n = 308) aOR 95% CI p-value aOR 95% CI p-value aOR 95% CI p-value aOR 95% CI p-value Sex Male 799 (69.5) 218 (71.2) – – – – Female 351 (30.5) 88 (28.8) – – – – Age (years) 36 [28–44] 36 [29–42] – – – 0.96 [0.93–0.99] 0.011 Smoking tobacco cigarettes in the previous 30 days No 442 (38.4) 130 (42.2) – – – 1 Yes 631 (54.8) 143 (46.4) – – – 0.48 [0.26–0.89] 0.021 Smoking mainly e-ciga- rettes 79 (6.9) 35 (11.4) – – – 0.52 [0.18–1.46] 0.215 Alcohol consumption in the previous 30 days No 346 (30.2) 88 (28.8) – – 1 – Yes 799 (69.8) 218 (71.2) – – 0.49 [0.26–0.91] 0.025 – Illegal cannabis use in the previous 30 days No – 1 1 – Yes – 0.44 [0.27–0.72] 0.001 1.99 [1.09–3.63] 0.026 – Education level higher than a secondary school diploma No 371 (33.1) 92 (30.9) – – 1 – Yes 749 (66.9) 206 (69.1) – – 2.6 [1.24–5.43] 0.011 – Employment situation Active employment 820 (70.8) 234 (75.5) – – – – Not in active employment 339 (29.2) 76 (24.5) – – – – Self-reported income levela Below average 381 (32.7) 72 (23.2) 1 1 – – Average 545 (46.7) 137 (44.1) 0.75 [0.44–1.30] 0.307 0.42 [0.24–0.73] 0.002 – – Above average 240 (20.6) 102 (32.8) 0.53 [0.28–0.99] 0.049 0.37 [0.19–0.7] 0.003 – – Body mass indexb < 25 757 (65.6) 223 (72.9) 1 – – – ≥ 25 397 (34.4) 83 (27.1) 1.8 [1.07–3.01] 0.026 – – – Fortin et al. Author details 1 University Paris 1 Sorbonne, Paris, France. 2 Aix Marseille Univ, Inserm, IRD, SESSTIM, Sciences Economiques & Sociales de la Santé & Traitement de l’Information Médicale, ISSPAM, Marseille, France. 3 Univ. Artois, CNRS, IESEG School of management, Univ. Lille, UMR 9221, Lille Economie Management (LEM), F‑62000 Arras, France. 4 University Paul Valéry Montpellier 3, CORHIS, Montpellier, France. Received: 20 May 2021 Accepted: 6 September 2021 Received: 20 May 2021 Accepted: 6 September 2021 Availability of data and materials The datasets generated and/or analyzed during the current study are not publicly available due to ongoing data treatment but are available from the corresponding author on reasonable request. Resultsh J Cannabis Res (2021) 3:46 Page 4 of 5 Table 2 Pattern of cannabidiol use in the whole study sample (n = 1166) CBD cannabidiol Number Percent Time since first CBD use (months) Less than 3 260 22.5 Between 3 and 12 255 22.1 Between 12 and 24 308 26.7 More than 24 331 28.7 Number of days CBD used in previous 30 days (days) 1–3 201 17.2 4–9 166 14.2 10–19 217 18.6 20–29 118 10.1 30 (i.e., every day) 464 39.8 CBD purchase locations in the previous 30 days On the internet 769 66.0 Specialized shop 231 19.8 From an acquaintance 56 4.8 Others 51 4.4 Self-cultivation 43 3.7 Tobacco shop 16 1.4 Principal mode of CBD use in the previous 30 days Smoking 701 60.7 Sublingual oil 214 18.5 Inhaling 122 10.6 Others (e-liquid, foodstuff, etc.) 117 10.1 Table 2 Pattern of cannabidiol use in the whole study sample (n = 1166) This may partly explain the lower preference for smoking CBD in UK users than in our French sample. To conclude, in our French user sample from the gen- eral population, we duplicated Moltke et al.’s findings for UK users that well-being, sleep, stress, and anxiety are the main reasons for using CBD. However, factors asso- ciated with reasons for use differed between both stud- ies as did patterns of use. Unlike Moltke et al., we did not find any gender effect. We agree with their conclusion that further research is needed to clarify whether CBD is effective in treating the health problems studied, and we also call for more research to highlight and better under- stand country-specific patterns of CBD use and corre- lates of use. Competing interests The authors declare that they have no competing interests. Acknowledgements g We thank all the study participants. Our thanks also to Jude Sweeney (Milan, Italy) for the English revision and copyediting of the manuscript. Discussion Ethical approval was given by the INSERM Ethics Committee (approval #20- 677 dated April 23, 2020). In accordance with French law, no consent was required since the survey was anonymous. Laws governing CBD in the UK are complex and vague, translating into a largely unregulated sector (Gibbs et al., 2019). In France, despite a European Union Court of Justice ruling during the survey period stating that CBD is not a narcotic drug (Court of Justice of the European Union, 2020), its status remains unclear (Bisiou, 2021). Consistent with Moltke et al.’s findings, we found that well-being was one of the most-cited primary reasons for using CBD in our French sample. Additionally, among those who declared this, reduced anxiety, improved sleep, and diminished stress were the expected effects most cited. Unlike Moltke et al., gender was not associ- ated with any reason for using CBD. Our French study population differed from the UK one in several ways: the male-female ratio was higher, time since first CBD use was longer, and a larger proportion of participants smoked CBD. While these differences may partly be due to recruitment methods, country-related specificities cannot be excluded. For example, law enforcement poli- cies in both countries seem to differ. In the UK, police preferentially target CBD in herbal form, while no such distinction between CBD products is made in France. Authors’ contributions DF, PC, SM, and YB designed the study. DF and TB analyzed the data and wrote the original manuscript. VDB performed the statistical analyses. PC, SM, and YB reviewed and amended the manuscript. The authors read and approved the final manuscript. References References Bisiou Y. Stupéfiants, cannabis et cannabidiol (CBD): la France sous pression de la CJUE. Dalloz. 2021;84;84. Court of Justice of the European Union. In case C-663/18, REQUEST for a preliminary ruling under Article 267 TFEU from the Cour d’appel Fortin et al. J Cannabis Res (2021) 3:46 Fortin et al. J Cannabis Res (2021) 3:46 Fortin et al. J Cannabis Res (2021) 3:46 Page 5 of 5 d’Aix-en-Provence (Court of Appeal, Aix-en-Provence, France), made by decision of 23 October 2018, received at the Court on 23 October 2018, in the criminal proceedings against B S, C A. [Internet]. 2020. Available from: https://curia.europa.eu/juris/document/document.jsf?text=& docid=233925&pageIndex=0&doclang=EN&mode=lst&dir=&occ=first &part=1&cid=15340648 Moltke J, Hindocha C. Reasons for cannabidiol use: a cross-sectional study of CBD users, focusing on self-perceived stress, anxiety, and sleep problems. J Cannabis Res. 2021;3:5. Walker LA, Koturbash I, Kingston R, ElSohly MA, Yates CR, Gurley BJ, et al. Can- nabidiol (CBD) in dietary supplements: perspectives on science, safety, and potential regulatory approaches. J Diet Suppl. 2020;17:493–502. Gibbs B, Yates A, Liebling J. CBD in the UK, Towards a responsible, innovative and high-quality cannabidiol industry [Internet]. Centre for Medicinal Cannabis; 2019. Available from: https://hempindustrydaily.com/wp-conte nt/uploads/2020/04/Report-_-CBD-in-the-UK-002.pdf Moltke J, Hindocha C. Reasons for cannabidiol use: a cross-sectional study of CBD users, focusing on self-perceived stress, anxiety, and sleep problems. J Cannabis Res. 2021;3:5. Moltke J, Hindocha C. Reasons for cannabidiol use: a cross-sectional study of CBD users, focusing on self-perceived stress, anxiety, and sleep problems. J Cannabis Res. 2021;3:5. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Lachenmeier DW, Habel S, Fischer B, Herbi F, Zerbe Y, Bock V, et al. Are side effects of cannabidiol (CBD) products caused by tetrahydrocannabinol (THC) contamination? 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https://openalex.org/W2003911778	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0076348&type=printable	English	null	Tacrolimus Does Not Affect Early Wound Healing in a Rodent Model of Bowel Anastomoses and Abdominal Wall Closure	PloS one	2,013	cc-by	4,753	Abstract Background: Use of immunosuppressant drugs has been associated with complications in wound healing. The calcineurin inhibitor tacrolimus is thought to have a relatively low complication rate, but preclinical research has yielded contradictory data, prompting the current comprehensive study Methods: Three groups of 33 male Wistar rats received a daily subcutaneous dose of 0,5, 2 or 5 mg/kg tacrolimus. A control group received saline. On day 0 a resection of 1 cm ileum and 1 cm colon was performed, and end-to-end anastomoses were constructed. Ten rats of each group were killed on day 3 and day 5 and the remaining animals on day 7. Both anastomoses and the wound in the abdominal wall were analyzed. Wound strength was the primary outcome parameter. Results: Mean strength of the abdominal wall increased significantly over time in all groups (p<0.0001). Both the breaking strength and the bursting pressure of the ileum and colon anastomoses followed the same pattern. No differences were observed between control and experimental groups. In addition, no consistent differences were found between groups regarding wound hydroxyproline content and the activities of matrix metalloproteinase-2 and -9. Conclusion: Tacrolimus does not affect early wound healing. Citation: Willems MCM, van der Vliet JA, Lomme RMLM, Hendriks T (2013) Tacrolimus Does Not Affect Early Wound Healing in a Rodent Model of Bowel Anastomoses and Abdominal Wall Closure. PLoS ONE 8(9): e76348. doi:10.1371/journal.pone.0076348 Editor: Giovanni Li Volti, University of Catania, Italy Received May 30, 2013; Accepted August 23, 2013; Published September 26, 2013 Received May 30, 2013; Accepted August 23, 2013; Published September 26, 2013 Copyright: © 2013 Willems et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: © 2013 Willems et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The research was funded by the UMCN St Radboud University. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. Abstract * E-mail: mwillems@flevoziekenhuis.nl this regime, tacrolimus is the newer agent, and extensive research has been carried out to establish its benefit over cyclosporine [1,8-10]. In contrast to most immunosuppressive drugs, tacrolimus, a macrolide derived from the fungus S tsukubaensis, is believed to have few or none adverse effects on wound healing. Still, as a calcineurin inhibitor, tacrolimus affects the first phase of T-cell activation. Because inflammatory T cells play a role in wound healing an effect of tacrolimus on wound healing is conceivable. Reported effects of tacrolimus in preclinical studies are contradictory, ranging from stimulation to inhibition of soft tissue repair [11-16]. A common drawback to these studies is the fact that, almost without exception, only one post-operative time point is evaluated, while wound healing is a complex process with different and overlapping phases. Tacrolimus Does Not Affect Early Wound Healing in a Rodent Model of Bowel Anastomoses and Abdominal Wall Closure Martine C. M. Willems, J. Adam van der Vliet, Roger M. L. M. Lomme, Thijs Hendriks Department of Surgery, Division of Vascular and Transplantation Surgery, Radboud University Medical Centre, Nijmegen, The Netherlands Martine C. M. Willems, J. Adam van der Vliet, Roger M. L. M. Lomme, Thijs Hendriks Department of Surgery, Division of Vascular and Transplantation Surgery, Radboud University Medical Centre, Nijmegen, The Ne Materials and Methods One hundred thirty-two male Wistar rats (body weight 240-260 g; Harlan, Horst, The Netherlands) were randomly divided into four groups of thirty-three animals. The animals were housed two per cage and allowed to become accustomed to laboratory conditions for one week before the start of the experiment. All animals had free access to water and standard rodent chow (Ssniff Specialdiäten GmbH, Soest, Germany). Three groups received tacrolimus (Prograft®, Astellas, Killorglin, Ireland) subcutaneously in daily dosages of 0.5 (group T0.5), 2.0 (group T2) and 5.0 (group T5) mg/kg/day from the day of operation until the end of the experiment. A control group received saline subcutaneously. Introduction Under different immunosuppressant regimes, wound healing disturbances are seen in 7-53% of kidney transplant recipients [1-4]. The premise that all immunosuppressant drugs have a negative effect on wound healing is widely accepted, although often scientific proof is lacking or inadequate. Clinical studies almost invariably use a regime of several immunosuppressant drugs, making it difficult to attribute adverse effects to a single component [4,5]. Thus, preclinical research is necessary to elucidate potential effects of individual drugs. This way we have demonstrated that everolimus, a m-TOR inhibitor, has a negative, dose- and time-dependent effect on experimental wounds in intestine and abdominal fascia [6,7]. Today, the typical standard regime of immunosuppressant drugs in solid organ transplantation consists of a calcineurin inhibitor such as cyclosporin or tacrolimus, an antiproliferative agent (azathioprine or mycophenolate mofetil) and a steroid. In The present comprehensive study has been performed to evaluate the effects of tacrolimus, used as a single drug in PLOS ONE \| www.plosone.org September 2013 \| Volume 8 \| Issue 9 \| e76348 September 2013 \| Volume 8 \| Issue 9 \| e76348 1 Tacrolimus Does Not Affect Early Wound Healing three different doses, and at multiple time points during healing of bowel anastomoses and abdominal fascia in rats. tissues and adhesions, and washed in saline. Bursting pressure and breaking strength were measured in the same segment as described previously [17]. In the abdominal wall the breaking strength was measured in the same way; from each segment of the abdominal wall, two separate strips of 1 by 2 cm were collected, with the suture line in the middle, and the breaking strength was measured in both. After biomechanical analysis, segments were cleaned from adhering tissue and standard sized samples containing the suture line were frozen in liquid nitrogen and stored at -80°C until further processing. Surgical Procedure On day 0, a midline laparotomy was performed and followed by a resection of 1 cm ileum 15 cm proximal to the ileocaecal junction and 1 cm colon 3 cm proximal to the rectal peritoneal reflection. End-to-end anastomoses were constructed with eight single-layer, inverting, interrupted 8-0 ethilon (Ethicon) sutures. The abdominal fascia was closed with an absorbable, polygalactin 3-0 suture, the skin was closed with staples. A heating pad was used to maintain body temperature at 38°C. The intestines were covered with gauze pads soaked with 0.9% NaCl to minimize desiccation. Fluid loss was compensated by administering 10 ml of 0.9% subcutaneously direct postoperative. The animals were weighed daily and observed for signs of illness. All operative procedures were performed by the same investigator (MW). Biochemical analysis and histology After lyophilisation, tissue samples were weighed, pulverized, and lyophilized again. Both hydroxyproline content and gelatinase activity were measured in control, T2 and T5 groups. The hydroxyproline content, as a measure of the collagen content, was measured by high-performance liquid chromatography after hydrolysis with 6-N-hydrochloric acid and derivatization with dabsyl-chloride. Ethic Statement This study was carried out in strict accordance with the National Dutch Act on Experimental Animals. The protocol (80320) was approved by the Animal Ethics Review Committee of the Radboud University, Nijmegen (Permit number DEC-2008-114). In order to minimize suffering all surgery was performed under general anesthesia using isoflurane 3%, in a mixture of oxygen and nitrous oxide. Postoperative analgesia was performed with buprenorphine, 0.02 mg/kg subcutaneously, twice daily for two days. The animals were killed by CO2/CO asphyxiation to reduce as much stress as possible. An individual animal welfare logbook was kept and reported back to the Animal Ethics Review Committee. Preparation of tissue extracts and procedures for gelatin zymography have been described previously [18]. The protein concentration of the extracts was measured using the bicinchoninic acid reagent. The various gelatinase activities were quantitated on the basis of lysed area and expressed as arbitrary units. Comparison of values obtained on different gels was performed by using collagenase type I (from Clostridium histolyticum; Sigma Chemical) as an internal standard. The presence of true matrix metalloproteinase (MMP) activity was confirmed by adding 10 mmol/L EDTA or 1,10 phenanthrolene to the buffers used after electrophoresis. Tacrolimus in whole blood was assayed using a PRO-TracTM ELISA kit from DiaSorin (Still water, Minnesota, USA). Sections of anastomoses originating from separate animals in the groups sacrificed after 7 days that had not been subjected to strength measurements were washed in 0.9% NaCl, spread out, and fixed immediately in a 4% phosphate-buffered formaldehyde solution. Subsequently, the samples were dehydrated and embedded in paraffin blocks. Sections of 4 mm in thickness were stained with hematoxylin and eosin (H&E). Statistical analysis To analyze differences in body weight and MMP-activity a Kruskal Wallis followed by Dunn’s test was used. Data of breaking strength, bursting pressure and hydroxyproline content were analysed with ANOVA followed by Tukey-Kramer post test. Figure 1. Postoperative course of body weight. Points represent average relative body weight, in relation to the weight prior to operation, for the control group (□) and the groups receiving tacrolimus: T0.5 (◼), T2 (Δ) and T5 (●). doi: 10 1371/journal pone 0076348 g001 Figure 1. Postoperative course of body weight. Points represent average relative body weight, in relation to the weight prior to operation, for the control group (□) and the groups receiving tacrolimus: T0.5 (◼), T2 (Δ) and T5 (●). doi: 10.1371/journal.pone.0076348.g001 (two after excessive weight loss and two of unknown reasons). Diarrhea, observed at least once during the experiment, was found in one thirty-second of the surviving rats in the control group and in 0/32, 2/31 and 7/29 (p=0.022) in the T0.5, T2 and T5 groups, respectively. Results Ten rats per group were killed on day 3 and day 5 each, and the remaining animals were killed on day 7. In the latter group, EDTA whole blood was sampled for tacrolimus assay (see below). Relaparotomy was performed by excision of a part of the abdominal wall of approximately 4 by 4 cm, including the suture line of the fascia. The anastomoses of ileum and colon were resected with adjacent bowel of approximately 4 cm in length and the suture line in the middle. The intestinal segments were carefully resected, including surrounding The mean trough level of tacrolimus, as measured in whole blood collected on day 7 after operation, was 0.3 ± 0.2 (SD) ng/ml in the control group and 4.9 ± 2.4, 10.1 ± 2.0 and 12.3 ± 5.6 ng/ml, respectively, in the T0.5, T2 and T5 groups. Eight animals died prematurely or were taken out of the experiment because of poor health: one each in the control and T0.5 groups (ileus), two in the T2 group (unknown reasons and excessive weight loss, respectively) and four in the T5 group PLOS ONE \| www.plosone.org September 2013 \| Volume 8 \| Issue 9 \| e76348 2 Tacrolimus Does Not Affect Early Wound Healing Figure 1. Postoperative course of body weight. Points represent average relative body weight, in relation to the weight prior to operation, for the control group (□) and the groups receiving tacrolimus: T0.5 (◼), T2 (Δ) and T5 (●). doi: 10.1371/journal.pone.0076348.g001 y g Wound strength Individual values for anastomotic bursting pressures in the intestine are given in Figure 2. They increased with time from day 3 onwards and, at all time points, median values were comparable in all four groups. From day 5 the bursting site was increasingly frequent outside the true anastomotic area (Table 1). An increase in the number of anastomoses bursting outside the suture line represents an increase in anastomotic strength. Altogether, this phenomenon occurred equally frequent in control and tacrolimus groups. All animals experienced a transient weight loss of approximately 10-15% of their body weight. From day 4 onwards they regained weight, those in the control group approximating their pre-operative weight at day 7 (Figure 1). However, the weight gain in animals receiving tacrolimus was less than in the control group. The relative weight in all experimental groups at day 7 was significantly (p<0.05) lower than in the control group. Wound breaking strength is depicted in Figure 3. After 3 days fascia strength was very low but increased rapidly thereafter. For all groups, the gain in fascia strength was very PLOS ONE \| www.plosone.org September 2013 \| Volume 8 \| Issue 9 \| e76348 3 Tacrolimus Does Not Affect Early Wound Healing Figure 2. Anastomotic bursting pressure. Individual values and medians (horizontal lines) in ileum and colon. A,B,C = ileum 3,5 and 7 days postoperative. D,E,F = colon 3, 5 and 7 days postoperative. X-axis: study groups. Open symbols denote rupture outside the suture line and closed symbols rupture inside the suture line. doi: 10.1371/journal.pone.0076348.g002 Figure 2. Anastomotic bursting pressure. Individual values and medians (horizontal lines) in ileum and colon. A,B,C = ileum 3,5 and 7 days postoperative. D,E,F = colon 3, 5 and 7 days postoperative. X-axis: study groups. Open symbols denote rupture outside the suture line and closed symbols rupture inside the suture line. doi: 10.1371/journal.pone.0076348.g002 significant (comparison of values at 3, 5 and 7 days by ANOVA: p<0.0001) and similar. At no time there were significant differences between the four groups. For the intestinal anastomoses, the gain in strength between day 3 and day 5 was less explicit. Still, for all groups anastomotic strength increased significantly (p<0.05) with time, values at day 7 always being higher (Tukey-Kramer, p<0.05) than those at day 3. Wound strength A significant difference between groups was only found for ileal anastomoses at day 7 where strength was higher in the T5 group than in the control group. Breaking always occurred within the suture line. the T5 group were lower than in the T2 group but equivalent to those in controls. In fascia, the hydroxyproline content was highest in the T5 groups. The results of the zymographic measurements of gelatinase activity, in ileal and colonic tissue at day 3 and 7, are summarized in Figure 5. The activities of proMMP-9 and proMMP-2 were similar in control and tacrolimus groups. Although MMP-9 activities were low and varied considerably between animals, they appeared higher in the tacrolimus groups, especially in ileum. For MMP-2, differences were seen in colon where tacrolimus apparently lowered activity at day 3 while increasing it after 7 days. Histology Wound hydroxyproline, as a measure for collagen content is given in Figure 4. Generally speaking, it increased with time, values at day 7 almost invariably being significantly (p<0.05) higher than those at day 3. In ileum, there were no differences between controls and experimental groups. In colon, values in A comprehensive examination of sections obtained from controls and the T0.5 and T2 groups failed to reveal any obvious architectural differences at day 7. Semiquantitative analysis also failed to indicate any differences between control PLOS ONE \| www.plosone.org September 2013 \| Volume 8 \| Issue 9 \| e76348 4 Tacrolimus Does Not Affect Early Wound Healing studies wound repair was analyzed at one time point only. Raptis et al. also used a low tacrolimus dose of 0.1 mg/kg/day and reported evidence of enhanced rodent colonic anastomotic healing, measured as bursting pressure at 4 and 8 days postoperatively [16]. Table 1. Anastomotic bursting site. Ileum Colon C 0/10 0/10 Day 3 T0.5 0/10 0/10 T2 0/9 0/10 T5 1/10 0/10 C 0/9 7/9 Day 5 T0.5 2/10 4/10 T2 5/10 8/9 T5 0/10 4/10 C 3/9 6/10 Day 7 T0.5 5/9 8/9 T2 2/9 8/9 T5 8/9 6/9 Numbers represent the frequency of the bursting site being without the actual suture line. doi: 10.1371/journal.pone.0076348.t001 In order to obtain data which would allow a comprehensive analysis of the possible effects of tacrolimus on soft tissue repair we analyzed wounds in intestine and abdominal fascia. Tacrolimus was administered in three doses which, dose- dependently, led to trough levels ranging from 5 to 12 ng/ml. These levels are in the range often reported in clinical studies [20]. Finally, the study included three time points, covering the inflammatory and early proliferative phases of the healing sequence. The first week after operation is particularly important because wound strength remains low during the first few days and increases from day 3 onwards. Any interference in this period will enhance chances on wound dehiscence. The results presented here are unambiguous. Tacrolimus, in three different clinically relevant dosages, does not interfere with and does not promote wound strength, which is the primary functional outcome parameter. This result holds for wounds in both small and large bowel and in the abdominal fascia at any of the three time points measured. For the bowel anastomoses, we measured two independent parameters for strength, the bursting pressure and the breaking strength. Discussion Although clinical studies of the effects on wound healing of single immunosuppressive drugs are lacking, wound complications after solid organ transplantation are often attributed to these drugs. Regimes including rapamycin derivates have been shown to be associated with more wound complications than those with calcineurin inhibitors. Our earlier experimental studies demonstrated a profound negative effect of everolimus on wound healing in the rat [6,7]. The present data clearly show that tacrolimus has no such effect and that surgical wounds demonstrate normal repair in the critical first week after operation, even during administration of doses leading to high trough levels of the drug. There is also no evidence for improvement of wound healing with tacrolimus. The introduction of new immunosuppressant agents has increased graft survival. However, at the same time, long term complications not related to graft function, become increasingly important, much of them due to development of cardiovascular disease or malignancies, related to the use of immunosuppressant drugs. The proportion of deaths attributed to malignancy in the first decade after transplantation is as high as 26% and rising [21]. Presumably, this phenomenon will result in a rise in future operations not related to the transplanted organ. Surgery needs to be executed under immunosuppressant therapy, possibly with increased chances of wound complications such as bowel leakage or incisional hernia. The present data emphasize the fact that tacrolimus, contrary to other drugs used for immunosuppression, does not have a negative effect on wound healing. This may be of clinical consequence in the direct post-transplant phase, as well as in patients on immunosuppressant therapy that have to undergo surgical procedures for other reasons. Such knowledge, obtained from preclinical studies as the present one, is very relevant for determining an optimal immunosuppressant regime in terms of composition, timing and dosage. Calcineurin inhibitors are notoriously known for their chronic toxicity and consequent chance of graft loss. Although cyclosporine and tacrolimus essentially inhibit the action of calcineurin in the same way, their side effects differ slightly. Tacrolimus is superior to cyclosporine in preventing acute rejection and improving graft survival which has resulted in an enormous increase in use of the agent since introduction in 1989 [10]. Tacrolimus has a greater effect on impairing the expression of alloantigen stimulated T-lymfocytes than does cyclosporine [19] but supposedly a less significant effect on wound healing. Histology The bursting pressure, which represents the ability to withstand intraluminal pressure, only reflects wound strength if the bursting site is within the suture line which will not always be the case after day 3. Therefore, it is very relevant to collect additional data for the breaking strength, which reflects wound strength over the entire period analysed here. Interestingly, undisturbed healing proceeded despite the fact that the rats in the experimental groups were clearly in a more catabolic state than those in the control group (Figure 1). and the tacrolimus groups with respect to histologic parameters such as mucosal repair, epithelial damage, wound area surface, degree of necrosis, and cellular infiltration. and the tacrolimus groups with respect to histologic parameters such as mucosal repair, epithelial damage, wound area surface, degree of necrosis, and cellular infiltration. Discussion Clinically, it is always used together with other drugs, therefore, its safety in terms of interference with wound repair must be assessed in a preclinical model. Preclinical data on the effects of tacrolimus on wound healing are few and contradictory. Doses of 2 mg/kg/day reportedly inhibit skin healing, but not colonic or ileal anastomotic healing [11,12,14,15]. Kiyama et al. even found that low doses (0.01-1 mg/kg/day) increased wound strength in the colon but not in the ileum [13]. In all these experimental September 2013 \| Volume 8 \| Issue 9 \| e76348 PLOS ONE \| www.plosone.org 5 Tacrolimus Does Not Affect Early Wound Healing . Wound breaking strength. Data represent mean and SEM in the control groups (white bars) an rs), T2 (grey bars) and T5 (black bars) tacrolimus groups. A= ileum anastomoses, B= colon anastomose /journal.pone.0076348.g003 NE \| plosone org 6 September 2013 \| Vol m Figure 3. Wound breaking strength. Data represent mean and SEM in the control groups (white bars) and the T0.5 (black & white bars), T2 (grey bars) and T5 (black bars) tacrolimus groups. A= ileum anastomoses, B= colon anastomoses, C = fascia. doi: 10.1371/journal.pone.0076348.g003 September 2013 \| Volume 8 \| Issue 9 \| e76348 PLOS ONE \| www.plosone.org 6 Tacrolimus Does Not Affect Early Wound Healing e 4. Wound hydroxyproline content. Data are expressed as hydroxyproline content per 5 mm tissue lengt and SEM for the controls (white bars) and the T2 (grey bars) and T5 (black bars) tacrolimus groups. A= ileum . : p<0.05 vs T2 group; #: p<0.05 vs control group. 1371/journal.pone.0076348.g004 Figure 4. Wound hydroxyproline content. Data are expressed as hydroxyproline content per 5 mm tissue length and represent mean and SEM for the controls (white bars) and the T2 (grey bars) and T5 (black bars) tacrolimus groups. A= ileum, B= colon, C = fascia. : p<0.05 vs T2 group; #: p<0.05 vs control group. doi: 10.1371/journal.pone.0076348.g004 Figure 4. Wound hydroxyproline content. Data are expressed as hydroxyproline content per 5 mm tissue length and represent mean and SEM for the controls (white bars) and the T2 (grey bars) and T5 (black bars) tacrolimus groups. A= ileum, B= colon, C = fascia. : p<0.05 vs T2 group; #: p<0.05 vs control group. doi: 10.1371/journal.pone.0076348.g004 September 2013 \| Volume 8 \| Issue 9 \| e76348 7 7 PLOS ONE \| www.plosone.org Tacrolimus Does Not Affect Early Wound Healing igure 5. References 1. Dean PG, Lund WJ, Larson TS, Prieto M, Nyberg SL et al. (2004) Wound-healing complications after kidney transplantations: A prospective, randomized comparison of sirolimus and tracolimus. Transplantation 77: 1555-1561. doi:10.1097/01.TP. 0000123082.31092.53. PubMed: 15239621. CD003961. Art. No. CD003961. doi: 10.1002/14651858.CD003961.pub2. PubMed: 16235347. 1. Dean PG, Lund WJ, Larson TS, Prieto M, Nyberg SL et al. (2004) Wound-healing complications after kidney transplantations: A prospective, randomized comparison of sirolimus and tracolimus. Transplantation 77: 1555-1561. doi:10.1097/01.TP. 0000123082.31092.53. PubMed: 15239621. doi: 10.1002/14651858.CD003961.pub2. PubMed: 16235347. p 11. Schäffer MR, Fuchs N, Proksch B, Bongartz M, Beiter T et al. (1998) T li i i d h li ibl l f d d it i 11. Schäffer MR, Fuchs N, Proksch B, Bongartz M, Beiter T et al. (1998) Tacrolimus impairs wound healing: a possible role of decreased nitric oxide synthesis. Transplantation 65: 813-818. doi: 10.1097/00007890-199803270-00008. PubMed: 9539093. 2. Kuppahally S, Al-Khaldi A, Weisshaar D, Valantine HA, Oyer P et al. (2006) Wound healing complications with de novo sirolimus versus mycophenolate mofetil-based regimen in cardiac transplant recipients. Am J Transplant 6: 986-992. doi:10.1111/j.1600-6143.2006.01282.x. PubMed: 16611334. 12. Kita J, Ogino Y, Kobayashi E, Fujimura A, Kogure H (1999) Effects of tacrolimus on small and large bowel anastomoses in the rat. Transplant Proc 31: 2789. doi:10.1016/S0041-1345(99)00568-0. PubMed: 10578292. 13. Kiyama T, Tajiri T, Tokunaga A, Yoshiyuki T, Barbul A (2002) Tacrolimus enhances colon anastomotic healing in rats. Wound Rep Regen 10:308-313 3. Bouzas-Mosquera A, Crespo-Leiro MG, Paniagua MJ, Naya C, Grille Z et al. (2008) Adverse effects of mammalian target of rapamycin inhibitors during the postoperative period after cardiac transplantation. Transplant Proc 40: 3027-3030. doi:10.1016/j.transproceed. 2008.09.021. PubMed: 19010181. 3. Bouzas-Mosquera A, Crespo-Leiro MG, Paniagua MJ, Naya C, Grille Z et al. (2008) Adverse effects of mammalian target of rapamycin inhibitors during the postoperative period after cardiac transplantation. Transplant Proc 40: 3027-3030. doi:10.1016/j.transproceed. 2008.09.021. PubMed: 19010181. 14. Wasserberg N, Tzakis AG, Santiago SF, Ruiz P, Salgar SK (2004) Anastomotic healing in a small bowel transplantation model in the rat. World J Surg 28: 69-73. doi:10.1007/s00268-003-7028-2. PubMed: 14639489. 4. Wszoła M, Kwiatkowski A, Ostaszewska A, Górski L, Kuthan R et al. (2013) Surgical site infections after kidney transplantation - where do we stand now? Transplantation 95: 878-882. doi:10.1097/TP. 0b013e318281b953. PubMed: 23511213. 15. Schäffer M, Fuchs N, Völker J, Schulz T, Kapischke M et al. (2005) Differential effect of tacrolimus on dermal and intestinal wound healing. J Invest Surg 18: 71-79. Discussion MMP activity in intestinal anastomoses. Columns represent mean values + SEM for controls (white bars) and the T2 grey bars) and T5 (black bars) tacrolimus groups. Data represent total activities, in arbitrary units, per 5-mm segment for proMMP-9 A), MMP-9 (B), proMMP-2 (C), and MMP-2 (D). : p<0.05 vs control group. oi: 10.1371/journal.pone.0076348.g005 LOS ONE \| www.plosone.org 8 September 2013 \| Volume 8 \| Issue 9 \| e76348 Figure 5. MMP activity in intestinal anastomoses. Columns represent mean values + SEM for controls (white bars) and the T2 (grey bars) and T5 (black bars) tacrolimus groups. Data represent total activities, in arbitrary units, per 5-mm segment for proMMP-9 (A), MMP-9 (B), proMMP-2 (C), and MMP-2 (D). *: p<0.05 vs control group. doi: 10.1371/journal.pone.0076348.g005 September 2013 \| Volume 8 \| Issue 9 \| e76348 PLOS ONE \| www.plosone.org 8 Tacrolimus Does Not Affect Early Wound Healing Author Contributions Tacrolimus, as a single drug, does not influence the repair sequence in soft tissues during the first week after operation. Conceived and designed the experiments: MW JvdV TH. Performed the experiments: MW RL. Analyzed the data: MW JvdV RL TH. Wrote the manuscript: MW JvdV RL TH. Conceived and designed the experiments: MW JvdV TH. Performed the experiments: MW RL. Analyzed the data: MW JvdV RL TH. Wrote the manuscript: MW JvdV RL TH. References doi:10.1080/08941930590926294. PubMed: 16036775. 5. Grim SA, Slover CM, Sankary H, Oberholzer J, Benedetti E et al. (2006) Risk Factors for Wound Healing Complications in Sirolimus- Treated Renal Transplant Recipients. Transplant Proc 38: 3520–3523. doi:10.1016/j.transproceed.2006.10.065. PubMed: 17175320. 16. Raptis D, Mantzoros I, Pramateftakis MG, Despoudi K, Zaraboukas T et al. (2012) The effects of tracolimus on colonic anastomotic healing in rats. Int J Colorectal Dis 27: 299-308. doi:10.1007/s00384-011-1337-y. PubMed: 22065109. doi:10.1016/j.transproceed.2006.10.065. PubMed: 17175320 6. van der Vliet JA, Willems MC, de Man BM, Lomme RM, Hendriks T (2006) Everolimus Interferes With Healing of Experimental Intestinal Anastomoses. Transplantation 82: 1477-1483. doi:10.1097/01.tp. 0000246078.09845.9c. PubMed: 17164720. 17. de Waard JWD, Wobbes T, de Man BM, van der Linden CJ, Hendriks T (1995) Postoperative levamisole may compromise early healing of experimental intestinal anastomoses. Br J Cancer 72: 456-460. doi: 10.1038/bjc.1995.355. PubMed: 7640232. 7. Willems MC, van der Vliet JA, de Man BM, van der Laak JAWM, Lomme RMLM et al. (2010) Persistent effects of everolimus on strength of experimental wounds in intestine and fascia. Wound Rep Reg 18: 98–104. doi:10.1111/j.1524-475X.2009.00558.x. PubMed: 20082683. 18. Siemonsma MA, de Hingh IHJT, de Man BM, Lomme RMLM, Verhofstad AA et al. (2003) Doxycycline improves wound strength after intestinal anastomosis in the rat. Surgery 133: 268-276. doi:10.1067/ msy.2003.27. PubMed: 12660638. 8. Kumar Anil, Heifets M, Fyfe B, Saaed MI, Moritz MJ et al. (2005) Comparison of steroid avoidance in tacrolimus/mycophenolate mofetyl and tacrolimus/sirolimus combination in kidney transplantation monitored by surveillance biopsy. Transplantation 80: 807-814. doi: 10.1097/01.tp.0000173378.28790.0b. PubMed: 16210969. 19. Vicari-Christensen M, Repper S, Basile S, Young D (2009) Tacrolimus: review of pharmacokinetics, pharmacodynamics, and pharmacogenetics to facilitate practitioners’ understanding and offer strategies for educating patients and promoting adherence. Prog Transplant 19: 277-284. PubMed: 19813492. 9. Kobashigawa JA, Miller LW, Russell SD, Ewald GA, Zucker MJ et al. (2006) Tacrolimus with mycophenolate mofetil (MMF) or sirolimus vs. Cyclosporine with MMF in cardiac transplant patients: 1-year report. Am J Transplant 6: 1377-1386. doi:10.1111/j.1600-6143.2006.01290.x. PubMed: 16686761. 20. Staatz CE, Tett SE (2004) Clinical Pharmacokinetics and Pharmacodynamics of Tacrolimus in Solid Organ Transplantation. Clin Pharmacokinet 43: 623-653. doi:10.2165/00003088-200443100-00001. PubMed: 15244495. 10. Webster AC, Woodroffe RC, Taylor RS, Chapman JR, Craig JC (2005) Tacrolimus versus cyclosporin as primary immunosuppression for kidney transplant recipients. Cochrane Database Syst Rev Issue 4: 21. Kapoor A (2008) Malignancy in kidney transplant recipients. Drugs 68 Supl l 1: 11-19. PubMed: 18442297. References PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org September 2013 \| Volume 8 \| Issue 9 \| e76348 9
https://openalex.org/W4365514948	https://link.springer.com/content/pdf/10.1007/s12144-023-04644-8.pdf	English	null	Body shame and problematic social networking sites use: the mediating effect of perfectionistic self-presentation style and body image control in photos	Current psychology	2,023	cc-by	9,791	Abstract Previous studies have shown that body shame and body image control in photos are associated with Problematic Social Networking Sites Use. The current study aims to build upon previous evidence by examining the role of perfectionis tic self-presentation in the relationship between body shame, on the one hand, and body image control in photos and Problematic Social Networking Sites Use, on the other hand. The sample included 695 participants (%F = 61.29; Mage = 21.01 ± 5.05; age range = 14–30). The hypothesized serial mediation effect of perfectionistic self-presentation style and body image control in photos in the association between body shame and Problematic Social Networking Sites Use was significant (indirect effect = 0.05; 95% CI: [0.02, 0.07] for young women; indirect effect = 0.02; 95% CI: [0.002, 0.04] for young men). The present study contributes toward the understanding of the possible predictors of Problematic Social Networking Sites Use and leads novel findings in the field of self-objectification research. Regardless of gender, young people who feel ashamed of their body may develop a perfectionistic self-presentation (by promoting a perfect image and hiding imperfections) as compensatory strategy. However, the gratification of the need for self-presentation through body image control in Social Networks photos activities could in turn lead to Problematic Social Networking Sites Use. Keywords Body shame · Body image · Perfectionistic self-presentation style · Problematic social Current Psychology (2024) 43:4073–4084 https://doi.org/10.1007/s12144-023-04644-8 Current Psychology (2024) 43:4073–4084 https://doi.org/10.1007/s12144-023-04644-8 Body shame and problematic social networking sites use: the mediating effect of perfectionistic self-presentation style and body image control in photos Giulia Fioravanti1 · Sara Bocci Benucci2 · Viola Vinciarelli3 · Silvia Casale1 Accepted: 2 April 2023 / Published online: 14 April 2023 © The Author(s) 2023 Accepted: 2 April 2023 / Published online: 14 April 2023 © The Author(s) 2023 Sara Bocci Benucci sara.boccibenucci@unifi.it 1 Department of Health Sciences, Psychology Unit, University of Florence, Via di San Salvi 12, Florence 50135, Italy 2 Department of Experimental and Clinical Medicine, University of Florence, Largo Brambilla, 3, Florence 50134, Italy 3 School of Psychology, University of Florence, Via della Torretta, 16, Florence 50037, Italy Body shame and problematic social networking sites use According to the dual-factor model (Nadkarni & Hoffmann, 2012), the need for self-presentation can be considered a motivational factor that can lead individuals to excessively use Social Networking Sites. The self-presentation style of individuals experiencing high levels of body shame might be characterized by the need to hide one’s own imperfec tions and shortcomings and the active promotion of a per fect image (i.e., perfectionistic self-presentation). This affirmation found empirical support in a previous study by Ferreira and colleagues (2017) who found that experience of shame lead to act self-presentation strategies as defen sive responses. In other words, shame experiences may trig ger striving to present a perfect body image. Accordingly, Nepon et al. (2016) found a strong positive correlation between self-image goals and perfectionistic self-presenta tion. Perfectionistic self-presentation style represents a state in which an individual presents him/herself to others in a way aimed at hiding imperfections (Hewitt et al., 2003). Individuals with a high need to present themselves as per fect might consider Social Networks a space in which they can have the opportunity to manage their self-presentation. In fact, a previous study showed that a self-presentation style characterized by the need to avoid showing or dem onstrating any perceived shortcomings is associated with As cultural beauty standards are often unattainable, inter nalization of such standards has been found to be positively associated with body shame (e.g., Kim et al., 2013). In fact, body shame has been defined as a psychological phenom enon that arises when individuals feel ashamed of their bod ies due to the perceived inability to satisfy cultural beauty standards (McKinley & Hyde, 1996). According to self- objectification theory (Fredrickson & Roberts, 1997), self- objectification occurs when women adopt a third-person perspective on the body, which leads them to look at their bodies as objects that they have to constantly monitor and judge, giving a high value to how they look to others instead of how they feel. The vigilant body surveillance activity (or body monitoring) leads individuals to have negative subjec tive experiences such as body shame. Although the majority of research has focused on investigating self-objectification in female populations (for a review see Daniels et al., 2020), some studies have also involved male samples, demonstrat ing that the theory’s basic tenets can also be applied to men (e.g., Calogero, 2009; Morrison et al., 2003; Strelan & Harg reaves, 2005a). Introduction face-to-face interactions, Social Networks let individuals control the information about themselves, thus enhancing the possibilities to manage self-presentation (Buffardi & Campbell, 2008). Self-presentation refers to “the process of controlling how one is perceived by other people” (Leary, 1995, p. 2), which is described as goal-driven behaviour (Leary & Kowalski, 1990). According to the uses and grati fication framework (Katz et al., 1973), different media are chosen by users to fulfil specific needs, and the need for self-presentation is one of the main reasons for using Social Networks (for an overview see Nadkarni & Hoffman, 2012). Adolescents and young adults are the populations who use Social Networking Sites the most (D’Arienzo et al., 2019; Kuss & Griffiths, 2017). Social networks are online platforms that allow individuals to create profiles, share and view content and interact with other users and their posts through “likes”, comments and private messages (Tiggemann & Vellissaris, 2020). Compared to traditional Given the high focus on visual self-presentation and appearance promoted by several photo-based Social Net works (i.e., Instagram), users might manage their self-pre sentations by hiding personal attributes that they consider unattractive, such as body size, or manage how they look by editing their pictures or selfies (Fox & Rooney, 2015). Unfor tunately, levels of body shame and the extent to which indi viduals control their body images in pictures before posting them on Social Networks appears to be strongly related to 3 School of Psychology, University of Florence, Via della Torretta, 16, Florence 50037, Italy 1 3 Current Psychology (2024) 43:4073–4084 4074 levels of self-objectification report desiring more muscular bodies (Oehlhof et al., 2009). According to self-objectifi cation theory, body shame is a central experience for those individuals who self-objectify their bodies, and research has documented negative outcomes related to body shame, such as eating disorders (e.g., Mustapic et al., 2015), depression (e.g., Grabe et al., 2007), body dysmorphic disorder (e.g., Weingarden et al., 2017) and psychological distress (e.g., Brownstone & Bardone-Cone, 2021). In regard to Problem atic Social Networking Sites Use, it has been shown that shame experiences and Problematic Social Networking Sites Use are strongly associated, and this link is mediated by the perceived benefits of computer-mediated commu nication (i.e., escapism, control over self-presentation and approval/acceptance). Introduction Meta-analyses showed that Problematic Social Networking Sites Use is associated with several issues, including depression (Cunningham et al., 2021), low self-esteem (Saiphoo et al., 2020), suicidal ideation (Nesi et al., 2021), lower well-being (Huang, 2020) and higher psychological distress (Shannon et al., 2022), and – as already mentioned – body shame. Building upon previous evidence, the current study focuses the attention on the potential role of perfectionistic- self-presentation as a behavioral strategy that might explain how body shame leads to body image control in photos which, in turn, leads to Problematic Social Networking Sites Use. Introduction In detail, social networking sites offer more control than face-to-face contact over self-presenta tion, which might be particularly appealing for individuals who see themselves as having negative and unattractive fea tures (see Caplan, 2005, 2007). It is therefore plausible to assume that those who experience body shame might use social networking sites not only as a way to escape from real-life issues and manage negative emotions (Casale & Fioravanti, 2017; Tangney & Dearing, 2002) but also for the enhanced control over self-presentation. Problematic Social Networking Sites Use (PSNSU) (Casale & Fioravanti, 2017; Gioia et al., 2020). Problematic Social Networking Sites Use can be defined as an excessive use of Social Networks characterized by being overly concerned about social networks and a strong motivation to use them which, in turn, impairs social lives, scholastic success, work lives and/or psychological health and well-being (Andreas sen & Pallesen, 2014). A recent research has enlightened the core symptoms associated with Problematic Social Net working Sites Use which can help in distinguishing between problematic and non-problematic social networking site use: (i) difficulties in controlling the use, (ii) thinking obses sively about going online; (iii) and a preference for online interactions (Svicher et al., 2021). Meta-analyses showed that Problematic Social Networking Sites Use is associated with several issues, including depression (Cunningham et al., 2021), low self-esteem (Saiphoo et al., 2020), suicidal ideation (Nesi et al., 2021), lower well-being (Huang, 2020) and higher psychological distress (Shannon et al., 2022), and – as already mentioned – body shame. Problematic Social Networking Sites Use (PSNSU) (Casale & Fioravanti, 2017; Gioia et al., 2020). Problematic Social Networking Sites Use can be defined as an excessive use of Social Networks characterized by being overly concerned about social networks and a strong motivation to use them which, in turn, impairs social lives, scholastic success, work lives and/or psychological health and well-being (Andreas sen & Pallesen, 2014). A recent research has enlightened the core symptoms associated with Problematic Social Net working Sites Use which can help in distinguishing between problematic and non-problematic social networking site use: (i) difficulties in controlling the use, (ii) thinking obses sively about going online; (iii) and a preference for online interactions (Svicher et al., 2021). Body shame and problematic social networking sites use While women desire to achieve the standard of beauty of a thin body promoted by society, men with high 1 3 Current Psychology (2024) 43:4073–4084 4075 Problematic Social Networking Sites Use (Casale et al., 2015). Problematic Social Networking Sites Use (Casale et al., 2015). Gender differences Gender differences are a relevant issue in social media and body image research. For what concerns the current study, previous studies have evidenced that women report higher levels of body shame than men (e.g., Dakanalis et al., 2015, 2017; Gioia et al., 2020), as well as higher levels of appearance control beliefs and body image control in pho tos (Boursier et al., 2020). Moreover, young women tend to invest more time in taking selfies as a means of promoting their best self-presentation, expressing their identities, and controlling their body images, and they apply more privacy restrictions to their images. Young men, instead, tend to control their body images in photos to improve their sexual attractiveness (Boursier et al., 2020; Boursier & Manna, 2019). To date, only one study (Gioia et al., 2020) has inves tigated gender differences concerning the risk of develop ing Problematic Social Networking Sites Use in individuals experiencing body shame. It was found that body shame strongly predicted photo investment and control, leading to problematic social networking in both young men and women, but with higher effects among young women. These results might confirm the self-objectification framework’s assumption that women compare their bodies with cultural body standards more than men, with consequent greater internalization and perception of these standards as a per sonal choice (Grabe et al., 2007; McKinley & Hyde, 1996). In the present study, we go a step further by arguing that body image control in photos might be a behavioural strat egy used by individuals who experience high levels of body shame to meet their needs to present an image to others that is less negatively impacted by their presumed body imper fections. This might also be consistent with the idealized virtual identity hypothesis (Back et al., 2010), which argues that some individuals display idealized attributes that do not depict their actual personalities on social networking sites. It is therefore plausible to assume that for individuals expe riencing body shame, social networking sites provide the opportunity to hide their imperfections and promote their bodies by controlling/manipulating their photos before post ing them. Body shame and problematic social networking sites use However, the opportunities provided by social networks to hide one’s own imperfections might, in turn, put a person at risk of developing Problematic Social Net working Sites Use. Previous observational studies showed an association between high levels of body-related concerns and the fre quency of manipulation of photos for online posting, espe cially in young individuals (e.g., McLean et al., 2015; Meier & Grey, 2013; Zheng et al., 2019), and findings from experi mental studies tend to support these results. Tiggemann and Zinoviev (2019) found that women who digitally manipu late their own photos report improvements in their body images after being exposed to enhancement-free images, which suggests an association between negative body image and photo manipulation behaviours. Similarly, Salomon and Brown (2020) demonstrate that the simple action of taking a selfie resulted in an increase in state self-objectification in those participants who were informed that the picture would have been posted on Facebook. In addition, a parallel line of research has shown a positive association between body appearance control in photos and the intensity of the use of Social Networks in adolescent samples (e.g., Manago et al., 2015; Tiggemann & Slater, 2017). Hypotheses As previously reported, previous studies found that body shame is positively associated with body image control in photos, which, in turn, is associated with Problematic Social Networking Sites Use (Gioia et al., 2020). However, the psychological processes that might explain how body shame — an affective state —leads to body image control in photos (a behaviour) have been less investigated. The cur rent study contributes to fill this gap by investigating the potential role of a motivational factor, that is the need for self-presentation, and in particular the need to appear perfect in the above mentioned links. Accordingly, a previous study showed that perfectionistic self-presentation is positively associated with Problematic Social Networking Sites Use (Casale et al., 2015). The goal of the present study was to provide an integration of these two research lines by testing a model that proposes that body shame generates the need to hide perceived imperfections (i.e., a perfectionistic self-pre sentation style), which leads to body image control in pho tos, which, in turn, leads to Problematic Social Networking Sites Use. In other words, we argue that for people report ing high levels of body shame, the use of Social Networks could represent a way of hiding imperfections/promoting a Very relevant to the current study, a recent study that combined these two lines of research showed a mediat ing role of body image control in photos in the association between body shame and Problematic Social Networking Sites Use in adolescents (Gioia et al., 2020). This study has shown that higher levels of body shame explain—at least in part— the strategies that an individual implements before and after having taken a picture prior to sharing it on Social Networks (i.e., control of the perspective, use of filters, digital correction, etc.), which in turn predicts Problematic Social Networking Sites Use levels. 1 3 4076 Current Psychology (2024) 43:4073–4084 Participants were recruited using advertisements on Social Networks, and they were informed that participation was voluntary and anonymous and that confidentiality was guaranteed. A web link directed the participants to the study website, and if they consented to participate, they were asked to answer some demographic information, questions about their use of Social Networks and to complete self- report questionnaires. Since it was not possible to submit the form without filling in all the required fields, the results did not present missing data. Demographic information Demographic information was collected, including respon dents’ age (in years), gender, marital and occupational status and level of education. Respondents were asked to indicate how many hours per day they use social networks and which Social Networks they use the most. Finally, par ticipants were asked to indicate their weight and height to calculate their BMI. Participants and procedure A sample of 695 participants (%F = 61.29; Mage= 21.01 ± 5.05 years; age range = 14–30) agreed to participate in the study. Regarding educational qualifications, 39.9% of the sample reported having a middle school diploma, 28.5%, a high school diploma, 17.0%, a bachelor’s degree, 12.1%, a mas ter’s degree, 2.3%, a higher degree (e.g., Ph.D.), and 0.3% had an elementary school diploma. In terms of occupation, the majority were students (63.9%), 19.9% were workers and 12.1% were working students. Concerning participants’ marital status, 52.8% were single, 33.7% reported having a noncohabiting partner, 13.2% had a cohabiting partner, and 0.3% declared “other” without specification. Hypotheses Data were collected between September and November 2021, and no remuneration was given. Informed consent was obtained from all participants. According to Italian law, parental consent was not required for participants over 14 years old. The study procedures were carried out in accordance with the Declaration of Hel sinki. The Institutional Review Board of the University of Florence approved the study. perfect image, and this opportunity is created by the ability to control and manage one’s own photos. However, these enhanced chances of controlling self-presentation might lead individuals to lose control over their use of Social Net works. The hypothesized model is displayed in Fig. 1.f yp p y g Due to the above-mentioned gender differences, we will explore if men and women differ on the study variables (expecting to find women reporting high levels of body shame and body image control in photos) and examine the validity of the proposed model separately for women and men (expecting to find higher effects among women). Finally, since a large number of studies (e .g., Smolak, 2009; Cheng et al., 2021) found that young individuals are at more risk of experiencing body shame and also report higher levels of Problematic Social Networking Sites Use age will be included as covariate in the model. The results of the current study may contribute to the understanding of Problematic Social Networking Sites use by identifying a potential explaining pathway: People who experience body shame may satisfy their need to appear perfect on social net work by posting photos in which they look attractive. The gratification of this need could act as a positive reinforce ment leading the individuals to lose control over their social network sites use. Statistical analyses Descriptive statistics, zero-order correlations between the study variables, and a one-way ANOVA for examining gender differences were computed using IBM Statistical Package for the Social Sciences (SPSS), version 23.0 (IBM Corp., Armonk, NY, USA). We then tested the hypoth esized conceptual model (serial mediation model) using the PROCESS macro for SPSS (Model 6) developed by Hayes (2013) separated for men and women, including age as a covariate. Bias-corrected bootstrap confidence intervals (CIs) derived from 5,000 bootstrap resamples are estimated to test for the significance of conditional direct and indirect effects. The effects are considered significant if the CI val ues do not include zero. Body shame Body shame was measured using the Italian version of the shame subscale (Dakanalis et al., 2017) from the Objecti fied Body Consciousness Scale (OBCS; Mckinley & Hyde, 1996). This subscale is composed of eight items and mea sures shame over one’s body that extends to the self as a result of a perceived failure to achieve cultural ideals and social standards of appearance. A simple item is “When I’m not the size I think I should be, I feel ashamed”. Partici pants are asked to respond on a 7-point Likert scale from 1 (strongly disagree) to 7 (strongly agree). Higher scores Fig. 1 The hypothesized media tion model, all the hypothesized relationships are positive Fig. 1 The hypothesized media tion model, all the hypothesized relationships are positive 1 3 1 3 4077 Current Psychology (2024) 43:4073–4084 indicate higher levels of body shame. Cronbach’s alpha in the current study was 0.85. indicate higher levels of body shame. Cronbach’s alpha in the current study was 0.85. core aspects of addictions (i.e., salience, mood modifica tion, tolerance, withdrawal, conflict, and relapse). Each item addresses experiences within a time frame of 12 months and is answered on a 5-point Likert scale ranging from 1 (very rarely) to 5 (very often). A sample item is “How often dur ing the last year have you felt an urge to use social media more and more?“. Higher scores indicate greater Problem atic Social Networking Sites Use. Cronbach’s alpha in the current study was 0.78. Body image control in photos In the present study, the revised Italian version (Boursier & Manna, 2019) of the 16-item Body Image Control in Photos – Revised (BICP-R) questionnaire (original version Pelosi et al., 2014) was used. Answers are rated on a five-point Likert scale, from 1 (never) to 5 (always), and the question naire assesses body image control in photos. The scale com prises five factors: selfie-related (e.g., “I prefer my image as it appears in self-portraits, because I know how to make it look better”), privacy filter (e.g., “I use privacy filters in order to show photos in which I appear more attractive only to certain people”), positive body image (e.g., “I post those photos which I hope will receive praise for my appearance”), sexual attraction (e.g., “I have posted provocative photos on Facebook in order to attract attention to myself”), and nega tive body image (e.g., “I feel awkward if I notice that some one has posted photos that show my body’s defects”). A total score can be computed. Higher scores indicate higher levels of body image control in photos. Cronbach’s alpha in the current study for the total BICP-R was 0.87. Descriptive statistics and correlations Regarding participants’ use of Social Networks, the total sample (n = 695) reported spending 3.24 ± 2.32 hours a day using Social Networks. The Social Networks they declared to use the most were Instagram (63.5%), TikTok (15.4%), YouTube (14.7%) and Facebook (3.6%). Descriptive statistics and correlations among the study variables are presented in Table 1. Body shame was posi tively correlated with perfectionistic self-presentation, body image control in photos and Problematic Social Network ing Sites Use. Having a higher BMI was also associated with higher levels of body shame. Perfectionistic self-pre sentation was positively associated with both body image control in photos and Problematic Social Networking Sites Use. Finally, body image control in photos was positively associated with Problematic Social Networking Sites Use. Age was significantly and negatively associated with all the study variables.f Perfectionistic self-presentation The Italian version (Borroni et al., 2016) of the 27-item Perfectionistic Self-Presentation Scale (Hewitt et al., 2003) was used to assess perfectionistic self-presentation style. Answers are rated on a seven-point Likert scale, from 1 (strongly disagree) to 7 (strongly agree), and the scale com prises 3 subscales: Perfectionistic Self-Promotion (e.g., “I try always to present a picture of perfection”) which con tains 10 items that reflect the need to appear perfect to oth ers; Nondisplay of Imperfection (e.g., “It would be awful if I made a fool of myself in front of others”), which includes 10 items that assess the need to avoid appearing imperfect to others. Nondisclosure of imperfection (e.g., “I should solve my own problems rather than admit them to others”), which comprises 7 items measuring the need to hide one’s imper fections. A total score can be computed. Higher scores indi cate higher levels of a perfectionistic self-presentation style. Cronbach’s alpha in the current study for the total PSPS was 0.91. Problematic social networking site use Problematic Social Networking Site Use was measured using the Italian version (Monacis et al., 2017) of the 6-item Bergen Social Media Addiction Scale (BSMAS; Andreassen et al., 2016). The BSMAS consists of six items reflecting Gender differences were computed using one-way ANOVA. As displayed in Table 2, women reported higher scores in body shame and body image control in photos than men, whereas no significant differences between women 1 3 Current Psychology (2024) 43:4073–4084 4078 Table 1 Descriptive statistics and Pearson’s correlations among the study variables M ± SD (1) (2) (3) (4) (5) (6) (1) Body Shame 3.65 ± 1.39 - (2) Perfectionistic Self-Presentation 106.58 ± 26.10 0.53 - (3) Body Image Control in Photos 36.62 ± 11.80 0.47 0.40 - (4) PSNSU 15.49 ± 5.28 0.31 0.34 0.46 - (5) BMI 21.72 ± 3.54 0.24 − 0.03 − 0.06 0.02 - (6) Age 21.01 ± 5.05 − 0.13 − 0.19 − 0.22 − 0.19 0.18 - Notes. BMI = Body Mass Index ** p < 0.001; * p < 0.05 and men were found for perfectionistic self-presentation and Problematic Social Networking Sites Use. Table 2 Gender differences among study variables Men Women M ± SD M ± SD F p η2 Body Shame 3.30 ± 1.13 3.86 ± 1.48 27.88 < 0.001 0.04 Perfection istic Self- Presentation 106.62 ± 22.85 106.51 ± 27.88 0.00 0.95 Body Image Control in Photos 33.50 ± 11.27 38.54 ± 11.71 31.13 < 0.001 0.04 Problem atic Social Network Site Use 15.09 ± 5.31 15.73 ± 5.24 2.46 0.12 Table 2 Gender differences among study variables (total indirect effect = 0.26; 95% CI: [0.18, 0.33]; direct effect = − 0.0008; 95% CI: [-0.38, 0.37]). Specifically, per fectionistic self-presentation style mediated the relation between body shame and Problematic Social Networking Sites Use (indirect effect = 0.11; 95% CI: [0.05, 0.18]) and body image control in photos mediated this relationship (indirect effect = 0.09; 95% CI: [0.06, 0.14]). The hypoth esized serial mediation effect of perfectionistic self-pre sentation style and body image control in photos on the association between body shame and Problematic Social Networking Sites Use was significant (indirect effect = 0.05; 95% CI: [0.02, 0.07]). Problematic social networking site use A significant negative effect of age was observed on perfectionistic self-presentation style (β = − 0.13, SE = 0.22, t = -3.27, p = 0.001) and on body image control in photos (β = − 0.18, SE = 0.10, t = -4.39, p = 0.001), while the effect of age on Problematic Social Networking Sites Use was not significant. The model explained 10% of the variance in Problematic Social Networking Sites Use. and men were found for perfectionistic self-presentation and Problematic Social Networking Sites Use. and men were found for perfectionistic self-presentation and Problematic Social Networking Sites Use. Mediation analysis C’ = direct effect; C = total effect. p < 0.05, p < 0.001 Fig. 3 Serial mediation model of perfectionistic self-presentation style and body image control in photos in the relationship between body shame and PSNSU in the male subsample. Values shown are standardized coeffi cients. C’ = direct effect; C = total effect. p < 0.05, *p < 0.001 Fig. 3 Serial mediation model of perfectionistic self-presentation style and body image control in photos in the relationship between body shame and PSNSU in the male subsample. Values shown are standardized coeffi cients. C’ = direct effect; C = total effect. p < 0.05, *p < 0.001 use strategies to control their body images in photos, such as engaging in photo manipulation activities, photo shar ing and selfie-related behaviours. However, it is important to note that the difference in body shame scores between women and men, despite the significativity, was not so high in effect size, indicating that men might also be at risk of developing self-objectification processes (Gioia et al., 2020; Vandenbosch & Eggermont, 2013). Next, the the validity of the hypothesized mediation model was tested separately for men and women. The first path confirms—regardless of gender—what was previously reported on the mediat ing role of body image control in photos in the relationship between body shame and Problematic Social Networking Sites Use (Boursier & Manna, 2019; Gioia et al., 2020). Individuals who experienced body shame tended to adopt image-centred coping strategies (Cash et al., 2005; Choma et al., 2009), such as body image control in photos on Social Networks, to improve their appearance and social accep tance (Fox & Vandemia, 2016). However, the gratification obtained may lead them to spend more time on Social Net works and related activities (i.e., spending much time editing their photos to post them on Social Networks) and develop ing Problematic Social Networking Sites Use. These results are not surprising in that this path was already highlighted in a recent study (Gioia et al., 2020). Our study adds to previ ous literature by also highlighting a second path— among women—which showed for the first time the mediating role of perfectionistic self-presentation in the relationship between body shame and Problematic Social Networking Sites Use. According to Hewitt et al. Mediation analysis Hayes’s (2013) SPSS macro PROCESS (Model 6), with a 95% bias corrected confidence interval (CI) based on 5,000 bootstrap samples, was used to examine the indirect effects of body shame on Problematic Social Networking Sites Use through perfectionistic self-presentation and body image control in photos in men and women separately. Age was included as a covariate in the model. The indirect effect was considered statistically significant if the CI did not contain zero. Among men, the results (see Fig. 3) showed that per fectionistic self-presentation style and body image control in photos mediated the relationship between body shame and Problematic Social Networking Sites Use (total indi rect effect = 0.17; 95% CI: [0.09, 0.26]; direct effect = 0.15; 95% CI: [0.16, 1.29]). There is therefore both a total indi rect effect and a significant direct relationship between body shame and Problematic Social Networking Sites Use.i Specifically, body image control in photos mediated the relationship between body shame and Problematic Social Networking Sites Use (indirect effect = 0.11; 95% CI: [0.06, 0.18]) whereas perfectionistic self-presentation did not mediate this relationship (indirect effect = 0.04; 95% CI: Among women, the results (see Fig. 2) showed that per fectionistic self-presentation style and body image control in photos completely mediated the relationship between body shame and Problematic Social Networking Sites Use Fig. 2 Serial mediation model of perfectionistic self-presentation style and body image control in photos in the relationship between body shame and PSNSU in the female subsample. Values shown are standardized coeffi cients. C’ = direct effect; C = total effect. p < 0.05, *p < 0.001 Fig. 2 Serial mediation model of perfectionistic self-presentation style and body image control in photos in the relationship between body shame and PSNSU in the female subsample. Values shown are standardized coeffi cients. C’ = direct effect; C = total effect. p < 0.05, *p < 0.001 Fig. 2 Serial mediation model of perfectionistic self-presentation style and body image control in photos in the relationship between body shame and PSNSU in the female subsample. Values shown are standardized coeffi cients. C’ = direct effect; C = total effect. p < 0.05, **p < 0.001 1 3 Current Psychology (2024) 43:4073–4084 4079 Fig. 3 Serial mediation model of perfectionistic self-presentation style and body image control in photos in the relationship between body shame and PSNSU in the male subsample. Values shown are standardized coeffi cients. Mediation analysis (2003), the perfection istic self-presentation style may act as a compensatory strat egy that individuals engage in to deal with feelings of shame and inadequacy for obtaining acceptance, and this was sup ported by empirical research that showed the role of per fectionistic self-presentation as a mediator between internal and external shame and the drive for thinness (Ferreira et al., 2015). The current study highlights for the first time that the experience of body shame is associated with perfection istic self-presentation, which, in turn, leads to Problematic Social Networking Sites Use. When individuals engage in a perfectionistic self-presentation style on Social Networks, they might satisfy the need to appear perfect online without [-0.009, 0.09]). The hypothesized serial mediation effect of perfectionistic self-presentation style and body image con trol in photos on the association between body shame and Problematic Social Networking Sites Use was significant (indirect effect = 0.02; 95% CI: [0.002, 0.04]). However, a significant direct effect of body shame on Problematic Social Networking Sites Use was also found. A significant negative effect of age on body image control in photos was found (β = − 0.13, SE = 0.12, t = -2.36, p = 0.019), while the effect of age on perfectionistic self-presentation style and Problematic Social Networking Sites Use was not signifi cant. The model explains 15% of the variance in Problem atic Social Networking Sites Use. Limitations and future directions Although this is the first study to investigate the role of perfectionistic self-presentation style in the association between body shame and Problematic Social Networking Sites Use, some limitations should be addressed. First, the cross-sectional design prevents to establish a cause-effect relationship and to clarify the direction of the relationship between body image issues and Prob lematic Social Networking Sites Use. In fact, according to the hypothesis of the bidirectional nature of the social networking-self-objectification relationship (Strelan & Harhreaves, 2005b), adolescents and young adults who experience body shame might develop Problematic Social Networking Sites Use due to the positive or negative rein forcement obtained on Social Networks, but they could also experience increases in their body image concerns due to the prolonged exposure to idealized body images on Social Networks (Fioravanti et al., 2022). As previously suggested (e.g., Boursier et al., 2020; Gioia et al., 2020), self-objecti fication and Problematic Social Networking Sites Use could mutually reinforce each other. The online environment allows users with appearance-related concerns to manage how their body image appears by editing pictures prior to posting them, and at the same time, these behaviours may lead individuals to overestimate the importance of their appearance, reinforcing self-objectification processes (Fox & Rooney, 2015; Gioia et al., 2020; McLean et al., 2015). Indeed, experimental designs have shown that exposure to appearance-focused Instagram profiles leads to greater body dissatisfaction among women and also increases the extent to which they define themselves and their self-worth by their physical appearance (Casale et al., 2019). The issue of reverse causation concerning the association between social networking sites use and negative consequences on psycho logical well-being was already pointed out (e.g., Hartanto et al., 2021). Future longitudinal and experimental studies are needed to ascertain directionality. However, the current study contributes to the existing debate about the psycho logical correlates of Problematic Social Networking Sites Use, by (i) confirming the association between body shame, body image control in photos and problematic social net working and (ii) evidencing for the first time the role of per fectionistic self-presentation style as a motivational factor The model tested showed some gender-related differ ences. While the link between body shame and Problematic Social Networking Sites Use was totally mediated among women, this study also highlights the direct effect of the for mer on the latter among men. Discussion In line with the Uses and Gratification Theory (Katz et al., 1973), adolescents and young adults may choose to use Social Networks to decrease their body shame and to satisfy their needs to self-present in a perfectionistic way to reach—at least online— gratification by displaying an ideal body by posting edited photos of themselves. This gratification may allow them to experience a decrease in body shame and may act as a rewarding experience in the increase of Problematic Social Networking Sites Use. norms (Hewitt et al., 2011). Finally, age did not predict Problematic Social Networking Sites Use among either male or female participants. This result was not surprising given that the sample was entirely composed of young peo ple and younger generations are at a greater risk of develop ing Problematic Social Networking Sites Use (e.g., Kuss & Griffiths, 2017). Discussion The current study explores for the first time the role of per fectionistic self-presentation in the relationship between body shame and Problematic Social Networking Sites Use in adolescents and young adults, contributing to the existing debate about predictors of Problematic Social Networking Sites Use. Specifically, the study tested a serial mediation model to explore the role of perfectionistic self-presenta tion style and body image control in photos as mediators in the relationship between body shame and Problematic Social Networking Sites Use. Since previous studies have indicated that women report higher levels of body shame, appearance control beliefs and body image control in pho tos than men (e.g., Dakanalis et al., 2015, 2017; Gioia et al., 2020; Boursier et al., 2020), we first explored gender differences among the study variables. The results were in line with previous research (Dakanalis et al., 2015, 2017; Gioia et al., 2020; Manago et al., 2015), with women report ing higher levels of body shame and body image control in photos than men. This is consistent with self-objectification theory (Fredrickson & Roberts, 1997), which postulates that women are at higher risk of internalizing a third-person per spective and experiencing their bodies as objects that lead them to feel ashamed when they do not meet (unrealistic) cultural beauty standards (McKinley, 1998). Women’s high awareness of cultural beauty standards may cause them to 1 3 Current Psychology (2024) 43:4073–4084 4080 showing imperfections and flaws (Hewitt et al., 2003), and Social Networks represent an ideal environment in which individuals can hide their true self (e.g., Michikyan et al., 2015). Most importantly, the serial mediating role of per fectionistic self-presentation style and body image con trol in photos in the association between body shame and Problematic Social Networking Sites Use emerged, and this path was confirmed among both young men and women. As expected, individuals who experience feelings of body shame could develop Problematic Social Networking Sites Use since Social Networks may represent an ideal environ ment to control one’s own self-presentation by promoting a perfect image and hiding imperfections through self-presen tation tactics, such as body image control in photos. 1 3 Conclusion The present study contributes to the understanding of the possible predictors of Problematic Social Networking Sites Use and demonstrates novel findings in the field of self-objectification research. Specifically, the previously unexplored role of perfectionistic self-presentation style in the association between self-objectification dimensions (i.e., body shame and body monitoring) and Problematic Social Networking Sites Use was confirmed in the current study. Young people who feel ashamed of their bodies may develop perfectionistic self-presentation as a compensatory strategy (by promoting a perfect image and hiding imper fections). The gratification of the need for self-presentation through body image control in social networking sites photo activities could in turn lead to Problematic Social Network ing Sites Use. This pathway was found to be relevant to both young women and young men, suggesting that self-objec tification experiences are not limited to young women, at least in the context of Social Networks. Second, this study used self-report measures that do not give us the possibility to exclude some response bias (e.g., social desirability) or misunderstanding of measures’ pur poses (Rosenman et al., 2011). Specifically, it is not possible to rule out that those participants with high perfectionistic self-presentation might have underestimated their levels of body shame or Problematic Social Networking Sites Use since previous research have discussed that perfectionis tic self-presentation style might lead individuals which are experiencing psychologically related issues to keep their distress hidden due to their tendency to present themselves perfectly (Flett & Hewitt, 2013). Third, the opportunistic sample of adolescents and young adults restricts the generalizability of the results. Our sam ple was primarily composed of women and individuals with a mean body mass index of 21.72 (which is classified as nor mal weight). Future research may include people across the whole spectrum of BMI to better understand whether these results are also generalized to underweight and overweight individuals. For example individuals with higher BMI could report higher scores in body shame as well as in Problematic Social Networking Sites Use, since previous studies have showed that these individuals are more dissatisfied with their body image and feel more avoidant in social situations (e.g., Annis et al., 2004). Therefore they could tend to com pensate these negative feelings with heavy and problematic use of Social Networks. Funding Open access funding provided by Università degli Studi di Firenze within the CRUI-CARE Agreement. Conclusion Data Availability The data that support the findings of this study are available from the corresponding author upon request. Limitations and future directions Men are usually less exposed than women to body-ideal images, and they are less cul turally socialized to alter their appearances to meet cultural beauty standards (Fox & Vandemia, 2016; McKinley, 2011). Consequently, it is possible that for men with high levels of body shame, social networking sites are used for reasons that are not necessarily related to self-presentation. For instance, Social Networks might be used an attempt to escape from or minimize negative moods and/or try to alleviate distressing feelings arising from body shame. This might be in line with the abundance of research showing a positive link between emotion dysregulation and Problematic Social Networking Sites Use (e.g., Hussain et al., 2021).l Regarding the influence of age in the tested models, age was negatively associated with body image control in pho tos among both men and women, suggesting that younger people are more frequently engaged in photo-editing activi ties on Social Networks, as previously reported (Boursier & Manna, 2019; Gioia et al., 2020). The effect of age on perfectionistic self-presentation was significant only among women. 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Cana dian Journal of School Psychology, 28(1), 12–27. https://doi. org/10.1177/0829573512468845 p g p Casale, S., & Fioravanti, G. (2017). Declarations Competing interests The authors have no competing interests to de clare that are relevant to the content of this article. In addition, we did not investigate the participants’ eth nicity or race, although researchers (e.g., Opara & Santos, 2019; Watson et al., 2019) suggested the importance to con sider the issue of intersectionality (i.e., how an individual’s multiple identities interact and intersect to shape personal experiences). Relevant to the current study, it is plausible to assume that individuals who live in one country but come from a different culture (for example African americans individuals) might face with a double internalization of cultural beauty standards, those from their original culture, and those of the dominant culture in which they live. There fore, the sociocultural pressures associated with cultural beauty standards could be more stronger, leading to more severe body image-related concerns. In the future, it will be important to collect data on a wider range of sociocultural demographic variables (such as race/ethnicity, gender, sex ual orientation, socioeconomic status/class, and education level) in order to examine how they influence each other in social media and body image research. Ethics approval The study procedures were carried out in accordance with the Declaration of Helsinki. The Institutional Review Board of the University of the authors approved the study. Informed consent Informed consent was obtained from all individual participants included in the study. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons. org/licenses/by/4.0/. 1 3 Current Psychology (2024) 43:4073–4084 4082 strategies Inventory. 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https://openalex.org/W2257823342	https://hal.univ-lorraine.fr/hal-02086232/file/Stefan_NatureComm_2015.pdf	English	null	Supramolecular amplification of amyloid self-assembly by iodination	Nature communications	2,015	cc-by	9,540	To cite this version: Arianna Bertolani, Lisa Pirrie, Loïc Stefan, Nikolay Houbenov, Johannes S Haataja, et al.. Supramolecular amplification of amyloid self-assembly by iodination. Nature Communications, 2015, 6 (1), 10.1038/ncomms8574. hal-02086232 Supramolecular amplification of amyloid self-assembly by iodination Arianna Bertolani, Lisa Pirrie, Loïc Stefan, Nikolay Houbenov, Johannes S Haataja, Luca Catalano, Giancarlo Terraneo, Gabriele Giancane, Ludovico Valli, Roberto Milani, et al. ARTICLE Received 5 Mar 2015 \| Accepted 19 May 2015 \| Published 30 Jun 2015 HAL Id: hal-02086232 https://hal.univ-lorraine.fr/hal-02086232v1 Submitted on 1 Apr 2019 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Results Si l Single crystal X-ray structure of p-iodo-phenylalanine. The halogen bond21 is the strong, speciﬁc and directional interaction involving halogen atoms as electrophilic species22. Increasing in strength with halogen atom polarizability, halogen bonds are most efﬁcient with iodine substituents. This rather new interaction is virtually unexplored in the ﬁeld of amino acid and peptide self-assembly. For this reason, to evaluate the possibility of exploiting the halogen bond in the self-assembly of amyloid peptides/proteins, we have ﬁrst studied the single crystal X-ray structure of the amino acid p-I-Phe. Amyloid ﬁbrils are characterized by their cross-b-sheet structures whereby bundles of b-strands form highly ordered ﬁlaments that run perpendicular to the ﬁbre axis11,13. The stability of amyloid structures can be attributed to noncovalent interactions, namely hydrogen bonds, p–p stacking and hydrophobic interactions occurring between both side chain and backbone atoms, which hold the b-sheets and strands together11. On the basis of the assumption that noncovalent interactions are key to the formation of the amyloid structure, and since halogen bonds have been recently appreciated as new tools for supramolecular engineering14, we decided to study the impact of introducing halogen-bond donor groups, for example, iodine atoms, into an already known amyloidogenic motif to study the potential of halogen bonding in controlling and promoting amyloid self-assembly. The crystal structure of p-I-Phe (Supplementary Fig. 1 and Supplementary Table 1) highlights the amphoteric behaviour of the iodine atom, which functions as Lewis acid along the extension of the C–I bond and as Lewis base at the belt perpendicular to it (Supplementary Fig. 2a). In fact, the crystal packing of this iodinated amino acid is characterized by two strands of amino acids running along the b crystallographic axis and connected through hydrogen bonds (Supplementary Fig. 2b). These strands are then laterally assembled into two-dimensional supramolecular sheets thanks to type II iodine iodine contacts23, that is, halogen bonds (I I distance 3.7517(4) Å, I I I angle 89.66), occurring at the strand surface (Supplementary Fig. 2c). Importantly, no sign of p–p stacking is observed, while C–H p interactions involving the benzyl hydrogens contribute to aromatic side-chain ordering. Despite the fact that inferring structural information on the amyloid state from high-resolution X-ray diffraction analysis of small-molecule models is difﬁcult, we introduced the p-I-Phe residue into the amyloidogenic core sequence DFNKF24 (NH2-Asp-Phe-Asn-Lys- Phe-COOH, Fig. ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms8574 A 1 A myloid ﬁbrils are a class of nanomaterials that can be assembled from a wide variety of peptides and proteins and have an array of functional and pathological roles in nature1. In the latter, the self-assembly process can be triggered by peptide mutations and leads to the formation of insoluble ﬁbrillar aggregates that are associated with Alzheimer’s, Parkinson’s, Creutzfeldt–Jakob and Huntington’s diseases2–6. Amyloid ﬁbrils also perform a number of physiological roles including being constituents of protective envelopes of ﬁsh and insect eggs, essential amphipathic materials of fungi and bacteria and a vital constituent of spider silk7. This wide variety of physiological functions of amyloids has inspired many nanocomposites thanks to the robustness of these ﬁbrillar materials, which are comparable to dragline silk and collagen, as well as biomaterial applications like tissue engineering8–11. In this context, it is important that the amyloidogenic peptide/protein self-assembles in a tunable and controlled manner to provide ﬁbrils of well-deﬁned architecture12. of the obtained hydrogels. This has been explained by the signiﬁcant electronic effects of ﬂuorine and the subsequent perturbation of the energetics of the p–p stacking interactions, although hydrophobic and steric effects could not be discounted20. Conversely, there is only one report on the ﬁbrillar nanostructures given by a diiodinated dipeptide17 but, to the best of our knowledge, no detailed study on the effect of iodination on promoting/inhibiting the self-assembly of amyloidogenic peptides. Under the hypothesis that introducing an iodine atom on the p-position of the benzyl side chain of an aromatic amino acid may result in the formation of halogen bonding thus promoting ﬁbrils’ formation, we studied the structure of p-iodo-phenylalanine (p-I-Phe) and the self- assembly behaviour of a series of iodinated derivatives of a known amyloidogenic core sequence. Results Si l 1), with the purpose of exploiting similar Although many modiﬁcations of amyloidogenic sequences have been utilized to tune their self-assembly behaviour12, halogenation has rarely been pursued and only on single amino acids or on a dipeptide15–17. The advantage of a strategy based on the introduction of halogen atoms on amyloidogenic motifs lies in the fact that halogenation is a minimal structural modiﬁcation, which, on the other hand, may induce a large difference in the peptide supramolecular behaviour as a consequence of the rich variety of noncovalent interactions given by halogen atoms18. It has been shown previously that incorporation of single halogen substituents on the aromatic side chain of amino acids enhances their self-assembly into amyloid-like ﬁbrils that promote hydrogelation in aqueous solvents19. In particular, ﬂuorinated amino acids were found to function better than chlorinated and brominated analogues in terms of assembly kinetics and rigidity O O O O O OH O OH H H2N NH2 NH2 R2 R2 R1 R1 DFNKF H H H H I Br Br CI CI I I I DFNKF(I) DF(I)NKF DF(I)NKF(I) DF(Br)NKF(Br) DF(CI)NKF(CI) hCT15–19 NH2 – Asp – Phe – Asn – Lys – Phe – COOH 1 2 3 4 5 O N H H N N H N Figure 1 \| Molecular formulae of the peptides used in this study. The natural peptide DFNKF was modiﬁed by halogenation on the phenylalanine residues in the para position. Modiﬁcations are shown in the peptide sequence in brackets where F(I) denotes 4-iodophenylalanine, F(Br) denotes 4-bromophenylalanine and F(Cl) denotes 4-chlorophenylalanine. O O O O O OH O OH H H2N NH2 NH2 R2 R1 hCT15–19 NH2 – Asp – Phe – Asn – Lys – Phe – COOH 1 2 3 4 5 O N H H N N H N hCT15–19 Figure 1 \| Molecular formulae of the peptides used in this study. The natural peptide DFNKF was modiﬁed by halogenation on the phenylalanine residues in the para position. Modiﬁcations are shown in the peptide sequence in brackets where F(I) denotes 4-iodophenylalanine, F(Br) denotes 4-bromophenylalanine and F(Cl) denotes 4-chlorophenylalanine. NATURE COMMUNICATIONS \| 6:7574 \| DOI: 10.1038/ncomms8574 \| www.nature.com/naturecommunications Macmillan Publishers Limited All rights reserved NATURE COMMUNICATIONS \| 6:7574 \| DOI: 10.1038/ncomms8574 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| 6:7574 \| DOI: 10.1038/ncomms8574 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. 2 2 \| \| / \| / & 2015 Macmillan Publishers Limited. All rights reserved. Supramolecular ampliﬁcation of amyloid self-assembly by iodination Arianna Bertolani1, Lisa Pirrie1,2, Loic Stefan1, Nikolay Houbenov3, Johannes S. Haataja3, Luca Catalano1, Giancarlo Terraneo1, Gabriele Giancane4, Ludovico Valli4, Roberto Milani2, Olli Ikkala3, Giuseppe Resnati1 & Pierangelo Metrangolo1,2 Amyloid supramolecular assemblies have found widespread exploitation as ordered nanomaterials in a range of applications from materials science to biotechnology. New strategies are, however, required for understanding and promoting mature ﬁbril formation from simple monomer motifs through easy and scalable processes. Noncovalent interactions are key to forming and holding the amyloid structure together. On the other hand, the halogen bond has never been used purposefully to achieve control over amyloid self-assembly. Here we show that single atom replacement of hydrogen with iodine, a halogen-bond donor, in the human calcitonin-derived amyloidogenic fragment DFNKF results in a super-gelator peptide, which forms a strong and shape-persistent hydrogel at 30-fold lower concentration than the wild-type pentapeptide. This is remarkable for such a modest perturbation in structure. Iodination of aromatic amino acids may thus develop as a general strategy for the design of new hydrogels from unprotected peptides and without using organic solvents. 1 Laboratory of Nanostructured Fluorinated Materials (NFMLab), Department of Chemistry, Materials, and Chemical Engineering ‘Giulio Natta’, Politecnico di Milano, Via Mancinelli 7, Milano I-20131, Italy. 2 VTT-Technical Research Centre of Finland, PO Box 1000, Espoo FI-02044, Finland. 3 Department of Applied Physics, Aalto University, PO Box 15100, Espoo FI-02150, Finland. 4 Dipartimento Beni Culturali, Universita` del Salento, Lecce I-73100, Italy. Correspondence and requests for materials should be addressed to P.M. (email: pierangelo.metrangolo@polimi.it). NATURE COMMUNICATIONS \| 6:7574 \| DOI: 10.1038/ncomms8574 \| www.nature.com/naturecommunications 1 1 & 2015 Macmillan Publishers Limited. All rights reserved. & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms8574 halogen bonds at the ﬁbril strand surface to promote ﬁbril–ﬁbril lamination effects. ﬂuorescence was observed upon mixing ThT with peptide solutions. This is well in accordance with the literature since previous studies have demonstrated that DFNKF does not bind this ﬁbril marker dye29. DFNKF is a short segment (residues 15–19) of the human calcitonin (hCT) hormone25, an amyloidogenic protein with 32 amino acids, whose amyloid formation is related to medullary carcinoma of the thyroid24. This particular pentapeptide is widely used for investigating the nanoscopic arrangement of a ﬁbril complex since DFNKF forms well-ordered ﬁbrils similar to the aggregates of hCT24,25. For this reason, we selected DFNKF as a simple model compound for ﬁbril formation and obtained its halogenated derivatives by introducing I, Br and Cl atoms at the p-position of the phenylalanine (Phe) benzene ring. To study the effect on ﬁbrillation of the introduction of halogen atoms on speciﬁc positions of the pentapeptide, we studied the 5-p-iodo- Phe derivative DFNKF(I), the 2-p-iodo-Phe derivative DF(I)NKF, the 2,5-bis-p-iodo-Phe derivative DF(I)NKF(I), as well as its bis- brominated and bis-chlorinated analogues DF(Br)NKF(Br) and DF(Cl)NKF(Cl), respectively (Fig. 1). Importantly, all of the studied peptides carry free amino (N) and carboxyl (C) termini. y Subsequently, a working concentration of 15 mM was chosen where all halogenated peptides formed clear and elastic gels within a reasonable time (r12 h). At this concentration, the speed of hydrogel formation followed the order DF(I)NK- F(I)4DF(I)NKFZDF(Br)NKF(Br)4DF(Cl)NKF(Cl)ZDFNKF(I), whereas DFNKF did not form a gel even after 30 days (Supplementary Table 3). The diiodinated peptide formed the most homogeneous and shape-persistent gel (Fig. 2b), whereas peptides DF(Cl)NKF(Cl) and DFNKF(I) showed phase separation of the ﬁbrous aggregates from the aqueous phase. The obtained hydrogels were all thermoreversible with gel–sol transition temperatures ranging from 65 C (DFNKF(I) to 119 C (DF(l)NKF(l); Fig. 2c). In particular, the observed transition temperatures followed the same order of the gel forming kinetics reported above. These results clearly show that, in general, halogenation of the DFNKF model peptide greatly promotes hydrogel formation, with the diiodinated peptide being the most efﬁcient. This efﬁciency decreases with the polarizability of the halogen atom, that is, DF(l)NKF(l)4DF(Br)NKF(Br)4 DF(CI)NKF(CI), and also depends on the speciﬁc position of the halogen atom in the peptide scaffold, that is, the peptide iodinated at the Phe in the second position is more efﬁcient than the one iodinated in the ﬁfth. & 2015 Macmillan Publishers Limited. All rights reserved. Hydrogelation properties of halogenated DFNKF derivatives. Hydrogelation is highly indicative of ﬁbrillation, therefore all peptides were in the ﬁrst instance assessed for their ability to form hydrogels26,27. All of the studied peptides were found to self- assemble into hydrogels over a minimum gelation concentration. Only the hydrogel of the wild-type peptide DFNKF has been previously reported, though of its N-acetylated form at the N terminus28. All the reported halogenated peptides are novel types of gelators and, importantly, formed gels (Fig. 2a and Supplementary Table 2) at concentrations much lower than that of their wild-type parent DFNKF, that is, 75 mM, with the super- gelator peptide DF(I)NKF that showed a minimum gelation concentration of o0.2% (2.5 mM; 30-fold lower than DFNKF). Characterization of the different halogenated peptide hydrogels was done by oscillatory rheology (ring-cast method) using the 15 mM concentration. At this low concentration, the wild-type peptide DFNKF only formed a viscous ﬂuid and was not studied further. The diiodinated peptide DF(I)NKF(I) was conﬁrmed to form the stiffest gel, which is reﬂected in its high elastic modulus (G04104 Pa and G044G00; Fig. 2d and Supplementary Fig. 3) and by the fact that the gel assumes the well-deﬁned shape of the mould (Fig. 2b). The G0 value for DF(I)NKF(I) is comparable to Attempts to study the kinetics of ﬁbril formation using Thioﬂavin T (ThT) were unsuccessful since no increase in 1 10 100 1,000 10,000 100,000 Storage modulus, G′ (Pa) Oscillation strain (%) Amplitude sweep DF(I)NKF(I) DF(I)NKF DF(Br)NKF(Br) DFNKF(I) DF(Cl)NKF(Cl) 1 10 100 1,000 10,000 100,000 0.1 Storage modulus, G′ (Pa) Angular frequency (rad s–1) Frequency sweep DF(I)NKF(I) DF(I)NKF DF(Br)NKF(Br) DFNKF(I) DF(Cl)NKF(Cl) Peptide DFNKF(I) DF(I)NKF DF(I)NKF(I) DF(Br)NKF(Br) DF(Cl)NKF(Cl) WT Halogenated Transition T (°C) 64.7–69.0 88.2–92.7 114.0–119.0 92.1–96.4 78.1–82.4 100 10 1 100 10 1 0.1 0.01 Figure 2 \| Characterization of hydrogels formed by halogenated DFNKF derivatives (a) Photograph of vials containing 15 mM peptide solutions upon aging 12 h at RT After this period, all halogenated peptides formed clear hydrogels whereas the wild-type peptide DFNKF (WT) remained as a viscous ﬂuid. (b) The hydrogel formed by DF(I)NKF(I) was the most shape-persistent of all gels. The gel was prepared using the ring-cast method at 15 mM concentration and imaged after 48 h. (c) Gel–sol transition temperatures of 15 mM halogenated peptide hydrogels formed in 48 h. & 2015 Macmillan Publishers Limited. All rights reserved. (d) Rheological characterization of the gels by frequency sweep studies whereby the storage modulus (G0) was recorded as a function of angular frequency (o). The peptide DF(I)NKF(I) was determined to be the strongest gel with G04104 Pa. (e) Amplitude sweep studies of peptide hydrogels showing G0 as a function of oscillation strain (g). All halogenated hydrogels show similar linear viscoelastic region (LVR) proﬁles. Peptide hydrogels at a concentration of 15 mM were utilized for the rheological measurements after 48 h from preparation. For the loss moduli, see the Supplementary Fig. 3. WT Halogenated 1 10 100 1,000 10,000 100,000 Storage modulus, G′ (Pa) Oscillation strain (%) Amplitude sweep DF(I)NKF(I) DF(I)NKF DF(Br)NKF(Br) DFNKF(I) DF(Cl)NKF(Cl) 100 10 1 0.1 0.01 1 10 100 1,000 10,000 100,000 0.1 Storage modulus, G′ (Pa) Angular frequency (rad s–1) Frequency sweep DF(I)NKF(I) DF(I)NKF DF(Br)NKF(Br) DFNKF(I) DF(Cl)NKF(Cl) 100 10 1 Storage modulus, G′ (Pa) ) a) Figure 2 \| Characterization of hydrogels formed by halogenated DFNKF derivatives (a) Photograph of vials containing 15 mM peptide solutions upon aging 12 h at RT After this period, all halogenated peptides formed clear hydrogels whereas the wild-type peptide DFNKF (WT) remained as a viscous ﬂuid. (b) The hydrogel formed by DF(I)NKF(I) was the most shape-persistent of all gels. The gel was prepared using the ring-cast method at 15 mM concentration and imaged after 48 h. (c) Gel–sol transition temperatures of 15 mM halogenated peptide hydrogels formed in 48 h. (d) Rheological characterization of the gels by frequency sweep studies whereby the storage modulus (G0) was recorded as a function of angular frequency (o). The peptide DF(I)NKF(I) was determined to be the strongest gel with G04104 Pa. (e) Amplitude sweep studies of peptide hydrogels showing G0 as a function of oscillation strain (g). All halogenated hydrogels show similar linear viscoelastic region (LVR) proﬁles. Peptide hydrogels at a concentration of 15 mM were utilized for the rheological measurements after 48 h from preparation. For the loss moduli, see the Supplementary Fig. 3. Figure 2 \| Characterization of hydrogels formed by halogenated DFNKF derivatives (a) Photograph of vials containing 15 mM peptide solutions upon aging 12 h at RT After this period, all halogenated peptides formed clear hydrogels whereas the wild-type peptide DFNKF (WT) remained as a viscous ﬂuid. & 2015 Macmillan Publishers Limited. All rights reserved. Figure 2 \| Characterization of hydrogels formed by halogenated DFNKF derivatives (a) Photograph of vials containing 15 mM peptide solutions upon aging 12 h at RT After this period, all halogenated peptides formed clear hydrogels whereas the wild-type peptide DFNKF (WT) remained as a viscous ﬂuid. (b) The hydrogel formed by DF(I)NKF(I) was the most shape-persistent of all gels. The gel was prepared using the ring-cast method at 15 mM concentration and imaged after 48 h. (c) Gel–sol transition temperatures of 15 mM halogenated peptide hydrogels formed in 48 h. (d) Rheological Figure 2 \| Characterization of hydrogels formed by halogenated DFNKF derivatives (a) Photograph of vials contain rogels formed by halogenated DFNKF derivatives (a) Photograph of vials containing 15 mM peptide solutions upon hydrogels formed by halogenated DFNKF derivatives (a) Photograph of vials containing 15 mM peptide solutions up d, all halogenated peptides formed clear hydrogels whereas the wild-type peptide DFNKF (WT) remained as a viscous ﬂ concentration and imaged after 48 h. (c) Gel sol transition temperatures of 15 mM halogenated peptide hydrogels formed in 48 h. (d) Rheological characterization of the gels by frequency sweep studies whereby the storage modulus (G0) was recorded as a function of angular frequency (o). The peptide DF(I)NKF(I) was determined to be the strongest gel with G04104 Pa. (e) Amplitude sweep studies of peptide hydrogels showing G0 as a function of oscillation strain (g). All halogenated hydrogels show similar linear viscoelastic region (LVR) proﬁles. Peptide hydrogels at a concentration of 15 mM were utilized for the rheological measurements after 48 h from preparation. For the loss moduli, see the Supplementary Fig. 3. 3 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms8574 that of Ac-LIVAGD, a hexamer that forms one of the best performing hydrogels in terms of ease of hydrogel formation and strength30,31. Interestingly, the trend of G0 values parallels those of gel formation efﬁciency and thermal stability whereby gels of peptides DF(I)NKF(I), DF(I)NKF and DF(Br)NKF(Br) are the stiffest and DFNKF(I) and DF(Cl)NKF(Cl) formed the weakest ones. similar to those of the diiodinated peptide DF(I)NKF(I) (Fig. 4a and Supplementary Fig. 4a), which, in turn, had the most complex structures with twisted ribbon-like ﬁbrils tens of nanometre wide and micrometre long. & 2015 Macmillan Publishers Limited. All rights reserved. These results highlight the importance of the speciﬁc position of the iodine atom in the peptide structure and therefore the subsequent speciﬁc inter- actions, which result in different amyloid ﬁbril topographies. That the ﬁbrillation and twisting is not due to drying is excluded by high-resolution cryo-TEM and electron tomography (ET) studies. This is exempliﬁed for the peptide DF(I)NKF(I) corresponding to the strongest hydrogel. It shows ﬁbrils with lateral sizes of 20–25 nm and helical twisting with periodicities between 250 and 140 nm (Fig. 4h and Supplementary Fig. 4b). Such periodicities correspond to the different maturation steps of amyloid ﬁbrils as previously reported for b-lactoglobulin ﬁbrils33. ET allowed three-dimensional visualization of the twisting morphology of a DF(I)NKF(I) ﬁbril imaged in solution (Fig. 4i). Morphology and microarchitecture of the self-assembled ﬁbrillar networks constituting the halogenated hydrogels were evaluated by confocal microscopy (Fig. 3)32. The network of the hydrogel of DF(I)NKF(I) appeared to be very ﬁlamentous with micrometre-long ﬁbrils assembled in bundles with a helical sense, which propagate along the same direction. These micrometre- long ﬁbrils become fewer and thinner on going from DF(I)NKF(I) to DF(I)NKF and DF(Br)NKF(Br), and disappear in the hydrogels of DF(CI)NKF(CI) and DFNKF(I) where only entangled matrices of small ﬁbrils and aggregates were observed. These observations ﬁt well with the rheology data where the strongest gels result from the entanglement of the longest ﬁbrils. In agreement with the confocal microscopy study, the hydrogel formed by the peptide DF(I)NKF(I) showed an intertwined network of long ﬁbrils in its transmission electron microscopy (TEM) image (Fig. 3f). Amyloid structure of ﬁbrils of halogenated peptides. There are three criteria that deﬁne a protein aggregate as an amyloid ﬁbril: green birefringence upon staining with Congo Red, ﬁbrillar morphology and b-sheet secondary structure34. Therefore, Congo red staining was carried out for all the peptides (at the same 15 mM concentration) and strong green-gold birefringence was observed under polarized light, which is a reporter for long-range cross-b structure (Supplementary Fig. 5). The nanostructure of the halogenated ﬁbrils was revealed by atomic force microscopy (AFM) (Fig. 4). The peptides were ﬁrst imaged after incubation in dilute aqueous conditions for 9 days (see also Supplementary Fig. 4) and Fig. 4a–c highlight the most strongly ﬁbrillating peptides DF(I)NKF(I), DF(I)NKF and DF(Br)NKF(Br). While only small spherical aggregates were observed for the wild-type peptide DFNKF (Supplementary Fig. 4f), all of the halogenated peptides showed pronounced ﬁbrillar structures. & 2015 Macmillan Publishers Limited. All rights reserved. Large differences were, however, observed between the monoiodinated peptides DFNKF(I) and DF(I)NKF: Peptide DFNKF(I) (Supplementary Fig. 4e) showed only small protoﬁbrils and aggregates, whereas the peptide DF(I)NKF (Fig. 4b and Supplementary Fig. 4c) showed large twisted ﬁbrils Further evidence of amyloid nature was obtained by Fourier transform infrared (FT-IR) spectroscopy. The FT-IR spectra of both the solution and the gel state in D2O were recorded (Supplementary Fig. 6). A solution of the wild-type peptide DFNKF showed no signiﬁcant amide I’ band, however, using a concentration at which it forms a gel (75 mM), this band became visible. This peak at around 1,630 cm 1 can be attributed to the peptide bond carbonyl upon formation of the amyloid b-sheet structure and subsequent gelation35. The spectra of the gel state DF(Br)NKF(Br) DF(I)NKF(I) DF(I)NKF DF(Br)NKF(Br) DF(Cl)NKF(Cl) DFNKF(I) DF(I)NKF(I) Figure 3 \| Fibrillar morphologies of halogenated hydrogels. (a–e) Confocal microscopy images of peptide hydrogels at a concentration of 15 mM upon aging 48 h at RTand after staining with Rhodamine B (scale bar, 100 mm). Panels a, b and c show bundles of long twisted ﬁbrils belonging to hydrogels of peptides DF(I)NKF(I), DF(I)NKF and DF(Br)NKF(Br), respectively. Panels d and e show much smaller ﬁbrils of hydrogels of peptides DF(Cl)NKF(Cl) and DFNKF(I), which intertwine and form a matrix-type structure. (f) TEM image of the dried hydrogel of peptide DF(I)NKF(I) showing an intertwined network of long ﬁbrils (scale bars, 0.5 mm and 5 nm). DF(I)NKF(I) DF(I)NKF DF(I)NKF(I) DF(Cl)NKF(Cl) DFNKF(I) DFNKF(I) Figure 3 \| Fibrillar morphologies of halogenated hydrogels. (a–e) Confocal microscopy images of peptide hydrogels at a concentration of 15 mM upon aging 48 h at RTand after staining with Rhodamine B (scale bar, 100 mm). Panels a, b and c show bundles of long twisted ﬁbrils belonging to hydrogels of peptides DF(I)NKF(I), DF(I)NKF and DF(Br)NKF(Br), respectively. Panels d and e show much smaller ﬁbrils of hydrogels of peptides DF(Cl)NKF(Cl) and DFNKF(I), which intertwine and form a matrix-type structure. (f) TEM image of the dried hydrogel of peptide DF(I)NKF(I) showing an intertwined network of long ﬁbrils (scale bars, 0.5 mm and 5 nm). NATURE COMMUNICATIONS \| 6:7574 \| DOI: 10.1038/ncomms8574 \| www.nature.com/naturecommunications Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS \| 6:7574 \| DOI: 10.1038/ncomms8574 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS \| 6:7574 \| DOI: 10.1038/ncomms8574 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. 4 & 2015 Macmillan Publishers Limited. All rights reserved. Electron tomography reconstructions were collected upon tilting of samples up to ±69 (scale bar, 25 nm). of the halogenated peptides showed this same amide I’ band consistent with the formation of amyloid ﬁbrillar species comprising b-sheet elements. These data conﬁrm that all the studied halogenated derivatives of DFNKF form ﬁbrils of the same amyloid nature to their wild-type parent. Interestingly, this 1,630 cm 1 peak is red-shifted in the spectra of DF(I)NKF(I) and DF(I)NKF, while it is not in the one of DFNKF(I) (Supplementary Fig. 6). This red-shift indicates a reduced electron density on the peptide bond and is consistent with the involvement of the carbonyl oxygen in halogen bonding36. others41. A direct correlation exists between the role of oxidative stress and protein ﬁbrillation42. However, how halogenation affects protein structure, folding and functioning is not yet known. To study to what extent a partial degree of halogenation may promote the ampliﬁed formation of amyloid ﬁbrils, we studied the co-assembly43 of the strongly self-assembling diiodinated peptide DF(I)NKF(I) with the wild-type peptide DFNKF. Mixing DFNKF and DF(I)NKF(I) at concentrations at which normally neither form gels alone (Fig. 5a), 15 and 0.75 mM (5%), respectively, resulted in a mixed hydrogel within 18 h. Characterization of the physical properties of the mixed hydrogel was performed by oscillatory rheology (ring-cast method) where the mixed hydrogel was found to be weaker than the hydrogel containing only DF(I)NKF(I) (Fig. 5b versus Fig. 2d). It was also observed that increasing the amount of the strong gelator DF(I)NKF(I) led to an increase in the elastic modulus value, whereas the gel containing 10% was stronger than the one containing 5% of DF(I)NKF(I). Interestingly, circular dichroism (CD) analysis of the mixed hydrogels showed the same structural features observed in the CD spectra of the pure iodinated peptides (see Supplementary Figs 8 and 9). Raman spectroscopy is a powerful tool to investigate the occurrence of the halogen bond37. For this reason, we followed the changes experienced by the C–I stretching band at around 167 cm 1 upon formation of the gel. Interestingly, compared with the bulk powders of the starting materials, in the dried gels of the iodinated peptides DF(I)NKF(I) and DF(I)NKF, this band shifted to lower frequency (see Supplementary Fig. 7). This shift is perfectly consistent with the weakening of the C–I bond as a consequence of its involvement in halogen bonding upon formation of the hydrogels37. & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms8574 0 0 4 0.4 0.8 1.2 μm 8 nm 0 0 0 0.1 0.2 0.3 0.4 0.5 0.6 4 0.4 0.8 1.2 μm μm Helical turn Half-pitch helical cut 8 nm 0 1 3 2 nm 0 0 4 0.4 0.8 25 nm 25 nm 250 nm 20 nm 20 nm 255 nm 150 nm 150 nm 1.2 μm 8 nm 0 0 4 0.4 0.8 1.2 μm 8 12 nm Figure 4 \| Nanoﬁbrillation of the halogenated pentapeptides. AFM image close-up insets of (a) peptide DF(I)NKF(I), (b) peptide DF(I)NKF, and (c) peptide DF(Br)NKF(Br) evaporated on mica substrates after 9 days incubation in aqueous solutions (scale bar, 1 mm). (d–f) Height proﬁles of ﬁbrils from a, b and c (cross-section lines highlighted in white). (g) Cross-sectional analysis on the top of a ﬁbril segment in a showing a helicoidal proﬁle along the longitudinal direction of the ﬁbrils (highlighted in black). (h) Cryo-TEM images of the dried hydrogel of DF(I)NKF, showing a network of long ﬁbrils (scale bar, 200 nm). (i) In situ electron tomography of half-pitch helicoidal peptide DF(I)NKF(I) vitriﬁed from aqueous solution. The twisted ﬁbril morphology is highlighted by arrows. Electron tomography reconstructions were collected upon tilting of samples up to ±69 (scale bar, 25 nm). 0 0 4 0.4 0.8 1.2 μm 8 nm 0 0 4 0.4 0.8 1.2 μm 8 12 nm 0 0.1 0.2 0.3 0.4 0.5 0.6 μm μm Helical turn Half-pitch helical cut 0 1 3 2 nm 0 0.1 0.2 0.3 0.4 0.5 0.6 μm 0 1 3 2 nm 25 nm 20 nm 255 nm 150 nm 25 nm 250 nm 20 nm 150 nm Figure 4 \| Nanoﬁbrillation of the halogenated pentapeptides. AFM image close-up insets of (a) peptide DF(I)NKF(I), (b) peptide DF(I)NKF, and (c) peptide DF(Br)NKF(Br) evaporated on mica substrates after 9 days incubation in aqueous solutions (scale bar, 1 mm). (d–f) Height proﬁles of ﬁbrils from a, b and c (cross-section lines highlighted in white). (g) Cross-sectional analysis on the top of a ﬁbril segment in a showing a helicoidal proﬁle along the longitudinal direction of the ﬁbrils (highlighted in black). (h) Cryo-TEM images of the dried hydrogel of DF(I)NKF, showing a network of long ﬁbrils (scale bar, 200 nm). (i) In situ electron tomography of half-pitch helicoidal peptide DF(I)NKF(I) vitriﬁed from aqueous solution. The twisted ﬁbril morphology is highlighted by arrows. & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS \| 6:7574 \| DOI: 10.1038/ncomms8574 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| 6:7574 \| DOI: 10.1038/ncomms8574 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms8574 (d) AFM of a sample of the mixed hydrogel containing 15 mM DFNKF and 1.5 mM DF(I)NKF(I) showed larger and more entangled ﬁbrils (scale bar, 400 nm). Figure 5 \| Mixed hydrogels formed by co-assembly of DFNKF and DF(I)NKF(I). (a) Photograph of vials after 48 h containing (I) 15 mM DFNKF, which did not form hydrogel; (II) 1.5 mM DF(I)NKF(I), which did not form hydrogel and some precipitation was observed; (III) 15 mM DFNKF:0.15 mM DF(I)NKF(I), which did not form hydrogel; (IV) 15 mM DFNKF:0.75 mM DF(I)NKF(I), which formed hydrogel within 48 h; (V) 15 mM DFNKF:1.5 mM DF(I)NKF(I), which formed hydrogel within 48 h. (b) Rheological characterization of mixed hydrogels by frequency sweep studies whereby the storage concentration of 15 mM after 48 h were utilized for the rheological measurements. (c) AFM of a 15 mM solution of wild-type peptide DFNKF showed small ﬁbrils and aggregates unable to network sufﬁciently to form a hydrogel. (d) AFM of a sample of the mixed hydrogel containing 15 mM DFNKF and 1.5 mM DF(I)NKF(I) showed larger and more entangled ﬁbrils (scale bar, 400 nm). Figure 6 \| Potential halogen bonds involving p-iodo-phenylalanine. IR and Raman spectroscopies are more consistent with the occurrence of type a interaction, however, other interactions reported cannot be discounted. (a) Orthogonal hydrogen and halogen bonds involving the carbonyl oxygen atom of the peptide bond36. (b) Type II iodine iodine contacts23. (c) Iodine p interactions49. (d) Triangular iodine synthon50. too small to crosslink and form a network (Fig. 5c). On the addition of 10% DF(I)NKF(I), the resulting ﬁbrils were much longer and intertwined to form a ﬁbrous network resulting in the hydrogelation of the wild-type peptide DFNKF solution (Fig. 5d and Supplementary Fig. 10a). The weakness of the resulting mixed hydrogel compared with that of DF(I)NKF(I) alone can be explained by the relative size and network formation of the ﬁbrils (Supplementary Fig. 10b). This result may have important implications in a biological setting since only small amounts of brominated or chlorinated peptides may be required to amplify the supramolecular self-assembly of an amyloid ﬁbril. Role of halogen bonding in hydrogels’ self-assembly. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms8574 1 100 10,000 0.1 1 10 100 Storage/loss moduli (Pa) Angular frequency (rad s–1) Frequency sweep 10% DF(I)NKF(I) G′ 10% DF(I)NKF(I) G″ 5% DF(I)NKF(I) G′ 5% DF(I)NKF(I) G″ 15 mM DFNKF 1.5 mM DF(I)NKF(I) 15 mM DFNKF 1 100 10,000 0.1 1 10 100 Storage/loss moduli (Pa) Angular frequency (rad s–1) Frequency sweep 10% DF(I)NKF(I) G′ 10% DF(I)NKF(I) G″ 5% DF(I)NKF(I) G′ 5% DF(I)NKF(I) G″ II III IV V I 15 mM DFNKF 1.5 mM DF(I)NKF(I) Figure 5 \| Mixed hydrogels formed by co-assembly of DFNKF and DF(I)NKF(I). (a) Photograph of vials after 48 h containing (I) 15 mM DFNKF, which did not form hydrogel; (II) 1.5 mM DF(I)NKF(I), which did not form hydrogel and some precipitation was observed; (III) 15 mM DFNKF:0.15 mM DF(I)NKF(I), which did not form hydrogel; (IV) 15 mM DFNKF:0.75 mM DF(I)NKF(I), which formed hydrogel within 48 h; (V) 15 mM DFNKF:1.5 mM DF(I)NKF(I), which formed hydrogel within 48 h. (b) Rheological characterization of mixed hydrogels by frequency sweep studies whereby the storage concentration of 15 mM after 48 h were utilized for the rheological measurements. (c) AFM of a 15 mM solution of wild-type peptide DFNKF showed small ﬁbrils and aggregates unable to network sufﬁciently to form a hydrogel. (d) AFM of a sample of the mixed hydrogel containing 15 mM DFNKF and 1.5 mM DF(I)NKF(I) showed larger and more entangled ﬁbrils (scale bar, 400 nm). 1 100 10,000 0.1 1 10 100 Storage/loss moduli (Pa) Angular frequency (rad s–1) Frequency sweep II III IV V I 10% DF(I)NKF(I) G′ 10% DF(I)NKF(I 5% DF(I)NKF(I) G′ 5% DF(I)NKF(I) 15 mM DFNKF 1.5 mM DF(I)NKF(I) 15 mM DFNKF Figure 5 \| Mixed hydrogels formed by co-assembly of DFNKF and DF(I)NKF(I). (a) Photograph of vials after 48 h containing (I) 15 mM DFNKF, which did not form hydrogel; (II) 1.5 mM DF(I)NKF(I), which did not form hydrogel and some precipitation was observed; (III) 15 mM DFNKF:0.15 mM DF(I)NKF(I), which did not form hydrogel; (IV) 15 mM DFNKF:0.75 mM DF(I)NKF(I), which formed hydrogel within 48 h; (V) 15 mM DFNKF:1.5 mM DF(I)NKF(I), which formed hydrogel within 48 h. (b) Rheological characterization of mixed hydrogels by frequency sweep studies whereby the storage concentration of 15 mM after 48 h were utilized for the rheological measurements. (c) AFM of a 15 mM solution of wild-type peptide DFNKF showed small ﬁbrils and aggregates unable to network sufﬁciently to form a hydrogel. & 2015 Macmillan Publishers Limited. All rights reserved. p p pp y g The resulting ﬁbril morphologies in the mixed hydrogels were studied by AFM (Fig. 5c,d). A 15 mM sample of the wild-type peptide DFNKF showed only a mixture of intertwined aggregates and small ﬁbrils, which is consistent with the observation that no gelation is observed at this concentration since the ﬁbrils appear Co-assembly of wild-type peptide with diiodinated analogue. Bromination and chlorination of proteins in vivo has been related to a series of oxidative stress-related diseases such as cystic ﬁbrosis38, atherosclerotic intima39, sepsis40 and asthma, among 5 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms8574 NATURE COMMUNICATIONS \| 6:7574 \| DOI: 10.1038/ncomms8574 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. Methods R t C Congo red staining. All the samples were monitored for green birefringence using an Olympus BX50 polarizing microscope with a SensiCam PCO camera used to display and enhance images. An 80% ethanol solution saturated with NaCl and Congo red was freshly prepared before each measurement. A piece of each peptide hydrogel was placed on a glass microscope, allowed to air dry and then stained with Congo red solution. Subsequently, excess Congo red solution was blotted off the slide and the samples were analysed using both bright and polarized light. Reagents. Congo red, Rhodamine B, NaCl and D2O were purchased from Sigma- Aldrich and used without further puriﬁcation. Peptides with conﬁrmed amino acid analysis (purity Z98%), were purchased from Biopeptek (Malvern, USA). The integrity of all peptides was conﬁrmed by ion spray mass spectrometry and the purity was determined by reverse phase high-pressure liquid chromatography. Hydrogel preparation. Hydrogels were prepared by dissolving each peptide (15 mM for halogenated peptides and 75 mM for the wild-type peptide DFNKF) in deionized water (18.2 Mo cm) or in D2O (Z99.9 atom % deuterium). The glass vials containing the 500 ml solutions were sealed, sonicated for 20 s, heated using a heat gun until complete dissolution of the peptides, before slow cooling to room temperature (RT) All the samples were stored at RT for 48 h before analysis. Infrared spectroscopy (FT-IR). Infrared spectra were recorded at RT using a Nicolet iS50 FT-IR spectrometer equipped with a DTGS detector. Peptides were analysed as solutions (after heating at 100 C to break any pre-formed ﬁbrils) or gels at 15 mM in D2O (for DFNKF 75 mM concentration was used). Spectra represent an average of 64 scans recorded in a single-beam mode with a 4 cm 1 resolution and corrected for the background. The second-derivative analyses of the spectra were performed using the Nicolet FTIR software, Omnic 9.0, with a 13-point and third polynomial order Savitzky and Golay function. Second- derivative spectra generated negative bands as compared with the original spectra, thus for comparison all the second-derivative spectra were multiplied by 1. Preparation of peptide solutions. Peptide solutions (40 mM) were freshly prepared in deionized water (18.2 Mo cm), sonicated for 20 s, and gently warmed to reach 90 C before ﬁltration through a 0.22 mm Millipore ﬁlter. The peptide solutions were stored in sealed vials at RT for varying time points before analysis. X-ray crystallography. Discussion In summary, we have demonstrated that iodination of a short amyloidogenic core sequence strongly promotes its ﬁbril formation ability and affects the structure of formed ﬁbrils. In particular, the 2-p-iodo-Phe derivative DF(I)NKF was demonstrated to be 30-fold more efﬁcient in forming hydrogels than the wild-type pentapeptide DFNKF. This is remarkable for such a modest perturbation in structure. Preliminary results indicate that the strong effect of iodination on peptide self- assembly is general and not limited to the core sequence reported here. In fact, iodination of the amyloidogenic core sequence KLVFF (residues 16–20 of Ab) as well as of the full-length hCT similarly promotes ﬁbril formation efﬁciency and self-assembly (data not shown). Iodination of aromatic amino acids may thus develop as a general strategy for the design of new hydrogels starting from unprotected peptides and without the use of organic solvents44,45. Rheology. Rheology experiments were performed using a TA instrument ARG2 Rheometer. A 20 mm stainless steel, parallel-plate geometry was used with a gap distance of 1,000 mm. Oscillatory frequency sweep studies were performed for a range of 0.1–100 rad s 1, using a 0.5% strain. Oscillatory amplitude sweep studies were conducted from 0.01 to 100% strain with an angular frequency of 1 rad s 1. The ring-cast method was used for hydrogel preparation at a peptide concentration of 15 mM. The peptide solution was sonicated for 20 s in a sealed glass vial before heating to 90 C to afford complete dissolution of the peptide. After cooling, the solutions were transferred into ring casts of 22 mm diameter and placed in tightly sealed tissue-culture dishes for 48 h. All the measurements were repeated a minimum of three times. Circular dichroism spectroscopy. All the CD experiments were carried out in deionized water (18.2 MO cm) in a 0.01 mm detachable quartz cuvette, using a JASCO J-815 CD spectrometer. Acquisitions were performed between 190 and 300 nm with a 0.1 nm data pitch, 1 nm bandwidth, 100 nm min 1 scanning speed and 1 s response time. All the spectra are an average of 10 scans and were corrected from a reference solution, comprising deionized water (18.2 MO.cm) alone. Discussion Raw data (y, in millidegree) were subsequently converted to mean residue ellipticity ([y] in deg cm2 dmol 1) for the sake of comparison, in accordance with the following formulae: The results reported in this paper are relevant in the ﬁeld of the programmed synthesis of amyloid supramolecular assemblies as well as in the context of amyloid-dependent diseases. In the former ﬁeld, we have demonstrated that single atom mutations of amyloidogenic core sequences by replacing hydrogen with iodine atoms greatly promote ﬁbril formation and self-assembly. Methods promoting the formation of mature ﬁbrils are advantageous for use, among others, as scaffolds for tissue engineering, controlled drug release, surgical reconstruction applications, microﬂuidic devices, biosensors and bioswitches7–11. As far as amyloid-dependent diseases are concerned, our results suggest that oxidative stress-induced halogenation of proteins might potentially be the triggering point for the transformation of a natively folded protein into a halogen- bonded and ﬁbrillar malfunctioning form. Research in this direction is currently being carried out in our laboratories and will be reported elsewhere. y½ ¼ y 10lcðn 1Þ ; ð1Þ ð1Þ where y is the observed ellipticity in millidegree, c is the concentration of the sample in mol l 1, (n 1) is the number of peptide bonds and l is the path length of the cuvette in centimetre. Confocal microscopy. Hydrogels were imaged using a Zeiss LSM 710 microscope with a He/Ne laser (lex ¼ 543 nm). The ﬂuorescent dye, Rhodamine B, was incorporated into an aged hydrogel (48 h) scaffold by the addition of 10 ml of the dye solution (0.1% w/v). Following complete absorption of the dye, the sample was excited at 543 nm and emitted light recorded using the E570LP emission ﬁlter. ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms8574 DF(Br)NKF(Br) and DF(Cl)NKF(Cl) are both more hydrophobic than DF(I)NKF (see Supplementary Table 6), which instead forms the second stiffest gel. Moreover, the two monoiodinated peptides, despite having similar hydrophobicity, display com- pletely different properties. For the same reasons discussed above, electrostatic and hydrophobic interactions also should not play an important role in the interﬁbrillar association observed in the halogenated gels. non-hydrogen atoms were reﬁned anisotropically and hydrogen atoms were ﬁxed geometrically and reﬁned isotropically (CCDC deposition number: 1049844). non-hydrogen atoms were reﬁned anisotropically and hydrogen atoms were ﬁxed geometrically and reﬁned isotropically (CCDC deposition number: 1049844). Mixed gels preparation. Mixed hydrogels were prepared using the same method as previously described. Brieﬂy, peptides were weighed into a glass vial and MilliQ water added. The glass vial was sealed and sonicated for 20 s before heating with a heat gun until complete solubilization of the peptides. The vials were allowed then to cool slowly to RT and stored at RT for 7 days before analysis. Thermal stability. The vials containing the hydrogels at 15 mM were inverted and ﬁxed at the bottom of an oil bath with stirring. The temperature was kept at 25 C for 10 min to equilibrate the system and then gradually increased from 25 to 140 C (at 1 C min 1). The temperature at which the gels break is reported as a range: the initial temperature corresponds to the fall of the ﬁrst drop and the ﬁnal one to the complete breakdown of the gel. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms8574 Although no direct structural evidence of halogen bonding between our peptide monomers has been obtained, yet, the reported results, IR and Raman in particular, provided corroborative evidence that iodinated peptides DF(I)NKF and DF(I)NKF(I) potentially use halogen bonds to efﬁciently self-assemble into mature amyloid ﬁbrils. In fact, the particular position of the iodinated residue in the peptide sequence greatly inﬂuences the efﬁciency of its self- assembly process, thus suggesting the involvement of the iodine atom in a speciﬁc noncovalent interaction (Fig. 6a), rather than nonspeciﬁc hydrophobic and/or steric effects. This is further supported by considerations of halogen atom polarizability since the dibrominated and dichlorinated derivatives DF(Br)NKF(Br) and DF(Cl)NKF(Cl) showed less tendency to form ﬁbrils than their diiodinated analogue DF(I)NKF(I). This result allows interactions involving halogen atoms as hydrogen bond acceptors to be ruled out as Cl and Br would give stronger interactions than I, which was not observed. Electrostatic interactions involving the ionizable side chains of the peptides, Asp and Lys, may also be ruled out as the formation of these halogenated hydrogels is largely pH-independent (see Supplementary Table 4). As a Figure 6 \| Potential halogen bonds involving p-iodo-phenylalanine. IR and Figure 6 \| Potential halogen bonds involving p-iodo-phenylalanine. IR and Raman spectroscopies are more consistent with the occurrence of type a interaction, however, other interactions reported cannot be discounted. (a) Orthogonal hydrogen and halogen bonds involving the carbonyl oxygen atom of the peptide bond36. (b) Type II iodine iodine contacts23. (c) Iodine p interactions49. (d) Triangular iodine synthon50. conﬁrmation of the minor role played by electrostatic attraction in the self-assembly of the reported halogenated peptides, increasing the ionic strength of the peptide solutions resulted in accelerated formation of the hydrogels (see Supplementary Table 5). It is also unlikely that aromatic p–p interactions are playing an important role in the self-assembly of the halogenated hydrogels, since this would not explain the polarizability depen- dence that we observed. Finally, hydrophobic interactions could not justify the observed trends in hydrogel properties because NATURE COMMUNICATIONS \| 6:7574 \| DOI: 10.1038/ncomms8574 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS \| 6:7574 \| DOI: 10.1038/ncomms8574 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. 6 & 2015 Macmillan Publishers Limited. All rights reserved. References 41. Wu, W. et al. Eosinophils generate brominating oxidants in allergen-induced asthma. J. Clin. Invest. 105, 1455–1463 (2000). 10. Mankar, S., Anoop, A., Sen, S. & Maji, S. K. Nanomaterials: amyloids reﬂect their brighter side. Nano Rev. 2, 6032–6043 (2011). 42. Mazzulli, J. R., Hodara, R., Lind, S. & Ischiropoulos, H. Oxidative stress and protein deposition diseases in protein misfolding, aggregation, and conformational diseases. Prot. Rev. 4, 123–133 (2006). g Cherny, I. & Gazit, E. 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From the polymorphism of amyloid ﬁbrils to their assembly mechanism and cytotoxicity. Adv. Protein Chem. 73, 217–233 (2006). 39. Hazen, S. L. 3-chlorotyrosine, a speciﬁc marker of myeloperoxidase? Catalyzed oxidation is markedly elevated in low density lipoprotein isolated from human atherosclerotic intima. J. Clin. Invest. 99, 2075–2075 (1997). 7. Hauser, C. A. E., Maurer-Stroh, S. & Martins, I. C. Amyloid-based nanosensors and nanodevices. Chem. Soc. Rev. 43, 5326–5345 (2014). 40. Gaut, J. P. Neutrophils employ the myeloperoxidase system to generate antimicrobial brominating and chlorinating oxidants during sepsis. Proc. Nat Acad. Sci. USA 98, 11961–11961 (2001). 8. Adamcik, J. & Mezzenga, R. Fibrils from a polymer physics perspective. Macromolecules 45, 1137–1150 (2012). 9. Li, C. & Mezzenga, R. The interplay between carbon nanomaterials and amyloid ﬁbrils in bio-nanotechnology. Nanoscale 5, 6207–6218 (2013). References Designed aromatic homo-dipeptides: formation of ordered nanostructures and potential nanotechnological applications. Phys. Biol. 3, S10–S19 (2006). g f 50. Lu, Y. et al. Triangular halogen trimers. A DFT study of the structure, cooperativity, and vibrational properties. J. Phys. Chem. A 109, 11956–11961 (2005). 18. Metrangolo, P., Pilati, T. & Resnati, G. Halogen bonding and other noncovalent interactions involving halogens: a terminology issue. Cryst. Eng. Comm. 8, 946–947 (2006). ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms8574 and 340 cm 1. Obtained data were subsequently normalized, for the sake of comparison, and plotted using Origin Pro 8. and 340 cm 1. Obtained data were subsequently normalized, for the sake of comparison, and plotted using Origin Pro 8. 20. Ryan, D. M., Anderson, S. B. & Nilsson, B. L. The inﬂuence of side-chain halogenation on the self-assembly and hydrogelation of Fmoc-phenylalanine derivatives. Soft Matter 6, 3220–3231 (2010). f 21. Desiraju, G. R. et al. Deﬁnition of the halogen bond (IUPAC Recommendations 2013). Pure Appl. Chem. 85, 1711–1713 (2013). Atomic force microscopy. Peptide solution (10 ml, 40 mM) or diluted gel was deposited onto freshly cleaved mica surface and air dried. AFM characterizations were performed on a Veeco Dimension 5,000 Scanning Probe Microscope with a Nanoscope V controller (Digital Instruments, Inc.). All the samples were prepared on mica substrate and measured without treatment. Al-coated silicon AFM tips (NSC 15/AIBS, MikroMasch, Estonia) with a tip radius of 10 nm were used to probe the surface proﬁles of the ﬁlms. Tapping-mode AFM imaging was used according to well-established procedures. All the images were post-treated with NanoScope Analysis 1.5 Software. pp 22. Metrangolo, P. & Resnati, G. Tracing iodine. Nat. Chem. 3, 260 (2011). 23. Metrangolo, P. & Resnati, G. Type II halogen halogen contacts are halogen bonds. IUCrJ 1, 5–7 (2014). 24. Berger, G., Berger, N., Guillaud, M. H., Trouillas, J. & Vauzelle, J. L. Calcitonin-like immunoreactivity of amyloid ﬁbrils in medullary thyroid carcinomas. An immunoelectron microscope study. Virchows Arch. A Pathol. Anat. Histopathol. 412, 543–551 (1988). 25. Arvinte, T., Cudd, A. & Drake, A. F. The structure and mechanism of formation of human calcitonin ﬁbrils. J. Biol. Chem. 268, 6415–6422 (1993). Transmission electron microscopy. High-resolution transmission electron microscopy imaging was carried out using JEM-3200Fsc ﬁeld emission microscope (JEOL) operated at 300 kV in bright-ﬁeld mode with Omega-type Zero-loss energy ﬁlter. The images were acquired with ULTRASCAN 4,000 CCD camera (GATAN) and with GATAN DIGITAL MICROGRAPH software, while the specimen temperature was maintained at 187 C. Dried TEM and ET samples were prepared by placing 4 ml of 40 mM DF(I)NKF(I) solution on 200 mesh carbon only grid (CFT200-Cu) and the excess removed with ﬁlter paper. 26. Steed, J. W. Supramolecular gel chemistry: developments over the last decade. Chem. Commun. 47, 1379–1383 (2011). 27. Meazza, L. et al. ARTICLE Halogen-bonding-triggered supramolecular gel formation. Nat. Chem. 5, 42–47 (2013). 28. Lakshmanana, A. et al. Aliphatic peptides show similar self-assembly to amyloid core sequences, challenging the importance of aromatic interactions in amyloidosis. Proc. Natl Acad. Sci. USA 110, 519–524 (2013). 29. Shtainfeld, A., Sheynis, T. & Jelinek, R. Speciﬁc mutations alter ﬁbrillation kinetics, ﬁber morphologies, and membrane interactions of pentapeptides derived from human calcitonin. Biochemistry 49, 5299–5307 (2010). Electron tomography. For image alignment purposes, the TEM grids were dipped in 11-mercapto-1-undecanol ligand-coated gold particle solution (d ¼ 3–10 nm) before sample deposition. ET tilt series were acquired with the SERIALEM- software package between tilt angles of ±69. Prealignment of tilt image series was done with IMOD and the ﬁne alignment and cropping with JPEGANIM software package48. The images were binned twice to reduce noise and computation time. Maximum entropy method reconstruction scheme was carried out with MEM software package48 on Linux cluster with regularization parameter value of d ¼ 5.0 10 2. Data visualization, volumetric graphics and analyses were performed with the UCSF CHIMERA package. The tomogram was ﬁltered with CHIMERA’s Gaussian ﬁlter with 1.5 voxel width. 30. Hauser, C. A. E. et al. Natural tri- to hexapeptides self-assemble in water to amyloid b-type ﬁber aggregates by unexpected a-helical intermediate 30. Hauser, C. A. E. et al. Natural tri- to hexapeptides self-assemble in water to amyloid b-type ﬁber aggregates by unexpected a-helical intermediate structures. Proc. Natl Acad. Sci. USA 108, 1361–1366 (2011). structures. Proc. Natl Acad. Sci. USA 108, 1361–1366 (2011 31. Mishra, A. et al. Ultrasmall natural peptides self-assemble to strong temperature-resistant helical ﬁbers in scaffolds suitable for tissue engineerin Nano Today 6, 232–239 (2011). 32. Smith, D. K. In Supramolecular Chemistry: From Molecules to Nanomaterials (John Wiley and Sons, Inc., 1999). y 33. Adamcik, J. et al. Understanding amyloid aggregation by statistical analysis of atomic force microscopy images. Nat. Nanotechnol. 5, 423–428 (2010). 34. Nilsson, M. R. Techniques to study amyloid ﬁbril formation in vitro. Methods 34, 151–160 (2004). Methods R t C The p-iodo-phenylalanine was suspended in water at RT and kept in an open vial under a hood. After 6 months, colourless crystalline needles appeared. Suitable crystals for XRD measurements were analysed without further manipulation. The crystals were measured using Mo-Ka radiation on a Bruker KAPPA APEX II diffractometer with a Bruker KRYOFLEX low tempera- ture device. The crystal structure was solved by direct method and reﬁned against F2 using SHELXL9746. Packing diagrams were generated using Mercury47. The Raman spectroscopy. Raman spectra were acquired at RT by using a Horiba Xplora MicroRaman instrument equipped with an Olympus BX-41 Microscope. An excitation wavelength of 785 nm was used. Laser power was attenuated by neutral density ﬁlters with a ﬁnal power density of B0.07 mW mm 2. The low wavenumber detection limit is 140 cm 1. Each spectrum was acquired with an exposure time of 5 s over 35 cycles. The raw data were ﬁrst corrected from the baseline, using the JASCO Nicolet FTIR software, Omnic 9.0, between 140 7 7 NATURE COMMUNICATIONS \| 6:7574 \| DOI: 10.1038/ncomms8574 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms8574 Competing ﬁnancial interests: The authors declare no competing ﬁnancial interests. ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms8574 Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ How to cite this article: Bertolani, A. et al. Supramolecular ampliﬁcation of amyloid self-assembly by iodination. Nat. Commun. 6:7574 doi: 10.1038/ncomms8574 (2015). Author contributions A.B. and L.P. performed most of the peptide self-assembly work. L.S. performed the IR spectroscopy and ionic strength-pH-dependence experiments. N.H. measured the samples by AFM. J.S.H. measured the samples by TEM. L.C. and G.T. performed the single crystal X-ray analysis. G.G. and L.V. performed the Raman spectroscopy. P.M., R.M., O.I., and G.R. conceived the experiments and contributed to results discussion. All the authors contributed to manuscript writing. Acknowledgements 19. Ryan, D. M., Doran, T. M., Anderson, S. B. & Nilsson, B. L. Effect of C-terminal modiﬁcation on the self-assembly and hydrogelation of ﬂuorinated Fmoc-Phe derivatives. Langmuir 27, 4029–4039 (2011). The European Research Council is acknowledged for the Starting Grant ERC-2012- The European Research Council is acknowledged for the Starting Grant ERC-2012- StG_20111012 FOLDHALO (Grant Agreement Number 307108) to P.M. Dr Claudia Piglia- celli (Politecnico di Milano) is acknowledged for help with the TEM experiment of Fig. 3f. The European Research Council is acknowledged for the Starting Grant ERC-2012- StG_20111012 FOLDHALO (Grant Agreement Number 307108) to P.M. Dr Claudia Piglia- celli (Politecnico di Milano) is acknowledged for help with the TEM experiment of Fig. 3f. NATURE COMMUNICATIONS \| 6:7574 \| DOI: 10.1038/ncomms8574 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| 6:7574 \| DOI: 10.1038/ncomms8574 \| www.nature. 8 & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS \| 6:7574 \| DOI: 10.1038/ncomms8574 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. & 2015 Macmillan Publishers Limited. All rights reserved. Additional information Accession codes: The X-ray crystallographic coordinates for the structure reported in this study have been deposited at the Cambridge Crystallographic Data Centre (CCDC), under deposition number 1049844. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Supplementary Information accompanies this paper at http://www.nature.com/ naturecommunications naturecommunications
https://openalex.org/W2970897407	https://ieeexplore.ieee.org/ielx7/6221039/8579633/08821306.pdf	English	null	A Miniature Dual-Biomarker-Based Sensing and Conditioning Device for Closed-Loop DBS	IEEE journal of translational engineering in health and medicine	2,019	cc-by	7,537	MAHBOUBEH PARASTARFEIZABADI AND ABBAS Z. KOUZANI , (Member, IEEE) School of Engineering, Deakin University, Geelong, VIC 3216, Australia CORRESPONDING AUTHOR: A. Z. KOUZANI (kouzani@deakin.edu.au) MAHBOUBEH PARASTARFEIZABADI AND ABBAS Z. KOUZANI , (Member, IEEE) School of Engineering, Deakin University, Geelong, VIC 3216, Australia CORRESPONDING AUTHOR: A. Z. KOUZANI (kouzani@deakin.edu.au) ABSTRACT In this paper, a dual-biomarker-based neural sensing and conditioning device is proposed for closing the feedback loop in deep brain stimulation devices. The device explores both local ﬁeld potentials (LFPs) and action potentials (APs) as measured biomarkers. It includes two channels, each having four main parts: (1) a pre-ampliﬁer with built-in low-pass ﬁlter, (2) a ground shifting circuit, (3) an ampliﬁer with low- pass function, and (4) a high-pass ﬁlter. The design speciﬁcations include miniature-size, light-weight, and 100 dB gain in the LFP and AP channels. This device has been validated through bench and in-vitro tests. The bench tests have been performed using different sinusoidal signals and pre-recorded neural signals. The in-vitro tests have been conducted in the saline solution that mimics the brain environment. The total weight of the device including a 3 V coin battery, and battery holder is 1.2 g. The diameter of the device is 11.2 mm. The device can be used to concurrently sense LFPs and APs for closing the feedback loop in closed-loop deep brain stimulation systems. It provides a tetherless head-mountable platform suitable for pre-clinical trials. INDEX TERMS Analog circuit, brain sensor, deep brain stimulation, fabrication, multiple biomarkers. NEUROVASCULAR DEVICES AND SYSTEMS NEUROVASCULAR DEVICES AND SYSTEMS Received 13 May 2018; revised 3 May 2019 and 8 August 2019; accepted 20 August 2019. Date of publication 30 August 2019; date of current version 13 September 2019. Digital Object Identifier 10.1109/JTEHM.2019.2937776 Digital Object Identifier 10.1109/JTEHM.2019.2937776 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see http://creativecommons.org/licenses/by/4.0/ A Miniature Dual-Biomarker-Based Sensing and Conditioning Device for Closed-Loop DBS MAHBOUBEH PARASTARFEIZABADI AND ABBAS Z. KOUZANI , (Member, IEEE) School of Engineering, Deakin University, Geelong, VIC 3216, Australia CORRESPONDING AUTHOR: A. Z. KOUZANI (kouzani@deakin.edu.au) Received 13 May 2018; revised 3 May 2019 and 8 August 2019; accepted 20 August 2019. Date of publication 30 August 2019; date of current version 13 September 2019. Digital Object Identifier 10.1109/JTEHM.2019.2937776 II. METHODOLOGY A. ACTION AND LOCAL FIELD POTENTIALS Any part of a neuron including soma, dendrites, axon, and axon terminals, contributes to the ionic processes in the extra- cellular space [23]. Extracellular potentials contain all the information from fast potentials to slow ﬂuctuations [23]. The fast potentials are called extracellular APs, and the slow ﬂuctuations are referred to as LFPs. APs include spikes and spike-induced after-hyperpolarization potentials. LFPs, on the other hand, are produced by spreading of APs through axons. High-frequency power of extracellular potentials pro- vides indirect access to the intracellular APs [24], mainly indicating the spiking activities [23]. Hence, it is feasible to measure the extracellular APs from the same electrodes as LFPs. FIGURE 1. Multiple-biomarker-based closed-loop DBS architecture. multiple biomarkers can perform better than one with only one biomarker [19]. Fig. 1 shows a closed-loop DBS system involving mul- tiple biomarkers. The overall system includes four com- ponents: (1) sensing/stimulating electrodes, (2) sensor and conditioner, (3) digitizer, feature extractor, and controller, and (4) stimulator. The ﬁrst component includes sensing and stimulating electrodes. The sensing electrodes are used to read the biomarkers from the brain and other part of the body. The sensed biomarkers are ampliﬁed and ﬁltered within the second component, sensor and conditioner, to produce biomarkers signals. The third component, digitizer-feature extractor-controller, samples the biomarkers signals, digitize them, and then extract key features out of them. The fea- tures are used as inputs to the controller which then forms control signals for modifying the stimulation parameters in the stimulator. The fourth component, stimulator, generates stimulation signals based on the input control signals. Finally, the stimulation signals are delivered to the brain via the stimulating electrodes. It should be noted that LFPs and APs are not directly used as biomarkers by themselves. They contain several frequency bands, and activities within those bands are used as potential biomarkers for symptoms of neurological disorders. These frequency bands include alpha (8-14 Hz), beta (∼13–30 Hz), slow-gamma (sG: 30-45 Hz), fast high-frequency oscilla- tions (fHFOs: 300-400 Hz), and spikes (>500 Hz). Alpha has been shown to be high in the limbic system of major depressive disorder patients, correlating with severity symp- tom [25]. Similarly, it is shown to be maximal in peduncu- lopontine nucleus (PPN) region of PD patients, correlating with improved gait performance [26]. In this situation, a closed-loop DBS should work towards suppression of alpha biomarker. I. INTRODUCTION devices can be used to measure more than one biomarker (e.g., local ﬁeld potentials (LFPs) and action potentials (APs)) [7]–[9], [13], [14], they have not implemented parallel acquisition of multiple biomarkers at the same time. There- fore, they optimize the stimulation parameters based on only one biomarker. Deep brain stimulation (DBS) devices are classiﬁed into open-loop and closed-loop groups based on their internal function. In the open-loop DBS, non-stop stimulation pulses are delivered into the brain regardless of the variations in the brain’s condition. In the closed-loop DBS, on the other hand, the stimulation pulses are adjusted and delivered into the brain according to the variations in the brain’s condition. Closing the feedback loop based on one biomarker may not always ameliorate a spectrum of disease motor signs. For example, patients with Parkinson’s disease (PD) may experience different primary (e.g. tremor, bradykinesia, rigid- ity, postural instability, etc.) or secondary (e.g. freezing, micrographia, mask-like expression, unwanted accelerations) motor symptoms [16]. However, not all of these symptoms are promoted by the same pathophysiological neuronal sys- tems. For example, rigidity and bradykinesia are function- ally discrete from tremor [17]. Therefore, they may require separate neurophysiological biomarkers to adequately cap- ture them [18]. Amelioration of a range of disease motor symptoms through only one biomarker could be a challenging task. Moreover, as discussed by Little and Brown [18], a single biomarker closed-loop control, may be efﬁcient for tackling only some impairments. Therefore, in order to alle- viate symptoms of a disease, a closed-loop system involving Most of the current DBS systems operate in an open- loop manner. Open-loop DBS may however produce some signiﬁcant functional (e.g. induction of paresthesia, invol- untary movements, worsening of gait or speech, gaze devi- ation or paralysis), as well as cognitive, and mood side effects [1]. Closed-loop DBS can alleviate these side effects through optimization of the stimulation parameters (pulse width, amplitude, and frequency) [1], [2]. For this purpose, a biomarker is continuously measured and analyzed to optimize stimulation pulses according to the brain’s clinical condition. Therefore, the risks of the brain over- or under-stimulation can be minimized [3]–[6]. The existing closed-loop DBS devices [7]–[15] employ only one biomarker as the input to their control module to adjust the stimulation parameters. While some of these 2000308 2000308 VOLUME 7, 2019 M. Parastarfeizabadi, A. Z. Kouzani: Miniature Dual-Biomarker-Based Sensing and Conditioning Device FIGURE 1. Multiple-biomarker-based closed-loop DBS architecture. I. INTRODUCTION dual-biomarker-based sensing and conditioning device. This device is portable and head-mountable, and will form the Sensor and Conditioner component of a closed DBS sys- tem. It will provide a great tool for use in tetherless head- mountable closed-loop DBS conﬁgurations for pre-clinical animal investigations. II. METHODOLOGY A. ACTION AND LOCAL FIELD POTENTIALS Beta, a well-known biomarker for PD [27], has prominent synchronization in the STN region of PD human and also animal models. A closed-loop DBS should work towards beta suppression, which causes improvement of rigidity, akinesia and bradykinesia [28], as well as the freez- ing of gait [29] symptoms in PD. In humans with Tourette’s syndrome, increased thalamic sG activity correlates with symptom relief following DBS [30]. In addition, suppression of sG oscillations is linked to DBS-induced tremor symp- tom reduction in PD patients [31]. In PD [32], [33] and epilepsy [34], [35], the existence of high-frequency oscilla- tions (HFOs) is frequently detected. Furthermore, HFOs has been observed in the STNs of patients suffering from essential tremor and dystonia [36]. Speciﬁc changes in neuronal spike ﬁring rate may be representative of seizure occurrence in epileptic patients [37], which could be used as a potential biomarker in a closed-loop DBS device. Use of multiple biomarkers provide more information to the controller. Therefore, a more accurate assessment of the patient clinical state is carried out. Typical biomarkers that have been used in closed-loop DBS include surface elec- tromyogram (sEMG), cortical neuro-signals such as elec- trocorticogram (ECoGs), subcortical neuro-signals such as LFPs (low-frequency oscillations) and APs (high-frequency oscillation), and neurochemical signals such as dopamine neurotransmitter release. A review by Arlotti et al. [20] con- cluded that, out of these biomarkers, LFP should be a primary choice because it contains more information needed for an ideal DBS biomarker. LFPs contain information about PD major symptoms such as bradkykinesia and rigidity. Besides, APs are considered to be a complementary biomarker for LFPs which together may enhance the outcome of DBS [21]. Johnson et al. [22] concluded that closed-loop systems that use multiple biomarkers, for example LFPs and APs, would provide more effective control in the system. LFPs and APs are typically weak bio-potentials and need ampliﬁcation and ﬁltering before being used in the feedback loop. The magnitude of these potentials can be in a range This paper focuses on measurement of two biomark- ers, LFPs and APs, simultaneously. It presents a miniature VOLUME 7, 2019 2000308 2000308 2000308 M. Parastarfeizabadi, A. Z. Kouzani: Miniature Dual-Biomarker-Based Sensing and Conditioning Device FIGURE 2. Front-end circuit diagram of the dual-biomarker-based sensing device. The LFPs and APs are differentially recorded from two contacts of a DBS microelectrode. The blue rectangles signify pre-amplifier and low-pass filter. II. METHODOLOGY A. ACTION AND LOCAL FIELD POTENTIALS The black rectangle demonstrates the ground shifting circuit. The red rectangles indicate the amplifier and low-pass filter. And, the green rectangle shows the high-pass filter circuit. FIGURE 2. Front-end circuit diagram of the dual-biomarker-based sensing device. The LFPs and APs are differentially recorded from two contacts of a DBS microelectrode. The blue rectangles signify pre-amplifier and low-pass filter. The black rectangle demonstrates the ground shifting circuit. The red rectangles indicate the amplifier and low-pass filter. And, the green rectangle shows the high-pass filter circuit. the APs similar to those of LFPs but with the band-width of 300 Hz - 6 kHz. from 10 µV to 1 mV depending upon the electrode type and position [38], [39]. The frequency range of the LFPs is variable between 1 to 500 Hz [39], [40], while that of APs is much higher, and mainly changes between 300 Hz −6 kHz [41]. Both the LFP and AP biomarkers can be differentially measured from a two-channel electrode as can be seen in Fig. 2. A low noise, low offset voltage, high common-mode rejection ratio (CMRR) precision instrumentation ampliﬁer (IA), AD8293G160, was used in the pre-ampliﬁcation stage of each channel. This IA has a ﬁxed gain of 160 V/V. By means of two external capacitors (C2 and C3 in LFP and C7 and C8 in AP paths), the IA also implements a 2-pole low-pass ﬁlter. An internal 320 k resistor forms a low- pass ﬁlter along with C2. Another 5 k internal resistor forms a second low-pass ﬁlter together with C3. Based on the ﬁlter equations, C2 and C3 were chosen as 12 nF and 0.8 uF, respectively, implementing a 40 Hz LPF in the LFP channel. C7 and C8 capacitors were calculated 82 pF and 5.3 nF, respectively, to create a 6 kHz LPF in the AP path. To achieve optimum performance of the AD8293G160 chip, a 0.1 µF capacitor (C1 in Fig. 2) was connected between the supply (VCC) and the ground (GND) lines. This capacitor suppresses the excessive noises on the VCC pin and prevents undesired offset voltage in the output of the chip. B. BENCH-TEST VALIDATION The functionality of the device was validated through apply- ing sinusoidal and pre-recorded neural signals. Three sinu- soidal signals with 25 µv amplitude and different frequencies (F1: 0.15 Hz, F2: 15 Hz, and F3: 150 Hz for LFP channel; F4: 15 Hz, F5: 500 Hz, and F6: 15 kHz for AP channel) were applied to the input of neural recorder using a signal generator. The F1 and F3 were chosen out of the low-pass and high-pass cut-off frequencies of the LFP channel. Similarly, F4 and F6 were chosen out of the working frequencies of the AP channel. To produce a 25 µv sine voltage, two voltage dividers were used at the input of each differential electrode. These voltage dividers were used to convert a 2 V AC signal to 45 µV and 20 µV AC signals for presenting to positive and negative inputs of the device, respectively. Hence, the device measured a 25 µV differential voltage (VIN+ - VIN-) at its input, and ampliﬁed it with a gain of 100,000 V/V. The observed outputs are presented in Fig 5 (A-F). As can be seen from Fig. 5 (A-F), the F1, F3, F4 and F6 were ﬁltered by the C. FABRICATION The neural recorder device was fabricated on a two-layer printed circuit board (PCB) with a circular conﬁguration as shown in Fig. 3. In terms of the dimensions, this device bene- ﬁts from a miniature size with a radius of 5.6 mm that enables head-mountable use with small laboratory animals such as mice. The complete device with battery, battery holder, and pins for connection to Plastic-One electrodes are shown in Fig. 3 (D). The weight of the device is only 0.21 g without the battery, 0.8 g with a CR1025 3 V coin battery, and 1.2 g with attached battery holder (see Fig. 3 (D) and (E)). To the best of our knowledge, this device is the lightest and smallest existing neural recorder designed with discrete components. B. CIRCUIT The phase response changes from roughly 100◦to 60◦, and 100◦to 50◦as the frequency is swept from 1 to 100 Hz and 100 to 10000 Hz in the LFP and AP channels, respectively. The phase response showing a positive phase shift in the lower frequencies and a negative phase shift in higher frequencies (+50 to -150). However, it smoothly changes from positive to the negative values with no fast variations, which indicates the stability of the designed device over different frequency ranges. Finally, the LFPs and APs can then be converted to digital values, through a single-supply, ADC for processing and adjustment of stimulation parameters. B. CIRCUIT Bode plot showing gain and phase responses of the LFP (A) and AP (B) channels. M. Parastarfeizabadi, A. Z. Kouzani: Miniature Dual-Biomarker-Based Sensing and Conditioning Device FIGURE 3. (A) Top view of the device. (B) Bottom view of the device. (C) Side view of the device. (D) Complete device. (E) Weight of the device without battery and battery holder. (F) Total weight of the complete device. FIGURE 3. (A) Top view of the device. (B) Bottom view of the device. (C) Side view of the device. (D) Complete device. (E) Weight of the device without battery and battery holder. (F) Total weight of the complete device. FIGURE 4. Bode plot showing gain and phase responses of the LFP (A) and AP (B) channels. ampliﬁers, and to enable driving a single-supply analog-to- digital converter (ADC). This circuit shifts the ground to the mid-supply voltage. It comprises a voltage divider (R4, R5, and C6 in Fig. 2) followed by a unity gain buffer (TSZ124). This circuit drives both the LFP and AP paths. Due to the use of a supply voltage of 3 V from a CR1025 coin battery, the signals are shifted with an offset of 1.5 V using this technique. FIGURE 4. Bode plot showing gain and phase responses of the LFP (A) and AP (B) channels. Following the ﬁrst stage, an ampliﬁer with a single pole active non-inverting LPF was used to further reject the signal artifacts and boost the LFP and AP signals to a 105 V/V overall gain factor. A TSZ124, a very high accuracy quad operational ampliﬁer chip, was used as the op-amps in the circuits of ampliﬁer and low-pass ﬁlter, and ground shifting circuit. This chip beneﬁts from a zero drift and micro-power properties to suit battery-operated experiments. In the ﬁnal stage of the analog part, there is a ﬁrst order passive HPF (with 2 Hz and 300 Hz cut-off frequencies for LFPs and APs, respectively) that removes the unwanted lower frequencies from the biomarkers. LFP and AP channels. The signal bandwidth is designed to be 2 – 40 Hz and 300 Hz – 6 kHz for the LFP and AP channels, respectively. The actual -3dB cut-off frequencies can be seen slightly affected due to the use of resistors and capacitors with 0.1-10% tolerances. The phase response of the LFP and AP channels are presented in red color in Fig. 4. B. CIRCUIT The dual-biomarker-based neural recording device was developed using discrete components to achieve higher ﬂex- ibility in modifying the design, lower production expenses, and shorter manufacturing time, compared with application speciﬁc integrated circuits (ASICs) [42]. The designed device consists of four major parts: (1) a pre-ampliﬁer combined with a low-pass ﬁlter, (2) an ampliﬁer combined with a low- pass ﬁlter, (3) a high-pass ﬁlter, and (4) a ground shifting circuit. A schematic of the circuit is presented in Fig. 2. This device has been speciﬁcally developed to be used in a closed-loop DBS system. It meets the DBS requirements on LFP and AP ampliﬁcation and artifact rejection conditions. Rossi et al. [43] deﬁned the requirements on LFPs as follows. A gain of 80 – 100 dB in the band-width of 2 – 40 Hz is needed to remove the 130 Hz (−40 dB) stimulation artifact from the LFP biomarker [43]. We have selected the requirements of A ground shifting circuit is developed to provide the single- supply operation to the instrumentation and operational 2000308 VOLUME 7, 2019 VOLUME 7, 2019 r-Based Sensing and Conditioning Device ottom view of the device. (C) Side view of the device. (D) Complete device. d battery holder. (F) Total weight of the complete device. ly analog-to- ground to the ider (R4, R5, er (TSZ124). Due to the use n battery, the his technique. a single pole ect the signal FIGURE 4. Bode plot showing gain and phase responses of the LFP (A) and AP (B) channels. eizabadi, A. Z. Kouzani: Miniature Dual-Biomarker-Based Sensing and Conditioning Device FIGURE 3. (A) Top view of the device. (B) Bottom view of the device. (C) Side view of the device. (D) Complete device. (E) Weight of the device without battery and battery holder. (F) Total weight of the complete device. s, and to enable driving a single-supply analog-to- onverter (ADC). This circuit shifts the ground to the ply voltage. It comprises a voltage divider (R4, R5, n Fig. 2) followed by a unity gain buffer (TSZ124). uit drives both the LFP and AP paths. Due to the use ply voltage of 3 V from a CR1025 coin battery, the re shifted with an offset of 1.5 V using this technique. wing the ﬁrst stage, an ampliﬁer with a single pole n-inverting LPF was used to further reject the signal FIGURE 4. III. EXPERIMENTAL RESULTS A. FREQUENCY RESPONSE The bode plot showing the gain and phase responses of the LFP and AP channels is presented in Fig. 4. The data was captured by a NI myDAQ device through its bode-plot soft- ware, and then plotted within MATLAB software. As shown in Fig. 4, the device has a ﬁxed gain of 100 dB in both the 2000308 2000308 VOLUME 7, 2019 M. Parastarfeizabad FIGURE 5. (A-F) The bench test outputs obtained from the LFP and AP channels for sinusoidal input signals. (G) Implemented bench test setup with pre-recorded neural signals. (H) LFP input applied to the voltage divider (VD), and the observed output from the LFP OUT pin. (F) AP input applied to the voltage divider and the observed output from the AP-OUT pin. M. Parastarfeizabadi, A. Z. Kouzani: Miniature Dual-Biomarker-Based Sensing and Conditioning Device FIGURE 6. (A) In-vitro setup used for measurements in the saline solution. (B) Saline bath and copper electrodes used in the experiments. (C) Implemented in-vitro experimental setup. (D) LFP input to the voltage divider (VD) and the observed output from the LFP OUT pin. (E) AP input to the VD and the observed output from the AP-OUT pin. FIGURE 6. (A) In-vitro setup used for measurements in the saline solution. (B) Saline bath and copper electrodes used in the experiments. (C) Implemented in-vitro experimental setup. (D) LFP input to the voltage divider (VD) and the observed output from the LFP OUT pin. (E) AP input to the VD and the observed output from the AP-OUT pin. FIGURE 5. (A-F) The bench test outputs obtained from the LFP and AP channels for sinusoidal input signals. (G) Implemented bench test setup with pre-recorded neural signals. (H) LFP input applied to the voltage divider (VD), and the observed output from the LFP OUT pin. (F) AP input applied to the voltage divider and the observed output from the AP-OUT pin. The result of the LFP channel for the ﬁrst 2 s is shown in Fig. 5 (H). The output has less variations to the input and well mimics the pattern. Fig. 5 (I) shows 100 ms of the input signal (S1) injected to the voltage dividers, and also the obtained output from the AP-OUT pin. As can be seen from the ﬁrst 50 ms, the S1 contains low frequency variations which have been combined with the high-frequency components. III. EXPERIMENTAL RESULTS A. FREQUENCY RESPONSE These low-frequency variations do not appear at the output of the AP channel. Both the LFP and AP outputs are cantered at 1.5 V with over 2.5 Vpk-pk ampliﬁed voltage. device due to being out of the neural recorders’ working area. Only the F2 and F5 were well ampliﬁed. Next, the procedure was repeated with pre-recorded neural data. The data was provided by Prof. Benoit Gosselin and Dr. Masoud Rezaei from Laval University, Canada. These signals were recorded from a 23 g mouse from the hippocam- pus area using a probe with approximately 8 M resistance. The pre-recorded neural signal was introduced to the inputs of the device through the arbitrary waveform generator (ARB) of NI myDAQ system (via the analog output (AO-0)). The bench test setup and results are shown in Fig. 5 (G-I). The raw neural signal contained mainly high-frequency compo- nents recorded with a sampling frequency of 20 kS/s. The NI myDAQ software provides the capability of sending a signal with other sampling rates. Using this feature, high-frequency signals can be presented as signals whose frequency has been reduced. Therefore, to verify the LFP channel, we set the sampling frequency to 600 S/s to create a neural signal corresponding to a LFP signal. On other hand, to verify the AP channel, the pre-recorded neural signal was injected into the device with its original sampling frequency of 20 kS/s. Since the NI myDAQ device is not able to send low-amplitude potentials, we ﬁrst ampliﬁed the original signal in MATLAB. Then, we used two different voltage dividers (2.2 M with 22 , and 2.2 M with 48 ) at the output of the NI myDAQ system to reduce the amplitude of the signal to µV level for driving the In- and In+ inputs of the neural recorder device. IV. DISCUSSIONS This paper presents a dual-biomarker based neural recording device designed for concurrent LFP and AP measurements. The LFP channel ampliﬁes the neural signals in the range of 2 – 40 Hz, and the AP channel boosts the signals in the range of 300 – 6000 Hz. A recent review by Amon and Alesch [47] discusses the recent advances and technical features of the electrodes and systems designed for DBS. Different electrode types are manufactured by three large companies including: (1) Medtronic, (2) Boston Scientiﬁc, and (3) St Jude. These electrodes have a contact length of 1.5-3 mm [47]. DBS-style macro-electrodes can be used to record LFPs. However, for measurements of APs, their contact length is orders of magni- tude larger than a typical microelectrode, and thus average the signals from a much larger area around the contact. Plastics One electrodes, with a bare electrode diameter of 75–250 µm, have been developed for both electrophysiological recordings and electrical stimulations. The very small surface contact area of this electrode tip (0.004-0.049 mm2, versus ∼6 mm2 in 3389 Medtronic DBS lead) will allow the extracellular APs to be collected from a much smaller area around the tip contact. In addition, this device is intended to record LFPs and APs in small animal primates (not humans). Usually in the laboratory animals because of several reasons (small brain size, costs, etc.), the use of Medtronic DBS lead (or other sim- ilar electrodes from other companies) is not preferred. In this case, usually microelectrodes or other electrodes with lower surface contact are desired. Plastics One electrodes have been frequently used in animal-based DBS experiments [48]. The phase response of the device in LFP and AP channels is shown in Fig. 4 (A) and (B), respectively. The direction of the phase shift (whether it is positive or negative) is usually determined by the type of the ﬁlters [52], where the low- pass ﬁlters produce a negative phase shift (lag-response of the output in relation to the input) and high-pass ﬁlters produce a positive phase shift (lead-response of the output in relation to the input). The phase shift of the current device ranges from near +50 to −150 degrees over different frequency ranges because of the combination of low-pass and high- pass ﬁlters. C. IN-VITRO VALIDATION The purpose of the in-vitro tests is to assess the sensing and conditioning abilities of the device in an environment that simulate a neuron-electrode interface in a neural record- ing system [44], [45]. The in-vitro setting includes a saline solution bath (0.9 % NaCl) prepared in the following steps: (1) solving of nine grams NaCl in one litter of distilled water, (2) gentle stirring of the solution for about ten sec- onds, (3) boiling of the solution for ﬁfteen minutes, and (4) allowing the solution to reach the room temperature. Fig. 6 (A-B) illustrates the setup used for the in-vitro experiments. A copper electrode (E1) delivers the original pre-recorded neural signal received from the NI myDAQ system into the saline bath. This signal then propagates in different directions within the solution. Two other copper electrodes (E2 and E3) are placed near E1 to capture the signals propagating in the solution [46]. The differentially captured signal on E2 and E3 is then ampliﬁed and ﬁltered by the device and the LFP and AP outputs are displayed on an oscilloscope for veriﬁcation. In order to verify the LFP channel, we set the sampling frequency of the original neural signal to 600 S/s to create 2000308 2000308 VOLUME 7, 2019 VOLUME 7, 2019 M. Parastarfeizabadi, A. Z. Kouzani: Miniature Dual-Biomarker-Based Sensing and Conditioning Device from a 3 V (30 mAh) lithium manganese dioxide coin battery (CR1025). Hence, the battery will last for 15 hours consid- ering the collective 2 mA current requirement of the two channels. a signal presenting LFPs. On other hand, to verify the AP channel, the pre-recorded neural signal was injected into the saline solution with its original sampling frequency of 20 kS/s. Due to the inability of the NI myDAQ system to output signals in µV level, one voltage divider was used at its output (AO-0) to provide the saline bath with the potential in µV level. Fig. 6 (C-E) represents the in-vitro setup and the results obtained for the LFP and AP channels. The results demonstrate the correct functionality of the device. As can be seen from Fig. 6 (D-E), the output follows the input pattern and is centered at the mid-supply voltage. One of the factors worth considering is the impedance of the recording electrodes in animal or human tissues. Higher electrode impedances can cause larger signal distortions at the input of the device [50]. IV. DISCUSSIONS However, it smoothly changes from positive to negative values with no fast variations in between, which shows the stability of the device over different frequency ranges. In a future application of the current neural recorder device in a closed-loop DBS paradigm, the phase shifts of the signals could be corrected if some temporal events in the brain (e.g. event based potentials) are considered as features to be extracted from the biomarkers [53]. This neural recording device has been designed using dis- crete components rather than ASICs. Therefore, we com- pare its features against discrete components based devices. Although ASICs may beneﬁt from a better performance in terms of size and power consumption, they suffer from low ﬂexibility and adaptability, extended design time and large prototyping costs [42]. In addition, ASICs are more appropriate for human-based long-term experiments rather than animal-based short-term trials. In the animal-based short-term trials, lower expenses and shorter design times are desirable [42], which can be provided by discrete- components-based design. Due to the delivery of DBS [43], the 130 Hz artifact of DBS must be rejected. It is recommended in the literature that the artifact to be rejected in hardware instead of soft- ware [43]. Software-based post-ﬁltering [49] degrades the signal quality due to the fact that neural signal amplitude is approximately ﬁve to six times smaller than that of the DBS artifact [43]. Therefore, manufacturing of a device with two separate channels for each of LFPs and APs are preferred over a 1-channel broadband neural recording device, despite the additional space and power consumption required. Our device can provide simultaneous dual-biomarker- based (LFP and AP) recordings, while most of the previous devices [10], [54]–[57] only record one biomarker at the same time. Although the gain and bandwidth of the current device is ﬁxed and non-adjustable compared to the devices reported in ref. [54], [56], [57], our device beneﬁts from a gain of 100 dB which is the highest gain achieved amongst the existing devices with a gain values between 35-100 dB. Moreover, in terms of the operating power supply, this device requires lower power voltage (3 V) compared to most of the This device beneﬁts from a miniature size (only 11.2 mm in diameter) and a light weight (only 1.2 g including battery and battery holder). These features enable head-mountable closed-loop DBS investigations on small laboratory animals. C. IN-VITRO VALIDATION Clinical impedance measurements of Plastics One electrodes show a range of 5 to 20 K vari- ations [51]. The impedance of this electrodes is considered to be low enough to prevent signal distortions. In addition, the high CMRR (140 dB) precision instrumentation ampli- ﬁer, AD8293G160, at the input of our neural recorder can handle signal distortions. This electrode, as recommended in the manufacturer website, is a suitable option for both electrophysiological recordings and electrical stimulations. IV. DISCUSSIONS The device is battery operated and consumes 2 mA current VOLUME 7, 2019 2000308 2000308 M. Parastarfeizabadi, A. Z. Kouzani: Miniature Dual-Biomarker-Based Sensing and Conditioning Device existing devices with a minimum of 3.3 – 4 V power supply requirement [54]–[57]. The requirement for higher supply voltages results in the use of larger and heavier batteries, which can be avoided in our design because of lower supply requirements. [3] B. Rosin et al., ‘‘Closed-loop deep brain stimulation is superior in amelio- rating parkinsonism,’’ Neuron, vol. 72, pp. 370–384, 2011. [3] B. Rosin et al., ‘‘Closed-loop deep brain stimulation is superior in amelio- rating parkinsonism,’’ Neuron, vol. 72, pp. 370–384, 2011. [4] C. Hamani, E. Richter, J. M. Schwalb, and A. M. Lozano, ‘‘Bilateral subthalamic nucleus stimulation for Parkinson’s disease: A systematic review of the clinical literature,’’ Neurosurgery, vol. 56, pp. 1313–1321, Jun. 2005. [5] R. Ramasubbu, S. Lang, and Z. H. T. Kiss, ‘‘Dosing of electrical parameters in deep brain stimulation (DBS) for intractable depression: A review of clinical studies,’’ Front Psychiatry, vol. 9, p. 302, Jul. 2018. In terms of the power consumption, the neural recorder by Irwin et al. [56] has the lowest power consumption per channel (681 µW). Our neural recorder consumes 3 mW power per channel from a small 3 V coin battery. In addition, it has a reasonable battery lifetime of 15 hours for a 30 mAh capacity battery. Use of larger capacity batteries will increase the operation time. For example, a 3 V 1120 mAh lithium-ion battery (similar to chestek et al. work [55]), would theoret- ically provide over 23 days of continuous neural recording with our device. [6] K. B. Hoang and D. A. Turner, ‘‘The emerging role of biomarkers in adaptive modulation of clinical brain stimulation,’’ Neurosurgery, vol. 85, no. 3, pp. E430–E439, 2019. [7] S. Stanslaski et al., ‘‘Design and validation of a fully implantable, chronic, closed-loop neuromodulation device with concurrent sensing and stimula- tion,’’ IEEE Trans. Neural Syst. Rehabil. Eng., vol. 20, no. 4, pp. 410–421, Jul. 2012. [8] J. Lee, H.-G. Rhew , D. R. Kipke, and M. P. Flynn, ‘‘A 64 channel programmable closed-loop neurostimulator with 8 channel neural ampli- ﬁer and logarithmic ADC,’’ IEEE J. Solid-State Circuits, vol. 45, no. 9, pp. 1935–1945, Sep. 2010. ACKNOWLEDGMENT [20] M. Arlotti, M. Rosa, S. Marceglia, S. Barbieri, and A. Priori, ‘‘The adaptive deep brain stimulation challenge,’’ Parkinsonism Rel. Disorders, vol. 28, pp. 12–17, Jul. 2016. We thank Prof. Benoit Gosselin and Dr. Masoud Rezaei from Laval University, Canada for providing us with pre-recorded neural data. [21] T. E. Özkurt et al., ‘‘High frequency oscillations in the subthalamic nucleus: A neurophysiological marker of the motor state in Parkinson’s disease,’’ Exp. Neurol., vol. 229, pp. 324–331, Jun. 2011. IV. DISCUSSIONS This device beneﬁts from a miniature physical size (11.2 mm in diameter, 0.6 cm in thickness) and is light in weight (1.2 g including battery and battery holder). Although this device might not be the smallest and lightest neural recorder, it is small and light enough to facilitate tetherless head-mountable battery-operated recording sessions on small freely-moving laboratory animals. [9] M. Azin, D. J. Guggenmos, S. Barbay, R. J. Nudo, and P. Mohseni, ‘‘A battery-powered activity-dependent intracortical microstimulation IC for brain-machine-brain interface,’’ IEEE J. Solid-State Circuits, vol. 46, no. 4, pp. 731–745, Apr. 2011. [10] R. C. Pinnell, J. Dempster, and J. Pratt, ‘‘Miniature wireless recording and stimulation system for rodent behavioural testing,’’ J. Neural Eng., vol. 12, no. 6, Aug. 2015, Art. no. 066015. [11] M. Arlotti, L. Rossi, M. Rosa, S. Marceglia, and A. Priori, ‘‘An exter- nal portable device for adaptive deep brain stimulation (aDBS) clinical research in advanced Parkinson’s Disease,’’ Med. Eng. Phys., vol. 38, pp. 498–505, May 2016. This neural recording device has been designed using discrete components rather than ASICs. Designing based on ASICs can help further reduce the device size and also minimize power consumption. Therefore, it is suggested as a future direction to design a multi-biomarker based neural recording device based on ASICs. [12] H.-G. Rhew, J. Jeong, J. A. Fredenburg, S. Dodani, P. G. Patil, and M. P. Flynn, ‘‘A fully self-contained logarithmic closed-loop deep brain stimulation SoC with wireless telemetry and wireless power management,’’ IEEE J. Solid-State Circuits, vol. 49, no. 10, pp. 2213–2227, Oct. 2014. [13] K. Abdelhalim, H. M. Jafari, L. Kokarovtseva, J. L. P. Velazquez, and R. Genov, ‘‘64-channel UWB wireless neural vector analyzer SOC with a closed-loop phase synchrony-triggered neurostimulator,’’ IEEE J. Solid- State Circuits, vol. 48, no. 10, pp. 2494–2510, Oct. 2013. V. CONCLUSION This paper presented a miniature (radius: 5.6 mm), light- weight (total weight: 1.2 g) tetherless, and self-contained dual-biomarker-based neural recording device. It was designed to be used in conjunction with a DBS device to create a closed-loop function for the adjustment of the stimu- lation parameters. LFPs and APs are sensed and conditioned simultaneously as the biomarkers for DBS pulse adjustments. This device performance was assessed through both bench and in-vitro tests. The bench tests were conducted using sinusoidal signals and pre-recorded neural signals. The in- vitro assessments were performed in the saline solution which is a brain environment simulator. 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https://openalex.org/W1999306148	https://digital.csic.es/bitstream/10261/155527/1/sensors-11-04840.pdf	English	null	Application of an E-Tongue to the Analysis of Monovarietal and Blends of White Wines	Sensors	2,011	cc-by	9,853	Sensors 2011, 11, 4840-4857; doi:10.3390/s110504840 Sensors 2011, 11, 4840-4857; doi:10.3390/s110504840 sensors ISSN 1424-8220 www.mdpi.com/journal/sensors OPEN ACCESS sensors ISSN 1424-8220 www.mdpi.com/journal/sensors OPEN ACCESS 1. Introduction Electronic tongues have been applied to many different fields in the last decades. However, it is in the food quality control and safety where the applicability of these biomimetic systems has been explored more [1]. Wine, which is a high added value product, is not an exception and to date one can find more than 60 papers in the bibliography describing different electronic tongues for classifying wines [2], quantifying parameters of interest for the wine-making industry [3] and even correlating with sensorial descriptors provided by an expert panel [4]. However, these electronic tongue systems are mainly optimized for the analysis of monovarietal wine samples [5,6]. The characterization, identification and even quantification of grape varieties in must and wine mixtures are of great interest to wine-makers [7]. Until now, most papers reporting the resolution of mixtures are based on the analysis of the residual grape DNA by using microsatellite markers [7-9]. This technique is highly reliable, but it requires several experimental steps (sample preparation, DNA extraction, microsatellite amplification and gel electrophoresis) and skilled personnel to carry out. Thus, it is interesting for winemakers to develop a simple and rapid method to characterize a must or wine blend. Recently, a review has covered the research done in the field of electronic and bioelectronic tongues for the analysis of wine samples [10]. However, one special point that was not covered enough in this review is the data fusion of various measurement techniques (potentiometry, amperometry, conductance, spectrophotometry, gas sensing). These systems are called hybrid electronic tongues because they merge variables of different nature. This approach was already reported in the end of nineties by Toko and coworkers as a powerful way to improve the performance of sensor technologies to the analysis of wines [11,12]. In this later work the combination of a taste sensor array [13] and a smell sensor array was applied to discriminate wines. This fact was reinforced by the formulation of the taste sense as the sum of taste, smell, texture, color, sound and temperature [14]. Since then, just four hybrid electronic tongues for wine have appeared in the bibliography, including those that also merge optical variables [4,15-17]. In our previous work [17], the application of a hybrid multiparametric system based on electrochemical and colorimetric measurements was presented. Keywords: hybrid electronic tongue; electrochemical microsensors; photonic lab on a chip; wine analysis; multivariate chemometric tools Keywords: hybrid electronic tongue; electrochemical microsensors; photonic lab on a chip; wine analysis; multivariate chemometric tools Application of an E-Tongue to the Analysis of Monovarietal and Blends of White Wines Manuel Gutiérrez 1, Andreu Llobera 1, Andrey Ipatov 1, Jordi Vila-Planas 1, Santiago Mínguez 2, Stefanie Demming 3, Stephanus Büttgenbach 3, Fina Capdevila 2, Carme Domingo 2 and Cecilia Jiménez-Jorquera 1,* 1 Instituto de Microelectrónica de Barcelona (IMB-CNM), CSIC. Campus UAB, 08193, Bellaterra, Spain; E-Mails: manuel.gutierrez@imb-cnm.csic.es (M.G.); andreu.llobera@imb-cnm.csic.es (A.L.); andrey.ipatov@imb-cnm.csic.es (A.I.); jordi.vila@imb-cnm.csic.es (J.V.-P.) 1 Instituto de Microelectrónica de Barcelona (IMB-CNM), CSIC. Campus UAB, 08193, Bellaterra, Spain; E-Mails: manuel.gutierrez@imb-cnm.csic.es (M.G.); andreu.llobera@imb-cnm.csic.es (A.L.); andrey.ipatov@imb-cnm.csic.es (A.I.); jordi.vila@imb-cnm.csic.es (J.V.-P.) 2 Estació de Viticultura i Enologia, Institut Català de la Vinya i el Vi (INCAVI). Plaça Àgora, 2-3, 08720, Vilafranca del Penedès, Spain; E-Mails: santiago.minguez@gencat.cat (S.M.); fcapdevila@gencat.cat (F.C.); carme.domingo@gencat.cat (C.D.) 2 Estació de Viticultura i Enologia, Institut Català de la Vinya i el Vi (INCAVI). Plaça Àgora, 2-3, 08720, Vilafranca del Penedès, Spain; E-Mails: santiago.minguez@gencat.cat (S.M.); fcapdevila@gencat.cat (F.C.); carme.domingo@gencat.cat (C.D.) 3 Institut für Mikrotechnik, Technische Universität Braunschweig, Alte Salzdahlumer Straβe 203, 38124, Braunschweig, Germany; E-Mails: s.demming@tu-bs.de (S.D.); s.buettgenbach@tu-bs.de (S.B.) * Author to whom correspondence should be addressed; E-Mail: cecilia.jimenez@imb-cnm.csic.es; Tel.: +34-93-594-7700; Fax: +34-93-580-1496. Received: 14 March 2011 / Accepted: 29 April 2011 / Published: 3 May 2011 Abstract: This work presents a multiparametric system capable of characterizing and classifying white wines according to the grape variety and geographical origin. Besides, it quantifies specific parameters of interest for quality control in wine. The system, known as a hybrid electronic tongue, consists of an array of electrochemical microsensors—six ISFET based sensors, a conductivity sensor, a redox potential sensor and two amperometric electrodes, a gold microelectrode and a microelectrode for sensing electrochemical oxygen demand—and a miniaturized optofluidic system. The test sample set comprised eighteen Catalan monovarietal white wines from four different grape varieties, two Croatian monovarietal white wines and seven bi- and trivarietal mixtures prepared from the Catalan varieties. Different chemometric tools were used to characterize (i.e., Principal Component Analysis), classify (i.e., Soft Independent Modeling Class Analogy) and quantify (i.e., Partial-Least Squares) some parameters of interest. The results demonstrate the usefulness of the multisensor system for analysis of wine. 4841 Sensors 2011, 11 Keywords: hybrid electronic tongue; electrochemical microsensors; photonic lab on a chip; wine analysis; multivariate chemometric tools Sensors 2011, 11 4842 Sensors 2011, 11 best models: Principal Component Analysis (PCA) combined with different strategies for characterization, Soft Independent Modeling Class Analogy (SIMCA) for classification and Partial-Least Squares (PLS) for quantification. Also in this work, the sample set is formed by 20 monovarietal white samples from six grape varieties. This group of samples was complemented with seven bi- and trivarietal wine mixtures. The analysis of these samples with the multiparametric system formed by the array of electrochemical microsensors and a colorimetric optofluidic system has demonstrated the feasibility of the system to classify white wines according to the grape varieties and to analyse some specific parameters. 2.1. Reagents and Solutions All reagents used were of high purity, analytical grade or equivalent. All solutions were prepared with de-ionized water. For ISFET calibration, solutions with ionic salts with concentrations between 10−2 and 10−4 M were prepared. In the case of those sensitive to cations (Na+, K+ and Ca2+), the corresponding chloride salts were considered. For the Cl− and NO3 − ions, solutions of NaCl and KNO3, respectively, were prepared. A solution containing 0.1 M KNO3 was used to activate the amperometric gold (Au) sensor. In order to calibrate the conductivity sensor, two different standard solutions from Crison (Barcelona, Spain), with nominal values of 1,413 and 147 µS/cm, were utilized. Two standard redox solutions from Panreac (Barcelona, Spain), with values of 468 mV and 220 mV, were used to calibrate the oxido-reduction potential (ORP) sensor. Finally, for the electrochemical oxygen demand (EOD) electrode, a solution containing 0.1 M NaOH and a glucose stock solution of 25 g/L were used. For the ISFET measurements, a reference solution containing an average concentration of the main species present in wine was prepared. The composition of this solution has been reported elsewhere [17]. 1. Introduction That was the first time in which a miniaturized optofluidic system was used as part of an electronic tongue for the analysis of white and red wines. Now, we present the next steps in which some improvements are incorporated to the system. First of all, the colorimetric analysis is performed using a multiple internal reflection (MIR) photonic chip fabricated by soft-lithography [18]. The main advantage of the MIR configuration is an increase in sensibility in comparison with the hollow prism configuration used in [17], thanks to the use of a longer optical path. This is especially important in the analysis of white wine, given the small absorbance values of this type of sample. Secondly, a flow cell has been fabricated in order to automate part of the measurements, concretely the potentiometric ones using Ion-Sensitive Field Effect Transistors (ISFETs). Besides, many different chemometric tools have been used in order to obtain the Sensors 2011, 11 one week. Two hours before the analysis, they were removed from the fridge to equilibrate at room temperature. During the analysis, the aliquots were successively opened and analyzed to prevent the possible changes of composition. Besides, two monovarietal white wines from Croatia based on Grasěvina and Zelenac grape varieties were included in the model and seven different wine mixtures were prepared from the Catalan samples, thus giving bivarietal and trivarietal wines, whose composition is shown in Table 1. These blends are typical for still white wines and sparkling wines by considering the oenological properties of these varieties: the Macabeu one gives light and fruity wines, the Xarel.lo gives wines with body and acidity and the Parellada, light and aromatic wines. Table 1. Composition of the seven wine mixtures prepared from the Catalan monovarietal samples. Mixture Macabeu Parellada Chardonnay Xarel·lo Sample (%) Sample (%) Sample (%) Sample (%) 1 17/07 33.33 79/07 33.33 - - 44/07 33.33 2 12/07 15 - - 01/07 85 - - 3 12/07 15 77/07 85 - - - - 4 12/07 15 - - - - 43/07 85 5 11/07 33.33 78/07 33.33 - - 45/07 33.33 6 10/07 66.66 80/07 33.33 - - - - 7 10/07 66.66 - - - - 45/07 33.33 Table 1. Composition of the seven wine mixtures prepared from the Catalan monovari mposition of the seven wine mixtures prepared from the Catalan monovarietal samples. Certain chemical parameters in these wines were determined by the INCAVI using standard methods. These methods are dictated by the European Union [19] and are well-established by the INCAVI laboratories. The routine protocol consists on one measurement for sample, without repetitions. Some of parameters, such as the volumetric alcoholic degree (VAD), the pH, the magnesium and calcium ions, are analyzed to evaluate the technological efficiency of the process, while others such as the total acidity are to meet legal limits. Some optical parameters were also determined by the INCAVI, such as the color of the wines by means of CIELab coordinates [20], the intensity of color and the tonality. These last two parameters correspond to the sum of absorbances at 620, 520 and 420 nm, and the ratio between the absorbance at 420 nm and 520 nm [19], respectively. 2.2. Wine Samples and Mixtures The set of samples consisted of 18 monovarietal (100%) white wines based on the following grape varieties: Macabeu (samples 09/07, 10/07, 11/07, 12/07 and 17/07), Parellada (samples 77/07, 78/07, 79/07, 80/07 and 81/07), Chardonnay (samples 01/07, 02/07, 03/07 and 04/07) and Xarel·lo (samples 43/07, 44/07, 45/07 and 63/07), all provided by the Catalan Institute of Vineyard and Wine (INCAVI). The index “07” indicates that the samples correspond to the 2007 vintage. All the samples have the same geographical origin (Penedes), except the Macabeu samples 11/07 and 12/07, which came from the Garraf zone. The winemaking process for all these samples is the same. It begins with the reception of the grapes in the winery. Processes of crushing, pressing and the addition of sulfites to the grape juice are performed successively. Then, a first clarification in order to eliminate the suspended solids and the alcoholic fermentation that transform the grape juice into wine are done. Once the wine is obtained, a second stage of clarification followed by stabilization and filtration steps and a second addition of sulfites are performed, before bottling the final product. Samples were provided by INCAVI in 750 mL bottles. Aliquots of 25 mL were collected from each wine sample in bottles protected against light and put into the refrigerator at 4 °C for a maximum of 4843 2.3. Hybrid Electronic Tongue A set of six ISFET sensors were fabricated using standard microelectronic technology [21]. One ISFET was used for measuring pH and the rest were modified with polymeric membranes sensitive to Na+, K+, Ca2+, Cl− and NO3 − ions. Polymeric membranes were based on photocurable polymers with commercial ionophores from Fluka (Buchs, Switzerland). The ionophores used in each case were: 4-tert-butylcalix[4]arenetetraacetic acid tetraethyl ester (Ionophore X) for Na+, valinomycin (Ionophore I) for K+, N,N,N′,N′-tetracyclohexyl-3-oxapentanediamide (Ionophore II, ETH 129) for Ca2+, tridodecylmethylammonium chloride for Cl− and tetraoctylammonium nitrate for NO3 −. All these ionophores are selective to the principal ion, but are not specific and they present a certain degree of cross-response to other ions in solution. Membrane composition, preparation and characterization has been presented elsewhere [22,23]. An Orion 90-02-00 double junction Ag/AgCl reference electrode (Thermo Electron, Waltham, MA, USA) with 0.1 M CH3COOLi solution in its outer chamber was Sensors 2011, 11 Sensors 2011, 11 4844 employed for all the potentiometric measurements. These were performed with the aid of a laboratory constructed data-acquisition system, which was connected to a computer through the RS-232 board. The readings were done employing specially designed software programmed with VisualBasic (Microsoft, Seattle, WA, USA). A flow cell (presented in Figure 1) to incorporate the ISFETs was made of a polymethyl-methacrylate (PMMA) plate with the help of a micro-milling machine (Stepfour GmbH, Salzburg, Austria). Different milled parts of the PMMA were glued together with methacrylic acid. For tight contact between the flow cell and the individual ISFET, a PDMS (polydimethylsiloxane) gasket formed in a special mould was used. To ensure that all the ISFET cells have no leakage three separate covers were made. The flow cell channel has 0.5 mm width and 0.5 mm high. Each ISFET has a 4 mm × 2 mm × 0.5 mm cell with a wall jet inlet configuration. The outlet from this cell is connected with inlet of the next ISFET, so all six ISFETs were placed sequentially. Standard 0.8 mm inner diameter tubing for flow injection analysis was connected to the inlet and outlet of the cell. The reference electrode (Orion) was mounted in a special flow-through cell connected to the output of the ISFET cell. Figure 1. (a) Picture of the flow cell employed for the ISFETs measurements and (b) a detailed scheme of an ISFET cell. Inlet Outlet ISFET PDMS gasket Supporting PMMA parts (b) Inlet Outlet ISFET PDMS gasket Supporting PMMA parts Inlet Outlet ISFET PDMS gasket Supporting PMMA parts (b) Sensors based on a Pt 4-electrode configuration were employed as conductivity sensor and ORP sensor. Their fabrication and characterization are reported elsewhere [24]. For signal conditioning and control data acquisition, a versatile and portable system developed at the IMB was used. This system was connected to the PC through the USB link. The control programs were based on LabView graphic language (National Instruments, Austin, TX, USA). T diff t ti i l d ti l A i l t d (b) (b) Inlet Outlet ISFET PDMS gasket Supporting PMMA parts (b) Inlet Outlet ISFET PDMS gasket Supporting PMMA parts Inlet Outlet ISFET PDMS gasket Supporting PMMA parts (b) Supporting PMMA parts Supporting PMMA parts Supporting PMMA parts PDMS gasket PDMS gasket PDMS gasket Sensors based on a Pt 4-electrode configuration were employed as conductivity sensor and ORP sensor. 2.4. Methodology Sample analysis was carried out under batch conditions, except for the ISFET set, which was performed under flow conditions. A complete analysis of a wine sample took around 15 min. No replications of each sample were done following the INCAVI protocol and also to get a rapid analysis and prevent changes of the wine sample. All the sensors that form the array were characterized before the analysis. The response characteristics are reported in [17], including the precision for the measurement of one wine sample. For the ISFET set, the output signals corresponded to the relative measurements of each sensor with respect to the reference solution, which was checked periodically. This is a common strategy to correct the possible drift of the sensors. The 4-electrode sensor was firstly chemically cleaned successively with ethanol 96%, H2SO4 6.0 M and de-ionized water. Calibration of the conductivity sensor was carried out using two standard solutions (1,413 and 147 µS/cm). For ORP sensor evaluation, a test with standard redox solutions of 220 and 468 mV (at 25 °C) versus the Ag/AgCl reference electrode was performed. Once the good behaviour of the sensors was confirmed, they were immersed in the wine sample and the signals were recorded every 30 seconds during 3 minutes. The Au microelectrode was firstly chemically cleaned as before, followed by an electrochemical activation carried out in 0.1 M KNO3 where the electrode was cycled from +0.8 to −2.2 V for at least 20 times. Then the electrode was immersed in the sample under studied and two cyclic voltammograms (CV) from +1.6 to −0.5 V at a scan rate of 0.1 V/s were run: the first one to stabilize the signal and the second one to obtain the information. Finally, the electrode was activated after measuring each wine samples by running five CV in 0.1 M KNO3. Regarding the electrochemical oxygen demand sensor (EOD) based on a CPE, previous studies in our laboratories have demonstrated the response of this electrode towards glucose and some other sugars. For this reason, analyses were performed using glucose as internal standard. Using the chronoamperometric mode and setting the potential at +600 mV, the CPE was immersed in NaOH 0.1 M. Then 50 µL of glucose 25 g/L was added. When the response became stable, 100 µL of the studied wine was added. Sensors 2011, 11 4845 Optical measurements were done using an MIR configuration fabricated in poly(dimethylsiloxane) (PDMS). The photonic lab on a chip comprises microlenses, self-alignment structures, microfluidic channels and air mirrors for highly sensitive absorbance measurements [18]. The light was emitted from broadband light source (Ocean Optics HL-2000, Redwood Shores, CA, USA). The readout multimode optical fibre was connected to a spectrometer (Ocean Optics HR-4000) with a spectral resolution of 2 nm. Using the Ocean Optics SpectraSuite software, the absorption of the sample from 200 to 1,100 nm wavelengths was recorded. Sensors 2011, 11 Their fabrication and characterization are reported elsewhere [24]. For signal conditioning and control data acquisition, a versatile and portable system developed at the IMB was used. This system was connected to the PC through the USB link. The control programs were based on LabView graphic language (National Instruments, Austin, TX, USA). Sensors based on a Pt 4-electrode configuration were employed as conductivity sensor and ORP sensor. Their fabrication and characterization are reported elsewhere [24]. For signal conditioning and control data acquisition, a versatile and portable system developed at the IMB was used. This system was connected to the PC through the USB link. The control programs were based on LabView graphic language (National Instruments, Austin, TX, USA). Two different amperometic microsensors were employed: a conventional Au microelectrode fabricated according to standard photolithographic techniques and a composite planar electrode (CPE) for sensing electrochemical oxygen demand developed by our group [25]. In both cases, the amperometric cell contained the working electrode, a Pt commercial electrode as counter electrode (Radiometer, Lyon, France) and a Ag/AgCl/10% (w/v) KNO3 reference electrode (Metrohm 0726 100, Herisau, Switzerland). A µ-Autolab potentiostat/galvanostat (Ecochemie, Utrecht, The Netherlands), using GPES 4.7 software package (General Purpose Electrochemical System) was used for all voltammetric and amperometric measurements. 2.5. Data Management Obtained data were treated using different multivariate methods. Principal Component Analysis (PCA) was used as the first technique to evaluate the characterization power of the system for monovarietal and mixture samples. The Soft Independent Modeling Class Analogy (SIMCA) method was also utilized to achieve a good classification model for the wine mixtures. The Partial Least Squares (PLS) regression was employed to perform the quantification of different parameters of the samples [26]. In this work, the PLS-1 algorithm was used in order to obtain more accurate predictions. For all these methods, the original values were previously autoscaled—all the variables were centered and set to a standard deviation equal to 1—to avoid variables from having a different influence on the model. Besides, all the obtained models were centered. For the PCA and SIMCA analyses, the cross-validation technique was used in order to ensure the performance of the generated model. In typical cross-validation, the training and validation sets of samples cross-over in successive iterations such that each sample has a chance of being validated against. Contrary, the test-set validation technique was used for the PLS regressions. In this case, a fixed calibration and prediction sets of samples are chosen. To control all these parameters and to perform the analyses, the Unscrambler v.9.1 informatics package (CAMO ASA, Oslo, Norway) was used. Sensors 2011, 11 Sensors 2011, 11 4846 between the experimental errors, confirming the reversibility of the photonic lab on a chip. Finally, for statistical purposes, for each wine type, the average of 10 consecutive scans was considered. 2.4. Methodology In this case, the output signals used to construct the model were the ratio of the wine signal to the glucose signal. In case of the photonic lab on a chip, measurements were carried out by filling the system with the sample to be analyzed. In order to obtain the spectra in absorbance units, de-ionized (DI) water was used as reference. Measurements were taken prior and after each wine to determine signal drifts due to non-specific adsorption at the walls. Throughout the experiments, the DI water reached the same value Table 2. Variables considered for constructing the models. Devices Variables ISFETs pH, Na+, K+, Ca2+, Cl− and NO3 − 4-bars electrode Au microelectrode Composite microelectrode Conductivity and ORP Current at 1.31 V, 1.01 V, 0.65 V and −0.38 V EOD Optofluidic system Absorbance values at 420, 520 and 620 nm 3.1. Input Variables for the Models Once all the samples were passed through the sensors, a data matrix was constructed with the different variables to be used as the input of the chemometric tool. These variables are fixed for ISFET sensors, conductivity, ORP and EOD sensors but the value of current and the absorbance depends on the specific response of the sensor to the samples. For this reason the election of the variables is an important step in a multivariable analysis. In other words, the goodness of the models generated by the algorithms depends essentially on the success of this choice. In the present study, the input data was composed by 16 variables as shown in Table 2. Table 2. Variables considered for constructing the models. Devices Variables ISFETs pH, Na+, K+, Ca2+, Cl− and NO3 − 4-bars electrode Au microelectrode Composite microelectrode Conductivity and ORP Current at 1.31 V, 1.01 V, 0.65 V and −0.38 V EOD Optofluidic system Absorbance values at 420, 520 and 620 nm Table 2. Variables considered for constructing the models. Table 2. Variables considered for constructing the models. Table 2. Variables considered for constructing the models. Devices Variables ISFETs pH, Na+, K+, Ca2+, Cl− and NO3 − 4-bars electrode Au microelectrode Composite microelectrode Conductivity and ORP Current at 1.31 V, 1.01 V, 0.65 V and −0.38 V EOD Optofluidic system Absorbance values at 420, 520 and 620 nm The input data of the six ISFETs correspond to the relative signal in mV of each ISFET with respect to the reference solution. It is also the case of the conductivity, ORP and EOD sensors, which give an absolute signal as a result. For the measurements with the Au microelectrode, cyclic voltammograms 4847 Sensors 2011, 11 (CV) were carried out. In Figure 2, the obtained CVs for four Catalan wines, one of each grape variety, are represented. The results show two redox peaks corresponding to the oxidation (+1.31 V) and reduction of Au (+0.65 V) from the electrode. Besides, two smaller peaks are observed at +1.01 V and at −0.38 V, which maybe correspond to the polyphenols content since their antioxidant capacity is well-known [27]. Therefore, the intensity of these four peaks was used as new variables. Also for the optofluidic system a spectrum of absorbance as function of the wavelength is obtained. In order to choose the variables, we applied the EU method that defines the color of wine at absorbance of 420, 520 and 620 nm [19]. With these variables, we obtained the maximum information about the wine samples. Figure 2. Cyclic voltammograms obtained with the Au microelectrode for four Catalan wines, one of each grape variety. Intensities for the indicated peaks were used as variables for the model. -30 -20 -10 0 10 20 30 40 50 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 E (V) vs. Ag/AgCl I (µA) Macabeu Parellada Chardonnay Xarel·lo I. +1.31 V I. -0.38 V I. +0.65 V I. +1.01 V -30 -20 -10 0 10 20 30 40 50 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 E (V) vs. Ag/AgCl I (µA) Macabeu Parellada Chardonnay Xarel·lo I. +1.31 V I. -0.38 V I. +0.65 V I. +1.01 V Macabeu Parellada Chardonnay Xarel·lo Macabeu Parellada Chardonnay Xarel·lo I. +1.31 V I. +1.31 V I. +1.01 V I. +1.01 V I (µA) I (µA) E (V) vs. Ag/AgCl E (V) vs. Ag/AgCl As an example of the values obtained by the different sensors, the response of three representative variables for the 20 monovarietal wines is represented in Figure 3. Figure 3. Response of sensors for the 20 monovarietal wines: (a) pH ISFET, (b) Au microelectrode at +1.01 V and (c) optofluidic system at 420 nm. Figure 3. Response of sensors for the 20 monovarietal wines: (a) pH ISFET, (b) Au microelectrode at +1.01 V and (c) optofluidic system at 420 nm. Sensors 2011, 11 0 5 10 15 20 25 30 35 40 Macabeu Parellada Chardonnay Xarel·lo Croatian ISFET pH (mV) (a) 0 5 10 15 20 25 30 35 40 Macabeu Parellada Chardonnay Xarel·lo Croatian ISFET pH (mV) (a) 0 2 4 6 8 10 12 Macabeu Parellada Chardonnay Xarel·lo Croatian Intensity at 1.01 V (µA) (b) 0 2 4 6 8 10 12 Macabeu Parellada Chardonnay Xarel·lo Croatian Intensity at 1.01 V (µA) (b) 4848 Sensors 2011, 11 Figure 3. Cont. 0 0.005 0.01 0.015 0.02 0.025 0.03 Macabeu Parellada Chardonnay Xarel·lo Croatian Absorbance at 420 nm (c) 0 0.005 0.01 0.015 0.02 0.025 0.03 Macabeu Parellada Chardonnay Xarel·lo Croatian Absorbance at 420 nm (c) For the pH ISFET [Figure 3(a)] similar values can be observed for the Macabeu and Parellada samples, and also comparable values for Chardonnay and Xarel·lo samples. However, values for the Croatian wines are significantly different due to the higher pH of these wines [28]. Figure 3(b) shows the intensity of current at +1.01 V using the gold microelectrode. The analysis of this graph shows homogeneous values for the Perallada group and more variability within the rest of grape varieties. For example, this is the case of the Macabeu samples, which form two groups with three and two samples, respectively. Finally, the results for the absorbance at 420 nm using the optofluidic system [Figure 3(c)] show again the differences between the Croatian wines and the rest. Besides, it can be observed some variability between the Catalan grape varieties with the highest values of absorbance for the Chardonnay group and the lowest for the Parellada group. Figure 3. Cont. 0 0.005 0.01 0.015 0.02 0.025 0.03 Macabeu Parellada Chardonnay Xarel·lo Croatian Absorbance at 420 nm (c) 0 0.005 0.01 0.015 0.02 0.025 0.03 Macabeu Parellada Chardonnay Xarel·lo Croatian Absorbance at 420 nm (c) Parellada Parellada Croatian Croatian For the pH ISFET [Figure 3(a)] similar values can be observed for the Macabeu and Parellada samples, and also comparable values for Chardonnay and Xarel·lo samples. However, values for the Croatian wines are significantly different due to the higher pH of these wines [28]. Figure 3(b) shows the intensity of current at +1.01 V using the gold microelectrode. The analysis of this graph shows homogeneous values for the Perallada group and more variability within the rest of grape varieties. Sensors 2011, 11 For example, this is the case of the Macabeu samples, which form two groups with three and two samples, respectively. Finally, the results for the absorbance at 420 nm using the optofluidic system [Figure 3(c)] show again the differences between the Croatian wines and the rest. Besides, it can be observed some variability between the Catalan grape varieties with the highest values of absorbance for the Chardonnay group and the lowest for the Parellada group. Sensors 2011, 11 Sensors 2011, 11 the PC 2 is composed basically by two variables of the Au microelectrode (intensities at +1.31 and +0.65 V). However, the three optical variables have a discrete contribution both to the first component (between 0.21 and 0.27) and to the second component (between 0.27 and 0.30). In other words, the electrochemical variables have a higher weight for the monovarietal white wine classification than the optical ones, and could be removed of this first model. Figure 4. PCA results for the 20 monovarietal wine samples: (a) scores plot and (b) loadings plot with a grey circumference which corresponds with the zone of low significance. -4 -3 -2 -1 0 1 2 3 4 -4 -2 0 2 4 6 8 PC 1 (41%) PC 2 (20%) Macabeu Parellada Chardonnay Xarel·lo Croatian Wines Zelenac Grasěvina (a) Catalan Wines -4 -3 -2 -1 0 1 2 3 4 -4 -2 0 2 4 6 8 PC 1 (41%) PC 2 (20%) Macabeu Parellada Chardonnay Xarel·lo Croatian Wines Zelenac Grasěvina (a) Catalan Wines -0.6 -0.4 -0.2 0 0.2 0.4 0.6 -0.5 -0.3 -0.1 0.1 0.3 PC 1 (41%) PC 2 (20%) ISFET Cl- Cond. EOD ISFET NO3 - ORP I. 1.01 V I. 1.31 V Abs. 420 nm pH I. 0.65 V ISFET K+ ISFET Na+ ISFET Ca2+ Abs. 620 nm I. -0.38 V (b) Abs. 520 nm -0.6 -0.4 -0.2 0 0.2 0.4 0.6 -0.5 -0.3 -0.1 0.1 0.3 PC 1 (41%) PC 2 (20%) ISFET Cl- Cond. EOD ISFET NO3 - ORP I. 1.01 V I. 1.31 V Abs. 420 nm pH I. 0.65 V ISFET K+ ISFET Na+ ISFET Ca2+ Abs. 620 nm I. -0.38 V (b) Abs. 520 nm next study performed with this system was the characterization of the 18 Catalan wine s PCA was performed using the raw data obtained from the selected variables. In Figure t principal components, which explain 52% of the total variance, are represented. In thi e four varieties are distributed in the four quadrants: Macabeu, Parellada, Chardon o. Sensors 2011, 11 However, the two Macabeu samples from the Garraf zone (Mac 12/07 and Mac 11 d f th f th P d d th f di ti i h d Th -4 -3 -2 -1 0 1 2 3 4 -4 -2 0 2 4 6 8 PC 2 (20%) Macabeu Parellada Chardonnay Xarel·lo Croatian Wines Zelenac Grasěvina (a) Catalan Wines -4 -3 -2 -1 0 1 2 3 4 -4 -2 0 2 4 6 8 PC 2 (20%) Macabeu Parellada Chardonnay Xarel·lo Croatian Wines Zelenac Grasěvina (a) Catalan Wines Macabeu Parellada Chardonnay Xarel·lo Croatian Wines Macabeu Parellada Chardonnay Xarel·lo Croatian Wines (a) (a) Grasěvina Grasěvina 8 Catalan Wines Catalan Wines -4 PC 1 (41%) -4 PC 1 (41%) -0.6 -0.4 -0.2 0 0.2 0.4 0.6 -0.5 -0.3 -0.1 0.1 0.3 PC 1 (41%) PC 2 (20%) ISFET Cl- Cond. EOD ISFET NO3 - ORP I. 1.01 V I. 1.31 V Abs. 420 nm pH I. 0.65 V ISFET K+ ISFET Na+ ISFET Ca2+ Abs. 620 nm I. -0.38 V (b) Abs. 520 nm -0.6 -0.4 -0.2 0 0.2 0.4 0.6 -0.5 -0.3 -0.1 0.1 0.3 PC 1 (41%) PC 2 (20%) ISFET Cl- Cond. EOD ISFET NO3 - ORP I. 1.01 V I. 1.31 V Abs. 420 nm pH I. 0.65 V ISFET K+ ISFET Na+ ISFET Ca2+ Abs. 620 nm I. -0.38 V (b) Abs. 520 nm 4 PC 1 (41%) 4 PC 1 (41%) -0.6 -0.4 -0.2 0 0.2 0.4 0.6 -0.5 -0.3 -0.1 0.1 0.3 PC 1 (41%) PC 2 (20%) ISFET Cl- Cond. EOD ISFET NO3 - ORP I. 1.01 V I. 1.31 V Abs. 420 nm pH I. 0.65 V ISFET K+ ISFET Na+ ISFET Ca2+ Abs. 620 nm I. -0.38 V (b) Abs. 520 nm -0.6 -0.4 -0.2 0 0.2 0.4 0.6 -0.5 -0.3 -0.1 0.1 0.3 PC 1 (41%) PC 2 (20%) ISFET Cl- Cond. EOD ISFET NO3 - ORP I. 1.01 V I. 1.31 V Abs. 420 nm pH I. 0.65 V ISFET K+ ISFET Na+ ISFET Ca2+ Abs. 620 nm I. -0.38 V (b) Abs. 520 nm (b) (b) The next study performed with this system was the characterization of the 18 Catalan wine samples. A new PCA was performed using the raw data obtained from the selected variables. In Figure 5(a) the two first principal components, which explain 52% of the total variance, are represented. 3.2. Characterization of the Monovarietal Samples In order to prove the capability of the system to differentiate monovarietal samples from different geographical origin, a PCA was performed using the 20 monovarietal wines: the 18 Catalan samples and the two Croatian samples. These Croatian wines are based on two characteristic grape varieties from the Slavonia zone: Grasěvina White and Zelenac. In Figure 4(a) is represented the scores plot with the two first principal components (PC), which explain 61% of the total variance. In this model, the first clear conclusion is that the Croatian wines are outliers of the population; they are completely different from the Catalan white wines. Grasěvina and Zelenac grape varieties are very distinguishable even between each other. On the contrary, the Catalan wines are intermixed forming a unique group. However, a slight separation is glimpsed between the Macabeu and Parellada samples placed in the negative PC1 axis and the Chardonnay and the Xarel·lo samples placed in the positive PC1 axis. It is interesting to analyze which is the weight of each original variable in the constructed model. We can observe in Figure 4(b) that all the used variables are located far from the origin (0, 0), except the EOD sensor that is situated in the border of the low-significant zone. The significance of the variables is directly related with the distance to the origin: the more away, the more importance in the model. Therefore, the pH ISFET has the biggest weight in the PC 1, which explains the 41% of the variance. The ORP sensor and the ISFETs for K+ and Ca2+ have also importance in the first PC, while 4849 Sensors 2011, 11 Macabeu wines correspond to the samples with lower values for the intensity of current at +1.01 V [Figure 3(b)]. Besides, some variability inside the Parellada, Chardonnay and Xarel·lo groups can be observed. As can be observed in Figure 5(b), the PC 3 of this model explains 14% of the variation of the Catalan wines and clearly separates the two Macabeu samples from Garraf of the rest of the Macabeu samples from Penedès. On the other hand, this third component does not discriminate between the Macabeu and Parellada wines, nor between the Chardonnay and Xarel.lo wines. In the loadings plot of this new model (data non-shown), the most important variables are again the electrochemical ones: pH ISFET and the variables of the Au microelectrode (intensities at +1.01, +0.65 and −0.38 V). However, the optical variables have more weight in comparison with the previous model, especially the absorbance at 620 nm that has a contribution of 0.32 in the first PC. Figure 5. PCA scores plots for the monovarietal Catalan wines using the values obtained with the hybrid system: (a) PC 1 (35%) vs. PC 2 (17%) and (b) PC 1 (35%) vs. PC 3 (14%). Sensors 2011, 11 In this scores plot, the four varieties are distributed in the four quadrants: Macabeu, Parellada, Chardonnay and Xarel·lo. However, the two Macabeu samples from the Garraf zone (Mac 12/07 and Mac 11/07) are separated of those from the Penedes zone and they form a new distinguished group. These two 4850 Sensors 2011, 11 -4 -3 -2 -1 0 1 2 3 4 -6 -4 -2 0 2 4 6 PC 1 (35%) PC 2 (17%) Xar 43 Xar 44 Xar 45 Xar 63 Char 01 Char 04 Char 03 Char 02 Mac 10 Mac 09 Mac 17 Mac 11 Mac 12 Par 77 Par 78 Par 79 Par 80 Par 81 (a) -4 -3 -2 -1 0 1 2 3 4 -6 -4 -2 0 2 4 6 PC 1 (35%) PC 2 (17%) Xar 43 Xar 44 Xar 45 Xar 63 Char 01 Char 04 Char 03 Char 02 Mac 10 Mac 09 Mac 17 Mac 11 Mac 12 Par 77 Par 78 Par 79 Par 80 Par 81 (a) -4 -3 -2 -1 0 1 2 3 4 -6 -4 -2 0 2 4 6 PC 1 (35%) PC 2 (17%) Xar 43 Xar 44 Xar 45 Xar 63 Char 01 Char 04 Char 03 Char 02 Mac 10 Mac 09 Mac 17 Mac 11 Mac 12 Par 77 Par 78 Par 79 Par 80 Par 81 (a) -3 -2 -1 0 1 2 3 4 -6 -4 -2 0 2 4 6 PC 1 (35%) PC 3 (14%) Macabeu Parellada Chardonnay Xarel·lo Mac 11 Mac 12 (b) -3 -2 -1 0 1 2 3 4 -6 -4 -2 0 2 4 6 PC 1 (35%) PC 3 (14%) Macabeu Parellada Chardonnay Xarel·lo Mac 11 Mac 12 (b) -4 -3 -2 -1 0 1 2 3 4 -6 -4 -2 0 2 4 6 PC 2 (17%) Xar 43 Xar 44 Xar 45 Xar 63 Char 01 Char 04 Char 03 Char 02 Mac 10 Mac 09 Mac 17 Mac 11 Mac 12 Par 77 Par 78 Par 79 Par 80 Par 81 (a) -4 -3 -2 -1 0 1 2 3 4 -6 -4 -2 0 2 4 6 PC 2 (17%) Xar 43 Xar 44 Xar 45 Xar 63 Char 01 Char 04 Char 03 Char 02 Mac 10 Mac 09 Mac 17 Mac 11 Mac 12 Par 77 Par 78 Par 79 Par 80 Par 81 (a) -4 -3 -2 -1 0 1 2 3 4 -6 -4 -2 0 2 4 6 PC 2 (17%) Xar 43 Xar 44 Xar 45 Xar 63 Char 01 Char 04 Char 03 Char 02 Mac 10 Mac 09 Mac 17 Mac 11 Mac 12 Par 77 Par 78 Par 79 Par 80 Par 81 (a) (a) (a) (a) Char 02 Char 02 Char 02 PC 1 (35%) PC 1 (35%) PC 1 (35%) -3 -2 -1 0 1 2 3 4 -6 -4 -2 0 2 4 6 PC 1 (35%) PC 3 (14%) Macabeu Parellada Chardonnay Xarel·lo Mac 11 Mac 12 (b) -3 -2 -1 0 1 2 3 4 -6 -4 -2 0 2 4 6 PC 1 (35%) PC 3 (14%) Macabeu Parellada Chardonnay Xarel·lo Mac 11 Mac 12 (b) 4851 Sensors 2011, 11 In order to compare the capability of our system to differentiate Catalan wine samples with the data from standard methods, a PCA using the chemical and optical data provided by the INCAVI was performed. The obtained scores plot along the first two components is shown in Figure 6. The white wine samples are aligned on the plot according to the grape variety along the first principal component, which explains 43% of the variance. Besides, the second component increases the dispersion of the Parellada samples, which seem to form two groups, as we observed in the PCA using the hybrid system [Figure 5(a)]. This PC 2 distinguishes Char 04/07 from the other Chardonnay samples and separates better the Xarel·lo group from the rest. Besides, the two Macabeu samples from the Garraf zone are also separated of the rest of the Macabeu group. In this case, these two samples are intermixed with the Chardonnay group. This fact confirms the differences within the Macabeu group depending on the geographical origin that are detected not only by the hybrid electronic tongue but also by data from standard methods of analysis. However, it is noticeable the highest power of wine samples classification of our system. Figure 6. PCA scores plot for the monovarietal Catalan wines using the chemical and optical parameters determined by standard methods of analysis. -3 -2 -1 0 1 2 3 -5 -3 -1 1 3 5 PC 1 (43%) PC 2 (21%) Xar 43 Xar 44 Xar 45 Char 01 Char 04 Char 03 Char 02 Mac 10 Mac 09 Mac 17 Mac 11 Mac 12 Par 77 Par 78 Par 79 Par 80 Par 81 -3 -2 -1 0 1 2 3 -5 -3 -1 1 3 5 PC 1 (43%) PC 2 (21%) Xar 43 Xar 44 Xar 45 Char 01 Char 04 Char 03 Char 02 Mac 10 Mac 09 Mac 17 Mac 11 Mac 12 Par 77 Par 78 Par 79 Par 80 Par 81 -3 -2 -1 0 1 2 3 -5 -3 -1 1 3 5 PC 1 (43%) PC 2 (21%) Xar 43 Xar 44 Xar 45 Char 01 Char 04 Char 03 Char 02 Mac 10 Mac 09 Mac 17 Mac 11 Mac 12 Par 77 Par 78 Par 79 Par 80 Par 81 3.3. Sensors 2011, 11 4852 Sensors 2011, 11 order to study in more detail the feasibility of the hybrid electronic tongue to distinguish between the monovarietal (100%) wines and the mixtures, the SIMCA classification method was applied. In Figure 7(b), the Coomans diagram (probability of 75%) obtained for the classification of the seven wine mixtures among the Macabeu and Xarel·lo models is depicted. These two groups are studied because they are well characterized by the system. In the x-axis of the Coomans diagram, the mixture distance to the Macabeu model is shown. Thus, mixtures that are placed between 0 and 1.75 on the x-axis belong to the Macabeu model. On the y-axis, the mixture distance to the Xarel·lo model is shown. Hence, mixtures that are placed between 0 and 1.75 on the y-axis belong to this group. Mixtures that are placed in the area corresponding to x, y > 1.75 do not belong to any of the represented models and can be distinguished from the monovarietal wine samples. Contrary, mixtures that are located in the area x, y < 1.75 of the plot belong to both represented models. As can be observed, the monovarietal Macabeu wines (blue diamonds) are located inside their model and are well separated of the rest of wine mixtures. It is important to say that all these mixtures have at least 15% of a Macabeu wine. However, the two Macabeu samples from the Garraf zone (Mac 11 and 12) are located outside the Macabeu group, which is formed by the Penedes samples, confirming that the hybrid system is capable of differentiating between them. At the same time, the monovarietal Xarel·lo wines (pink squares) are also inside their own model and are well distinguished of the rest of wine mixtures, in general. The case of the Xar 43/07 and the mixture with 85% Xarel·lo is remarkable in that, with a y value of 1.70 and 1.71 respectively, are just in the border of the Xarel·lo model. It is important to say that although the probability of the classification model is 75%, this is a first step to evaluate the feasibility of the system in the resolution of wine mixtures. Figure 7. (a) Interpolation of the multivarietal mixtures in the PCA model; (b) Coomans diagram for the Macabeu and Xarel·lo models with a probability of 75%. Sensors 2011, 11 Characterization of the Bi- and Trivarietal Samples Xar 44 Xar 44 Xar 44 Xar 43 Xar 43 Xar 43 Par 81 Par 81 Par 81 Xar 45 Xar 45 Xar 45 5 Char 02 Char 02 Char 02 Char 03 Char 03 Char 03 Mac 11 Mac 11 Mac 11 3.3. Characterization of the Bi- and Trivarietal Samples Once the model using raw data from the sensor variables was constructed, the values obtained for the seven mixtures were interpolated in the PCA. The situation of these samples inside the model is shown in Figure 7(a). As can be observed, trivarietal mixtures, which are formed by 33.33% of each variety, tend to be near the origin (0,0). Bivarietal samples with 85% of one variety are inside the group of the major variety, being almost impossible to distinguish them from the monovarietal wines. However, the mixtures that content 66.66% Mac 10/07 and 33.33% Parellada or Xarel·lo seem to be halfway between the two groups, as if there is a gradation in terms of percentage of each variety. In Sensors 2011, 11 The relative standard errors of the calibration set (RSEC) and the prediction set (RSEP) were calculated in order to compare the precision of the different PLS regressions. The RSEC values were between 1.0 and 5.0% for all the determinations and the RSEP values were between 2.2 and 7.6%, without excluding any value. Figure 7. Cont. 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 8 9 Mixture distance to Macabeu Mixture distance to Xarel·lo Macabeu 100% Xarel·lo 100% Bivarietal Mixtures Trivarietal Mixtures (b) p = 75% p = 75% Mac 11 Mac 12 Xar 43 85% Xar 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 8 9 Mixture distance to Macabeu Mixture distance to Xarel·lo Macabeu 100% Xarel·lo 100% Bivarietal Mixtures Trivarietal Mixtures (b) p = 75% p = 75% Mac 11 Mac 12 Xar 43 85% Xar 3.4. Quantification of Chemical and Optical Parameters Figure 7. Cont. Figure 7. Cont. Figure 7. Cont. 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 8 9 Mixture distance to Macabeu Mixture distance to Xarel·lo Macabeu 100% Xarel·lo 100% Bivarietal Mixtures Trivarietal Mixtures (b) p = 75% p = 75% Mac 11 Mac 12 Xar 43 85% Xar 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 8 9 Mixture distance to Macabeu Mixture distance to Xarel·lo Macabeu 100% Xarel·lo 100% Bivarietal Mixtures Trivarietal Mixtures (b) p = 75% p = 75% Mac 11 Mac 12 Xar 43 85% Xar Mixture distance to Macabeu Mixture distance to Macabeu 3.4. Quantification of Chemical and Optical Parameters Next, a PLS was realized in order to assess if the system was able to quantify some quality parameters of the samples already analyzed with standard methods. For the regression, the calibration set was formed by 14 samples: four Macabeu samples (10/07, 11/07, 12/07 and 17/07), four Parellada samples (78/07, 79/07, 80/07 and 81/07), three Chardonnay samples (01/07, 03/07 and 04/07) and three Xarel·lo samples (43/07, 45/07 and 63/07). The rest of the samples were used as the prediction data set. The relative standard errors of the calibration set (RSEC) and the prediction set (RSEP) were calculated in order to compare the precision of the different PLS regressions. Sensors 2011, 11 -4 -3 -2 -1 0 1 2 3 4 -6 -4 -2 0 2 4 6 PC 1 (35%) PC 2 (17%) Xarel·lo Chardonnay Macabeu Mac 11 Mac 12 Parellada (a) Trivarietal Mixtures 33% Xar 85% Char 85% Xar 33% Par 85% Par Mac 10 -4 -3 -2 -1 0 1 2 3 4 -6 -4 -2 0 2 4 6 PC 1 (35%) PC 2 (17%) Xarel·lo Chardonnay Macabeu Mac 11 Mac 12 Parellada (a) Trivarietal Mixtures 33% Xar 85% Char 85% Xar 33% Par 85% Par Mac 10 -4 -3 -2 -1 0 1 2 3 4 -6 -4 -2 0 2 4 6 PC 1 (35%) PC 2 (17%) Xarel·lo Chardonnay Macabeu Mac 11 Mac 12 Parellada (a) Trivarietal Mixtures 33% Xar 85% Char 85% Xar 33% Par 85% Par Mac 10 -4 -3 -2 -1 0 1 2 3 4 -6 -4 -2 0 2 4 6 PC 1 (35%) PC 2 (17%) Xarel·lo Chardonnay Macabeu Mac 11 Mac 12 Parellada (a) Trivarietal Mixtures 33% Xar 85% Char 85% Xar 33% Par 85% Par Mac 10 (a) (a) Chardonnay Chardonnay 4853 Sensors 2011, 11 Figure 7. Cont. 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 8 9 Mixture distance to Macabeu Mixture distance to Xarel·lo Macabeu 100% Xarel·lo 100% Bivarietal Mixtures Trivarietal Mixtures (b) p = 75% p = 75% Mac 11 Mac 12 Xar 43 85% Xar 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 8 9 Mixture distance to Macabeu Mixture distance to Xarel·lo Macabeu 100% Xarel·lo 100% Bivarietal Mixtures Trivarietal Mixtures (b) p = 75% p = 75% Mac 11 Mac 12 Xar 43 85% Xar 3.4. Quantification of Chemical and Optical Parameters Next, a PLS was realized in order to assess if the system was able to quantify some quality parameters of the samples already analyzed with standard methods. For the regression, the calibration set was formed by 14 samples: four Macabeu samples (10/07, 11/07, 12/07 and 17/07), four Parellada samples (78/07, 79/07, 80/07 and 81/07), three Chardonnay samples (01/07, 03/07 and 04/07) and three Xarel·lo samples (43/07, 45/07 and 63/07). The rest of the samples were used as the prediction data set. Sensors 2011, 11 4854 The data of optical and chemical parameters for the prediction set obtained with our system and with standard methods are shown in Tables 3 and 4, respectively. The interpolated values are in good agreement with the data obtained using standard methods. The relative errors are below 10% for all the predictions, except for the determination of glycerol in the 44/07 Xar sample. Especially good results are obtained for VAD, pH and magnesium predictions, with errors below 5%. Besides, the inclusion of the three optical variables permits to quantify with a high precision the intensity of color and the tonality of the wine. As can be observed, for these two parameters the errors are in general below 5%. Table 4. Quantification with PLS technique of some chemical parameters of white wine samples with the system. Standard method’s data were provided by INCAVI and relative error refers to these data. Sample Standard method Multiparametric system Relative error (%) VAD (%) Macabeu 09/07 9.59 9.86 2.82 Parellada 77/07 9.37 9.38 0.07 Chardonnay 02/07 11.90 12.30 3.32 Xarel·lo 44/07 13.86 13.77 −0.62 Total acidity (g/L) Macabeu 09/07 5.9 5.7 −3.7 Parellada 77/07 7.7 8.4 9.5 Chardonnay 02/07 8.0 7.8 −2.7 Xarel·lo 44/07 5.7 5.8 1.0 pH Macabeu 09/07 3.03 3.01 −0.79 Parellada 77/07 2.93 2.83 −3.55 Chardonnay 02/07 3.10 3.14 1.16 Xarel·lo 44/07 3.34 3.18 −4.76 Calcium (mg/L) Macabeu 09/07 71 70 −0.7 Parellada 77/07 69 72 5.1 Chardonnay 02/07 59 62 5.7 Xarel·lo 44/07 43 45 4.0 Magnesium (mg/L) Macabeu 09/07 48 46 −4.9 Parellada 77/07 43 43 −0.1 Chardonnay 02/07 65 64 −2.2 Xarel·lo 44/07 46 45 −2.8 Glycerol (g/L) Macabeu 09/07 6.1 6.5 6.0 Parellada 77/07 4.8 4.9 2.0 Chardonnay 02/07 7.6 7.3 −4.3 Xarel·lo 44/07 6.3 7.1 12.3 Sensors 2011, 11 The RSEC values were between 1.0 and 5.0% for all the determinations and the RSEP values were between 2.2 and 7.6%, without excluding any value. Table 3. Quantification with PLS technique of some optical parameters of white wine samples with the system. Standard method’s data were provided by INCAVI and relative error refers to these data. Sample Standard method Multiparametric system Relative error (%) Intensity of color Macabeu 09/07 0.0540 0.0565 4.67 Parellada 77/07 0.0520 0.0544 4.56 Chardonnay 02/07 0.0700 0.0706 0.87 Xarel·lo 44/07 0.0960 0.103 7.29 Tonality Macabeu 09/07 4.777 4.829 1.09 Parellada 77/07 4.777 4.952 3.66 Chardonnay 02/07 5.272 5.403 2.48 Xarel·lo 44/07 3.000 2.873 −4.23 Sensors 2011, 11 4855 Sensors 2011, 11 to obtain the maximum sample information. Then, this obtained data is treated with powerful chemometric tool to extract useful chemical information. The qualitative results using PCA technique confirm that this system is capable of distinguishing the samples according to the grape origin and even to the geographical origin. Using the SIMCA classification technique, the ability of the system to differentiate between the monovarietal samples and their mixtures has been demonstrated with a probability of 75%. In addition, the application of the PLS technique to the collected data, permits one to quantify some chemical and optical parameters with relative errors smaller than 10% in general, obtaining especially good results for the prediction of VAD, pH, magnesium and tonality. Even though the electrochemical variables seem to be more significant for these analyses, the optical ones cannot be undervalued because they are essential for some applications, like the correct differentiation of the Catalan wine set and the quantification of optical parameters. In conclusion, good results are obtained both for discrimination and quantification methodologies that confirm the viability of the multisensor system. Ongoing experiments are focused on processing a higher number of samples, from different varieties, vintage years and origin to improve the reliability of the system. Moreover, an important effort will be performed to analyze qualitatively bi- and trivarietal samples and even to quantify the proportion of each grape variety presents in the wine mixture. Besides the integration of sensors in the same substrate in order to obtain a compact system that could be applied to flow measurements will permit more automated and feasible analysis. Acknowledgements We acknowledge funding from the Spanish R & D National Program (MICINN Project TEC2007-68012-C03-01/03), the CIDEM network GTQ-Tecnio and the Juan de la Cierva scholarship (Manuel Gutiérrez). The technical assistance of Nidia Santamaría, Alfredo Cadarso and Héctor Cabezas is highly appreciated. 4. 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https://openalex.org/W4383960893	https://e-journal.sttexcelsius.ac.id/index.php/excelsisdeo/article/download/96/113	Indonesian	null	KEBENARAN ALKITAB MENDEWASAKAN UMAT ALLAH MENURUT II TIMOTIUS 3:14-16	Excelsis Deo	2,022	cc-by-sa	7,984	UTH MAKES GOD'S PEOPLE GROW ACCORDING TO II TIMOTHY 3:14-16 Abstract: The Bible is the Word of God, the book of all books, and the greatest miracle in the history of mankind. The benefits that we can get when we study it are truly incalculable. The Bible is the ultimate authority and true source of truth. In his letter to Timothy, the apostle Paul emphasized the importance of God's Word. He wrote, “You must hold fast to the things you have learned and about which you have been persuaded to believe.” The “things” that Paul mentioned were Bible truths that moved Timothy to faith in the good news. Research Methods: The method used in this research is a qualitative method of literature with a descriptive approach and the objectives to be achieved by the author in this study are (1) to explain the meaning of the Bible etymologically, namely, the Bible is useful for educating people in the truth. After the Bible corrects behavior, He is also willing to lead people in the direction God wants, as long as we approach Him (2) provide evidence of Bible truth. All Scriptures that are inspired by God are indeed useful for teaching, for rebuking, for correcting behavior and for educating people in the truth' (3) explaining the importences of the Bible on the faith of believers who grow to maturity first, the Bible is useful for teaching, second, God's Word Useful for declaring error, Third Bible is useful for Correcting behavior, Fourth Bible Useful for educating people in righteousness. g p p g Keywords: Bible, Teaching, Error, Correction, Behavior, Educate, Truth Keywords: Bible, Teaching, Error, Correction, Behavior, Educate, Truth Abstrak: Alkitab adalah Firman Tuhan, buku dan mukjizat terbesar dalam sejarah umat manusia. Manfaat yang bisa kita dapatkan ketika kita mempelajarinya sungguh tak terhitung. Alkitab adalah otoritas tertinggi dan sumber kebenaran yang sejati. Dalam suratnya kepada Timotius, rasul Paulus menekankan pentingnya Firman Tuhan. Dia menulis, “Kamu harus berpegang teguh pada hal-hal yang telah kamu pelajari dan yang tentangnya kamu telah dibujuk untuk percaya.” ”Hal-hal” yang Paulus sebutkan adalah kebenaran Alkitab yang menggerakkan Timotius kepada iman akan kabar baik. Metode Penelitian : Metode yang digunakan dalam penelitian ini adalah metode kualitatif kepustakaan dengan pendekatan deskriptif dan tujuan yang ingin dicapai oleh penulis dalam penelitian ini adalah (1) menjelaskan pengertian alkitab secara etimologis yaitu alkitab berguna untuk mendidik manusia dalam kebenaran. Jurnal Excelsisi Deo: Vol. 6 No.1 Juni 2022 Jurnal Excelsisi Deo: Vol. 6 No.1 Juni 2022 Kata Kunci: Alkitab, Mengajar,Kesalahan,perbaiki,Kelakuan,mendidik, Kebenaran Jurnal Excelsis Deo: Teologi, Misiologi, dan Pendidikan\| 99 KEBENARAN ALKITAB MENDEWASAKAN UMAT ALLAH MENURUT II TIMOTIUS 3:14-16 Yulian Anouw1* 1Universitas Kristen Papua (UKIP) *Email: Anouwyulian55@gmail.com UTH MAKES GOD'S PEOPLE GROW ACCORDING TO II TIMOTHY 3:14-16 Setelah Alkitab mengoreksi perilaku, Dia juga bersedia memimpin orang ke arah yang Tuhan inginkan, selama kita mendekati-Nya (2) memberikan bukti kebenaran Alkitab. Semua Kitab Suci yang diilhamkan Tuhan memang berguna untuk mengajar, untuk menegur, untuk mengoreksi tingkah laku dan untuk mendidik manusia dalam kebenaran' (3) menjelaskan pentingnya Alkitab terhadap iman orang percaya yang bertumbuh menjadi dewasa terlebih dahulu, Alkitab adalah berguna untuk pengajaran, kedua, Firman Tuhan Berguna untuk menyatakan kesalahan, Alkitab Ketiga berguna untuk Memperbaiki perilaku, Alkitab Keempat Berguna untuk mendidik orang dalam kebenaran. Jurnal Excelsis Deo: Teologi, Misiologi, dan Pendidikan\| 99 Jurnal Excelsisi Deo: Vol. 6 No.1 Juni 2022 1Henry C. Thiessen, Teologi Sistematika (Malang: Gandum Mas,1995), 66. 2Charles C. Ryrie, Teologi Dasar I (Yogyakarta: Yayasan Andi, 1991), 103. 3Ibid.,68. 100 \| Jurnal Excelsis Deo: Teologi, Misiologi, dan Pendidikan 4S. Wismoady Wahono, Di Sini Kutemukan (Jakarta: Gunung Mulia,1998), 22. PENDAHULUAN Dasar I sebagai be-rikut: “Mereka yang menentang atau ingin mengurangi pen- tingnya ketidak keliruan Alkitab sering membuat per-nyataan ini: “Karena Alkitab tidak jelas mengajarkannya, demikian pula kami”. Paling sedikit pernyataan ini menempatkan mereka yang menekankan pentingnya ketidakkeliruan Alkitab pada posisi mau melebihi Alkitab. Maksimalnya mereka ingin menegaskannya bahwa ketidak keliruan bukanlah ajaran Alkitab.2 Alkitab marupakan kitab unik dan luarbiasa, oleh karena keunikannya ini, maka penulis sangat tertarik untuk menulis skripsi dengan judul, Kebenaran Alkitab Mendewasakan Umat Allah Menurut II Timotius 3:16. Selain itu penulisan ini juga dilator belakangi oleh beberapa masalah antara lain: Pertama, ada keraguan tentang kebenaran Alkitab sebagai manifestasi dari wahyu Tuhan, seperti yang dikatakan Henry C. Thiessen dalam bukunya Systematic Theology sebagai berikut: Mengakui bahwa penalaran ini tidak dapat memberikan keyakinan yang lebih dalam daripada keyakinan bahwa sangat mungkin bahwa Alkitab adalah bentuk wahyu ilahi. . Karena walaupun Tuhan adalah kasih dan kasih itu dipakai dalam Ketuhanan, selain pernyataan yang jelas dengan maksud itu tidak tahu apakah Tuhan yang pengasih juga mengasihi orang berdosa, karena jika demikian, maka kasih tidak lagi kasih, belas kasihan tidak lagi jadilah rahmat dan kasih karunia bukan lagi kasih karunia. Ketiga, adanya usaha untuk memusnahkan Alkitab dan membatasi pengajaran-pengajarannya bagi kaum awam pada masa pemerintahan Romawi, salah satunya ialah Kaisar Diocletianus melalui dekrit pada tahun 303 M. Hal ini dinyatakan oleh Henry C. Thiessen dalam bukunya yang berjudul Systematic Theology sebagai berikut: 'Kaisar Diocletian menuntut agar setiap jilid Alkitab dibakar. Dia membunuh begitu banyak orang Kristen dan menghancurkan Alkitab sehingga dia merasa telah berhasil menghancurkan Alkitab sepenuhnya ketika orang-orang Kristen begitu menganggur selama beberapa waktu. Diocletian berkata, "Kekristenan telah binasa dan penyembahan para dewa telah diilhami.3 Kemauan harus dilestarikan dalam ketiganya, karena manusia kehilangan semua haknya untuk memperoleh kasih, rahmat, dan anugerah Tuhan. Tapi alasan ini tetap berharga karena bisa membangkitkan harapan bahwa Allah berkenaan melalui kebutuhan manusia.1 Keempat Alkitab mempunyai otoritas untuk memimpin orang kepada kebenaran (II Tim 3:16). Alkitab itu dapat memberikan pengertian untuk mendapat keselamatan melalui iman kepada Yesus Kristus. Semua yang tertulis di dalam Alkitab, diilhami oleh Allah dan berguna untuk mengajarkan yang benar, untuk menegur dan membetulkan yang salah, dan Kedua, Adanya pendapat yang mengatakan bahwa terdapat kekeliruan dalam Alkitab seperti yang dinyatakan oleh Charles C.Ryrie dalam bukunya Teologi 100 \| Jurnal Excelsis Deo: Teologi, Misiologi, dan Pendidikan Jurnal Excelsisi Deo: Vol. 6 No.1 Juni 2022 Jurnal Excelsis Deo: Teologi, Misiologi, dan Pendidikan\| 101 HASIL DAN PEMBAHASAN Untuk menyatakan kebenaran tentang sesuatu hal, harus ada fakta yang kongkrit, maka diakui bahwa sesuatu itu benar adanya. Demikian pula sama halnya dengan Alkitab. Jika Alkitab diakui dan dinyatakan sebagai Firman Allah, maka Alkitab sendiri harus mempunyai bukti yang nyata. Oleh karena itu, dalam bab akan membahas dan memberikan bukti kebenaran Alkitab sebagai Firman Allah, dengan beberapa pokok penting sebagai berikut. Alkitab adalah nama kumpulan buku yang diakui sebagai kanonik, dan diakui sebagai Firman Tuhan oleh gereja Kristen. Nama ini didasarkan pada penggunaan kata Yunani biblia (jamak buku) untuk seluruh buku seolah-olah hanya satu buku.9 METODE PENELITIAN biblia dalam bentuk jamak yang berarti buku atau buku. Barcley M. Newman dalam bukunya kamus Yunani-Indonesia menyebutkannya demikian, “biblos, kitab yang suci, kata sifat ini pada umumnya digunakan untuk menunjukkan kepada buku- buku (kitab-kitab).7 Metode yang digunakan dalam penulisan ini adalah metode kualitatif kepustakaan dengan pendekatan deskriptif. Adapun teknik pengumpulan data yang digunakan penulis adalah kajian kepustakaan dengan menggali terhadap kata kunci berhubungan konsep mesianik dalam II Timotius 3:16 dan melihat relevansinya dengan orang Percaya masa kini.5 Dalam bahasa Inggris, Bible berasal dari kata bible yang diambil dari bahasa Inggris Yunani biblia, hingga pengertiannya sama yakni buku-buku (kitab-kitab). Jadi Alkitab merupakan sebua kumpulan buku yang telah dikanonkan. Tentang hal ini W. R. F. Browning dalam bukunya Dictionary of the Bible memberikan penjelasan sebagai berikut: "Bible, kata bahasa Inggris "bible" berasal dari kata Yunani biblia, yang berarti buku. Bentuk jamak menunjukkan fakta bahwa Alkitab Kristen bukanlah keseluruhan, tetapi koleksi.8” Pada Ensklopedi Alkitab masa kini menjelaskan hal yang sama sebagai berikut 7Ibid., 28. 8W. R. F. Bruning, Kamus Alkitab (Jakarta: Gunung Mulia, 2007), 13. 9Ibid., 28. Jurnal Excelsisi Deo: Vol. 6 No.1 Juni 2022 kembali kepercayaan mereka secara tuntas4. Kalau orang-orang yang ingin mempelajari Alkitab itu pertanyaan-pertanyaan tersebut tepat untuk memahami isi Alkitab, Alkitab dapat memberikan pemahaman untuk mendapatkan keselamatan melalui iman kepada Yesus Kristus. Segala sesuatu yang tertulis dalam Alkitab, diilhami oleh Tuhan dan berguna untuk mengajarkan apa yang benar, untuk menegur dan mengajar orang untuk hidup sesuai dengan kehendak Tuhan. Dengan Alkitab, mereka yang melayani Tuhan dapat diperlengkapi dengan sempurna untuk segala macam perbuatan baik, mengoreksi kesalahan. Keenam, Adanya paham yang menentang sifat Alkitab tentang bebas dari kesalahan dan tak mungkin bersalah. Seperti yang dijelaskan oleh Willian W. Manzies & Stanley M. Horton dalam bukunya Doktrin Alkitab, sebagai berikut“Ketidakpercayaan paham humanisme merupakan sumber sebenarnya dari berbagai keberatan terhadap sifat tak mungkin bersalah dari Alkitab. Alasan-alasan mereka tidak baru. Para penulis kuno seperti Irenius, Tertulianus dan Agustinus menentang beberapa pan-dangan mereka dan dengan berbuat demikian mereka mengaku bahwa mereka percaya Alkitab. Para pem-baharu seperti Swingli, Calvin, dan Luther juga menerima sepenuhnya wibawa Alkitab. Selama bertahun-tahun, para kritikus yang tidak percaya telah membuat daftar penting tentang apa yang mereka sebut inkonsistensi dalam Alkitab, dan beberapa dari mereka berpendapat bahwa Alkitab mengandung kesalahan yang tidak dapat disangkal. Adapun tujuan yang ingin dicapai penulis dalam penelitian ini adalah (1) menjelaskan Definisi Alkitab secara etimologi? (2) memberikan bukti-bukti tentang kebenaran Alkitab. (3) menjelaskan pentingnya Alkitab bagi iman orang percaya yang semakin bertumbuh menjadi dewasa. Jika Alkitab dipahami dan diakui sebagai Firman Allah, serta diterapkan dalam kehidupan ini, maka iman orang akan bertumbuh dan dilengkapi dalam tujuan pekerjaan yang baik.percaya Kelima, Pada abad 18 munculnya studi kritis terhadap Alkitab. Studi tersebut selalu mulai dengan mempertanyakan segala sesuatu yang diterima secara umum, terutama gereja dan Kristen, ketika seluruh dunia menunjukkan tanda-tanda keme-rosotan yang hebat. Seperti yang dinyatakan oleh S.Wismoady Wahono dalam bukunya disini kutemukan adalah sebagai berikut: Perubahan-perubahan sosial dan politik terjadi dimana-mana. Bersamaan dengan itu terjadi juga perubahan di bidan ide dan pemikiran, munculnya pertanyaan- pertanyaan filosofis serta metode-metode ilmia yang baru. Semuanya harus dipelajari, karena prinsip-prinsip baru mulai berlaku. Alkitab juga tidak dan harus ditinjau! Pertanyaan yang kemudian muncul adalah: kalau hal itu benar, bagai mana bisa terjadi seperti itu? Apakah Alkitab memang berbeda dari buku-buku lain? Jalan satu-satunya untuk menemukan jawabannya adalah mempelajari Alkitab itu sendiri. Orang-orang yang mengajukan per-tanyaan-pertanyaan seperti tersebut diatas bukanlah orang luar. Jurnal Excelsisi Deo: Vol. 6 No.1 Juni 2022 Mereka adalah orang-orang Kristen sendiri yang tidal sengan-sengan untuk mengkaji Jurnal Excelsis Deo: Teologi, Misiologi, dan Pendidikan\| 101 Jurnal Excelsisi Deo: Vol. 6 No.1 Juni 2022 102 \| Jurnal Excelsis Deo: Teologi, Misiologi, dan Pendidikan 5Mestika Zed. Metode Penelitian Kepustakaan (Jakarta: Yayasan Pustaka Obor Indonesia, 2004), 3-5. 6Ibid., 31. Jurnal Excelsisi Deo: Vol. 6 No.1 Juni 2022 Allah yang dituangkan secara tertulis untuk manusia. merupakan kata sifat. Sehingga penyusunan kalimatnya menjadi segala apa yang tertulis dalam Alkitab diilhami oleh Tuhan, seperti yang dijelaskan oleh B. F. Drewes, Wilfrit Haebech & Heinrich Vonsiebnthal dalam bukunya Kunci Bahasa Yunani Perjanjian Baru, sebagai berikut setiap bagian Kitab Suci = segala sesuatu yang tertulis dalam Kitab Suci. kata sifat yang diilhami oleh Tuhan; kata benda predikatif, segala sesuatu yang tertulis dalam kitab suci diilhami oleh Tuhan, dan mungkin sebuah atribut, segala sesuatu yang tertulis dalam kitab suci yang diilhami oleh Tuhan adalah juga.10 10 B. F. Drewes, Wilfrit Haebech & Heinrich Vonsiebenthal Kunci Bahasa Yunani Perjanjian Baru, Jakarta: Gunung Mulia, 2006, hal. 233 Definisi Istilah Alkitab Secara Etimologi Alkitab merupakan kitab suci yang diakui sebagai Firman Allah oleh umat nasrani, sebagaimana tercantum dalam Kamus Besar Bahasa Indonesia, sebagai berikut: “Alkitab adalah kitab suci agama Kristen yang terdiri dari Perjanjian Lama dan Perjanjian Baru.6 Dikarenakan pengertian Alkitab dari bahasa aslinya adalah buku (kitab), maka perlu diketahui bahwa pengertian sebuah buku terkandung pada tulisannya. Berhubungan dengan penyataan tersebut, maka Alkitab diakui sebagai kitab yang bernilai dan bermutu tinggi bagi orang percaya. Karena Alkitab berisikan Firman Namun bila dikaji secara etimologi, Alkitab berasal dari kata Yunani biblion bentuk tunggal artinya buku atau buku. Dan 5Mestika Zed. Metode Penelitian Kepustakaan (Jakarta: Yayasan Pustaka Obor Indonesia, 2004), 3-5. 6Ibid., 31. 102 \| Jurnal Excelsis Deo: Teologi, Misiologi, dan Pendidikan Jurnal Excelsisi Deo: Vol. 6 No.1 Juni 2022 11 Tafsiran Alkitab Wyliffe , Malang: Gandum Mas, 2001, hal. 895 12Ibid., 420. 13 George Sandison, Bible Answers for 1000 Difficulty Questions (Malang: Gandum Mas, 2006), 8. Jurnal Excelsisi Deo: Vol. 6 No.1 Juni 2022 pemeliharaan, wahyu kepada dan melalui para nabi, pencarian atau penghakiman. Demikian juga semua yang termasuk dalam kategori “Kitab Suci” yaitu semua kata-kata yang terdapat dalam Alkitab (Kitab Suci) adalah suatu hasil karya Allah melalui hembusan atau Roh-Nya. Seperti yang dinyatakan dalam Ensklopedi Alkitab Masa Kini, dengan berdasarkan II Timotius 3:16, sebagai berikut: Alkitab diilhamkan – Kepastian. Mengandung otoritas ilahi dan pengaruhnya dapat terwujud nyata bagi manusia. Seperti yang dinyatakan oleh George Sandison dalam bukunya, Bible answers for 1000 difficult Questions, mengutip perkatan dan keyakinan bapak Moody sebagai berikut: “Bapak Moody evangelis Amerika terhebat pernah ditanyai apakah anda menganggap kalau Alkitab diilhamkan, jawabannya singkat dan tepat: yakin Alkitab telah diilhamkan, karena Alkitab mengilhami.13” Jawaban ini adalah salah satunya dan banyak orang akan mengulanginya. Namun bukan berarti bahwa pengilhman berdasarkan tanggapan pendengarnya, atau berdasarkan pengalaman subjektif penulisnya, tetapi berdasarkan bukti bahwa "semua tulisan dihembuskan oleh Tuhan" inilah arti harafiah yang diterjemahkan 'diilhami'. Jadi poin terpenting adalah terinspirasi itu saja untuk hal-hal yang disebutkan disini. Pernyataan tersebut berlaku untuk “Kitab Suci” secara keseluruhan “Berdasarkan ayat Dalam teologi ini biasanya menggunakan kata 'ilham' (ilham) untuk menyatakan asal usul dan kualitas ketuhanan Kitab Suci. Secara aktif kata benda itu mengungkapkan pekerjaan Tuhan yang 'menembus' secara lahiriah untuk menghasilkan Kitab Suci; secara pasif, keadaan Kitab Suci telah dihembuskan dan itu terjadi dengan cara ini. Kata itu juga lebih umum digunakan untuk pengaruh ilahi, yang memungkinkan orang-orang pewahyuan, nabi, pemazmur, orang bijak dan rasul, untuk mewartakan dan juga menuliskan Sabda Allah.12 Orang yang tidak rohani tidak memiliki hubungan dengan Roh dan tidak memiliki kemampuan untuk memahami dan menafsirkan Kitab Suci bagaimanapun pengetahuannya. Jadi manusia alamiah tidak dapat menuliskan kata-kata dalam Alkitab sebagai Firman Allah. Oleh karena itu Rasul Petrus dengan jelas menegaskan bahwa: “tidak pernah nubuat yang dihasilkan oleh kehendak manusia, tetapi oleh dorongan Roh Kudus orang-orang berbicara atas nama Allah” (II Pet. 1:21). Para nabi mengatakan begini: “Firman Tuhan yang datang kepadaku” (Hos. 1:1; Yoel 1:1). Bukti Bukti Alkitab Sebagai Firman Allah Menurut II Timotius 3:16 Jika manusia hanya mengaku bahwa setiap catatan yang tertulis dalam Alkitab adalah suatu ungkapan ilahi yang terbukti asli, dapat dipercaya dan sangat sah sebagai bentuk ungkapan itu, tanpa menunjukan bukti tentang kebenarannya, maka masih menjadi orang-orang yang dikelabui oleh tulisan-tulisan kuno yang tak berwibawa atau merupakan cerita fiktif belaka. Oleh karena itu tugas jangan hanya menghafal. Setiap kata-kata yang tertulis didalam Alkitab tetapi berusaha untuk memberikan penjelasan seakurat mungkin tentangnya. Sesuai kata pemazmur: “tetapi yang kesukaannya ialah Taurat Tuhan, dan merenungkan Taurat itu siang dan malam” (Maz. 1:2). Betapa aku mencintai hukum-Mu, aku tahu sepanjang hari (Maz. 1:97) alasannya, jadi pada bagian berikut, penulis akan memberikan bukti kebenaran Alkitab sebagai Firman Tuhan berdasarkan kutipan dari Rasul Paulus dalam II Timotius 3:16 yang berbunyi “Semua tulisan yang diilhami Allah memang berguna untuk pengajaran, untuk menegur kesalahan, mengoreksi perilaku dan untuk mendidik orang dalam kebenaran. Namun kata diilhamkan pneustos dapat di artikan sebagai “dihembuskan” , seperti yang dijelaska dalam Tafsiran Alkitab Wycliffe, sebagai berikut: “terinspirasi oleh Tuhan dalam bahasa Inggris adalah kata yang berarti dihembuskan oleh Tuhan, Alkitab datang ke tingkat otoritas ilahi sepenuhnya karena itu adalah kebenaran mutlak dan karena itu berguna. Ada versi terjemahan tertentu yang menerjemahkan istilah segala sesuatu dengan tulisannya masing-masing, dan keduanya dapat dijelaskan karena penjelasannya sama: jika setiap tulisan, maka pasti semua tulisan juga.11 Menurut Kamus Singkat Bahasa Iberani “dihembuskan atau diilhamkan disebut “ruakh” artinya roh angin, Roh atau nafas. Sebagai contoh (Kej. 1: 2) ‘ruakh’ Roh (Roh Allah), sedangkan (Yunus 1:4)‘ruakh’ angin. Hembusan atau Roh Allah dengan secara aktifnya mengelaurkan daya kuasa ilahi. Sehingga menghasilkan sesuatu, apakah itu dalam penciptaan, Khususnya pada kalimat “Segala tulisan yang diilhamkan Allah” dalam bahasa Yunani “pasa grafe theopneutos katatiap nas dari Kitab Suci, sejajar dengan segala yang tertulis dalam Kitab Suci, sementara berarti diilhamkan Allah 11 Tafsiran Alkitab Wyliffe , Malang: Gandum Mas, 2001, hal. 895 11 Tafsiran Alkitab Wyliffe , Malang: Gandum Mas, 2001, hal. 895 Jurnal Excelsis Deo: Teologi, Misiologi, dan Pendidikan\| 103 Jurnal Excelsisi Deo: Vol. 6 No.1 Juni 2022 Keunikkan Sifat Alkitab Istilah keunikkan , kata dasarnya “unik” Menurut Kamus Besar Bahasa Indonesia, “unik” berarti “terpisah dalam bentuk atau jenisnya, berbeda dengan yang lain” atau tidak persamaan dengan yang lain.14 Sedangkan kata “sifat” artinya rupa atau keadaan yang tampak pada sesuatu benda. Kedua pengertian tersebut menunjukkan kepada sesuatu hal atau benda yang memangnya ada dan taraf 14Ibid., 1247. 14Ibid., 1247. 104 \| Jurnal Excelsis Deo: Teologi, Misiologi, dan Pendidikan 104 Jurnal Excelsisi Deo: Vol. 6 No.1 Juni 2022 bukunya Teologi Sistematika sebagai berikut: “Para raja Romawi segera sadar bahwa orang-orang Kristen melandaskan kepercayaan mereka pada Alkitab. Oleh karenanya mereka berusaha untuk menumpas atau memusnahkan Alkitab. Melalui sebuah dekrit pada Tahun 303 Masehi, Kaisar Diocletianus menuntut agar setiap jilid Alkitab dibakar. Ia membunuh begitu banyak orang Kristen dan menghancurkan begitu banyak Alkitab, sehingga itu merasa telah berhasil memusnahkan Alkitab secara tuntas, ketika orang-orang Kristen bersembunyi dan tidak memperlihatkan selama beberapa waktu. Diocletianus menyuruh membuat sebuah medali yang bertuliskan,“ Kekristenan telah binasa dan penyembahan dewa-dewa telah dipertunjukkan.16 keberadaannya lebih unggul dari sesuatu yang pernah ada. Dalam hal membahas masalah keunikkan sifat Alkitab, maka harus memberikan fakta jelas yang membedakannya. Apakah faktanya? Kalau menempuh jalan singkat dan mengatakan, “Alkitab memiliki keunggulan yang luar biasa, adalah sebuah jawaban yang tepat. Tetapi masih ada terdapat orang-orang yang di zaman modern ini masih mempersoalkan keunggulannya. Seperti yang dijelaskan oleh S. Wisma Adi Wahono dalam bukunya, saya menemukan di sini, sebagai berikut: “Apakah Alkitab itu? Pertanyaan ini biasanya dijawab dengan mengatakan: Alkitab adalah buku dari semua buku. Alkitab adalah kitab suci yang berisi Firman Tuhan dan Wahyu Tuhan. Jawaban seperti itu memang bagus, tetapi orang yang hidup di zaman kemajuan seperti sekarang ini sering kali menimbulkan ambiguitas dan kebingungan ketidak-jelasan.15 Oleh karena itu jalan lain yang harus ditempuh untukmencari jawaban yang tepat adalah mempelajari dan mengerti secara benar akan sifat-sifat Alkitab yang unik dan yang membedakan dari kitab-kitab apapun di dunia ini. Adapun dua keunikan sifat Alkitab yang akan dibahas pada bagian berikut ini. Kemudian pada masa Reformasi, Roma Katolik berupaya untuk membatasi pembacaan dan pemahaman Alkitab bagi kaum awam dengan dalih bahwa hanya orang terkemuka yang dapat memahaminya, bahkan mereka pun membuat hukum tertentu yang melarang proses penerbitan Alkitab, tentang hal ini dapat dijelaskan oleh Henry C. Keunikkan Sifat Alkitab Thiessen dalam bukunya teologi sistematika, sebagai berikut: “Pada masa Reformasi yaitu ketika Alkitab diterjemakan ke dalam bahasa yang dapat dipahami umum, gereja yang resmi membatasi secara ketat pembacaan Alkitab dengan alasan bahwa kaum awam tidak mampu mengartikan isi Alkitab. Orang awam tidak diizinkan membaca dan menafsirkan isi Alkitab itu sendiri. Banyak orang yang harus berkorban jiwa karena mereka merupakan pengikut Kristus yang percaya pada Alkitab. Bahkan pada masa itu 15Wismoady Wahono, Disini kutemukan (Jakarta: Gunung Mulia), 2004), 17. Alkitab tidak bisa dimusnahkan Alkitab adalah Firman Allah tertulis yang ada dan walaupun kerap kali ada orang- orang tertentu yang berusaha untuk memusnahkan Alkitab dari muka bumi tetapi usaha itu selalu gagal, karena terbukti sampai hari ini. Firman Allah tetap ada, tentang hal ini Henry C. Thiessen menyatakan dalam 16Ibid.,68-69. 16Ibid.,68-69. Jurnal Excelsis Deo: Teologi, Misiologi, dan Pendidikan\| 105 Jurnal Excelsis Deo: Teologi, Misiologi, dan Pendidikan\| 105 Jurnal Excelsisi Deo: Vol. 6 No.1 Juni 2022 106 \| Jurnal Excelsis Deo: Teologi, Misiologi, dan Pendidikan Jurnal Excelsisi Deo: Vol. 6 No.1 Juni 2022 dibuat hukum-hukum tertentu yang melarang penerbitan Alkitab.17” kekerasan badani. Walaupun pengantaraan pribadi dari penatua pada akhirnya akan menentukan kekuasaan dari surat-suratnya, bisa saja dibatalkan oleh pengaruh yang ada sekarang pada Diotlefes (III Yoh. 9-10).18 Alasan yang dipakai oleh gereja resmi (gereja Roma Katolik) itu kurang tepat. Karena memahami dan menafsirkan keberan Firman Tuhan itu bukan hanya menggunakan kemampuan mental dan pengetahuan yang diperoleh secara alamiah, akan tetapi oleh dorongan Roh. Seperti yang dinyatakan oleh Rasul Paulus sebagai berikut: “Dan karena kami menafsirkan hal-hal rohani kepada mereka yang mempunyai Roh, kami berkata- kata tentang karunia-karunia Allah dengan perkataan yang bukan diajarkan kepada kami dengan hikmat manusia, tetapi oleh Roh”. (I Kor. 2:13) Ini salah satu alasan yang dipakai oleh Para reformator. Karena para reformator ingin menafsirkan Kitab Suci secara benar dan juga menghendaki agar isinya diketahui dan dimengerti semua orang termasuk kaum awam sekalipun. Inilah tujuan para reformator supaya kepercayaan dan tradisi- tradisi kekristenan itu ditundukkan pada otoritas utama Alkitab. Selain itu hadir pula seorang bernama Ditrefes yang bersikap anti- kekristenan dengan tidak sengan-sengan menyerang dan menolak Yohanes murid Tuhan Yesus bahkan mengucilkannya. Hanya karena berambisi untuk menghalangi pemberitaan Firman Allah. Penyataan ini dijelaskan dalam Ensklopedi Alkitab Masa Kini, sebagai berikut: “Diotrefes seorang yang keras kepala dan mempunyai cita-cita yang angkuh sekali. Ia tidak mau mengakui penatua (Yoh) bahkan menyerang penatua itu secara terbuka, ia melarang menerima penatua-penatua itu dan mengucilkan mereka yang tetap menerimanya, atau dengan jalan ilk i t Diotrefes berkelakuan buruk dan bertindak menghalangi jalan pemberitaan Injil dengan kata-kata yang tidak bermakna.(III Yoh. 9-10). Dalam AlkitabYunani- Indonesia menyebutkan “sebab ia berbicara tanpa makna dengan melontarkan kata kata yang kasar”. Diotrefes berbicara dengan kasar, oleh karena ia telah memiliki cita-cita yang angkuh. Dalam Kamus bahasa Yunani- Indonesia “filoproteuon artinya ingin mendahului atau ingin menjadi pemimpin”. Dan makna lain yang dapat disejajarkan dengan Filoproteuon adalah kesombongan. Dimana ada sifat kesombongan makin lama makin muncul kekacauan sehingga kekacauan menghasilkan penceraian. Penceraian itu adalah puncak dari akar kata sifat kesombongan. Sekalipun Diotrefes dengan bersikap keras dan berusaha menghalangi jalannya pemberitaan Injil, akan tetapi ia tidak berhasil. Baik keputusan Negara Romawi maupun peraturan-peraturan kegerejaan tidak pernah berhasil memusnahkan Alkitab. Makin keras usaha memusnahkan Alkitab makin luas pula Alkitab itu tersebar. Robrt Backhouse mengutip kata Napoleon dalam bukunya 5000 Kata Hikmat sebagai berikut: Alkitab bukanlah sembarang buku, melainkan sebuah Makhluk yang Hidup, dengan kekuatan 18Ibib., 249. 17Ibid., 69. Jurnal Excelsisi Deo: Vol. 6 No.1 Juni 2022 untuk manak-lukkan siapa pun yang menentangnya.19 penyelamatan itu adalah tanpa syarat dan tiada tuntutan atau perintah yang dilampirkan dan dapat digunahkan sebagai syarat yang kepadanya kasih karunia itu bergantung. Seperti yang tertera dalam (Yoh. 3:16), demikian: “karena begitu besar kasih Allah akan dunia ini, sehingga Ia telah mengaruniakan anak-Nya yang tunggal, supaya setiap otang yang percaya kepada- Nya tidak binasa, melainkan beroleh hidup yang kekal.” Kesatuan berita kitab-kitab dalam Alkitab Jurnal Excelsisi Deo: Vol. 6 No.1 Juni 2022 106 \| Jurnal Excelsis Deo: Teologi, Misiologi, dan Pendidikan Jurnal Excelsisi Deo: Vol. 6 No.1 Juni 2022 19 Robert Backhouse, 5000 Kata Hikmat (Yogyakarta: Andi, 2006), 13. Jurnal Excelsis Deo: Teologi, Misiologi, dan Pendidikan\| 107 Jurnal Excelsisi Deo: Vol. 6 No.1 Juni 2022 Pentatehuk (Lima kitab Taurat Musa). Penta- tehuk adalah kelima kitab pertama dalam Alkitab, adalah Kejadian, Keluaran, Imamat, Bilangan dan Ulangan. Nama itu berasal dari kata Yunani artinya, lima gulungan kitab. penyelamatan yang khas itu akan menuntut sebagai lapangan tempat terjadinya satu sejarah umum manusia. Itulah sebabnya kutuk perjanjian yang dipatahkan itu diberitakan kepada sepanjang manusia yang jatuh sebagai wakil dari umat manusia seluruhnya, dilunakkan (ayat 16-19) dengan asas kasih karunia yang umum.21 Pentateukh dikenal sebagai hukum Musa (dalam Bahasa Ibrani ‘Tora’). Pentateukh dipandang sebagai bagian Alkitab yang paling suci bagi orang Yahudi. Dan biasanya gulungan Tora ini diberi tempat terhormat di dalam sinagoge. Kedua, kitab-kitab sejarah terdiri dari dua belas kitab yaitu: Yosua, Hakim-hakim, Rut, 1 dan 2 Samuel, 1 dan 2 Raja-raja, 1 dan 2 Tawarikh, Ezra, Nehemia dan Ester. Seperti yang dijelaskan oleh Andrew E. Hill dan John H. Walton dalam bukunya Survei Perjanjian Lama, demikian:“Pengaturan kitab-kitab sejarah Perjanjian Lama dalam Akitab bahasa Inggris meliputi Yosua, Hakim-hakim, Rut, 1 dan 2 Sa-muel, 1 dan 2 Raja-Raja, 1 dan 2 Tawarikh, Ezra, Nehemia dan Ester.22 Ketiga, Kitab Puisi (Syair)) terdiri dari lima kitab yaitu: Ayub, Mazmur, Amsal, Pengkhotbah dan Kidung Agung. Keempat, Kitab Para Nabi. Ada enam belas nabi disebut namanya, dan memiliki tujuh belas kitab yang berisi visi dan nubuatan mereka. Ratapan, versi Yunani dikenal sebagai “Ratapan Yeremia”. Empat nabi Besar dan duabelas Nabi kecil. Kitab nabi- nabi besar adalah Yesaya, Yeremia, Ratapan, Yehezkiel dan Daniel. Sedangkan kitab nabi- nabi kecil adalah, Hosea, Yoel, Amos, Obaja, Yunus, Mikha, Nahum, Habakuk, Sefanya, Hagai, Zakaria dan Maleakhi. Seorang Nabi adalah seorang yang berbicara atas nama Perjanjian Lama terus-menerus mengacu ke depan tentang keselamatan yang akan mencapai puncaknya dalam gambar Hamba yang menderita, seperti yang dinubuatkan oleh Nabi Yesaya dalam pasal 53:5 bahwa: “Tetapi dia tertikam oleh karena pemberontakan, dia diremukkan oleh karena kejahatan; ganjaran yang mendatangkan keselamatan bagi ditimpakan kepadanya” Pasal ini memberikan serangkain gambaran mengenai hamba Allah dan misi pelayanan- Nya. Hamba yang menderita mengacu kepada pribadi wakil Israel yang sebenarnya, yang masih akan datang. Alkitab secara keseluruhan dianggap hanya memiliki satu tujuan dari Allah yaitu, tujuan penyelamatan bagi manusia. 21Y Mahengkeng, Tafsiran Alkitab Masa Kini (Jakarta: Komunikasi Bina Kasih/ OMF, 2005), 86. 22Andrew E. Hill dan John H. Walton, Survei Perjanjian Lama (Malang: Gadum Mas, 2004), 117. Kesatuan berita kitab-kitab dalam Alkitab Alkitab yang ditulis oleh sekitar empat puluh penulis yang dari latar belakang budaya yang berbeda. Enam puluh enam kitab dalam Alkitab ditulis sekitar waktu seribu enam ratus (1600) tahun, namun amanat intinya satu. Apakah amanat inti tersebut? Pesan inti dari Alkitab adalah janji Tuhan kepada umat manusia untuk setiap zaman. Dan tentang hal ini telah dijelaskan dalam Encyclopedia of the Bible Today, sebagai berikut: “Pesan inti dari Alkitab adalah sejarah sejarah; melalui kedua Perjanjian ini mekar, dan dalam hal ini tiga elemen utama dapat dilihat: Pembawa keselamatan, jalan keselamatan, dan ahli waris keselamatan.20 Kedua Perjanjian (Perjanjian Lama dan Perjanjian Baru) mengaku bahwa janji penyelamatan adalah anugera Allah kepada manusia. Dari sejak kejatuhan manusia pertama (Adam dan Hawa) kedalam dosa di taman Eden merupakan titik tolak pemikiran Allah bagi penetapan janji tersebut. Seperti yang diungkapkan dalam (Kej 3:15), tentang ayat ini Tafsiran Alkitab Masa Kini Jilid 1 menafsirkan: “Keturunannya akan meremukkan kepalamu, dan engkau akan meremukkan tunitnya, ‘Engkau’ yang akan masih bertanding di hari depan yang masih jauh, dengan malalui ular menunjuk kepada Iblis. Karena itu yang dimaksudkan dengannya (tumitnya) itu juga bukan beni perempuan secara kolektif, tapi juga adalah perorangan (Kristus). Karena ayat ini memperluas pengumuman yang baru saja dibuat tentang pengutukan Iblis, maka pokok pertentangan antara peremukan kepala dan tumit itu adalah demikian, bahwa yang pertama adalah mematikan, sedang yang kedua tidak. Tapi terang bahwa kemenangan yang menyelamatkan itu akan mengandung di dalamnya penderitaan. Program Keselamatan bersumber dari Allah, dengan berdasarkan anugera-Nya. Alkitab memberikan pernyataan-pernyataan yang makin lama makin jelas tentang bagaimana Allah menyediakan dasar keselamatan, menawarkannya dan bagaimana Dia sen-diri pada diri-Nya adalah satu-satunya keselamatan manusia. Keselamatan bersumber dari Allah, dengan berdasarkan anugera-Nya. Alkitab memberikan pernyataan-pernyataan yang makin lama makin jelas tentang bagaimana Allah menyediakan dasar keselamatan, menawarkannya dan bagaimana Dia sendiri pada diri-Nya adalah satu-satunya keselamatan manusia. Jadi janji di 20 Ibid., 30. 20 Ibid., 30. Jurnal Excelsis Deo: Teologi, Misiologi, dan Pendidikan\| 107 Jurnal Excelsisi Deo: Vol. 6 No.1 Juni 2022 108 \| Jurnal Excelsis Deo: Teologi, Misiologi, dan Pendidikan Perjanjian Baru Alkitab Perjanjian Baru terdiri dari dua puluh tujuh kitab. Kedua puluh tujuh kitab itu dapat dibagi juga dalam lima bagian besar: Pertama, kitab-kitab Injil. Apabila kita membuka halaman depan dari Perjanjian Baru maka menemui empat kitab yang dikenal dengan empat kitab injil yaitu: Matius, Markus, Lukas, dan Yohanes. Keempat kitab ini merupakan kelompok tersendiri. Kedua, kitab sejarah, terdiri dari satu kitab yaitu: Kisah Para Rasul. Ketiga, surat-surat kiriman Rasul Paulus, setelah kitab Kisah Para Rasul , terdapat tiga belas kitab kiriman Paulus. Ketiga belas kitab dibagi dalam dua bagian yaitu: pertama, surat-surat am atau umum terdiri dari sembilan kitab, ialah “Roma, 1 dan 2 Korintus, Galatia, Efesus, Pilipi, Kolose, dan 1 dan 2 Tesaloni. Kedua, empat surat-surat Penggembalaan. Surat-surat ini tidak ditujukan kepada jemaat-jemaat Kristen, tetapi masin-masin dikirim kepada perorangan. Dua surat Pertama dituliskan kepada Timotius, yaitu anak kekasih Paulus. Yang ketiga kepada Titus . keempat, kepada Filemon. Keempat, surat-surat kiriman kepada orang-orang Kristen Iberani, ada delapan surat yaitu: Iberani, Yakobus, 1 dan 2 Petrus, 1, 2 dan 3 Yohanes dan Yudas. Kelima, Wahyu, adalah kitab terhakir dalam Alkitab. Kitab tersebut ini merupakan kitab nubuat atau penyataan Allah kepada Rasul Yohanes. Kitab-kitab baik yang terdapat dalam Perjanjian Lama maupun dalam Pentingnya Alkitab bagi orang percaya menurut II Timotius 3 : 16 Alkita memiliki otoritas ilahi dan keunikkan sifatnya telah terwujud dalam kehidupan orang percaya. Alkitab memainkan peranan penting dalam segala hal. Relevansinya bukan hanya di kalangan orang percaya, tapi juga bagi masyarakat. Namun penjelasan dalam bab ini, penulis hanya berfokus pada pengaruh Alkitab bagi setiap orang percaya menurut II Timotius 3:16. Dalam ayat tersebut ada empat M yang hendak menjadi pokok pembahasan di sepanjang bab ketiga ini adalah, mengajar, menyatakan , memperbaiki dan mendidik. Pembagian Kitab-kitab dalam Alkitab Alkitab terdiri dari enam puluh enam kitab dan dibagi dalam dua bagian besar yaitu, Perjanjian Lama dengan Per-janjian Baru. Kedua Perjanjian itu dapat dibagi juga dalam beberapa bagian, seperti yang dijelaskan pada bagian berikut ini: Perjanjian Lama Alkitab Perjanjian Lama terdiri atas tiga puluh sembilan kitab, dapat dibagi juga dalam empat bagian besar yaitu: Pertama, 108 \| Jurnal Excelsis Deo: Teologi, Misiologi, dan Pendidikan 108 Jurnal Excelsisi Deo: Vol. 6 No.1 Juni 2022 Jurnal Excelsisi Deo: Vol. 6 No.1 Juni 2022 Allah, dan disebut sebagai jurubicara atau penyambung lidah Allah. Sebagai contoh, mereka berkata “beginilah Firman Tuhan” (Yer. 1:9), dan “Firman Tuhan datang kepada” (Yer. 47 :1; Yehz. 17:1). Perjanjian Baru diakui dan diterima sebagai Firman Allah. (dikanonikasikan). Kitab-kitab Perjanjian Lama menjadi satu, sehingga menjadi pedoman hidup dan kepercayaan orang-orang Yahudi. Akhirnya Tuhan Yesus dan para rasul menerima kitab itu sebagai Firman Tuhan. Jadi umat Tuhan dalam Perjanjian Baru menerima Perjanjian Lama sebagai Firman Tuhan. Seluruh Alkitab adalah apa yang dilihat umat Allah sebagai kumpulan yang dengan baik dan benar menyaksikan pekerjaan Allah di dalam Kristus. Oleh karena itu, buku-buku ini dipandang sebagai alat Roh Kudus untuk bersaksi tentang Kristus. Alkitab Berguna Untuk Mengajar Istilah ‘mengajar’ dalam bahasa Yunani didaskalia feminim, artinya mengajarkan atau pengajaran. Menurut Kamus Besar Bahasa Indonesia “Mengajar, Jurnal Excelsis Deo: Teologi, Misiologi, dan Pendidikan\| 109 Jurnal Excelsisi Deo: Vol. 6 No.1 Juni 2022 24 Ibid., 22. 110 \| Jurnal Excelsis Deo: Teologi, Misiologi, dan Pendidikan Jurnal Excelsisi Deo: Vol. 6 No.1 Juni 2022 110 \| Jurnal Excelsis Deo: Teologi, Misiologi, dan Pendidikan Jurnal Excelsisi Deo: Vol. 6 No.1 Juni 2022 belum, sekalipun gembala sidang mengajak kepada anggota jemaatnya bahwa semua umat harus menerima dan melakukannya Firman Tuhan. Dan dalam situasi berkhotbah masih belum bisah mengenal perkembangan kualitas iman seseorang. Justru Gembala sidang harus ada kesempatan untuk mengajar. Kesempatan mengajar merupakan suatu kesempatan bagi semua orang, yaitu dimana gembala sidang atau pengajar mengajukan pertanyaan-pertanyaan dan memberikan kesempatan kepada orang lain untuk berbicara. Kesempatan yang baik ialah di kelas Sekolah Minggu, dimana bisa menemukan bermacam-macam orang Kristen. Seperti J Seth Msweli dan Donald Crider dimana mereka menganggap sekolah Minggu begitu penting bagi setiap anggota jemaat baik anak-anak mau pun orang dewasa seperti yang disampaikan lewat bukunya Gembala sidang dan Pelayanannya sebagai berikut: “Sekolah Minggu adalah kelompok-kelompok pemahaman Alkitab pada hari Minggu, bukan hanya untuk anak- anak tetapi jua untuk orang dewasa”.25 Kelas Sekolah Minggu untuk setiap anggota jemaat itu sangat penting, karena melaluinya adalah suatu kesempatan yang baik untuk mengajarkan tentang, peristiwa-peristiwa, tokoh-tokoh dan pengalaman hidupnya, serta menghafalkan ayat-ayat Firman Allah. Selain sekolah Minggu kelas katekisasi untuk orang Kristen baru. Gembala sidang juga harus mengajar di kebaktian pemuda dengan mengajukan pertanyaan-pertanyaan sesuai dengan topik yang diangkat. Ajarkan, agar belum, sekalipun gembala sidang mengajak kepada anggota jemaatnya bahwa semua umat harus menerima dan melakukannya Firman Tuhan. Dan dalam situasi berkhotbah masih belum bisah mengenal perkembangan kualitas iman seseorang. Justru Gembala sidang harus ada kesempatan untuk mengajar. Kesempatan mengajar merupakan suatu kesempatan bagi semua orang, yaitu dimana gembala sidang atau pengajar mengajukan pertanyaan-pertanyaan dan memberikan kesempatan kepada orang lain untuk berbicara. Kesempatan yang baik ialah di kelas Sekolah Minggu, dimana bisa menemukan bermacam-macam orang Kristen. Seperti J Seth Msweli dan Donald Crider dimana mereka menganggap sekolah Minggu begitu penting bagi setiap anggota jemaat baik anak-anak mau pun orang dewasa seperti yang disampaikan lewat bukunya Gembala sidang dan Pelayanannya sebagai berikut: “Sekolah Minggu adalah kelompok-kelompok pemahaman Alkitab pada hari Minggu, bukan hanya untuk anak- anak tetapi jua untuk orang dewasa”.25 Kelas Sekolah Minggu untuk setiap anggota jemaat itu sangat penting, karena melaluinya adalah suatu kesempatan yang baik untuk mengajarkan tentang, peristiwa-peristiwa, tokoh-tokoh dan pengalaman hidupnya, serta menghafalkan ayat-ayat Firman Allah. Selain sekolah Minggu kelas katekisasi untuk orang Kristen baru. Gembala sidang juga harus mengajar di kebaktian pemuda dengan mengajukan pertanyaan-pertanyaan sesuai dengan topik yang diangkat. Jurnal Excelsisi Deo: Vol. 6 No.1 Juni 2022 Ajarkan, agar mereka mengetahui akan hal apa yang harus diketahui, dan mereka melakukan akan hal apa yang harus dilakukan. 25Seth Moweli dan Donald Crider, Gembala Sidang dan Pelayanan (Bandung: Kalam Hidup, 2003), 204. Jurnal Excelsisi Deo: Vol. 6 No.1 Juni 2022 memberikan pengajaran kepada. 23 “Dalam Perjanjian Lama istilah ini terutama disebut ‘laqakh’ artinya diterima, seperti penjelsan kamus singkat Ibrani Indonesia diterjemahkan “mengambil” atau “diterima” dari naska aslinya ‘liqikhi’ arti harfiah yang biasa gunahkan ‘pengajaran’, (Ay. 11:2). Sebagai contoh (Mat. 7: 28) ‘didache’ artinya ajaran atau pengajaran. Mengajar merupakan suatu tugas dalam memberikan pelajaran tertentu kepada orang lain. Pergeseran pada Alkitab II Timotius 3:16 mendapatkan sebuah istilah mengajar, yang diikuti oleh istilah “bermanfaat” (berguna, dalam Alkitab Kabar Baik) berarti Alkitab berguna untuk mengajarkan orang lain (umat Allah). Alkitab bukan hanya digunakan untuk berkhotbah, tapi juga berguna untuk mengajar. Karena berkhotbah itu hampir berbeda dengan mengajar. Khotbah lebih bersifat memberitakan, sedangkan mengajar lebih cenderung ke pelayanan pastoral Kedua pekerjaan ini harus dilakukan oleh seorang hamba Tuhan terutama oleh gembala sidang sebagai tugas yang mulia. Sebab pekerjaan gembala sidang bukan hanya sebatas pada berkhotbah, tetapi juga dalam mengajar. Kedua-duanya mempunyai fungsi masing- masing dalam menolong anggota jemaatnya. Dalam Perjanjian Baru terdapat dua kata yang berhubungan dengan istilah tersebut dan mempunyai pengertian yang sama, dan digunakan oleh Paulus sebanyak sembilan belas kali, lima belas diantaranya terdapat pada surat-surat penggembalaan, pertama, didaskalia itu mencakup baik pekerjaan mengajar maupun isi ajaran itu sendiri. Tafsiran Alktab Wycliffe Perjanjian Baru mengatakannya, sebagai berikut:“ditekankan didalam surat-surat penggembalaan sembilan belas antara dua puluh pemakaian kata ini di dalam Perjanjian Baru yang terdapat dalam surat- surat Paulus dari sembilan belas tersebut, lima belas diantaranya terdapat didalam surat-surat penggembalaan kata mengajar kira-kira terdapat tiga kata didalam kitab II Timotius, seperti (II Tim. 2:22) didaxai aorit infinitif didasko mengajar atau mengajarkan, (II Tim. 2:24) didaktikon artinya cakap megajar, (II Tim. 3:16 didaskalia mengajar.” Istilah kedua yang sama dengan diatas adalah didache. Menurut Ensklopedi Alkitab masa kini Jilid 1 menjelaskan mengenai kata tersebut demikian: “dipakai lebih sering dalam bagian terbanyak pasal Pejanjian Baru. Kata ini juga dapat berarti baik pekerjaan mengajar mau pun isi ajaran itu sendiri.24 Lihatlah pelayanan Yesus, ketika Ia memberitakan Injil di se-kitar Galilea, Markus 9:35 “Demikianlah Yesus berkeliling ke semua kota dan desa; Ia mengajar dalam rumah-rumah Ibadat dan memberitakan Injil kerajaan sorga.” Yesus mengajar dan berkhotbah merupakan suatu teladan yang baik bagi seorang hamba Tuhan atau Gembala sidang, agar melakukan kedua pekerjaan tersebut. Perbedaan Pengertian antara kedua pekerjaan ini. Pada saat gembala sidang berkhotbah, ia sendiri yang berbicara dan belum tentu juga apa mereka mengerti atau 24 Ibid., 22. Jurnal Excelsis Deo: Teologi, Misiologi, dan Pendidikan\| 111 Jurnal Excelsisi Deo: Vol. 6 No.1 Juni 2022 yang hebat. Firman Allah sungguh memperbaiki karakter-karakter kita yang rusak untuk menjadi lebih baik lewat penyerahan hidup. Oleh karena itu Alktab baik Perjanjian Lama maupun Perjanjian Baru menekankan bahwa umat Allah harus berbeda sikap kelakuannya dengan orang- orang dunia lainnya. Maka Alkitab menyoroti kepada perubahan tingkalaku kearah yang lebih baik seperti yang tercantum dalam surat pertama kepada Timotius 1:18-19, disini demikian bunyinya: “Tugas ini kuberikan kepadamu Timotius anakku, sesuai dengan apa yang telah dinubuatkan tentang dirimu supaya dikuatkan oleh nubuat itu engkau memperjuangkan perjuangan yang baik dengan iman dan hati nurani yang murni. Beberapa orang telah menolak hati nurani yang murni itu dan karena itu kandaslah Iman mereka” memisahkan jiwa dan roh yang paling erat sekalipun, dan sanggup membedakan pikiran dan hati yang sulit dibedakan oleh manusia. Dengan cara inilah Firman Allah mengungkapkan manusia seutuhnya, terutama dalam kaitan dengan sikap hati dan imannya. J. Stephen Lang mengatakan dalam bukunya Pedoman Lengkap Janji- janji Alkitab, sebagai berikut: “Sebab apapun yang dikatakan Allah kepada selalu penuh dengan kuasa yang hidup: Firman Allah lebih tajam dari pada pedan bermata dua yang paling tajam yang dapat dengan cepat menembusi pikiran dan keinginan yang paling dalam sehingga memperlihatkan diri yang sebenarnya. Ia mengetahui seluk beluk di mana pun juga.”26 J. Stephen Lang mengatakan dalam bukunya Pedoman Lengkap Janji- janji Alkitab, sebagai berikut: “Sebab apapun yang dikatakan Allah kepada selalu penuh dengan kuasa yang hidup: Firman Allah lebih tajam dari pada pedan bermata dua yang paling tajam yang dapat dengan cepat menembusi pikiran dan keinginan yang paling dalam sehingga memperlihatkan diri yang sebenarnya. Ia mengetahui seluk beluk di mana pun juga.”26 Firman Setelah anggota jemaat (umat Allah) mengetahui yang telah diajarkan, maka sesuatu hal itu telah nyata bagi mereka. Menyatakan kesalahan, dalam bahasa Yunani elegmos kata Yunani tersebut mengandung dua pengertian dalam bahasa Indonesia. Karena pengertian “menyatakan” adalah, proses dalam menunjukkan sesuatu, sedangakan “kesalahan” artinya prihal salah atau kekeliruan yang terjadi. Jadi elegmos artinya proses untuk menunjukkan sesuatu yang dianggap sudah salah atau keliru. Alkitab itu berguna dan masih berproses di dalam diri orang percaya, dan berkuasa menyelidiki bahkan mengubah hidup seseorang, seperti yang tertulis dalam (Ibr. 4:12,13) adalah sebagai berikut sebab Firman Tuhan itu hidup dan kuat dan lebih tajam dari pada pedang bermata dua mana pun; ia menusuk amat dalam sampai memisahkan jiwa dan roh sendi-sendi dan sum-sum ia sanggup membedakan pertimbangan dan pikiran hati. Dan tidak ada suatu makhluk pun yang tersembunyi dihadapan-Nya, sebab segala sesuatu telanjang dan terbuka dihadapan mata Dia, yang kepada-Nya harus memberikan pertanggungan jawab Firman Allah yang diilhami mengandung kuasa ilahi yang sanggup menyatakan kesalahan dan Jurnal Excelsis Deo: Teologi, Misiologi, dan Pendidikan\| 111 Jurnal Excelsisi Deo: Vol. 6 No.1 Juni 2022 26J. Stephen Lang, Pedoman Lengkap Janji- 112 \| Jurnal Excelsis Deo: Teologi, Misiologi, dan Pendidikan 112 \| Jurnal Excelsis Deo: Teologi, Misiologi 26J. Stephen Lang, Pedoman Lengkap Janji- janji Alkitab (Bandung: Kalam Hidup, 2001), 21. 26J. Stephen Lang, Pedoman Lengkap Janji- janji Alkitab (Bandung: Kalam Hidup, 2001), 21. Alkitab Berguna Untuk Memperbaiki kelakuan Rasul Paulus telah mengarahkan perhatian kepada pentingnya mempertahankan standar tingka laku yang benar disamping mempertahankan keyakinan yang benar. Pedoman Lengkap Pendalaman Alkitab mengatakan “memperjuangkan perjuangan yang baik dengan iman dan hati nurani yang murni.” Jadi harus ada perbedaan antara iman dan kelakuan (perbuatan). Iman adalah apa yang yakini tentang Kristus. Hati nurani adalah hati yang bersih atau tidak mengizinkan nurani dikotor dengan kebiasaan-kebiasaan berdosa yang bertentangan dengan doktrin Alkitab. Jika doktrin yang benar (doktrin Alkitab) tidak ditaati, maka sesungguhnya doktrin Setelah Alkitab menyatakan kesalahan, maka ia juga bersedia untuk memperbaikinya.IstilahYunani panorthosis perbaikan kelakuan. Diterjamahkan dalam bahasa Indonesia, sebagai perbaiki, membetulkan sesuatu yang dianggap salah menjadi labih baik atau rapi, dari semulanya. Inilah tujuan utama dalam perbaikan sesuatu hal atau benda apa pun. Hasil yang baik menyenangkan hati dan menjadi kebahagiaan bagi diri dan sering bagi keluarga dan lingkungannya. Pentingannya Perbaikan kelakuan, Karena tanpa ada proses perbaikan, maka kelakuan yang salah malah sering menjadi kerusakan Jurnal Excelsisi Deo: Vol. 6 No.1 Juni 2022 27John Hunter, Kehidupan Kristen yang Sukses (Bandung: Kalam Hidup, 1994), 27. Jurnal Excelsis Deo: Teologi, Misiologi, dan Pendidikan\| 113 en yang 27. 28Cahya R, Pola hidup Kristen (Malang: Gandum Mas, 2002), 282. Jurnal Excelsisi Deo: Vol. 6 No.1 Juni 2022 tersebut ditolak dan menjadi “iman yang mati”, sehingga kemajuan iman pun “kandas”. Jika tingka laku tidak benar, maka hal itu akan menghancurkan keyakinan yang benar. perbuatan kebenaran, perbuatan keadilan dan perbuatan kemurahan. Jika iman yang tidak menghasilkan perbuatan yang benar iman itu mati.28” Tentang kedua hal tersebut diatas mana yang lebih penting, iman atau perbuatan. Bagi orang yang belum diselamatkan didahulukan dengan iman, tetapi bagi yang telah diselamatkan perbuatan yang diutamakan. Iman yang benar kepada Yesus Kristus menyenangkan hati Allah (Ibr. 11:6) demikian bunyinya: tetapi tanpa iman tidak mungkin orang berkenan kepada Allah. sebab barang siapa berpaling kepada Allah, ia harus percaya bahwa Allah ada, dan bahwa Allah memberi upa kepada orang- orang yang sungguh-sungguh mencari Dia John Hunter mengatakan dalam bukunya kehidupan Kristen yang sukses, sebagai berikut: “Banyak orang Kristen yang layani adalah orang-orang Kristen sejati yang sudah mengalami pengalaman rohani yang sangat penting . Mereka sudah percaya kepada Kristus sebagai Juruselamat pribadi mereka dan memang mereka bersungguh-sungguh dalam soal. Ketika Alkitab menembus ke dalam diri manusia, maka ia akan menyatakan kelakuan yang kurang menyenangkan mengasihi dan melayani Kristus. Mereka tidak puas hanya dengan diampuni tetapi setelah mereka meneliti dan mengakui dosa-dosa mereka, mereka masih mendapatkan diri mereka dalam kelemahan dan kegagalan yang belum teratasi. Kepercayaan mereka memang benar tetapi kelakuan mereka tidak seperti yang mereka harapkan.27 Bagi orang yang mempunyai iman seperti ini, adalah diciptakan didalam Kristus Yesus untuk melakukan pekerjaan yang baik , seperti dalam (Efe. 2:10), sebagai berikut:“Karena ini buatan Allah, diciptakan di dalam Kristus Yesus untuk melakukan pekerjaan yang baik, yang dipersipkan Allah sebelumnya. Ia mau supaya hidup didalamnya.”Demikian juga dalam surat Yakobus dapat melengkapi kedua hal yaitu iman dan perbuatan baik, sebagai berikut: “Apa gunanya saudara- saudaraku jika orang mengataan bahwa ia mempunyai iman, pada hal ia tidak mempunyai perbuatan? Dapatkah iman itu menyelamatkan dia? Jika seorang suadara atau saudari tidak mempunyai pakaian dan kekurangan makanan sehari-hari, dan seorang dari antara kamu berkata: “Selamat jalan knakanlah kain panas dan makanlah Kebanyakan orang telah mengetahui rahasia untuk menjadi orang Kristen, tetapi mereka masih belum mengetahui rahasia untuk berkelakuan sebagai orang kudus. Kelakuan sebagai orang percaya dapat terlihat dalam aspek kehidupan sebagai bukti iman. Jika iman yang benar senantiasa mengahasilkan perbuatan yang benar. Seperti yang dijelaskan dalam buku Pola Hidup Kristen, sebagai berikut: “Iman Kristen yang benar harus terlihat dalam perbuatan yang benar 28Cahya R, Pola hidup Kristen (Malang: Gandum Mas, 2002), 282. Jurnal Excelsisi Deo: Vol. 6 No.1 Juni 2022 Jurnal Excelsis Deo: Teologi, Misiologi, dan Pendidikan\| 113 Jurnal Excelsisi Deo: Vol. 6 No.1 Juni 2022 dalam soal-soal pokok iman. dan dalam ajaran sehat”. Maksud Paulus agar pendidikan atau pembinaan bagi setiap orang percaya itu sangat penting. Karena melalui didikan (pembinaan), maka umat Allah akan bertumbuh dengan baik dan dapat mempertahankan iman kepada Kristus, sehingga mereka dapat menginjak pada posisi kedewasaan secara rohani. percaya itu sangat penting. Karena melalui didikan (pembinaan), maka umat Allah akan bertumbuh dengan baik dan dapat mempertahankan iman kepada Kristus, sehingga mereka dapat menginjak pada posisi kedewasaan secara rohani. sampai kenyang!” tapi ia tidak memberikan kepadanya apa yang perlu bagi tubuhnya apa gunanya itu? Demikian juga halnya dengan iman: jika iman itu tidak disertai perbuatan, maka iman itu pada hakekatnya adalah mati” (Yak. 2:14-17). Iman Kristen yang benar senantiasa menghasilkan buah yang baik. Seperti kasih, kemurahan dan lain sebagainya. Jadi iman dan perbuatan yang benar bergandengan. 114 \| Jurnal Excelsis Deo: Teologi, Misiologi, dan Pendidikan KESIMPULAN Dalam pembahasan masalah tentang nilai kebenaran dan Alkitab bagi umat manusia yang terutama kepada umat Allah menurut II Timotius 3:16, maka kini menjadi kesempatan untuk menyimpulkan masalah tersebut. Pengaruh alkitab bagi orang percaya menurut II Timotius 3: 16 yang pertama, Alkitab berguna untuk mengajar, kedua, Firman Allah Berguna untuk menyatakan kesalahan, Ketiga Alkitab berguna untuk Memperbaiki kelakuan, Keempat Alkitab Berguna untuk mendidik orang dalam kebenaran. Dengan adanya alasan-alasan dasar tersebut diatas, maka tidak meragukan lagi untuk mengatakan dengan jelas: bahwa “Alkitab adalah Firman Allah. Alkitab orang Kristen secara etimologi adalah sebuah kitab yang bersifat unik dan luar biasa. Nilai kebenaran dan pengaruh Alkitab bagi umat Allah telah terbukti, sehingga hampir seluruh umat Kristen terasa akan kuasa pengaruhnya. Oleh karena itu dengan hati yang penuh kesadaran percaya dan mengakui bahwa seluruh isi Alkitab itu adalah Firman Allah. Pengakuan tersebut bukan asal mengungkapkannya, tetapi, sudah mempunyai dasar keyakinan akan hal tersebut seperti yang telah membahas diatas. Bawah “Segala tulisan diilhamkan Allah” atau “dihembuskan oleh Allah” jadi setiap kata yang mengambil bagian di dalam Alkitab telah diilhami. Pengilhaman Allah bukan jatuh pada para penulisnya, tetapi melalui para penulis. Seperti menurut ayat pokok II Tim. 3:16) “pasa grafe” “segala” atau “setiap tulisan” atau “nast” hal ini menunjukan kepada tulisannya. Alkitab berguna untuk mendidik orang dalam kebenaran. Setelah Alkitab memperbaiki kelakuan, maka Ia juga bersedia untuk memimpin orang kearah yang dikehendaki Allah, asalkan mendekati kepadanya. Jurnal Excelsisi Deo: Vol. 6 No.1 Juni 2022 Tulisan yang diilhamkan Allah memang bermanfaat untuk mengajar, untuk menyatakan kesalahan, untuk memperbaiki kelakuan dan untuk mendidik orang dalam kebenaran’ yang Pertama melihat; Keunikkan Sifat Alkitab, Kedua, Alkitab tidak bisa dimusnahkan, Ketiga, Kesatuan berita kitab-kitab dalam Alkitab, keempat, Pembagian Kitab-Kitab dalam Alkitab. “Bagi- Nya kemuliaan, sekarang sampai selama-lamanya. Alkitab Berguna Untuk mendidik orang dalam kebenaran Pengaruh Alkitab bagi orang percaya bukan hanya terbatas pada memperbaiki sikap kelakuan yang salah. Namun Alkitab masih terus berperang memainkan peranan penting untuk mendidik (membina) orang percaya ke arah yang dikehendakinya. Penjelasan lebih lanjutnya sebagai berikut: Pengertian istilah mendidik (pembinaan) bagi umat Allah, Istilah Yunani ‘paideia’ yang diterjemahkan dengan “disiplin” atau “didikan”. Istilah tersebut menurut Kamus Besar Bahasa Indonesia me-ngatakan, “memelihara, memberi pelatihan ajaran, tuntunan, pimpinan” mengenai kelakuan hidup dan kecerdasan pikiran”. Jadi “mendidik orang dalam kebenaran” menunjuk kepada latihan atau pembinaan yang harus diadakan di jalan kebenaran atau di dalam iman. Istilah paidia yang terdapat dalam surat-surat Paulus mencakup pada pengajaran atau pembinaan bagi orang percaya baik didikan orang tua terhadap anak-anak, seperti (Ef. 6:4) “didiklah mereka dalam ajaran Tuhan, maupun seorang hamba Kristus terhadap umat Allah, seperti (I Tim. 4:6) “terdidik Pokok utama dalam pembinaan bagi umat Allah, Pembinaan umat Allah adalah pembinaan yang berpusat pada Kristus dan di dalam Kristus serta ke arah Dia, Kristus. Seperti Paulus mengatakan dalam, (Ef. 4:14-16) “sehingga bukan lagi anak-anak yang diombang ambingkan oleh rupa-rupa angina pengajaran, oleh Permainan palsu manusia dalam kelicikan mereka yang menyesatkan, tetapi dengan teguh berpegang kepada kebenaran didalam kasih bertumbuh didalam segala hal kearah Dia, Kristus, yang adalah Kepala. Dari pada-Nyalah seluruh tubuh yang rapi tersusun dan diikat menjadi satu oleh pelayanan semua bagiannya, sesuai dengan kadar pekerjaan tiap-tiap anggota – menerima pertumbuhannya dan membangun diri dalam kasih. ”Sama hal juga Petrus menekankan kepada orang percaya “bertumbuhlah dalam kasih karunia dan dalam pengenalan akan Tuhan dan Juruselamat, Yesus Kristus” (II Pet. 3:18). Kepada Kristus, yang adalah awal, proses, dan penggenapan dari keselamatan. 114 \| Jurnal Excelsis Deo: Teologi, Misiologi, dan Pendidikan Jurnal Excelsisi Deo: Vol. 6 No.1 Juni 2022 DAFTAR PUSTAKA Subagyo, Andreas Bambang. Pengantar Riset Kuantitatif dan Kualitatif. Bandung: Kalam Hidup, 2004. E. Hill, Andrew dan Walton, John H. Survei Perjanjian Lama. Malang: Gadum Mas, 2004. Louis. Berkhof. Theologi Sitematika. Jakarta: Lembaga Informed Injili Indonesia, 1996. B. F. Drewes, Wilfrit Haebech & Heinrich Vonsiebenthal unci. Bahasa Yunani Perjanjian Baru, Jakarta: Gunung Mulia, 2006. Charles. C. Ryrie.Teologi Dasar I, Yogyakarta: Yayasan Andi, 1991 Bukti-bukti kebenaran Alkitab berdasarkan perkataan Rasul Paulus dalam II Timotius 3:16 yang berbunyi “Segala Bukti-bukti kebenaran Alkitab berdasarkan perkataan Rasul Paulus dalam II Timotius 3:16 yang berbunyi “Segala George, Sandison. Bible Answers for 1000 Difficulty Questions. Malang: Gandum Mas 2006, Jurnal Excelsis Deo: Teologi, Misiologi, dan Pendidikan\| 115 Jurnal Excelsisi Deo: Vol. 6 No.1 Juni 2022 116 \| Jurnal Excelsis Deo: Teologi, Misiologi, dan Pendidikan Jurnal Excelsisi Deo: Vol. 6 No.1 Juni 2022 William W. Wenzies & Stanley M. Horton. Doktrin Alkitab. Gandum Mas, 1998. Henry C. Thiessen, Teologi Sistematika, Vernon D. Doerksen, peny. Malang: Gandum Mas, 1995 Heath, Stanley. Teologi Pendidikan Anak Dasar Pelayanan kepada Anak. Bandung: Yayasan Kalam Hidup, 2005. Irawati, Enny. Peranan Mahasiswa dan Lembaga dalam Membangun Toleransi Keragaman Budaya di Sekolah Tinggi Teologi Biblika Jakarta. Jurnal Anthroplogi Sosial dan Budaya Anthropos, 6 (1), 2020. J. Stephen, Lang. Pedoman Lengkap Janji- janji Alkitab. Bandung: Kalam Hidup, 2001. John, Hunter. Kehidupan Kristen yang Sukses. Bandung: Kalam Hidup, 1994. Robert, Backhouse, 5000 Kata Hikmat, Yogyakarta: Andi, 2006 S. Wismoady Wahono. Di Sini Kutemukan. Jakarta: Gunung Mulia, 2009 Seth Moweli dan Donald, Crider. Gembala Sidang dan Pelayanan. Bandung: Kalam Hidup, 2003. Mahengkeng Y. Tafsiran Alkitab Masa Kini. Jakarta: Komunikasi Bina Kasih/ OMF, 2005. R, Cahya. Pola hidup Kristen. Malang: Gandum Mas, 2002. Witoro, Johanes. Peranan Orang Tua Terhadap Tingkat Penggunaan Media Online Remaja di Jakarta .Universitas Negeri Malang: Jurnal Inspirasi Pendidikan 9 (2), 2019. Wismoady,Wahono Disini kutemukan. Jakarta: Gunung Mulia, 2004. Bruning, W. R. F. Kamus Alkitab. Jakarta: Gunung Mulia, 2007. 116 \| Jurnal Excelsis Deo: Teologi, Misiologi, dan Pendidikan
https://openalex.org/W2145520758	https://bmcpublichealth.biomedcentral.com/counter/pdf/10.1186/1471-2458-10-472	English	null	High risk behavior for HIV transmission among former injecting drug users:a survey from Indonesia	BMC public health	2,010	cc-by	6,147	Abstract Background: Injecting drug use is an increasingly important cause of HIV transmission in most countries worldwide, especially in eastern Europe, South America, and east and southeast Asia. Among people actively injecting drugs, provision of clean needles and opioid substitution reduce HIV-transmission. However, former injecting drug users (fIDUs) are often overlooked as a high risk group for HIV transmission. We compared HIV risk behavior among current and former injecting drug users (IDUs) in Indonesia, which has a rapidly growing HIV- epidemic largely driven by injecting drug use. Methods: Current and former IDUs were recruited by respondent driven sampling in an urban setting in Java, and interviewed regarding drug use and HIV risk behavior using the European Addiction Severity Index and the Blood Borne Virus Transmission Questionnaire. Drug use and HIV transmission risk behavior were compared between current IDUs and former IDUs, using the Mann-Whitney and Pearson Chi-square test. Results: Ninety-two out of 210 participants (44%) were self reported former IDUs. Risk behavior related to sex, tattooing or piercing was common among current as well as former IDUs, 13% of former IDUs were still exposed to contaminated injecting equipment. HIV-infection was high among former (66%) and current (60%) IDUs. Conclusion: Former IDUs may contribute significantly to the HIV-epidemic in Indonesia, and HIV-prevention should Conclusion: Former IDUs may contribute significantly to the HIV-epidemic in Indonesia, and HIV-prevention should therefore also target this group, addressing sexual and other risk behavior. HIV to others [2,3]. To our knowledge, no studies on fIDUs have been reported from low- or middle-income countries. RESEARCH ARTICLE Open Access © 2010 Iskandar et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. High risk behavior for HIV transmission among former injecting drug users:a survey from Indonesia Shelly Iskandar1,2, Diba Basar2, Teddy Hidayat1, Ike MP Siregar1, Lucas Pinxten2,3, Reinout van Crevel2,4, Andre JAM Van der Ven 2,4, Cor AJ De Jong5 Iskandar et al. BMC Public Health 2010, 10:472 http://www.biomedcentral.com/1471-2458/10/472 Iskandar et al. BMC Public Health 2010, 10:472 http://www.biomedcentral.com/1471-2458/10/472 Correspondence: shelly_bdg@yahoo.com 1Department of Psychiatry, Faculty of Medicine, Padjajaran University/Hasan Sadikin Hospital, Bandung, Indonesia Full list of author information is available at the end of the article Background Worldwide, injecting drug use is estimated to account for just less than one-third of new infections outside sub-Saharan Africa [1]. HIV-prevention programs for injecting drug users (IDUs) therefore put emphasis on people actively injecting drugs, especially through needle exchange or opioid replacement. Besides active IDUs, people who have a previous history of injecting drug use (former IDUs) are probably also an important risk group for HIV transmission. However, relatively little is known about this group. Sporadic studies from western countries have shown that former IDUs (fIDUs) may have a high risk of becoming HIV-infected or spreading Injecting drug use increased dramatically in the late ‘90s in Indonesia, acting as the main force driving the HIV-epidemic. Among the general population, the pre- valence of HIV-infection is still low (0.3%), but up to 50% or more of IDUs are already HIV-infected [4]. Drug use is illegal in Indonesia, and harm reduction pro- grams, although officially supported by the Indonesian government, only reach a minority of IDUs. Apart from sharing needles, sexual risk behavior is also common among drug users [5]. In three large cities in Indonesia, over two thirds of IDUs were sexually active, of whom many reported having multiple partners (48%) and sex with female sex workers (40%) in the preceding year. * Correspondence: shelly_bdg@yahoo.com 1Department of Psychiatry, Faculty of Medicine, Padjajaran University/Hasan Sadikin Hospital, Bandung, Indonesia Full list of author information is available at the end of the article Iskandar et al. BMC Public Health 2010, 10:472 http://www.biomedcentral.com/1471-2458/10/472 Page 2 of 7 entering the study twice, physical marks such as tattoos, scars, or birth marks were recorded. Consistent condom use was only reported by 10% of sexually active IDUs [5]. We know of no reported data concerning HIV risk behavior among fIDUs in Indonesia, and fIDUs receive very little attention in current prevention programs in general. This may seriously limit the success of HIV- prevention focusing on drug injection, as a considerable number of IDUs change from injection to non-injection drug administration or completely abstain from illicit drug use [6]. The prevalence of HIV may be high in fIDUs. Transmission of blood borne viruses may con- tinue to occur through sexual behavior and/or by con- taminating equipment that is subsequently used by others for drug use, tattooing and/or piercing [2,7,8]. Only those candidates who were or had previously been IDUs were eligible to be included in the study. Setting and patients From June to September 2008, 210 IDUs were recruited in Bandung, the capital of West-Java and epicenter of the epidemic of injecting drug use in Indonesia. Respon- dent driven sampling, a form of peer recruitment, was used for recruitment of IDUs from the community [11]. With help from local non-governmental organizations involved in outreach to IDUs, three cIDUs and three fIDUs from different parts of Bandung were selected to act as ‘seeds’ for RDS and invited to a community clinic which has a specific program for IDUs. Following their inclusion in the study, these six seeds were asked to recruit two other persons injecting drugs, either in the last six months (cIDUs) or longer ago (fIDU), by giving individually numbered coupons. IDUs presenting at the community clinic with the coupons, were asked them- selves to recruit two other (current or former) IDUs. This process of recruitment continued until the desired sample size was achieved. Numerical simulations have shown that respondent driven sampling estimates con- verge to the true values even if the seeds are not drawn as desired [12]. g The interviewers used two validated questionnaires: the European Addiction Severity Index (EuropASI) and the Blood Borne Virus Transmission Questionnaires (BBV-TRAQ). The EuropASI is an adaptation of the Addiction Severity Index (fifth version). It is a semi- structured interview which takes about one hour, cover- ing issues that may contribute to patients’ substance- abuse problems, such as medical status, employment/ support status, drug/alcohol use, legal status, family social relationship, and psychiatric problems [14]. Parti- cipants are asked if they ever used a number of listed drugs regularly (more than 3 times or 2 consecutive days a week). For regularly used drugs, further informa- tion is recorded including the first time the particular drug was used, the duration of use in a life time, the fre- quency of drug use in the previous 30 days, and drug route of administration [14]. ASI has shown excellent reliability and validity across a range of types of patients and treatment settings in many countries [15]. For the translation into Bahasa Indonesia, WHO translation procedures were used [16]. As a part of the RDS process, an incentive was offered for participating in the interview ($3) and for recruiting two injecting drug using peers ($2 per eligible peer recruited). Background Two outreach workers from non-governmental harm reduction organizations, both with a previous history of drug use, confirmed that the respondents were indeed IDUs. To this purpose they looked for possible needle tracks, asked each respondent to demonstrate how he/ she injected drugs, and to clarify specific ‘slang’ used by IDUs. All IDUs who passed this screening then provided informed consent. The study was approved by the regio- nal medical-ethical committee (The Health Research Ethics Committee, Faculty of Medicine, Padjadjaran University/Dr. Hasan Sadikin General Hospital Ban- dung) and conducted within the context of program on prevention and treatment of HIV in the context of injecting drug use in Indonesia [13]. Furthermore, fIDUs may also play an important role in transmitting HIV infections to the general population because, compared with current IDUs (cIDUs), fIDUs have more sexual contact with people who do not use drugs [9,10]. Hopefully, a better characterization of for- mer IDUs may contribute to improve HIV-prevention. Therefore, the aim of the present study was to explore the characteristics and the risk behavior of former IDUs in Indonesia in comparison with current IDUs. Assessment The interview was done at the community health center by trained interviewers who assured all participants that their anonymity would be strictly maintained. All parti- cipants who completed the interview session received a coupon for free HIV, HBV, HCV and syphilis testing at Hasan Sadikin hospital, Bandung. If found positive for HIV, participants were offered CD4-cell counts, chest X-ray and if needed, antiretroviral and/or syphilis treat- ment, all free of charge. Setting and patients After the initial seeds were recruited, only those people who presented coupons were permitted to participate in the study. The study was completely anon- ymous, but to prevent the same participant from The BBV-TRAQ questionnaire assesses how often injecting drug users participate in specific injecting, sex- ual and other risk-practices that may expose them to blood-borne viruses. The instrument consists of Iskandar et al. BMC Public Health 2010, 10:472 http://www.biomedcentral.com/1471-2458/10/472 Page 3 of 7 34 questions divided in three sub-scales which measure frequency of current risk behavior related to blood to blood transfer (20 questions); sexual practices (8 ques- tions); and other skin penetration activities (6 questions) in the previous month. With respect to possible blood to blood transfer, information is collected about contact with contaminated needles and syringes, other drug injecting equipment sharing and involvement of other people in the drug preparation and injecting process. Questions related to sexual risk behavior address unpro- tected vaginal, anal, oral, and manual sex with other people, with or without lubricant, and during menstrua- tion or not. Other questions address skin penetration risk behavior (tattooing and piercing), and shared use of toothbrush, razor, and personal hygiene equipment. The administration time for the instrument is short (around 15 minutes), and it has been shown good reliability and validity [17,18]. after non-injecting drug administration, and the period of injecting drugs averaged 7 (± 4) years. Three quarters of the participants had been tested for HIV and among those tested 63% reported to be HIV-infected. There was no significant association between the cumulative years of drug injecting and HIV-status (P = 0.47). One third of those who were HIV-infected reported to have developed AIDS. Although cIDUs had injected for a longer period than fIDUs, HIV-infected individuals in the latter group more commonly reported having AIDS (44% vs 18%; P = 0,01). The most used substance by all participants was her- oin. Ninety four percent of the total participants had at some point used heroin regularly (at least three times a week or for two consecutive days in a week for more than 6 months). The other most used substances were cannabis, benzodiazepines, and alcohol. Data analysis and statistics A former IDU (fIDU) was defined as a person who reported to have injected an illicit drug at some point in his/her life, but not to have injected any drugs in the six months prior to the interview (17, 18). A current IDU (cIDU) was defined as a person who reported that he/ she had injected any type of illicit drug in the six months prior to the interview [6,10]. Data were analyzed both descriptively and inferentially. Descriptive data are presented in terms of percentage, mean, and standard deviation. Subjects engaging in at least one risk taking behavior in a subscale of the BBV-TRAQ were regarded as taking risks in that domain. Data were analyzed infer- entially for differences between fIDUs and cIDUs. Pear- son Chi-Square was used for dichotomous data and the Mann-Whitney test for non-parametric continuous data. All tests were two-sided, with a P-value of 0.05 or less considered to indicate statistical significance. Analyses were performed with the use of SPSS, version 11.5. In the last 30 days, cIDUs had typically used more drugs than fIDUs but neither cIDUs nor fIDUs reported total abstinence in the last 30 days. The most used sub- stances by fIDUs in the last 30 days were alcohol, licit or illicit methadone/buprenorphine while cIDUs mostly used licit or illicit methadone/buprenorphine, heroin, benzodiazepines, and cannabis (table 2). Licit or illicit use of methadone or buprenorphin cannot be differen- tiated with the ASI. Risk behavior related to transmission of blood-borne viruses Blood to blood transfer risk behavior was reported by 76% of cIDUs and 13% of fIDUs (X2 = 82,73; p < 0,01) (table 3). Risk behavior of ninety cIDUs (76%) was related to sharing of contaminated drug injecting equipment. Current IDUs often reported behavior associated with a very high risk of transmission of blood-borne pathogens, including injecting with another person’s used needle or syringe (reported by 15% of cIDUs), re-use of a needle or syringe taken out of a shared disposal/sharps container without using bleaching (9%), and sharp injuries from another per- son’s used needle/syringe (15%). cIDUs also reported behavior associated with a somewhat lower risk of transmission. For example, they had exposure to con- taminated drug injecting equipment included shared use of a tourniquet (43%); injecting a drug prepared with water previously used by another person (41%); handling another person’s used needle or syringe when wounded at his or her hand (34%); wiping his/her own injection site with an object that had been used by Setting and patients More than three quarters of participants had ever used or still used dif- ferent drugs at the same time (poly drug use) and 70% of the total IDUs had ever used or still used ampheta- mine or methamphetamine regularly (median 2 years (range less than 1 year until 15 years). Characteristics of IDUs in Bandung BMC Public Health 2010, 10:472 http://www.biomedcentral.com/1471-2458/10/472 Page 4 of 7 Table 1 Sociodemographic characteristics of former and current injecting drug users Total group (N = 210) fIDUs (N = 92) cIDUs (N = 118) P Age, mean 27,8 (3,8) 28,1 (4,0) 27,5 (3,8) 0,64 Male gender 92% 89% 95% 0,12 Drug use 0,35 Age of first drug use, mean 14,0 (2,8) 14,2 (3,3) 13,8 (2,2) 0,31 Age of first drug injection, mean 18,0 (3,1) 18,4 (3,1) 17,8 (3,1) 0,17 Years of injecting in life time, mean 7,1 (3,8) 5,5 (3,6) 8,4 (3,4) < 0,01 Marital Status 0,35 Married/remarried 30% 36% 25% Widowed 2% 3% 2% Separated/divorced 11% 6% 14% Never married 57% 55% 59% Employment in the past 3 years 0,13 Full time 41% 48% 36% Part-time 37% 36% 39% Student 5% 5% 4% Unemployed or housewife 17% 11% 21% Education 0,48 Junior high school or less 6% 4% 7% Senior high school 87% 87% 87% Undergraduate or higher 7% 7% 6% HIV-AIDS Ever HIV-tested 75% 71% 78% 0,23 HIV-infected# 63% 66% 60% 0,47 Have developed AIDS## 30% 44% 18% 0,01 All data are presented in percentage unless stated otherwise # n = 145; no data available for 12 subjects; ## n = 83; no data for 8 HIV-positive subjects odemographic characteristics of former and current injecting drug users All data are presented in percentage unless stated otherwise # n = 145; no data available for 12 subjects; ## n = 83; no data for 8 HIV-positive subjects All data are presented in percentage unless stated otherwise # n = 145; no data available for 12 subjects; ## n = 83; no data for 8 HIV-positive subjects sucking or licking and other handling of another per- son’s used needle/syringe. Interestingly, benzodiazepine use was more common among IDUs engaging in risky injecting behavior, 56% vs. 39% among cIDUs (X2 = 2,26; P = 0,19) and fIDUs (33% vs 9%; X2 = 5,99; P = 0,03). Injecting risk behavior was not associated with use of alcohol, cannabis or methadone/buprenorphin (data not shown). another person (27%); touching his/her own injection site soon after ‘assisting’ another person with their injection (26%); and injecting a drug that was prepared immediately after ‘assisting’ another person with their injection but without washing hands between activities (26%). egular use (more than 3 times or 2 consecutive days a week). ## ≥3 drinks in 1-2 hours, ≥3 times or 2 consecutive days a week. g ( y ) , y Note: Less than three participants used inhalant, hallucinogens or cocaine in the last 30 days. # regular use (more than 3 times or 2 consecutive days a week). ## ≥3 drinks in 1-2 hours, ≥3 times or 2 consecutive days a w Note: Less than three participants used inhalant hallucinogens or cocaine in the last 30 days Note: Less than three participants used inhalant, hallucinogens or cocaine in the last 30 days. # regular use (more than 3 times or 2 consecutive days a week). ## ≥3 drinks in 1-2 hours, ≥3 times or 2 consecutive Note: Less than three participants used inhalant, hallucinogens or cocaine in the last 30 days. Characteristics of IDUs in Bandung Characteristics of IDUs in Bandung A total of 210 IDUs were recruited, of whom 194 were men (92%), 92 were fIDUs (44%), and 118 were cIDUs (56%). Thirty-three out of 92 fIDUs (35.9%) were invited by cIDUs, while 34 of 118 cIDUs (30.3%) were invited by fIDUs, showing extensive social linking between the two groups. Most of the demographic characteristics of fIDUs and cIDUs did not differ, except for the length of injecting drug and percentage of those who developed AIDS (table 1). The mean age was 28 (±4) years and most participants had graduated from senior high school and had been employed at some point in the last 3years. They had started using drugs at a young age (14 (± 3) years). Injection of drugs had typically started 4 years Iskandar et al. regular use (more than 3 times or 2 consecutive days a week) ≥3 drinks in 1-2 hours, ≥3 times or 2 consecutive days a week. Note: Less than three participants used inhalant, hallucinogens or cocaine in the last 30 days. Table 3 Risk behavior among former (fIDUs) and current injecting drug users (cIDUs) in the last 30 days Risk Behavior fIDUs (N = 92) cIDUs (N = 118) X2 P Table 3 Risk behavior among former (fIDUs) and current injecting drug users (cIDUs) in the last 30 days Risk Behavior fIDUs (N = 92) cIDUs (N = 118) X2 P Blood-blood transfer, n (%) 13% 76% 82,73 <0,01 Suck or lick a filter which had been used by another person 4% 14% 5,81 0,02 Inject a drug prepared with water which had been used by another person 0% 41% 45,30 <0,01 Been injected by another person who had already injected or assisted in someone else’s injection 0% 20% 21,13 <0,01 Receive an accidental needle-stick/prick from another person’s used needle/syringe 3% 15% 7,56 <0,01 Re-use a needle/syringe taken out of a shared disposal/sharps container 0% 9% 8,26 <0,01 Sexual risk behavior, n (%) 42% 56% 3,57 0,06 Engage in unprotected vaginal sex with another person 35% 47% 2,98 0,08 Engage in unprotected vaginal sex with another person during menstruation 11% 20% 2,90 0,09 Engage in unprotected anal sex with another person 7% 8% 0,10 0,76 Tattoo or piercing, n (%) 52% 53% 0,02 0,90 Tattooed by someone who was not a professional tattooist 4% 15% 6,56 <0,01 Been pierced by someone who was not a professional piercer 10% 14% 0,96 0,327 Use another person’s toothbrush 3% 14% 7,45 <0,01 Table 3 Risk behavior among former (fIDUs) and current injecting drug users (cIDUs) in the last 30 days Engage in unprotected vaginal sex with another person Tattooed by someone who was not a professional tattooist Been pierced by someone who was not a professional piercer Tattooed by someone who was not a professional tattooist Been pierced by someone who was not a professional piercer Use another person’s toothbrush Use another person’s toothbrush with previous local and national data [4]. The prevalence of AIDS was higher among fIDUs; concern about their general health or the development of AIDS may cause them to move from injecting drugs [6]. Sexual risk behavior of some form was reported by 56% of cIDUs and 42% of fIDUs (X2 = 3,57; P = 0,06). The most common sexual risk behavior for cIDUs and fIDUs was unprotected vaginal sex (47% respectively 35%, NS); reported unprotected anal sex was much lower (8% respectively 7%, NS). No statistical significant differences were found between former and current IDUs regarding oral and manual sex (data not shown). Table 3 Risk behavior among former (fIDUs) and current injecting drug users (cIDUs) in the last 30 days Risk Behavior fIDUs (N = 92) cIDUs (N = 118) X2 P Excessive alcohol use was more common in IDUs enga- ging in risky sexual behavior, both among cIDUs (51% vs 33%; X2 = 3,86; P = 0,06) and fIDUs (46% vs 11%; X2 = 14,14; P < 0.01). Other risk behavior such as tat- tooing and piercing, which confer a much lower risk of HIV transmission compared to needle-sharing, was reported by half of all respondents (52% of fIDUs and 53% of cIDUs). Injecting with needles or syringe from other people (15%) or from shared disposal/sharps containers without bleaching (9%) was reported by a substantial proportion of IDUs, although lower compared to previous research in Indonesia (24% - 80%) [5,20]. The high rate of needle stick injuries is no surprise given the fact 40% of IDUs fail to discard used needles safely [21]. Risk related to injecting drugs was especially high in cIDUs, but fIDUs are experience certain things (e.g. needle sticks or shar- ing of injecting equipment) which may have a low risk of HIV-transmission, but which may pose a significiant risk of transmitting HCV. This is important, as the pre- valence of HCV is very high among IDUs in this setting; among 633 HIV patients with a history of IDU 87.7% were HCV-infected [19]. Characteristics of IDUs in Bandung While fIDUs did not inject anymore, 13% still had some risk of blood borne pathogens transmission, especially through accidental needle stick injuries and Table 2 Drug use among former and current injecting drug users Kind of drug # Life time drug use Drug use in last 30 days fIDUs (n = 92) cIDUs (n = 118) P fIDUs (n = 92) cIDUs (n = 118) P Any use of alcohol 91% 97% 0,14 42% 59% 0,03 Alcohol, over threshold ## 66% 70% 0,66 26% 42% 0,02 Heroin 99% 100% 0,44 0% 79% < 0,01 Methadone or buprenorphine 33% 65% < 0,01 12% 53% < 0,01 Other opiates 22% 23% 0,87 1% 5% 0,14 Benzodiazepines 66% 79% 0,06 12% 52% < 0,01 Amphetamine 52% 53% 0,89 1% 9% 0,03 Cannabis 84% 87% 0,55 13% 47% < 0,01 Ecstasy (MDMA) 44% 37% 0,40 9% 14% 0,28 More than one substance 78% 83% 0,38 12% 55% < 0,01 # l ( th 3 ti 2 ti d k) ## ≥3 d i k i 1 2 h ≥3 ti 2 ti d k Table 2 Drug use among former and current injecting drug users Table 2 Drug use among former and current injecting drug users Kind of drug # Life time drug use Iskandar et al. BMC Public Health 2010, 10:472 http://www.biomedcentral.com/1471-2458/10/472 Page 5 of 7 Table 3 Risk behavior among former (fIDUs) and current injecting drug users (cIDUs) in the last 30 days Risk Behavior fIDUs cIDUs g We would like to thank Prof. Tri Hanggono Ahmad, Dean of the Medical Faculty Padjadjaran University for encouraging and accommodating research in Padjadjaran University. We also thank the interviewers, Rinske de Graaff Stoffers, Mark Holland, staffs and ORC from Salam Primary Health Center. Rumah Cemara, Bahtera, PKBI, and Grapiks are thanked for their valuable help. Discussion h This cross-sectional study from Indonesia shows a high prevalence of HIV-infection among relatively young and well-educated former and current IDUs. Current IDUs obviously had much higher risks of viral transmission related to injecting drugs, but also former IDUs had risks related to blood to blood transmission. Impor- tantly, both groups engaged in substantial risks related to sexual transmission of HIV. Almost half of respon- dents in our study were former IDUs, showing that this group may contribute significantly to HIV transmission. Injecting drug use is the main factor driving the HIV- epidemic in Indonesia. In a large cohort of HIV patients in our setting, two thirds had a history of IDU [19]. In line with this finding, the prevalence of HIV-infection among fIDUs and cIDUs in this study was high (66% respectively 60%), similar or slightly higher compared Both among current and former IDUs, sexual risk behavior may contribute significantly to HIV transmis- sion. Sexual risk behavior was equally high in both groups, which is in contrast with previous studies reporting associations between injecting drug use and unsafe sex [22,23]. Condom use among IDUs has found to be inconsistent and especially low with sex workers and other risk groups for HIV transmission [20]. As IDUs often have multiple sex partners, including sex workers, HIV transmission may easily spread to people outside the IDU community. Heroin was the most frequently used drug among IDUs in this sample but many reported use of cannabis, benzo- diazepines and alcohol as well, in line with reports from China, Thailand, Ukraine, Lithuania, and Poland [24]. Iskandar et al. BMC Public Health 2010, 10:472 http://www.biomedcentral.com/1471-2458/10/472 Iskandar et al. BMC Public Health 2010, 10:472 http://www.biomedcentral.com/1471-2458/10/472 Page 6 of 7 The use of methadone or buprenorphine among cIDUs was higher compared to fIDUs. The EuropASI question- naire does not allow us to verify whether this was as part of substitution treatment or not. Both drugs are officially registered in Indonesia for substitution treatment but illegal use is quite common. Buprenorphine injection has been reported in many countries including Indonesia, and can be regarded as a response to inadequate care, rather than simply as misuse [25]. Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. Received: 28 January 2010 Accepted: 10 August 2010 Published: 10 August 2010 Received: 28 January 2010 Accepted: 10 August 2010 Published: 10 August 2010 Authors’ contributions SI made and carried out the research protocol, performed the statistical analysis, and drafted the manuscript. DB and LP participated in making the research protocol. TH and IS participated in the design of the study. RvC, AvV, CdJ conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. This study suffers form the limitations of a cross-sec- tional study in a population which is difficult to reach, and the question is therefore how representative the samples are. By using RDS, we tried to minimize this risk [12,29]. Numerical simulations have shown that the possible bias, even if the seeds are not drawn randomly, is extremely small (0.3%) for all sample sizes greater than 200 [12]. Still, some IDUs who are not in the social networks with these participants can not be recruited through respondent driven sampling [30]. Conclusions Neaigus A, Gyarmathy VA, Zhao M, Miller M, Friedman SR, Des Jarlais DC: Sexual and other noninjection risks for HBV and HCV seroconversions among noninjecting heroin users. J Infect Dis 2007, 195(7):1052-1061. 7. Neaigus A, Gyarmathy VA, Zhao M, Miller M, Friedman SR, Des Jarlais DC: Sexual and other noninjection risks for HBV and HCV seroconversions among noninjecting heroin users. J Infect Dis 2007, 195(7):1052-1061. 8. Tortu S, McMahon JM, Pouget ER, Hamid R: Sharing of noninjection drug- use implements as a risk factor for hepatitis C. Subst Use Misuse 2004, 39(2):211-224. 8. Tortu S, McMahon JM, Pouget ER, Hamid R: Sharing of noninjection drug- use implements as a risk factor for hepatitis C. Subst Use Misuse 2004, 39(2):211-224. 9. Gyarmathy VA, Neaigus A, Miller M, Friedman SR, Des Jarlais DC: Risk correlates of prevalent HIV, hepatitis B virus, and hepatitis C virus infections among noninjecting heroin users. J Acquir Immune Defic Syndr 2002, 30(4):448-456. Conclusions 2. Des Jarlais DC, Arasteh K, Perlis T, Hagan H, Abdul-Quader A, Heckathorn DD, McKnight C, Bramson H, Nemeth C, Torian LV, et al: Convergence of HIV seroprevalence among injecting and non-injecting drug users in New York City. AIDS 2007, 21(2):231-235. 2. Des Jarlais DC, Arasteh K, Perlis T, Hagan H, Abdul-Quader A, Heckathorn DD, McKnight C, Bramson H, Nemeth C, Torian LV, et al: Convergence of HIV seroprevalence among injecting and non-injecting drug users in New York City. AIDS 2007, 21(2):231-235. In conclusion, this study from Indonesia shows that for- mer IDUs, when compared to current IDUs, may have similar high HIV prevalence rates and high sexual risk behavior. Specific programs focusing on the reduction of sexual risk behavior are needed among former and cur- rent IDUs, in order to prevent further transmission to the general community. Drug-use treatment and inter- ventions that examine the relationship between drug use and sexuality should be conducted. In addition, earlier HIV treatment may lower transmission among IDUs by reducing the ‘community viral load’[31]. We have recently shown that patients with a history of injecting drug use in our setting have a similar clinical and virolo- gical response to anti-retroviral treatment compared to non-IDUs [19]. Finally, evidence-based prevention should also be targeted at schoolchildren and young adolescents as injecting drug use starts at an early age in this setting. 3. Darke S, Ross J, Teesson M: Twelve-month outcomes for heroin dependence treatments: does route of administration matter? Drug Alcohol Rev 2005, 24(2):165-171. 4. NAC: Country Report on The Follow Up to The Declaration of Commitment on HIV/AIDS: UNGASS Reporting Period 2006-2007. Committee NA 2007. 5. Pisani E, Dadun , Sucahya PK, Kamil O, Jazan S: Sexual behavior among injection drug users in 3 indonesian cities carries a high potential for HIV spread to noninjectors. J Acquir Immune Defic Syndr 2003, 34(4):403-406. 6. Des Jarlais DC, Arasteh K, Perlis T, Hagan H, Heckathorn DD, McKnight C, Bramson H, Friedman SR: The transition from injection to non-injection drug use: long-term outcomes among heroin and cocaine users in New York City. Addiction 2007, 102(5):778-785. 6. Des Jarlais DC, Arasteh K, Perlis T, Hagan H, Heckathorn DD, McKnight C, Bramson H, Friedman SR: The transition from injection to non-injection drug use: long-term outcomes among heroin and cocaine users in New York City. Addiction 2007, 102(5):778-785. 7. Financial support SI received fellowship from Radboud University, The Netherlands. Financial support was provided by ‘IMPACT’ (Integrated Management of Prevention And Care and Treatment of HIV/AIDS), a collaborative research and implementation program of Padjadjaran University, Bandung, Indonesia; Maastricht University and Radboud University, Nijmegen, the Netherlands; and Antwerpen University, Belgium. IMPACT is funded by the European Commission (SANTE/2005/105-033), and contracted by CORDAID. Author details 1Department of Psychiatry, Faculty of Medicine, Padjajaran University/Hasan Sadikin Hospital, Bandung, Indonesia. 2Health Research Unit, Faculty of Medicine, Padjadjaran University/Dr. Hasan Sadikin Hospital, Bandung, Indonesia. 3Department of Health Education and Health Promotion, Faculty of Health, Medicine and Life Sciences, Maastricht University, Maastricht, the Netherlands. 4Department of General Internal Medicine and Nijmegen Institute for Inflammation, Infection and Immunity, Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands. 5Nijmegen Institute for Scientist-Practitioners in Addiction (NISPA), Nijmegen, the Netherlands. Alcohol abuse was common, and was associated with risky sexual behavior, as has been reported previously [26]. 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Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: 29. Platt L, Wall M, Rhodes T, Judd A, Hickman M, Johnston LG, Renton A, Bobrova N, Sarang A: Methods to recruit hard-to-reach groups: comparing two chain referral sampling methods of recruiting injecting drug users across nine studies in Russia and Estonia. J Urban Health 2006, 83(6 Suppl):i39-53. 30. Trotter RT II, Bowen AM, Potter JM Jr: Network models for HIV outreach and prevention programs for drug users. NIDA Res Monogr 1995, 151:144-180. 31. 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https://openalex.org/W2574092430	https://vestnik.mgimo.ru/jour/article/download/430/430	Russian	null	Theoretical Aspects of Analysis of International Environmental Security	Vestnik MGIMO-universiteta	2,015	cc-by	4,175	МИРОВАЯ ПОЛИТИКА МИРОВАЯ ПОЛИТИКА ТЕОРЕТИЧЕСКИЕ АСПЕКТЫ АНАЛИЗА МЕЖДУНАРОДНОЙ ЭКОЛОГИЧЕСКОЙ БЕЗОПАСНОСТИ Ю.А. Русакова Московский государственный институт международных отношений (университет) МИД России. Россия, 119454, Москва, пр. Вернадского, 76. Международная экологическая безопасность является сегодня в выс шей степени актуальной проблемой, от решения которой зависит сохране ние окружающей среды пригодной для проживания человечества. Отсутствие такой среды делает бессмысленным решение всех остальных проблем совре менности. Автор статьи рассматривает теоретические аспекты решения проблемы экологической безопасности. В частности, анализируется пробле ма негативных социальных экстерналиев и связанная с ней «трагедия общин». Эти проблемы создают принципиальные препятствия для реализации эколо гической безопасности на глобальном уровне. Традиционно с проблемой экстер налиев в экологической сфере боролись с помощью экономических инструмен тов, обязывая производителей данных экстерналиев оплачивать их в форме дополнительных налогов. Однако практика показывает, что экономические решения экологической безопасности малоэффективны. Автор предлагает аль тернативные, неэкономические подходы, связанные с укреплением и развитием системы институтов постоянных международных переговоров по вопросам экологической безопасности и стимулированием экологического самосознания. Решение острейших экологических проблем невозможно без смены по литической философии правящих элит в большинстве государств, отказа от реалистской политики и традиционной ставки на силовые методы решения проблем в пользу поиска компромиссов и сотрудничества в интересах решения глобальных, общечеловеческих, в том числе экологических проблем. Необходимо постоянное утверждение – политически и в международном праве – обязатель ных гуманистических и экологических норм и правил для сосуществования двух сотен государств в мире, их развития и реализации главного, общечеловеческого (биологического) интереса. Международная экологическая безопасность является сегодня в выс шей степени актуальной проблемой, от решения которой зависит сохране ние окружающей среды пригодной для проживания человечества. Отсутствие такой среды делает бессмысленным решение всех остальных проблем совре менности. Автор статьи рассматривает теоретические аспекты решения проблемы экологической безопасности. В частности, анализируется пробле ма негативных социальных экстерналиев и связанная с ней «трагедия общин». Эти проблемы создают принципиальные препятствия для реализации эколо гической безопасности на глобальном уровне. Традиционно с проблемой экстер налиев в экологической сфере боролись с помощью экономических инструмен тов, обязывая производителей данных экстерналиев оплачивать их в форме дополнительных налогов. Однако практика показывает, что экономические решения экологической безопасности малоэффективны. Автор предлагает аль тернативные, неэкономические подходы, связанные с укреплением и развитием системы институтов постоянных международных переговоров по вопросам экологической безопасности и стимулированием экологического самосознания. Решение острейших экологических проблем невозможно без смены по у Решение острейших экологических проблем невозможно без смены по литической философии правящих элит в большинстве государств, отказа от реалистской политики и традиционной ставки на силовые методы решения проблем в пользу поиска компромиссов и сотрудничества в интересах решения глобальных, общечеловеческих, в том числе экологических проблем. ТЕОРЕТИЧЕСКИЕ АСПЕКТЫ АНАЛИЗА МЕЖДУНАРОДНОЙ ЭКОЛОГИЧЕСКОЙ БЕЗОПАСНОСТИ Необходимо постоянное утверждение – политически и в международном праве – обязатель ных гуманистических и экологических норм и правил для сосуществования двух сотен государств в мире, их развития и реализации главного, общечеловеческого (биологического) интереса. Ключевые слова: экологическая безопасность, реализм, экстерналии, меж дународные переговоры, новая экологическая философия. 162 Ю.А. Русакова Ю.А. Русакова этой парадигмой в целом рассматривается в на стоящее время как: П П онятие «международная экологическая безопасность» возникло и широко вошло в политический оборот во второй поло вине ХХ в. в результате бурного экономического и технологического развития человечества после Второй мировой войны и возникших во многом в связи с этим объективных потребностей в лик видации негативных последствий для экологии и самого существования человечества. - результат противоречий политического, общественного и экономического развития на национальном и международном уровне, кото рые привели к появлению множества не только положительных, но и, прежде всего, отрица тельных экстерналий. Речь, прежде всего, идет о темпоральных отрицательных экстерналий, имеющих такие глобальное последствия, как из менение климата, ухудшение качества воздуха, воды и т.п. у Теоретические проблемы и подходы к изу чению различных аспектов международной экологической безопасности возникли и ста ли исследоваться практически одновременно с возникновением глобальных экологических проблем во второй половине ХХ в. параллельно в научных и политических кругах различных стран. Но первые наблюдения ученых можно от нести к более раннему периоду. В США, напри мер, еще в 1933 г. известный геолог Ч. Шухерт писал о геологических последствиях деятельно сти человека, а в СССР внимание к ним пока ещё в самых общих чертах стал привлекать в 30-х и 40-х гг. академик В.И. Вернадский, сформули ровавший концепцию ноосферы как результата взаимодействия человечества, геологических сил природы и творчества [2]. - наиболее актуальная проблема, требующая объединенных усилий всего мирового сообще ства, и не допускающая отсрочки или медлен ного решения. На 70-ой сессии Генеральной Ассамблеи ООН, например, все важнейшие инициативы лидеров ведущих государств и министров иностранных дел непосредственно были обращены к этой теме. Так, в выступлении президента РФ В.В. Путина) на 70-ой сессии Ге нассамблеи ООН отчетливо прозвучала, во-пер вых, взаимосвязь между геополитическими и экологическими проблемами международной безопасности и, во вторых, не только готовность, но и примеры конкретных шагов России в эко логической области; Позже, в 50-е и особенно в 70-е гг. после появления модели Форрестора и Медоуза, а также работ Римского клуба обеспокоенность неконтролируемым ростом производства и про мышленных отходов нарастала, превратившись в признанную угрозу международной безопас ности вообще и экологической безопасности, в частности. С конца ХХ в. ТЕОРЕТИЧЕСКИЕ АСПЕКТЫ АНАЛИЗА МЕЖДУНАРОДНОЙ ЭКОЛОГИЧЕСКОЙ БЕЗОПАСНОСТИ проблемы междуна родной экологической безопасности становятся частью постоянной повестки дня международ ного сообщества и международных отношений как в широком контексте безопасности, так и в узких ее аспектах. - как проблема международной безопас ности, требующая особого политического внимания и значительных материальных и финансовых ресурсов. Президент Казахстана Н. Назарбаев, например, на юбилейной сес сии ООН вновь предложил целенаправленно выделять 1% военных расходов на эти цели), а президент Белоруссии А. Лукашенко ясно сфор мулировал проблему «кризиса международной системы безопасности», ставшего следствием нерешенных геополитических и экологических проблем международной безопасности; у В 70-е гг. стали формироваться первые тео ретические концепции предотвращения угроз экологической безопасности, которые условно разделились на внешние и внутренние в зави симости прежде всего от последствий (между народных, техногенных, глобальных и др. – для внешних угроз) и характера самих угроз – ис точников этих угроз. Так, источниками угроз экологической безопасности техногенного ха рактера стали признаваться не силы природы, а деятельность (или бездеятельность) человека и государства. Это, в свою очередь, привело к появлению целой группы общественных отно шений, целью которых стала профилактика и борьба с угрозами экологической безопасности в самых различных областях – экономической, военной, социальной и международной. р у р - как труднорешаемая в принципе полити ческая и экономическая проблема, требующая особенно серьезных международных усилий на самом высоком политико-дипломатическом уровне, темпы и эффективность решения кото рой, как показывает практика, заведомо отстают от темпов обострения угроз международной эко логической безопасности). Эта политико-дипло матическая специфика экологических проблем создаёт объективные трудности даже в тех слу чаях, когда не существует принципиальных по литических разногласий в понимании решения проблем. - как труднорешаемая в принципе полити ческая и экономическая проблема, требующая особенно серьезных международных усилий на самом высоком политико-дипломатическом уровне, темпы и эффективность решения кото рой, как показывает практика, заведомо отстают от темпов обострения угроз международной эко логической безопасности). Эта политико-дипло матическая специфика экологических проблем создаёт объективные трудности даже в тех слу чаях, когда не существует принципиальных по литических разногласий в понимании решения проблем. В эти же годы в целом в мировом обще ственном мнении и научных кругах сложился достаточно устойчивый консенсус относительно того, что общие причины деградации окружа ющей среды и нерационального использования природных ресурсов и возникновение в этой связи угрозы международной безопасности прежде всего связаны: у р Ко второму десятилетию ХХI в. в мировом общественном мнении в основных чертах сло жилась парадигма международной экологиче ской безопасности, в основе которой лежали исследования и политические реалии второй половины ХХ – начала ХХI вв. ТЕОРЕТИЧЕСКИЕ АСПЕКТЫ АНАЛИЗА МЕЖДУНАРОДНОЙ ЭКОЛОГИЧЕСКОЙ БЕЗОПАСНОСТИ Международная экологическая безопасность в соответствии с р во-первых, с общей неэффективностью, существующей в механизмах регулирования глобального рынка, ориентированного на из 163 Мировая политика внешние эффекты (экстерналии), общественные блага, транзакционные издержки, отсутствие знаний и неопределённость информации, не дальновидность политико-экономической эли ты. Общепринято считать, например, что «в концептуальном плане провалы рынка в охра не окружающей среды связаны, прежде всего, с практически невозможным адекватным учетом таких экстерналий, как социальные издержки общества от деградации окружающей среды». влечение максимальной прибыли, что, в свою очередь, связано нередко с нанесением ущерба природной среде; во-вторых, с отсутствием эффективных механизмов международного регулирования, неразвитостью соответствующей нормативно- правовой базы и несогласованности действий отдельных государств, международных органи заций и международных акторов; у р р в-третьих, со слабым развитием, либо даже отсутствием вообще, средств и собственно институциональных механизмов регулирования природопользования в отдельных государствах и у отдельных акторов мировой политики. Ска занное означает, что даже в тех случаях, когда признается актуальность решения экологи ческих проблем, у целого ряда государств нет средств и способов их решения, и без междуна родной помощи они не способны их решить в принципе. Становление института иностранной помощи в экологической области стало проис ходить только после того, как в 2000 г. все 191 государств договорились о том, чтобы в качестве седьмой (из 8) целей ООН объявить «обеспече ние экологической устойчивости». Что касается социальных экстерналий, то их невозможно адекватно учесть, в связи с чем цены на природные блага занижены. Транзакци онные издержки также могут быть достаточно велики по отношению к ожидаемым выгодам (расходы, связанные с получением информации, ведением многосторонних переговоров, обеспе чением соблюдения переговоров и т.д.). О существенной проблеме для рынка – «экологической близорукости» – сказал еще Ф. Энгельс: «Не будем, однако, слишком обо льщаться нашими победами над природой. Каждая из этих побед имеет, правда, в первую очередь те последствия, на которые мы рассчи тывали, но во вторую и третью очередь совсем другие, непредвиденные последствия, которые очень часто уничтожают значение первых» [8]. По своей природе рынок ориентируется на по лучение быстрых результатов, большой прибыли и не учитывает долгосрочный ущерб и будущие выгоды. у Это признание, однако, до сих пор мало ска залось на повышении эффективности усилий отдельных стан и всего мирового сообщества в области международной экологической безопас ности. Что касается, например, первой причи ны, то можно по-прежнему утверждать наличие у рынка особенностей в экологической сфере. «Экологические товары представляют собой ти пичные общественные блага и внешние эффек ты, которые трудно продать индивидуальному потребителю» [9]. ТЕОРЕТИЧЕСКИЕ АСПЕКТЫ АНАЛИЗА МЕЖДУНАРОДНОЙ ЭКОЛОГИЧЕСКОЙ БЕЗОПАСНОСТИ Фактически современное между народное право установило международно-пра вовую обязанность сотрудничества государств независимо от их политических, экономических и социальных систем во имя общего благососто яния народов, в интересах всего человечества [4]. лишь часть издержек, связанных с потравой вы паса. Хардин делает следующий вывод: «В этом и состоит трагедия. Каждый замкнут в рамках системы, побуждающей его беспредельно увели чивать свое стадо, — в мире, который является ограниченным. Конечным пунктом, к которо му все спешат, является катастрофа, — каждый преследует свой интерес в обществе, верящем в свободу общин» [14]. у Трагедия общин была известна до Хардина. Задолго до него Аристотель писал: «К тому, что составляет предмет владения очень большого числа людей, прилагается наименьшая забота. Люди заботятся всего более о том, что принад лежит лично им и менее заботятся они о том, что является общим». В 1833 г. Уильям Форстер Ллойд разработал теорию общин, согласно кото рой предсказывалось, что люди будут склонны расточительно использовать имущество, нахо дящееся в их совместном владении [15].Более чем за десять лет до статьи Хардина Эйч Скотт Гордон убедительно продемонстрировал схожую логику в другом классическом труде: «Исследо вание экономической теории общей собствен ности: рыболовство». Гордон описывает ту же динамику, что и Хардин: «Выясняется, что ста рое изречение, согласно которому общая соб ственность означает ничейную собственность, содержит некоторую истину. Богатство, которым свободно распоряжаются все, никем не ценится, потому что тот, кто настолько безумен, чтобы выжидать, пока не наступит подходящее время для использования ресурса, обнаружит лишь то, что все уже использовано другими… Рыба, имеющаяся в море, не имеет ценности для ры бака, поскольку он не уверен в том, что, если сегодня он воздержится от лова, она будет ждать его завтра» [13]. р р Вторая модель – региональные организации и союзы, способные консолидировать авторитет и ресурсы целого ряда стран, например, Евро союза. Третья модель – создание новых механизмов двустороннего и регионального сотрудничества, «выработка общих подходов “снизу”. «Сперва – внутри сложившихся региональных структур – ЕС, НАФТА, АТЭС, АСЕАН и других, а затем – путем диалога между нами. Именно из таких “кирпичиков” может сложиться более устойчи вый характер мировой экономики» [11]. Следует сказать, что единая экологическая стратегия возможна только в рамках единой стратегии развития, точнее – устойчивого раз вития, когда интересы всех государств в той или иной мере учитываются основными политиче скими игроками, а не в рамках традиционно су ществующей системы, «правила игры» которой создаются, определяются и контролируются ве дущими мировыми державами. ТЕОРЕТИЧЕСКИЕ АСПЕКТЫ АНАЛИЗА МЕЖДУНАРОДНОЙ ЭКОЛОГИЧЕСКОЙ БЕЗОПАСНОСТИ Действия, направленные на охрану природной среды, часто не дают в итоге какой-либо материализованный продукт. Они могут привести, например, к недопущеннию или предотвращению ущерба экосистемам, народно му хозяйству и населению, или оказать воздей ствие на стабилизацию или улучшение качества среды обитания людей, что, однако, далеко не всегда видно общественности и далеко не всегда оправдывает вложенные средства. Представляется, что необходимо более по дробно остановиться на неизбежности возник новения и развития локальных экстерналиев. Локальные экстерналии возникают на огра ниченной территории, когда деятельность ка кого-либо предприятия приводит к внешним издержкам среди местного населения, других предприятий и т.п. В научной литературе данная проблема известна как «трагедия общин». После публикации в 1968 г. в журнале «Science» статьи Гарретта Хардина [14], выражение «трагедия об щин» стало широко использоваться для обозна чения процесса деградации окружающей среды, которой следует ожидать в ситуациях, когда множество индивидов совместно используют некий ограниченный ресурс. Средства, вложенные в природоохранную деятельность, выпадают из активов, так как положительный эффект (положительные экс терналии) получают безвозмездно другие пред приятия (а, иногда, и конкуренты) и население. Например, восстановление лесного массива ока зывает благотворное влияние на окружающую среду, речной сток, предотвращает эрозию почв и т.д., но не приносит ни непосредственной при были на вложенные затраты, ни общественно- политических дивидентов. Для иллюстрации логической структуры своей модели Хардин обращался к читателю с предложением вообразить пастбище, «открытое для всех». Затем он анализировал особенности такой ситуации с точки зрения рационального владельца стада. Каждый владелец стада получа ет непосредственную выгоду от принадлежащих ему животных и несет отложенные издержки из-за того, что общинное пастбище постепенно приходит в негодность, будучи вытоптанным его собственными животными и животными, принадлежащими другим владельцам. Каждый владелец стада мотивирован увеличивать стадо и выгонять на пастбище дополнительных живот ных, поскольку он удерживает непосредствен ные выгоды, связанные с владением ими, и несет В экологической сфере мы часто становимся свидетелями «недостаточной» эффективности механизмов или даже «провалов» рынка. Эти же «провалы» являются по своей сути потен циалом будущих экологических угроз и даже катаклизмов. Конкретными источниками та ких «провалов», как уже говорилось, являются: 164 Ю.А. Русакова Ю.А. Русакова зультаты возможны только в том случае, когда в совместные действия вовлечено большинство ведущих государств, а принцип международного сотрудничества, закрепленный в международ ном праве, становится практическим принци пом политического сотрудничества. «Значение принципа сотрудничества во имя экономиче ского и социального прогресса всех народов в деле поддержания международного мира и без опасности (в том числе экологической) трудно переоценить. Список литературы М.: МГИМО(У), 2010. 12. Экономическая дипломатия в условиях глобализации / под общ.ред. Л.М. Капицы. М.: МГИМО(У), 2010. 13 Gordon The Economic Theory of a Common-Property Research: The Fishery 1954 Экономическая дипломатия в условиях глобализации / под общ.ред. Л.М. Капицы. М.: МГИМО(У), 201 Gordon. The Economic Theory of a Common-Property Research: The Fishery 1954. Hardin G. The Tragedy of the Commons // Science. 13 December 1968: Vol. 162 no. 3859 pp. 1243-1248 14. Hardin G. The Tragedy of the Commons // Science. 13 December 1968: Vol. 162 no. 3859 pp. 1 14. Hardin G. The Tragedy of the Commons // Science. 13 December 1968: Vol. 162 no. 3859 pp. 1243-1248 15. William Foster Lloyd. Population and Development Review Vol. 6, No. 3 (Sep., 1980), pp. 473-496 g y pp 15. William Foster Lloyd. Population and Development Review Vol. 6, No. 3 (Sep., 1980), pp. 473-496 Мировая политика Мировая политика пользовать как уже сложившееся направленные на это культурные традиции, так и развивать и пропагандировать культуру устойчивого разви тия. Данная культура должна определить новое отношение к природе, которым и будет опреде ляться характер экономической, технической, инженерной и др. видов деятельности. Культура устойчивого развития должна создать механиз мы и экономические, и социальные, и правовые для перехода к устойчивому развитию. стических и экологических норм и правил для сосуществования двух сотен государств в мире, их развития и реализации главного, общечело веческого (биологического) интереса. р Важно отметить, что сильно возрастает роль переговоров как инструментов регулирования международных отношений, что стало заметным во второй половине ХХ века и абсолютно необ ходимым с конца прошлого столетия. Прежде всего, отчетливо прослеживается зависимость роста глобальных проблем и интенсификации международных переговоров. Можно констати ровать, что по мере увеличения значения гло бальных проблем, в том числе экологических, будет усиливаться значение механизмов между народного сотрудничества, а также численности и интенсивности международных переговоров, развитие существующих и неизбежное появле ние новых структур и механизмов международ ного сотрудничества. Культура устойчивого развития непосред ственно связана с понятием «экологической ци вилизации» [7]. Целью перехода современного общества к устойчивому развитию является гармонизация социально-экономических от ношений, технологического и экологического развития, т.е. достижение основного показателя экологической цивилизации. «Можно считать, что понятие «экологическая цивилизация» ха рактеризует качественную специфику будущего общества, его состояние, тогда как «устойчивое развитие» – динамику этого общества, особен ности его связей с природной средой» [7]. б Объективно этот процесс усиливает и дру гая тенденция – изменение в соотношении сил в мире, появление новых центров силы, которые прежде активно не участвовали в таких между народных переговорах. Прежде всего, речь идет о новых политико-экономических гигантах – Китае и Индии. Решение острейших экологических проблем невозможно без смены политической филосо фии правящих элит в большинстве государств, отказа от реалистской политики и традиционной ставки на силовые методы решения проблем в пользу поиска компромиссов и сотрудничества в интересах решения глобальных, общечелове ческих, в т.ч. экологических проблем. Необходи мо постоянное утверждение – политически и в международном праве – обязательных гумани Таким образом, наиболее реалистичным подходом к решению проблем низкой специ фикации прав собственности на глобальные общины и высоких транзакционных издержек является международные переговоры и укреп ление ценностей «экологической цивилизации». Русакова Юлия Анатольевна – аспирант МГИМО(У) МИД России. E-mail: vestnik@mgimo.ru. ТЕОРЕТИЧЕСКИЕ АСПЕКТЫ АНАЛИЗА МЕЖДУНАРОДНОЙ ЭКОЛОГИЧЕСКОЙ БЕЗОПАСНОСТИ Эта сверхтрудная задача сегодня кажется иллюзорной, но время говорит в пользу того, что она все настойчивее переходит из области фило софско-гуманитарной в политико-прикладную. Как отмечают исследователи МГИМО, «сложив шиеся обстоятельства диктуют необходимость выработки единой общепланетарной стратегии и синхронизированной политики развития, что представляется чрезвычайно трудной задачей в свете названных социально-экономических различий между странами и регионами мира. Решению именно этой задаче и подчинена ди пломатия устойчивого развития» [12]. О Преодоление «трагедии общин» связано с интернализацией экстерналиев. «Методами ин тернационализации внешних эффектов могут быть всевозможные платежи, налоги и другие рычаги как административного, так и эконо мического характера» [6]. Интернализовать негативные внешние эффекты можно путем введения корректирующего налога (так назы ваемого, налога Пигу) или путем приватизациий общественных благ. Для решения экологических проблем и фор мирования экологической мировой политики необходимы как увеличение внимание к эко логической проблематике на уровне государств, так и создание прочных стабильных наднацио нальных структур. Отдельное направление заключается в том, что такие инициативы должны претендовать на формирование новой системы ценностей. Для того, чтобы избежать экологической катастрофы и сохранить природу для последу ющих поколений человеку предстоит на основе новых реалий преобразовать не только преиму щественно разрушительный тип деятельности, но и всю систему культурных ценностей. В настоящее время теоретически существует три наднациональные модели, которые можно взять за основу [10]. Первая модель – глобальная, использующая все (или большинство) мировое сообщество, в которой задействованы ведущие международ ные институты, прежде всего ООН. На смену стратегиям, которые ориентиро ваны исключительно на развитие экономики, должна прийти стратегия, которая в центр ста вит человека. Для этого важно правильно ис у р Применительно к такой глобальной пробле ме как климат, очевидно, что позитивные ре 165 Список литературы Бобылев С.Н., Ходжаев А.Ш. Экономика природопользования. М.: Изд-во ИНФРА, 2004.с.84 2. Вернадский В.И. Научная мысль как планетное явление /Отв. Ред. А.Л.Яншин.- М.; Наука,1991 г. 2. Вернадский В.И. Научная мысль как планетное явление /Отв. Ред. А.Л.Яншин.- М.; Наука,1991 г. 3. Иностранная помощь: монография/ под общ. ред. Л.М. Капицы. М.: МГИМО-Университет, 2013. с.562. 4. Клюканова Л.Г. Экологический аспект интеграционных процессов в ЕС и СНГ (международно-правовой анализ) / СПб.: Издательство Юридического института (Санкт-Петербург). 2001. р щ р ф д щ р д ц р , 4. Клюканова Л.Г. Экологический аспект интеграционных процессов в ЕС и СНГ (международно-правовой анализ) / СПб.: Издательство Юридического института (Санкт-Петербург). 2001. 4. Клюканова Л.Г. Экологический аспект интеграционных процессов в ЕС и СНГ (международно-правовой анализ) / СПб.: Издательство Юридического института (Санкт-Петербург). 2001. 5. Кравченко С.А. Словарь новейшей социологической лексики: теории, понятия, персоналии (с англ. эквивалентами). М.: МГИМО(У), 2011. С. 364. 5. Кравченко С.А. Словарь новейшей социологической лексики: теории, понятия, персоналии (с англ. эквивалентами). М.: МГИМО(У), 2011. С. 364. 6. Макар С.В., Основы экономики природопользования. - М.: ИМПиЭ??? 7. Мамедов Н.М. Культурные предпосылки становления экологической цивилизации / Электронное периодическое научное издание «Вестник Международной академии наук. Русская секция». 2012. № 1. 7. Мамедов Н.М. Культурные предпосылки становления экологической цивилизации / Электронное периодическое научное издание «Вестник Международной академии наук. Русская секция». 2012. № 1. 8. Маркс, К. Сочинения / К. Маркс, Ф. Энгельс. - 2-е изд. - М.: Госполитиздат, 1961. Т. 20. С. 495-496. 9 П Н В Р К К Э й СПб И 8. Маркс, К. Сочинения / К. Маркс, Ф. Энгельс. - 2-е изд. - М.: Госполитиздат, 1961. Т. 20. С. 495-496. 9. Пахомова Н.В., Рихтер К.К., Экономика природопользования и охраны окружающей среды.- СПб.:Изд. 9. Пахомова Н.В., Рихтер К.К., Экономика природопользования и охраны окружающей среды.- С СПбГУ, 2000. 10. Подберезкин А.И. «Национальный человеческий капиталъ»./ Т. II. Эволюция идеологии российской политической элиты (1990-2011 гг.)/ Издательство «МГИМО-Университет», 2012. 10. Подберезкин А.И. «Национальный человеческий капиталъ»./ Т. II. Эволюция идеологии российской политической элиты (1990-2011 гг.)/ Издательство «МГИМО-Университет», 2012. 11. Путин В.В. Новый интеграционный проект для Евразии – будущее, которое рождается сегодня // Известия. 2011. 4 октября. 11. Путин В.В. Новый интеграционный проект для Евразии – будущее, которое рождается сегодня // Известия. 2011. 4 октября. р 12. Экономическая дипломатия в условиях глобализации / под общ.ред. Л.М. Капицы. М.: МГИМО(У), 2010. 13. Gordon. The Economic Theory of a Common-Property Research: The Fishery 1954. 12. Экономическая дипломатия в условиях глобализации / под общ.ред. Л.М. Капицы. References 1. Bobylev S.N., Khodzhaev A.Ş. Environmental economics. Moscow: Publishing House INFRA, 2 2. Vernadsky V.I .Scientific Thought as a Planetary Phenomenon. Moscow.; Science, 1991. 3. Foreign aid: monograph / Ed. by L.M. Kapitsa. Moscow: MGIMO-University, 2013. 4. Klyukanova L.G. The environmental aspect of the integration process in the EU and the CIS (international legal analysis) / SPb.: Publishing Law Institute (St. Petersburg). 2001. 5. Kravchenko S.A. Dictionary of contemporary sociological vocabulary: theories, concepts, personalities. Moscow, 2011. 364 p. 6. Makar S.V. Fundamentals of Environmental Economics. - M.: IMPiE. 7. Mamedov N.M. The cultural background of formation of ecological civilization / Electronic periodical edition "Herald of the International Academy of Sciences. Russian Section". 2012. № 1. 8. Marx K. Works. 2nd ed. M oscow.: Gospolitizdat, 1961. Vol. 20. pp. 495-496. 9. Pakhomov N. Richter K.K., Environmental Economics and Environmental a medium, St. Petersburg. Univ. St. Petersburg State University, 2000. 9. Pakhomov N. Richter K.K., Environmental Economics and Environmental a medium, St. Petersburg. Univ. St. Petersburg State University, 2000. Padbyarozkin AI "National Human Capital". Vol. II. The evolution of the ideology of the Russian political eli 1990-2011 gg.). Moscow: MGIMO-University, 2012. 11. Putin VV. A new integration project for Eurasia - a future that is born today // Izvestia. 2011 Octob 12. Economic diplomacy in the conditions of globalization / under obsch. red. LM Kapitsa. M. 2010. 13. Gordon. The Economic Theory of a Common-Property Research: The Fishery 1954. 13. Gordon. The Economic Theory of a Common-Property Research: The Fishery 1954. 14. Hardin G. The Tragedy of the Commons // Science. December 13, 1968: Vol. 162 no. 3859 pp. 1243-1248 15. William Foster Lloyd. Population and Development Review Vol. 6, No. 3 (Sep., 1980), pp. 473-496 14. Hardin G. The Tragedy of the Commons // Science. December 13, 1968: Vol. 162 no. 3859 pp. 1243-1248 15. William Foster Lloyd. Population and Development Review Vol. 6, No. 3 (Sep., 1980), pp. 473-496 15. William Foster Lloyd. Population and Development Review Vol. 6, No. 3 (Sep., 1980), pp. 473-496 Об авторе усакова Юлия Анатольевна – аспирант МГИМО(У) МИД России. E-mail: vestnik@mgimo.ru. 166 Ю.А. Русакова Ю.А. Русакова THEORETICAL ASPECTS OF ANALYSIS OF INTERNATIONAL ENVIRONMENTAL SECURITY Yu.A. Rusakova Moscow State Institute of International Relations (University), 76 Prospect Vernadskogo, Moscow, 119454, Russia. Abstract: International environmental security is a very hot contemporary issue of world politics, which in a large part defines the future of our environment. Dealing with this issue is of outmost importance since its failure will render all other issues and challenges as negligible. The article examines the theoretical aspects of solving the problem of environmental security. In particular, it analyzes the problem of negative social externalities, and the related concept of "tragedy of the commons." These problems create a fundamental obstacle to the implementation of environmental security at the global level. Traditionally, the problem of externalities in the environmental field have been approached economically, states and their manufacturers were to pay for the externalities in the form of additional taxes. However, experience shows that the economic tools of dealing with environmental security are not effective. The author suggests alternative non-economic approaches: strengthening and developing the system of permanent institutions of international negotiations on environmental security and promotion of environmental awareness. l h l bl bl h h f h l l h l h Solving the acute environmental problems is impossible without a change of the political philosophy of the ruling elites in most states. Key words: ecological safety, realism, externalities, international negotiations, a new environmental philosophy. Juliya A. Rusakova – post-graduate student (University) of the MFA of Russia. E-mail: vestnik@mgimo.ru. About the author 167
https://openalex.org/W2483562524	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0159335&type=printable	English	null	Chronic Kidney Disease, Fluid Overload and Diuretics: A Complicated Triangle	PloS one	2,016	cc-by	7,419	Results Copyright: © 2016 Khan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Out of 312 patients, 64 (20.5%) were hypovolemic while euvolemia and hypervolemia were observed in 113 (36.1%) and 135 (43.4%) patients. Overall 144 patients were using diuret- ics among which 98 (72.6%) were hypervolemic, 35 (30.9%) euvolemic and 11 (17.2%) were hypovolemic. The mean decline in estimated GFR of entire cohort was -2.5 ± 1.4 ml/ min/1.73m2 at the end of follow up. The use of diuretics was significantly associated with decline in eGFR. A total of 36 (11.5%) patients initiated renal replacement therapy (RRT) and need of RRT was more profound among diuretic users. Data Availability Statement: Individual data points are available upon request due to ethical restrictions by Medical Research Advisory Committee [Jawatankuasa Etika Penyelidikan Manusia USM (JEPeM-USM)] as stated in “Rule Number 3” (Privacy and Confidentiality of data) of “Ethical Considerations” and these rules are available on its official website (www.jepem.kk.usm.my) by downloading “Template for Research Protocol”. Furthermore, confidentiality of medical information of patients is strictly required by ethical board and following statement was also consented to the patients during data collection. However, individual OPEN ACCESS Despite promising role of diuretics to manage fluid overload among chronic kidney disease (CKD) patients, their use is associated with adverse renal outcomes. Current study aimed to determine the extent of renal deterioration with diuretic therapy. Citation: Khan YH, Sarriff A, Adnan AS, Khan AH, Mallhi TH (2016) Chronic Kidney Disease, Fluid Overload and Diuretics: A Complicated Triangle. PLoS ONE 11(7): e0159335. doi:10.1371/journal. pone.0159335 RESEARCH ARTICLE Methods A total 312 non-dialysis dependent CKD (NDD-CKD) patients were prospectively followed- up for one year. Fluid overload was assessed via bioimpedance spectroscopy. Estimated GFR (eGFR) was calculated from serum creatinine values by using Chronic Kidney Dis- ease- Epidemiology Collaboration (CKD-EPI) equation. Editor: Jaap A. Joles, University Medical Center Utrecht, NETHERLANDS Received: March 28, 2016 Accepted: June 30, 2016 Published: July 21, 2016 Editor: Jaap A. Joles, University Medical Center Utrecht, NETHERLANDS Editor: Jaap A. Joles, University Medical Center Utrecht, NETHERLANDS Received: March 28, 2016 Accepted: June 30, 2016 Published: July 21, 2016 Editor: Jaap A. Joles, University Medical Center Utrecht, NETHERLANDS Editor: Jaap A. Joles, University Medical Center Utrecht, NETHERLANDS Received: March 28, 2016 Accepted: June 30, 2016 Published: July 21, 2016 Yusra Habib Khan1,2, Azmi Sarriff1, Azreen Syazril Adnan2, Amer Hayat Khan1, Tauqeer Hussain Mallhi1,2 1 Discipline of Clinical Pharmacy, School of Pharmaceutical Sciences, University Sains Malaysia, Penang, 11800, Malaysia, 2 Chronic Kidney Disease Resource Centre, School of Medical Sciences, Health Campus, University Sains Malaysia, Kubang Kerain, 16150, Kelantan, Malaysia * yusrahabib@ymail.com (YHK); drazreenadnan@gmail.com (ASA) * yusrahabib@ymail.com (YHK); drazreenadnan@gmail.com (ASA) a1111 Chronic Kidney Disease, Fluid Overload and Diuretics: A Complicated Triangle Yusra Habib Khan1,2, Azmi Sarriff1, Azreen Syazril Adnan2, Amer Hayat Khan1, Tauqe Hussain Mallhi1,2 Introduction data points can be provided to the researchers who meet the criteria for access to confidential data via approval from the Medical Research Advisory Committee and the Chronic Kidney Disease Resource Center, School of Medical Sciences University Sains Malaysia; contact person (In-charge Data and Records) zamli@usm.my. Chronic kidney disease (CKD) is a global health concern that substantially increases the risk of mortality and the use of specialized health care [1]. Progressive loss of renal function causes reduced sodium filtration and inappropriate suppression of tubular reabsorption that ulti- mately lead to volume expansion [2]. Fluid overload frequently manifests in patients with mod- erate to particularly late stages of CKD and has been associated with hypertension, congestive heart failure (CHF), left ventricular hypertrophy (LVH) as well as edema. In such cases, diuret- ics are frequently prescribed to control blood pressure and for symptomatic relief of fluid over- load [3, 4]. However, the role of diuretics remains quite controversial in CKD patients. Apart from their beneficial effects, these agents also decrease glomerular filtration rate (GFR) and cause metabolic disturbances that in turn increases risk of cardiovascular events [5, 6]. Funding: The authors have no support or funding to report. Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. Various guidelines suggest the use of loop (GFR<30ml/min/1.73m2) and thiazide diuretics (GFR >30ml/min/1.73m2) in CKD patients [7]. Unfortunately, randomized controlled trials demonstrating clinical benefits and subsequent harms of diuretic therapy in mild to moderate CKD patients do not exist. Observational studies with small sample size and short duration have shown that diuretics decrease blood pressure (BP) and improve edema in CKD patients but their use, particularly at higher doses, is associated with rise in serum creatinine and several metabolic complications [4,8–9]. The clinical assessment of fluid overload is relatively difficult and diuretics are mostly pre- scribed in clinical settings on the basis of high blood pressure and physical signs of edema. Although edema can roughly estimate excess extravascular volume but it is of limited value in assessing excess intravascular volume. Moreover, several liters of water should be retained before physical signs of edema become visible [10]. Other techniques to assess fluid status include ultrasonic evaluation of inferior vena cava diameter but it is subjected to interpatient and interoperator variability. Methodology Study location and Participants Diuretics Use in Chronic Kidney Disease Introduction Biomarkers such as brain natriuretic peptide (BNP) and N-termi- nal pro brain natriuretic peptide (NT-pro BNP) can reflect changes in fluid status but both are influenced by presence of cardiovascular disease (CVD) and are also accumulated in CKD patients, rendering these methods inappropriate for evaluation of fluid status in CKD patients [11]. Recently few studies have used bioimpedance spectroscopy i.e. Body Composition Monitor- ing (BCM) for assessment of fluid status in CKD patients and have shown association of fluid overload with decline in renal function in non-dialysis dependent (NDD) CKD patients [2, 11– 13]. However, all except one have not addressed the use of diuretics and its association with both volume overload and decline in renal function [4]. In order to overcome this clinical issue, we conducted a prospective observational study to assess association of diuretics use with severity of fluid overload and loss of renal function/decline in eGFR. The purpose of current study was not to devalue the potential benefits of diuretic therapy among CKD patients. We intended to see the extent of eGFR decline and odds of RRT initiation among NDD-CKD patients receiving diuretics. The findings of the present study could be hypothesis-generating, forming evidence to be considered during future research. Conclusions The use of diuretics was associated with adverse renal outcomes indicated by decline in eGFR and increasing risk of RRT initiation in our cohort of NDD-CKD patients. Therefore, it is cautiously suggested to carefully prescribe diuretics by keeping in view benefit versus harm for each patient. 1 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0159335 July 21, 2016 Ethical consideration Current study was approved by ethical committee [Jawatankuasa Etika Penyelidikan-Manusia (JEPeM] of Hospital University Sains Malaysia. All the patients were asked to sign a consent form before participating in study. Identity of each patient was kept confidential and patients were anonymized during data analysis. Study location and Participants Current study was conducted at a tertiary care hospital in North-Eastern part of Malaysia. All patients visiting outpatient nephrology clinic with confirm diagnosis of CKD (stage 3 to 5ND i.e. non dialysis) according to K/DOQI were invited to participate in the study. Subjects with active infection, decompensated liver disease (liver cirrhosis, ascites), acute kidney injury and 2 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0159335 July 21, 2016 Diuretics Use in Chronic Kidney Disease Fig 1. Methodological flow chart of study. doi:10.1371/journal.pone.0159335.g001 Fig 1. Methodological flow chart of study. doi:10.1371/journal.pone.0159335.g001 Fig 1. Methodological flow chart of study. doi:10.1371/journal.pone.0159335.g001 doi:10.1371/journal.pone.0159335.g001 autoimmune disorders i.e. systemic lupus erythematous (SLE) were excluded on account of rapid decline in eGFR (>5ml/min/1.73m2). Moreover, participants with pregnancy, malignan- cies, prosthesis, pacemakers, implanted metal devices (vales, stents or sutures), disabilities and impaired skin integrity were excluded from study due to pre- requirements of BCM (Fig 1). Subjects with uncontrolled hypertension (BP >180/110 mmHg), urinary tract obstruction, unstable CKD (having >15% increase in serum creatinine over last 3 months), congestive heart failure, allergy to sulfa drugs and using diuretics or NSAIDs at the time of beginning of study were excluded at the preliminary stage (before physical examination) of patient recruitment. autoimmune disorders i.e. systemic lupus erythematous (SLE) were excluded on account of rapid decline in eGFR (>5ml/min/1.73m2). Moreover, participants with pregnancy, malignan- cies, prosthesis, pacemakers, implanted metal devices (vales, stents or sutures), disabilities and impaired skin integrity were excluded from study due to pre- requirements of BCM (Fig 1). Subjects with uncontrolled hypertension (BP >180/110 mmHg), urinary tract obstruction, unstable CKD (having >15% increase in serum creatinine over last 3 months), congestive heart failure, allergy to sulfa drugs and using diuretics or NSAIDs at the time of beginning of study were excluded at the preliminary stage (before physical examination) of patient recruitment. Diuretics Use in Chronic Kidney Disease using one single calibrated manual sphygmomanometer. Comorbidities were defined as follow: diabetes; repeated determination of fasting plasma glucose (>6.4mmol/L), high blood pressure; an average systolic/diastolic blood pressure of 140/90mm Hg or prescription of anti-hyper- tensive medications, hyperlipidemia; low density lipoprotein (LDL)level of >100 mg/dl and cholesterol level >200mg/dl, Cardiovascular disease (CV); CV disease includes coronary artery disease or ischemic heart disease (blockage of coronary artery by artherosclerosis that results in reduced blood supply to heart) and includes stroke, angina and myocardial infarction. Body mass index (BMI) was subsequently calculated as weight/height (kg/m2). Following an over- night fasting, 5 ml of blood was withdrawn to measure full blood count, serum creatinine, cal- cium, phosphorous, albumin and lipid profile. Estimated GFR (eGFR) was calculated from serum creatinine values by using Chronic Kidney Disease- Epidemiology Collaboration (CKD-EPI) renal function predictive equation [14]. Severity of leg edema was graded on a 4-point scale as: 0 (none), 1 (mild), 2 (moderate), 3 (severe) via assessment chart used in hospi- tal. After routine examination, patient then underwent measurement of fluid status by means of Bioimpedance spectroscopy followed by their scheduled check-up at clinic. Both patients and consulting physicians were unaware of BCM results. Prescription of diuretics was then checked via Hospital online prescription database by entering patient code. All patients were followed up at outpatient nephrology clinic at 3 months interval to ascertain vital status and renal function for a period of one year. Measurement of Fluid status A multi frequency (5–1000kHz) bioimpedance spectroscopy device (Body composition Moni- tor, BCM, Fresenius Medical Care, Germany) was used to assess fluid status. The principle of measuring current flow through body is dependent on applied frequency. At low frequencies, current predominantly passes through extracellular space while at higher frequencies it passes through both intracellular and extracellular water. This device has been intensively validated against different gold standards in general and hemodialysis population [10,15,16]. However few studies have shown its validation in NDD-CKD population [11–13,17]. The measurements were performed after a 5 minute resting period with patient lying in the supine position. Elec- trodes were attached to one foot and one hand at the ipsilateral side. Metal and electronic devices that might interact with transmission were removed before start of procedure. Further- more, the procedure was performed after ensuring that patient had not consumed a heavy meal at least within 4 to 5 hour, had not exercised within 12 hours prior to test and had not consumed any beverages including alcohol and caffeine within 24 hours before the test. In the present study, absolute overhydration/fluid overload (OH), intracellular water (ICW), extracel- lular water (ECW) and total body water (TBW) were calculated based on bioelectrical imped- ance analysis following the model of Moissl et al [18]. The value of overhydration (OH) as calculated by BCM was used as an indicator of fluid overload. In general population, the 90th percentile of OH is +1.1 L. Accordingly, OH > 1.1L was classified as fluid overload (hypervole- mia). Hypovolemia was defined as OH value lower than 10th percentile -1.1 L. An OH value between ± 1 was defined as euvolemia i.e. normal hydration status [19, 20]. Bioimpedance anal- ysis (BIA) was done twice i.e. at the time of beginning of study and at the end of follow-up (1 year). Data collection Patient demographics were recorded from hospital database and by in person interviewing. A physical examination was performed to measure height, weight and blood pressure. Blood pressure was recorded as mean of three consecutive measurements with 5 minutes interval, 3 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0159335 July 21, 2016 Results Baseline demographics and clinical characteristics of study participants for each category of fluid status are shown in Table 1. After applying exclusion criteria, a total of 312 clinically sta- ble NDD-CKD patients (mean age 64.5 ± 6.43, 57% male) were enrolled in current study. All patients had moderate to severe CKD with mean eGFR 21.4 ± 9.2 ml/min/1.73 m2 (32%, 36% and 31% in CKD stage 3, 4, 5ND respectively). About 81% of study participants were hyperten- sive and 64% were diabetics. Pre-existing and documented cardiovascular and cerebrovascular disease were noted in 29% and 10% patients, respectively. Distribution of absolute overhydration (OH) in entire cohort is shown in Fig 2. On the basis of OH value, 64 (20.5%) patients were hypovolemic while euvolemia and hypervolemia were observed in 113 (36.2%) and 135 (43.3%) patients, respectively. Majority of the hypovolemic and euvolemic patients belonged to CKD stage 3 followed by CKD stage 4 while CKD stage 5 was more prevalent in hypervolemic group. Hyperlipidemia was found to be equally distrib- uted among three categories of fluid status while all other comorbidities were more prevalent among hypervolemic patients. With the exception of β blockers, there was statistically signifi- cant difference in medication use of three groups, with hypervolemic patients receiving more medications than the two other groups. Inter-comparison between three groups showed that there was a step-wise increase in levels of extracellular water, total body water, systolic blood pressure, urinary protein excretion, glycated hemoglobin, serum uric acid, blood urea nitrogen (BUN) and leg edema score from hypovolemic to hypervolemic group. On the other hand, a stepwise decline was observed in lean tissue mass, serum hemoglobin, cholesterol and albumin from hypervolemic to hypovolemic group. Diuretics Use in Chronic Kidney Disease total number of patients served as denominator. Relevant denominator was stated before pro- portion, where it varied. Comparison of categorical variables between two groups was done by using Chi-Square test (if at least 80 percent of cells have expected frequencies of 5 or more) or Fisher`s Exact test (if less than 80 percent of cells have expected frequencies of 5 or more). Comparison of continuous variables was done by a Student’s t-test when comparing two groups and by ANOVA when comparing more than two groups. Decline in kidney function was assessed by eGFR slope, defined as regression coefficient between eGFR and time. Univariate correlation between eGFR decline and potential explanatory variables was assessed by Pearson Correlation analysis. Correlations were analyzed to determine strength of relationship between continuous variables. Significant variables in univariate were then entered into multivariate regression analysis to identify variables that are independently associated with decline in eGFR. By keeping overhydration (OH), blood pressure and baseline eGFR as continuous variable, univariate and multivariate cox regression analysis was done to find haz- ard ratio (HR) of diuretic users and non-users in terms of adverse renal outcomes (initiation of RRT, decline in eGFR). For all analysis a p-value of <0.05 was considered statistically signifi- cant. All categorical data were entered by coding 0 indicating absence/ no while 1 showing presence/yes. Data were analyzed by using SPSS 20.0.0. Statistical analysis The accordance of quantitative data with normal distribution was examined with the Kolmo- gorov-Smirnov test. Continuous data were presented as mean (standard deviation). Categorical data were presented as frequency (proportion) for which frequency served as numerator and 4 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0159335 July 21, 2016 Diuretics Use in Chronic Kidney Disease Table 1. Clinical characteristics of CKD patients stratified by OH values. Diuretics and Fluid Overload A total of 153 (49.0%) patients received diuretics based on physician assessment of leg edema, blood pressure and cardiovascular complications. During follow-up period, diuretics were dis- continued in 9 patients for clinical reasons; therefore these patients were excluded from final analysis leaving 144 (46.1%) patients receiving diuretics until end of follow-up. Baseline eGFR of patients receiving diuretics versus non-users is shown in Table 2. Out of 144 patients pre- scribed with diuretics, majority of the patients (72.6%) were hypervolemic while euvolemia and 5 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0159335 July 21, 2016 Demographics Overall N = 312 Hypovolemic N = 64 Euvolemic N = 113 HypervolemicN = 135 p-value Age (years) 64.5 ± 6.43 67.0 ± 7.68 62.3 ± 5.81 65.1 ± 6.73 0.2 Male gender 178 (57.0%) 28 (43.8%) 57 (50.4)% 93 (68.9%) 0.001 Body mass index (kg/m2) 24.1 ± 5.4 24.3 ± 3.8 23.7 ± 3.8 24.6 ± 3.9 0.5 Current Smoker 96 (30.7%) 20 (31.2%) 27 (23.8%) 49 (36.2%) 0.1 Current alcohol drinkers 0 0 0 0 Comorbidities Hypertension 252(80.7%) 44(68.7%) 89(78.7%) 119(88.1%) 0.01 Diabetes mellitus 200 (64.1%) 28 (43.7%) 66 (58.4%) 106 (78.5%) <0.001 Hyperlipidemia 166 (53.2%) 35 (54.6%) 55 (48.6%) 76 (56.2%) 0.8 Cardiovascular disease 90 (28.8%) 14 (21.8%) 30 (26.5%) 46 (34.0)% 0.04 Cerebrovascular disease 33 (10.6%) 3.0 (4.6%) 12 (10.6%) 20 (14.8%) 0.003 CKD staging Stage 3 101 (32.4%) 34 (53.1%) 50 (44.2%) 17 (12.6%) <0.001 Stage 4 113 (36.2%) 22 (34.4%) 42 (37.2%) 49 (36.3%) 0.03 Stage 5ND (non-dialysis) 98 (31.4%) 8.0 (12.5%) 21 (18.6%) 69 (51.1%) <0.001 eGFR ml/min/1.73m2 21.4 ± 9.2 27.1 ± 9.9 27.0 ± 10.3 16.4 ± 9.6 0.03 Systolic BP(mmHg) 140.4 ± 21.3 133.5 ± 15.4 138.2 ± 18.7 147.8 ± 10.1 <0.001 Diastolic BP(mmHg) 74.5 ± 10.1 75.4 ± 9.1 73.3 ± 10.3 75.2 ± 11.4 0.6 Leg edema score>1 72 (23.0%) 3 (4.6%) 13 (11.5%) 56 (41.4%) <0.001 Body composition parameters Lean tissue index(kg/m2) 13.9 ± 3.1 14.2 ± 2.8 13.5 ± 2.5 13.8 ± 2.7 0.04 Fat tissue index (kg/m2) 9.6 ± 3.5 10.3 ± 4.1 9.7 ± 3.8 9.4 ± 3.5 0.07 Total body water (TBW) L 34.9 ± 5.4 33.0 ± 5.8 34.3 ± 5.6 37.4 ± 7.6 0.001 Intracellular water (ICW) L 18.2 ± 3.4 17.6 ± 3.4 18.4 ± 3.3 18.5 ± 4.2 0.7 Extracellular water(ECW) L 16.7 ± 3.7 15.4 ± 2.4 15.9 ± 2.9 18.9 ± 4.1 <0.001 ECW: ICW ratio 0.91 ± 0.2 0.87 ± 0.2 0.86 ± 0.2 1.02 ± 0.2 0.001 Fluid overload (OH) L 1.0 (0.5–2.3) -1.6 (-1.2 - -2.3) 1.0(-0.4–0.9) 2.9 (2.2–4.2) <0.001 Laboratory proﬁle Na (mmol/L) 138.54 ± 3.35 138 ± 3.4 138.40 ± 3.2 140.67 ± 3.0 0.4 K (mmol/L) 4.36 ± 0.53 4.36±0.59 4.32 ± 0.48 4.96 ± 0.45 0.3 Urea (mmol/L) 10.21 ± 5.77 8.81 ± 3.69 8.84 ± 4.78 12.07 ± 6.31 0.04 Uric acid (mg/dl) 7.9 ± 1.5 7.6 ± 1.4 7.9 ± 1.9 8.2 ± 1.8 0.02 Ca-phosphate product (mg2/dl2) 37.3 37.7 36.5 37.9 0.6 Albumin (g/dl) 4.2 ± 0.4 4.4 ± 0.5 4.3 ± 0.6 4.0 ± 0.3 0.03 Fasting blood sugar (g/dl) 101 101 99 106 0.4 Glycated hemoglobin (%) 5.7 5.6 5.6 6.1 0.03 Cholesterol (mg/dl) 182 (155–212) 188 (164–216) 183 (160–212) 175 (141–204) 0.03 Triglycerides (mg/dl) 119 (83–169) 126 (90–167) 109 (80–172) 115 (75–161) 0.425 Urine protein >1+ * 162 (51.9%) 25 (39.0%) 48 (42.5%) 89 (65.9%) <0.001 Medication Beta blockers (BB) 110 (35.3%) 18 (28.1%) 38 (33.6%) 54 (40%) 0.318 Calcium channel blockers (CCB) 190 (60.9%) 26 (40.6%) 60 (53.1%) 104 (77%) 0.02 RAAS blockers 177 (56.7%) 24 (37.5%) 63 (55.8%) 90 (66.6%) 0.04 Diuretics Therapy 144 (46.1%) 11 (17.2%) 35 (30.9%) 98 (72.6%) Mono Diuretic therapy 108 (34.6%) 11 (14%) 35 (32.7%) 62 (45.9%) • Loop diuretics 69 (22.1%) 7 (10.9%) 10 (8.8%) 52 (38.5%) • Thiazide Diuretics 39 (12.5%) 4 (6.3%) 25 (22.1%) 10 (7.4%) (Continued) Table 1. Diuretics Use in Chronic Kidney Disease Table 1. (Continued) Demographics Overall N = 312 Hypovolemic N = 64 Euvolemic N = 113 HypervolemicN = 135 p-value Multiple diuretic therapy 36 (11.5%) 0 0 36 (15.6%) Values of categorical variables are presented as percentages (%) whereas continuous variables are shown as mean ± SD or median with interquartile range. P-value for continuous variables is calculated by one way ANOVA or Kruskal-Wallis H test, as appropriate, p-value for categorical variables is calculated by X2 test P-value are calculated by comparing three groups (hypovolemic, hypervolemic and euvolemic) Assessment of urine protein was done via dipstick test Mono diuretic therapy: either loop or thiazide diuretic, Multiple diuretic therapy: concomitant use of more than one diuretic RAAS: renin-angiotensin aldosterone system blockers (include ACEI &ARBS) doi:10 1371/journal pone 0159335 t001 doi:10.1371/journal.pone.0159335.t001 hypovolemia were observed in 35 (30.9%) and 11 (17.2%) patients, respectively. Loop diuretics were predominantly prescribed in hypervolemic patients [52 (38.5%)] while thiazide diuretics were received by euvolemic patients [25 (22.1%)]. Multiple diuretic therapies were observed in 36 (15.6%) patients and all of them were hypervolemic. Clinical characteristics of CKD patients stratified by OH values. PLOS ONE \| DOI:10.1371/journal.pone.0159335 July 21, 2016 6 / 13 Diuretics Use in Chronic Kidney Disease Table 2. Baseline value of eGFR in different fluid status categories with respect to diuretic use. Fluid status category eGFR ml/min/1.73m2 Diuretics users eGFR ml/min/1.73m2 Diuretics non-users P-value Hypovolemic 28.9 ± 9.6 28.5 ± 10.1 0.26 Euvolemic 28.4 ± 10.8 29.8 ± 11.1 0.07 Hypervolemic 19.9 ± 8.2 21.5 ± 8.4 0.42 doi:10 1371/journal pone 0159335 t002 Table 2. Baseline value of eGFR in different fluid status categories with respect to diuretic use. to multivariate regression analysis in order to find out independent determinants of GFR to multivariate regression analysis in order to find out independent determinants of GFR decline (Table 4). Systolic blood pressure, Diabetes mellitus, fluid overload, proteinuria, diuret- ics use and baseline eGFR were found to be significantly associated with GFR decline in current study. to multivariate regression analysis in order to find out independent determinants of GFR decline (Table 4). Systolic blood pressure, Diabetes mellitus, fluid overload, proteinuria, diuret- ics use and baseline eGFR were found to be significantly associated with GFR decline in current study. Renal replacement therapy (RRT) was initiated in 36 (11.5%) patients at the end of follow- up period. It is worthwhile to mention that among patients who initiated RRT, 30 patients were using diuretics. Majority of these patients (n = 28) initiated hemodialysis while peritoneal dialysis was chosen by 8 patients. With respect to CKD staging, out of 36 patients who pro- gressed to RRT, 19 (63.3%) belonged to CKD stage 5 while 11 patients (36.7%) had CKD stage 4. Fortunately, none of the patient in CKD stage 3 initiated RRT. Mortality was observed in two cases and it is interesting to note that these patients belonged to CKD stage 5 hypervolemic group receiving diuretic therapy. Cardiogenic shock was the documented cause of fatality in both cases. We did not observe any death among patients with CKD stage 3 and stage 4. We further performed multivariate regression analysis to evaluate risk of initiation of dialy- sis and eGFR decline with respect to diuretic use. The unadjusted and adjusted risk of com- mencing RRT in diuretic users and non-users is shown in Table 5. Regardless of fluid status category, diuretic users had increased risk of RRT initiation and eGFR decline than non-users. Clinical Outcomes In order to assess the impact of diuretics on clinical outcomes of patients, we categorized patients on the basis of diuretic use (Table 3) and assessed outcomes in terms of decline in eGFR, initiation of RRT and death. At the end of follow-up, the mean decline in eGFR of entire cohort was -2.5 ± 1.4 ml/min/1.73m2. Inter-comparison between diuretic users and non-users showed that decline in eGFR and initiation of RRT were more profound among diuretic users compared to non-users (Table 3). Possible and well documented progression factors of GFR decline along with diuretic use were tested by Pearson`s correlation. Factors that showed significant correlation were subjected Fig 2. Distribution of absolute overhydration (0H) in 312 NDD-CKD patients, ranging between -2.1–4.4 L (82± 1.79). d i 10 1371/j l 0159335 002 Fig 2. Distribution of absolute overhydration (0H) in 312 NDD-CKD patients, ranging between -2.1–4.4 L (82± 1.79). doi:10.1371/journal.pone.0159335.g002 doi:10.1371/journal.pone.0159335.g002 7 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0159335 July 21, 2016 p-value calculated by student t-test for continuous variables and chi-square test for categorical variables between diuretic users and non-users S-cr: serum creatinine, ΔeGFR: change in estimated glomerular ﬁltration rate. RRT: renal replacement therapy Diuretics Use in Chronic Kidney Disease Table 4. Determinants of eGFR decline in entire cohort (n = 312). Variables Univariate Multivariate Beta Coefﬁcient (β) R2 p-value Beta Coefﬁcient (β) R2 Incremental R2 p-value Age -0.358 0.128 <0.001 -0.241 0.058 0.058 0.067 Male sex -0.517 0.267 0.001 -0.283 0.080 0.136 0.082 Smoking 0.224 0.050 0.081 0.137 0.018 0.006 0.253 Systolic Blood pressure -0.530 0.281 0.003 -0.368 0.135 0.104 0.012 Diabetes mellitus -0.715 0.511 <0.001 -0.427 0.182 0.123 0.005 Cardiovascular disease -0.217 0.047 <0.001 -0.092 0.008 0.042 0.142 Hyperlipidaemia 0.364 0.132 0.083 0.211 0.044 0.003 0.360 Fluid overload (OH) -0.792 0.627 0.001 -0.497 0.247 0.122 0.003 Proteinuria -0.699 0.489 <0.001 -0.562 0.315 0.041 <0.001 Diuretics -0.583 0.340 <0.001 -0.384 0.147 0.062 0.016 Baseline eGFR 0.738 0.545 <0.001 0.544 0.295 0.071 <0.001 Table 4. Determinants of eGFR decline in entire cohort (n = 312). Multivariate Model R2 = 0.768 Multivariate Model R2 = 0.768 Dichotomous variables: male sex (0: female/1: male), smoking (0: non-smokers/1: smokers), diabetes mellitus (0: absent/1: present), cardiovascular disease (0: absent/1: present), Hyperlipidemia (0: absent/1: present), Proteinuria (0: absent/1: present), diuretics (0: non-users/1: users) Multivariate regression analysis was performed by enter method Dichotomous variables: male sex (0: female/1: male), smoking (0: non-smokers/1: smokers), diabetes mellitus (0: absent/1: present), cardiovascular disease (0: absent/1: present), Hyperlipidemia (0: absent/1: present), Proteinuria (0: absent/1: present), diuretics (0: non-users/1: users) Multivariate regression analysis was performed by enter method doi:10.1371/journal.pone.0159335.t004 hypervolemic indicating approximately half of NDD-CKD patients are not getting adequate care, despite of their regular visits to nephrology clinic where much attention is paid to volume status. These findings suggest that current clinical and technical tools for aiding physicians to diagnose fluid status and achieve euvolemia are not sufficient. An additional non-invasive tool such as BCM can be useful for routine clinical use in hospitals and would effectively aid diag- nosis of fluid overload in NDD-CKD patients. A total of 144 (46.1%) patients received diuretics at baseline. Apart from management of hypertension and peripheral edema, diuretics are prescribed to control fluid overload [3]. It is interesting to note that diuretics were found to be associated with adverse renal outcomes in our study cohort. Poor renal outcomes among diuretic users might be contributed to several factors. Firstly, maximum diuretic use was observed in hypervolemic patients. As evident from the term “hypervolemia”, these patients were having higher ECW as compared to the other two groups. Discussion Current study evaluated fluid status in a cohort of 312 NDD-CKD patients by using noninva- sive technique of bioimpedance spectroscopy (BIS). By using cutoff values of absolute overhy- dration (OH), study subjects were divided into 3 categories of fluid status i.e. hypovolemia, euvolemia and hypervolemia. A total of 135 (43.2%) patients met the definition criteria of hypervolemia that is less than to what reported in different studies. Reason for low prevalence of hypervolemia in our study might be attributed to difference in cutoff values of fluid overload. On the basis of absolute OH range (0.9–4.1L), maximum prevalence of hypervolemia (65%) was reported by Tsai et al [12]. We did not include 0.9 to 1.1 L in our cutoff range. Similarly in other studies hypervolemia was reported to be 52% and 54.6% based on OH > 7% and OH/ ECW 0.15 respectively [11, 13]. Although the prevalence of hypervolemia reported in our study is less than to what reported in other studies, still 43% of our study subjects were Table 3. Comparison of outcomes between diuretic users and non-users. Outcomes Total cohort N = 312 Diuretic users N = 144 Non-users N = 168 p-value S-cr measurements 4 (3–7) 5 (3–7) 5 (3–7) eGFR 23.7±7.1 22.3 ± 7.4 25.1 ± 6.8 <0.001 ΔeGFR (ml/min/1.73m2) -2.5±1.4 -3.5 ± 1.6 -1.6 ± 0.77 0.02 RRT 36 (11.5%) 30 (20.8%) 6 (3.5%) <0.001 Death 2 (0.6%) 2 (1.4%) 0 Table 3. Comparison of outcomes between diuretic users and non-users. Outcomes Total cohort N = 312 Diuretic users N = 144 Non-users N = 168 p-value S-cr measurements 4 (3–7) 5 (3–7) 5 (3–7) eGFR 23.7±7.1 22.3 ± 7.4 25.1 ± 6.8 <0.001 ΔeGFR (ml/min/1.73m2) -2.5±1.4 -3.5 ± 1.6 -1.6 ± 0.77 0.02 RRT 36 (11.5%) 30 (20.8%) 6 (3.5%) <0.001 Death 2 (0.6%) 2 (1.4%) 0 p-value calculated by student t-test for continuous variables and chi-square test for categorical variables between diuretic users and non-users S-cr: serum creatinine, ΔeGFR: change in estimated glomerular ﬁltration rate. RRT: renal replacement therapy doi:10.1371/journal.pone.0159335.t003 Table 3. Comparison of outcomes between diuretic users and non-users. PLOS ONE \| DOI:10.1371/journal.pone.0159335 July 21, 2016 8 / 13 Continuous variables: age, systolic blood pressure, ﬂuid overload, baseline eGFR Continuous variables: age systolic blood pressure ﬂuid overload baseline eGFR g y p Dichotomous variables: male sex (0: female/1: male), smoking (0: non-smokers/1: smokers), diabetes mellitus (0: absent/1: present), cardiovascular disease (0: absent/1: present), Hyperlipidemia (0: absent/1: present), Proteinuria (0: absent/1: present), diuretics (0: non-users/1: users) Multivariate regression analysis was performed by enter method stolic blood pressure, ﬂuid overload, baseline eGFR Continuous variables: age, systolic blood pressure, ﬂuid overload, baseline eGFR Multivariate Model R = 0.768 Hypervolemia itself causes eGFR decline by independently effecting vascular and endothelial cells leading to artheosclerosis and arterial stiffness [21]. Moreover, fluid over- loaded patients have increased extravascular volume and decreased intravascular volume Table 5. Hazard ratio of diuretic users and non-users in relation to renal outcomes. Renal outcomes Unadjusted univariate HR (95% CI) p-value Adjusted multivariate HR (95% CI) p-value Initiation of RRT Non-users (Reference) 1 1 Diuretic users 3.4 (1.29–2.56) <0.001 2.5 (1.69–3.12) 0.03 Decline in eGFR Non-users (Reference) 1 1 Diuretic users 5.6 (2.57–4.83) <0.001 3.8 (2.32–4.61) 0.01 Model adjusted for ﬂuid status, systolic BP, baseline eGFR as continuous variable CV disease, proteinuria, diabetes mellitus were adjusted as categorically doi:10.1371/journal.pone.0159335.t005 Table 5. Hazard ratio of diuretic users and non-users in relation to renal outcomes. Renal outcomes Unadjusted univariate HR (95% CI) p-value Adjusted multivariate HR (95% CI) p-value Initiation of RRT Non-users (Reference) 1 1 Diuretic users 3.4 (1.29–2.56) <0.001 2.5 (1.69–3.12) 0.03 Decline in eGFR Non-users (Reference) 1 1 Diuretic users 5.6 (2.57–4.83) <0.001 3.8 (2.32–4.61) 0.01 Model adjusted for ﬂuid status, systolic BP, baseline eGFR as continuous variable CV disease, proteinuria, diabetes mellitus were adjusted as categorically Table 5. Hazard ratio of diuretic users and non-users in relation to renal outcomes. PLOS ONE \| DOI:10.1371/journal.pone.0159335 July 21, 2016 9 / 13 Diuretics Use in Chronic Kidney Disease leading to decrease blood flow towards kidney [22, 23]. In addition, a recent study has shown strong association between fluid overload and proteinuria. As proteinuria significantly causes decline in eGFR, this might add to another possibility of poor renal outcomes in hypervolemic patients [24]. Secondly, systolic blood pressure (SBP) was significantly higher among these patients causing more use of other classes of anti-hypertensives. Both RAAS and CCB alter intra-glomerular hemodynamics leading to nephrotoxicity [25, 26]. However, the attribution of hypervolemia, proteinuria, higher SBP and use of anti-hypertensives was adjusted in all sta- tistical analysis (Tables 3–5). Despite adjustment of these confounders, diuretics were found to be independently associated with poor renal outcomes indicated by decline in eGFR and initia- tion of RRT in NDD-CKD patients (Tables 4 and 5). Previously, a number of evidences have reported that diuretic therapy is either detrimental to renal function or it significantly allows quantifiable renal impairment [4, 9, 27]. Secondary analysis of major hypertensive trials have shown that diuretics cause significant increase in serum creatinine values [28–30]. Data analysis of NHANES III survey reported that increase creatinine level is directly proportional to diuretic prescriptions [31]. Similar results were reported by Hawkins and Houston in their retrospective analysis of United States Renal Data System (USRDS). By using data fusion methodology, authors found that increase diuretic dis- tribution is directly associated with ESRD incidence [32]. Similar few small sample size obser- vational studies conducted in CKD patients have also reported rise in serum creatinine with diuretic use [9]. To the best of our knowledge, our study is first hospital based clinical study demonstrating association of diuretic use with decline in eGFR and initiation of RRT in NDD-CKD patients. The exact mechanism by which diuretics cause renal injury is not clear. Apart from metabolic disturbances, diuretics directly cause apoptosis in distal tubular cells of nephron [33]. Hypoka- lemia that is the major metabolic disturbance of both thiazide and loop diuretics leads to renal hypertrophy and tubulointerstitial fibrosis. Concomitant administration of more than one diuretic causes massive volume loss that results in renal vasoconstriction with increased tubular uptake of sodium chloride (NaCl) and decreased urine output (UO). Prolonged vasoconstric- tion then leads to tubular dysfunction and necrosis [34]. Moreover, co-administration of diuretics with other anti-hypertensives especially vasodilators (calcium channel blockers) results in rapid fluctuations of BP causing pre-renal azotemia [33]. The purpose of current study is not to the challenge the potential benefits of diuretics in NDD-CKD patients. There is a well reported evidence for potential benefits of diuretics as not only anti-hypertensive agents but they also significantly reduce risk of cardiovascular and cere- brovascular diseases. However, we made an attempt to highlight the potential risks of diuretics with an intent to underscore clinicians’ attention towards dark side of diuretic therapy that can be potentially fatal for patients in long run. Need of the hour is careful selection of hypervole- mic NDD-CKD patients where benefit of using diuretic therapy outweighs subsequent risks and use of other equally rational treatments (CCB, RAAS blockers). Diuretics Use in Chronic Kidney Disease that BCM gives appropriate results of fluid status in CKD patients. Furthermore, fluid overload might lead to underestimation of creatinine levels and therefore gives misleading creatinine values in hypervolemic patients. Use of diuretics was recorded categorically, not as time vary- ing dose of medication. Our study lacks information regarding salt consumption and dietary intake, as both of them affect fluid status and diuretic efficacy; therefore it might affect study results. Lastly, the follow-up period was only 12 months. A longer follow-up period will give better understanding of outcomes of diuretics especially in terms of decline in eGFR and dis- ease progression. Despite mentioned limitations, current study is strengthened by its prospec- tive nature and being first study in Asia that shows association between diuretic use and decline in renal function in clinical settings. Author Contributions Conceived and designed the experiments: YHK AS ASA. Performed the experiments: YHK ASA. Analyzed the data: YKH AHK THM. Contributed reagents/materials/analysis tools: THM YHK. Wrote the paper: YHK THM. Final approval of manuscript: AS ASA. Conclusions In conclusion, current study demonstrates that diuretic use is an independent predictor of adverse renal outcomes in NDD-CKD patients causing decline in eGFR and increasing the risk of RRT initiation. Unless a contradictory data from randomized controlled trial discourages above findings, it is cautiously concluded that irrespective of fluid overload, diuretics cause adverse renal outcomes. Future interventional studies or double blinded randomized con- trolled studies with large sample size are needed to rule out the association between diuretic use (type and dose) and renal outcomes in NDD-CKD patients, as this complicated triangle is present in majority of NDD-CKD patients. Such studies should be designed to include time averaged defined daily dose of diuretics. PLOS ONE \| DOI:10.1371/journal.pone.0159335 July 21, 2016 Limitation Some potential limitations of current study are needed to be addressed. Being an observational study, confounding by indication was the main limitation. Efforts were made to minimize bias by adjusting confounders in all statistical analysis. 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https://openalex.org/W2969335915	https://pure.mpg.de/pubman/item/item_3155610_3/component/file_3155612/shh2371.pdf	English	null	Ancient DNA from the skeletons of Roopkund Lake reveals Mediterranean migrants in India	Nature communications	2,019	cc-by	13,907	1 Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA 02138, USA. 2 The Max Planck-Harvard Research Center for the Archaeoscience of the Ancient Mediterranean, Cambridge, MA 02138, USA. 3 Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. 4 Department of Archaeology, Max Planck Institute for the Science of Human History, D-07745 Jena, Germany. 5 Broad Institute of Harvard and MIT, Cambridge, MA 02142 USA, USA. 6 Department of Human Evolutionary Biology, Harvard University, Cambridge, MA 02138, USA. 7 Deccan College, Pune 411006, India. 8 Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115, USA. 9 Institutes of Energy and the Environment, The Pennsylvania State University, University Park, PA 16802, USA. 10Department of Anthropology, The Pennsylvania State University, University Park, PA 16802, USA. 11 The Max Planck-Harvard Research Center for the Archaeoscience of the Ancient Mediterranean, D-07745 Jena, Germany. 12Anthropological Survey of India, North West Regional Centre, Dehradun 248195, India. 13CSIR Centre for Cellular and Molecular Biology, Hyderabad, Telangana 500007, India. 14Birbal Sahni Institute of Palaeosciences, Lucknow, Uttar Pradesh 226007, India. 15Gautam Budh Health Care Foundation, Noida, Uttar Pradesh 201301, India. 16Department of Anthropology, University of California, Santa Barbara, CA 93106, USA. 17These authors jointly directed this work: Ayushi Nayak, Douglas J. Kennett, Kumarasamy Thangaraj, David Reich, Niraj Rai. Correspondence and requests for materials should be addressed to D.R. (email: reich@genetics.med.harvard.edu) or to N.R. (email: nirajrai@bsip.res.in) NATURE COMMUNICATIONS \| (2019) 10:3670 \| https://doi.org/10.1038/s41467-019-11357-9 \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11357-9 N Results N estled deep in the Himalayan mountains at 5029 m above sea level, Roopkund Lake is a small body of water (~40 m in diameter) that is colloquially referred to as Skeleton Lake due to the remains of several hundred ancient humans scattered around its shores (Fig. 1)1. Little is known about the origin of these skeletons, as they have never been subjected to systematic anthropological or archaeological scrutiny, in part due to the disturbed nature of the site, which is frequently affected by rockslides2, and which is often visited by local pilgrims and hikers who have manipulated the skeletons and removed many of the artifacts3. There have been multiple proposals to explain the origins of these skeletons. Local folklore describes a pilgrimage to the nearby shrine of the mountain goddess, Nanda Devi, undertaken by a king and queen and their many attendants, who —due to their inappropriate, celebratory behavior—were struck down by the wrath of Nanda Devi4. It has also been suggested that these are the remains of an army or group of merchants who were caught in a storm. Finally, it has been suggested that they were the victims of an epidemic5. Bioarcheological analysis of the Roopkund skeletons. We obtained genome-wide data from 38 individuals by extracting DNA from powder drilled from long bones, producing next- generation sequencing libraries, and enriching them for approximately 1.2 million single nucleotide polymorphisms (SNPs) from across the genome6–9, obtaining an average coverage of 0.51 × at targeted positions (Table 1, Supplementary Data 1). We also obtained PCR-based mitochondrial haplogroup deter- minations for 71 individuals (35 of these were ones for whom we also obtained genome-wide data that conﬁrmed the PCR-based determinations) (Table 2, Supplementary Note 1). We generated stable isotope measurements (δ13C and δ15N) from 45 indivi- duals, including 37 for whom we obtained genome-wide genetic data, and we obtained direct radiocarbon dates for 37 individuals for whom we also had both genetic and isotope data (Table 1). g p In this study, we also present an osteological assessment of health and stature performed on a different set of bones from Roopkund; this report was drafted well before genetic results from Roopkund were available but was never formally published (an edited version of the original report is presented here as Supplementary Note 2). Ancient DNA from the skeletons of Roopkund Lake reveals Mediterranean migrants in India Ancient DNA from the skeletons of Roopkund Lake reveals Mediterranean migrants in India Ancient DNA from the skeletons of Roopkund Lake reveals Mediterranean migrants in India Éadaoin Harney1,2,3, Ayushi Nayak4,17, Nick Patterson5,6, Pramod Joglekar7, Veena Mushrif-Tripathy 7, Swapan Mallick3,5,8, Nadin Rohland3, Jakob Sedig 3, Nicole Adamski3,8, Rebecca Bernardos3, Nasreen Broomandkhoshbacht 3,8, Brendan J. Culleton9,10, Matthew Ferry3,8, Thomas K. Harper10, Megan Michel3,8,11, Jonas Oppenheimer3,8, Kristin Stewardson3,8, Zhao Zhang3, Harashawaradhana12, Maanwendra Singh Bartwal12, Sachin Kumar13,14, Subhash Chandra Diyundi 15, Patrick Roberts 4, Nicole Boivin4, Douglas J. Kennett16,17, Kumarasamy Thangaraj13,17, David Reich2,3,5,8,17 & Niraj Rai13,14,17 Situated at over 5,000 meters above sea level in the Himalayan Mountains, Roopkund Lake is home to the scattered skeletal remains of several hundred individuals of unknown origin. We report genome-wide ancient DNA for 38 skeletons from Roopkund Lake, and ﬁnd that they cluster into three distinct groups. A group of 23 individuals have ancestry that falls within the range of variation of present-day South Asians. A further 14 have ancestry typical of the eastern Mediterranean. We also identify one individual with Southeast Asian-related ancestry. Radiocarbon dating indicates that these remains were not deposited simulta- neously. Instead, all of the individuals with South Asian-related ancestry date to ~800 CE (but with evidence of being deposited in more than one event), while all other individuals date to ~1800 CE. These differences are also reﬂected in stable isotope measurements, which reveal a distinct dietary proﬁle for the two main groups. 1 1 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11357-9 ARTICLE Results The analysis suggests that the Roopkund individuals were broadly healthy, but also identiﬁes three individuals with unhealed compression fractures; the report hypothesizes that these injuries could have transpired during a violent hailstorm of the type that sometimes occurs in the vicinity of Roopkund Lake, while also recognizing that other scenarios are plausible. The report also identiﬁes the presence of both very To shed light on the origin of the skeletons of Roopkund, we analyzed their remains using a series of bioarcheological analyses, including ancient DNA, stable isotope dietary reconstruction, radiocarbon dating, and osteological analysis. We ﬁnd that the Roopkund skeletons belong to three genetically distinct groups that were deposited during multiple events, separated in time by approximately 1000 years. These ﬁndings refute previous sug- gestions that the skeletons of Roopkund Lake were deposited in a single catastrophic event. ~ 1000 km ~ 25 km b c a Roopkund lake Start Finish Roopkund Fig. 1 Context of Roopkund Lake. a Map showing the location of Roopkund Lake. The approximate route of the Nanda Devi Raj Jat pilgrimage relative to Roopkund Lake is shown in the inset. b Image of disarticulated skeletal elements scattered around the Roopkund Lake site. Photo by Himadri Sinha Roy. c Image of Roopkund Lake and surrounding mountains. Photo by Atish Waghwase b a Roopkund lake c Fig. 1 Context of Roopkund Lake. a Map showing the location of Roopkund Lake. The approximate route of the Nanda Devi Raj Jat pilgrimage relative to Roopkund Lake is shown in the inset. b Image of disarticulated skeletal elements scattered around the Roopkund Lake site. Photo by Himadri Sinha Roy. c Image of Roopkund Lake and surrounding mountains. Photo by Atish Waghwase NATURE COMMUNICATIONS \| (2019) 10:3670 \| https://doi.org/10.1038/s41467-019-11357-9 \| www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11357-9 D Skeletal codes No. libraries produced Population label Sex Mitochondrial DNA haplogroup (based on Sequenom genotyping) Mitochondrial DNA haplogroup (based on mt capture) Y-chromosome haplogroup Proportion of endogenous human DNA before capture (best library) 1240k coverage (average) No. of SNPs hit on autosomes C-to-T damage rate at terminal bases (average) X-chromosome contamination point estimate (for males with > 200 SNPs) Mitochondrial DNA match rate to consensus sequence Calibrated radiocarbon datesc δ13C (‰)d δ15N (‰)d R01 3 Roopkund_A M M1a1c M33d H1a2a1 0.014 0.868 570995 0.071 .. 0.996 890–982 CE −19.40 −7.69 R04 4 Roopkund_A F M3C1 M3c1a .. 0.005 0.579 441880 0.049 .. Results 0.997 773–940 CE −16.32 −9.77 R06 4 Roopkund_A F M3c2 M3c2 .. 0.003 0.199 196393 0.046 .. 1.000 773–940 CE −19.00 −9.24 R08 1 Roopkund_A M M3a2 M3a2 H1a1d2 0.007 0.577 403739 0.047 0.013 1.000 773–940 CE −18.94 (−18.88) −9.69 (−9.85) R10 1 Roopkund_A F M3 M3 .. 0.006 0.223 203058 0.055 .. 1.000 773–890 CE −19.74 −9.99 R11 1 Roopkund_A F U U2c1 .. 0.003 0.105 111184 0.049 .. 1.000 775–890 CE −11.45 −8.71 R15 1 Roopkund_A M .. M30c E1b1b1 0.004 0.304 271560 0.065 0.008 1.000 717–889 CE −15.93 −10.29 R17 1 Roopkund_A F .. M5a .. 0.004 0.133 136268 0.059 .. 0.998 770–945 CE −10.74 −9.58 R19 1 Roopkund_A M M3a1 M4 Jb 0.006 0.044 50278 0.057 .. 0.994 770–887 CE −14.47 (−14.42) −9.39 (−9.63) R20 1 Roopkund_A M HV HV14 R2a3a2b2c 0.017 1.476 591844 0.041 0.004 0.998 689–876 CE −16.27 −9.13 R25 1 Roopkund_A M M5a U1a1a H3b 0.002 0.118 125762 0.036 .. 1.000 770–887 CE −17.18 −10.36 R43 1 Roopkund_A M M30 M30 J2a1 0.016 0.295 251527 0.045 .. 0.999 885–980 CE −18.46 (−18.07) 7.95 (−8.23) R44 1 Roopkund_A M M3a1 M3a1 H1a1d2 0.011 0.105 110441 0.045 .. 0.976 775–961 CE −18.22 (−18.27) −9.85 (−9.69) R45 1 Roopkund_A F .. .. .. 0.034 0.861 521678 0.033 .. 1.000 773–890 CE −16.53 −8.41 R51 1 Roopkund_A F X X2p .. 0.011 0.481 405124 0.058 .. 0.999 694–875 CE −18.62 (−18.16) −8.25 (−8.4) R55 1 Roopkund_A M J1b1a1 J1b1a1 .. 0.009 0.590 452228 0.044 −0.001 1.000 894–985 CE −10.13 −8.90 R57 1 Roopkund_A M P4b1 R30b2a .. 0.008 0.602 470065 0.047 −0.001 1.000 770–887 CE −18.66 (−18.42) −8.22 (−8.33) R61 1 Roopkund_A M M3a1 M3a1 .. 0.007 0.133 145489 0.064 .. 0.999 675–769 CE −10.10 −8.24 R62 1 Roopkund_A F U2e3 U4d3 .. 0.009 0.340 313369 0.055 .. 1.000 726–885 CE −18.00 (−18.10) −8.58 (−7.9) R64 1 Roopkund_A F M4″67 M30 + 16234 .. 0.007 0.035 40150 0.045 .. 0.997 687–870 CE −17.08 −8.92 R65 1 Roopkund_A M U2a1 U8b1a1 .. 0.005 0.349 328001 0.055 −0.002 1.000 773–890 CE −10.21 −10.09 R68 1 Roopkund_A F U7 U7a2 .. 0.008 0.565 446699 0.041 .. 0.999 889–971 CE −16.74 (−16.50) −10.19 (−10.21) R69 1 Roopkund_A M H H13a2a .. 0.009 0.370 342426 0.057 0.005 1.000 778–988 CE −18.59 −9.33 R02 4 Roopkund_B M H H6b1 J1a3a 0.036 0.782 578890 0.057 .. Results 0.997 1668–1945 CE −18.69 −10.89 R03 2 Roopkund_B F T1 T1a .. 0.024 0.028 31880 0.039 .. 0.938 1706–1915 CE −18.67 −11.15 R13 1 Roopkund_B M H1 H1 R1a1a1b1a2b 0.056 1.547 706651 0.059 0.002 0.997 1681–1939 CE −18.93 −10.76 R16 1 Roopkund_B F H1 H1c .. 0.006 0.409 352584 0.051 .. 1.000 1682–1932 CE −19.23 −9.21 R18 1 Roopkund_B M H H60a G2a2b2a1a1c1a2 0.031 1.349 614489 0.069 0.004 0.995 1675–1943 CE −19.41 (−19.10) −9.95 (−10.02) R22 1 Roopkund_B M N2 W1 R1b1ab 0.005 0.049 56291 0.056 .. 1.000 .. .. .. R39 1 Roopkund_B M N X2d T1a2 0.018 0.492 379935 0.035 0.005 0.995 1691–1925 CE −18.60 (−18.19) −10.77 (−10.61) R40 1 Roopkund_B M H H12 E1b1b1b2 0.040 1.077 541763 0.041 0.006 0.997 1706–1915 CE −19.23 −9.62 R42 1 Roopkund_B F J1b J1b .. 0.019 0.514 346216 0.031 .. 1.000 1656-… CE −19.72 −10.07 R46 1 Roopkund_B F HV .. .. 0.017 0.627 524922 0.060 .. 0.997 1668–1945 CE −18.97 −8.91 R48 1 Roopkund_B M M2a1a H1b .. 0.034 1.371 728448 0.043 0.005 0.998 1681–1939 CE −18.79 −9.79 R49 1 Roopkund_B F H12 H12a .. 0.026 0.837 584656 0.035 .. 1.000 1661-… CE −19.56 −8.93 R53 1 Roopkund_B M H1 H1 .. 0.008 0.605 476797 0.037 0.006 0.999 1680–1939 CE −19.22 −10.46 R66 1 Roopkund_B M K K1a .. 0.050 0.026 30592 0.025 .. 0.940 1675–1943 CE −18.95 −9.96 R54 1 Roopkund_C M M24 M24a O1b1a1a1b 0.011 0.489 419098 0.047 0.022 1.000 1653-… CE −19.25 (−18.32) −9.98 (−9.74) ndrial DNA haplogroups that are inconsistent between the capture and PCR-based methods are indicated osome calls that should be interpreted with caution due to low coverage onﬁdence interval, rounded to nearest 5 years. Intervals that extend beyond the year 1950 CE are indicated with “..” 11 individuals generated at the Max Planck Institute for the Science of Human History in Jena are reported in parentheses; the other data were generated at the Yale Analytical and Stable Isotope Center Sample ID Skeletal codes No. libraries produced Population label Sex Mitochondrial DNA haplogroup (based on Sequenom genotyping) Mitochondrial DNA haplogroup (based on mt capture) Y-chromosome haplogroup Proportion of endogenous human DNA before capture (best library) 1240k coverage (average) No. of SNPs hit on autosomes C-to-T damage rate at terminal bases (average) X-chromosome contamination point estimate (for males with > 200 SNP ) Mitochondrial DNA match rate to consensus sequence Calibrated radiocarbon datesc δ13C (‰)d δ15N (‰)d R39 N 2706, 9540, 12705, 8701, 11719, 14766, 16223 I3403 X2d R40 H 2706, 9540, 12705 I3404 H12 R41 T1 16223, 14766, 11719, 8701, 12705, 9540, 2706, 16126, 15043, 4491 .. .. R42 J1b 709, 1888, 4216, 12633, 16126, 8697, 9540, 14905, 15607, 8701, 15452, 11251, 12633, 16223 I3405 J1b R43 M30 4216, 16126, 3010, 9540, 16612, 12705, 8701, 12406, 15452, 16069, 11251, 16223 I3406 M30 R44 M3a1 12007, 15043 I3407 M3a1 R45 .. .. I6934 .. R46 HV 15043, 16126, 482, 4703 I6935 .. R47 H 2706, 9540, 12705, 8701, 11719, 14766 .. .. R48 M2a1a 15670, 207, 4703 I6936 H1a R49 H12 2706, 9540, 12705, 16223 I6937 H12a R50 U4 11467, 12308 .. .. R51 X 6221, 9540, 8701 I6938 X2p R52 M6 15043, 5082, 5301 .. .. R53 H1 2706, 3010, 9540, 12705, 8701, 11719, 14766, 16223 I6939 H1 R54 M24 15043, 13359, 15607 I6940 M24 R55 J1b1a1 4216, 12007, 16126, 3010, 9540, 12612, 12705, 8701, 15452, 16069, 16172, 11251, 16223 I6941 J1b1a R56 M 15043 .. .. R57 P4b1 12007, 15043 I6942 R30b R59 D4 15043, 3010, 5178, 8414 .. .. R60 M4″67 12007, 15043 .. .. R61 M3a1 15043, 16126, 482, 4703 I6943 M3a1 R62 U2e3 16223, 1811, 8701, 12705, 9540, 12308, 11467 I6944 U4d3 R63 U2e3 11467, 12308, 9540, 12705, 8701, 1811, 16223 .. .. R64 M4″67 12007, 15043 I6945 M30 R65 U2a1 11467, 12308, 9540, 12705, 8701, 10609, 1811, 16223 I6946 U8b1 R66 K 11467, 12308, 8701, 1811, 16223 I6947 K1a R67 M 15043 .. .. R68 U7 11467, 12308, 9540, 12705, 8701, 14569, 1811, 16223 I7035 U7a2 R69 H 709, 2706, 9540, 12705, 8701, 11719, 14766, 16223 I7036 H13a R72 T 4216, 16126, 9540, 12705, 8701, 16223 .. .. R73 U 3741, 12308, 11467 .. .. Table 2 Mitochondrial DNA haplogroup determination for 71 individuals Skeletal codes mt-DNA haplogroup (determined via multiplex PCR analysis) Mutational differences from rCRS (determined via multiplex PCR analysis) Whole-genome ID mt-DNA haplogroup (determined via whole-genome sequencing) Population label (determined via whole-genome sequencing) R01 M1a1c 15043, 3384, 7094, 11215 I2868 M33d Roopkund_A R02 H 2706, 12705, 11719, 14766, 16223 I2869 H6b1 Roopkund_B R03 T1 16294, 16223, 12633, 11251, 15452, 8701, 15607, 1888, 14905, 11215, 9540, 8697, 16126, 12633, 4216, 709 I2870 T1a Roopkund_B R04 M3C1 15043, 482, 16294 I2871 M3c1a Roopkund_A R05 M2c 15043, 4216 .. .. .. Sample ID Skeletal codes No. libraries produced Population label Sex Mitochondrial DNA haplogroup (based on Sequenom genotyping) Mitochondrial DNA haplogroup (based on mt capture) Y-chromosome haplogroup Proportion of endogenous human DNA before capture (best library) 1240k coverage (average) No. of SNPs hit on autosomes C-to-T damage rate at terminal bases (average) X-chromosome contamination point estimate (for males with > 200 SNP ) Mitochondrial DNA match rate to consensus sequence Calibrated radiocarbon datesc δ13C (‰)d δ15N (‰)d R06 M3c2 15043, 16126, 482 I2872 M3c2 Roopkund_A R07 U4b2 11467, 8701 .. .. .. R08 M3a2 15043, 16126, 482, 5783, 10727 I3342 M3a2 Roopkund_A R09 U2b2 1888, 11467, 12308, 2706, 12705, 8701, 1811 .. .. .. R10 M3 15043, 16126 I3343 M3 Roopkund_A R11 U 11467, 12308, 8701, 3714, 13188 I3344 U2c1 Roopkund_A R12 M4″67 12007, 15043 .. .. .. R13 H1 16223, 14766, 11719, 12705, 9540, 3010, 2706 I3345 H1 Roopkund_B R14 N1b 9540, 8701, 1598 .. .. .. R15 .. .. I3346 M30c Roopkund_A R16 H1 16223, 11719, 5301, 3434, 12705, 9540, 3010, 2706 I3348 H1c Roopkund_B R17 .. 16223, 14766, 11719, 8701, 12705, 9540, 2706 I3349 M5a Roopkund_A R18 H 15043, 482, 4703 I3350 H60a Roopkund_B R19 M3a1 9540, 12705, 8701, 11719, 14766, 16223 I3351 M4 Roopkund_A R20 HV 9540, 12705, 8701, 11719, 14766, 16223 I3352 HV14 Roopkund_A R21 HV 709, 16126, 207, 9540, 8701 .. .. .. R22 N2 8701, 11719, 14766, 16223 I3401 W1 Roopkund_B R23 HV 15043, 9540, 8701, 12361 .. .. .. R24 N1a1b1 1888, 15043, 7094, 7859, 11215, 8701, 16172, 13104, 16223 .. .. .. R25 M5a 709, 11083, 15043, 8502, 16274, 12810 I3402 U1a1a Roopkund_A R26 M2a 709, 1888, 15043 .. .. .. R28 M5 9540, 12705, 8701, 16223 .. .. .. R29 R2 15043, 16126, 5301 .. .. .. R31 M6 1888, 15043 .. .. .. R32 M5 15043 .. .. .. R33 M 12007, 15043, 5301, 3714, 13104, 16223, 16294 .. .. .. R34 M4″67 1888, 11467, 12308, 2706, 9540, 12705, 8701, 1811 .. .. .. R35 U2b 15043 .. .. .. R36 M9a2 16126, 9540, 12705, 8701, 1811, 16223 .. .. .. R37 HV 11467, 12308, 9540, 12705, 8701, 1811, 16223 .. .. .. R38 U2e 6221, 6371, 9540, 8701 .. .. .. R39 N 2706, 9540, 12705, 8701, 11719, 14766, 16223 I3403 X2d Roopkund_B R40 H 2706, 9540, 12705 I3404 H12 Roopkund_B R41 T1 16223, 14766, 11719, 8701, 12705, 9540, 2706, 16126, 15043, 4491 .. .. .. R42 J1b 709, 1888, 4216, 12633, 16126, 8697, 9540, 14905, 15607, 8701, 15452, 11251, 12633, 16223 I3405 J1b Roopkund_B R43 M30 4216, 16126, 3010, 9540, 16612, 12705, 8701, 12406, 15452, 16069, 11251, 16223 I3406 M30 Roopkund_A R44 M3a1 12007, 15043 I3407 M3a1 Roopkund_A R45 .. .. I6934 .. Roopkund_A R46 HV 15043, 16126, 482, 4703 I6935 .. Roopkund_B R47 H 2706, 9540, 12705, 8701, 11719, 14766 .. .. .. Sample ID Skeletal codes No. libraries produced Population label Sex Mitochondrial DNA haplogroup (based on Sequenom genotyping) Mitochondrial DNA haplogroup (based on mt capture) Y-chromosome haplogroup Proportion of endogenous human DNA before capture (best library) 1240k coverage (average) No. of SNPs hit on autosomes C-to-T damage rate at terminal bases (average) X-chromosome contamination point estimate (for males with > 200 SNP ) Mitochondrial DNA match rate to consensus sequence Calibrated radiocarbon datesc δ13C (‰)d δ15N (‰)d 11467, 12308, 9540, 12705, 8701, 14569, 1811, 16223 I7035 709, 2706, 9540, 12705, 8701, 11719, 14766, 16223 I7036 4216, 16126, 9540, 12705, 8701, 16223 .. 3741, 12308, 11467 .. 11467, 12308 .. 16126, 12308 .. 11467, 12308 .. Table 2 Mitochondrial DNA haplogroup determination for 71 individuals Skeletal codes mt-DNA haplogroup (determined via multiplex PCR analysis) Mutational differences from rCRS (determined via multiplex PCR analysis) Whole-genome ID mt-D via w R01 M1a1c 15043, 3384, 7094, 11215 I2868 M33d R02 H 2706, 12705, 11719, 14766, 16223 I2869 H6b1 R03 T1 16294, 16223, 12633, 11251, 15452, 8701, 15607, 1888, 14905, 11215, 9540, 8697, 16126, 12633, 4216, 709 I2870 T1a R04 M3C1 15043, 482, 16294 I2871 M3c1 R05 M2c 15043, 4216 .. .. R06 M3c2 15043, 16126, 482 I2872 M3c2 R07 U4b2 11467, 8701 .. .. R08 M3a2 15043, 16126, 482, 5783, 10727 I3342 M3a2 R09 U2b2 1888, 11467, 12308, 2706, 12705, 8701, 1811 .. .. R10 M3 15043, 16126 I3343 M3 R11 U 11467, 12308, 8701, 3714, 13188 I3344 U2c1 R12 M4″67 12007, 15043 .. .. R13 H1 16223, 14766, 11719, 12705, 9540, 3010, 2706 I3345 H1 R14 N1b 9540, 8701, 1598 .. .. R15 .. .. I3346 M30 R16 H1 16223, 11719, 5301, 3434, 12705, 9540, 3010, 2706 I3348 H1c R17 .. 16223, 14766, 11719, 8701, 12705, 9540, 2706 I3349 M5a R18 H 15043, 482, 4703 I3350 H60a R19 M3a1 9540, 12705, 8701, 11719, 14766, 16223 I3351 M4 R20 HV 9540, 12705, 8701, 11719, 14766, 16223 I3352 HV14 R21 HV 709, 16126, 207, 9540, 8701 .. .. R22 N2 8701, 11719, 14766, 16223 I3401 W1 R23 HV 15043, 9540, 8701, 12361 .. .. R24 N1a1b1 1888, 15043, 7094, 7859, 11215, 8701, 16172, 13104, 16223 .. .. R25 M5a 709, 11083, 15043, 8502, 16274, 12810 I3402 U1a1a R26 M2a 709, 1888, 15043 .. .. R28 M5 9540, 12705, 8701, 16223 .. .. R29 R2 15043, 16126, 5301 .. .. R31 M6 1888, 15043 .. .. R32 M5 15043 .. .. R33 M 12007, 15043, 5301, 3714, 13104, 16223, 16294 .. .. R34 M4″67 1888, 11467, 12308, 2706, 9540, 12705, 8701, 1811 .. .. R35 U2b 15043 .. .. R36 M9a2 16126, 9540, 12705, 8701, 1811, 16223 .. .. R37 HV 11467, 12308, 9540, 12705, 8701, 1811, 16223 .. .. R38 U2e 6221, 6371, 9540, 8701 .. .. Sample ID Skeletal codes No. libraries produced Population label Sex Mitochondrial DNA haplogroup (based on Sequenom genotyping) Mitochondrial DNA haplogroup (based on mt capture) Y-chromosome haplogroup Proportion of endogenous human DNA before capture (best library) 1240k coverage (average) No. of SNPs hit on autosomes C-to-T damage rate at terminal bases (average) X-chromosome contamination point estimate (for males with > 200 SNP ) Mitochondrial DNA match rate to consensus sequence Calibrated radiocarbon datesc δ13C (‰)d δ15N (‰)d 3 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11357-9 Table 2 Mitochondrial Skeletal codes mt-DNA h (determine analysis) R01 M1a1c R02 H R03 T1 R04 M3C1 R05 M2c R06 M3c2 R07 U4b2 R08 M3a2 R09 U2b2 R10 M3 R11 U R12 M4″67 R13 H1 R14 N1b R15 .. R16 H1 R17 .. R18 H R19 M3a1 R20 HV R21 HV R22 N2 R23 HV R24 N1a1b1 R25 M5a R26 M2a R28 M5 R29 R2 R31 M6 R32 M5 R33 M R34 M4″67 R35 U2b R36 M9a2 R37 HV R38 U2e R39 N R40 H R41 T1 R42 J1b R43 M30 R44 M3a1 R45 .. R46 HV R47 H R48 M2a1a R49 H12 R50 U4 R51 X R52 M6 R53 H1 R54 M24 R55 J1b1a1 R56 M R57 P4b1 R59 D4 R60 M4″67 R61 M3a1 R62 U2e3 R63 U2e3 R64 M4″67 R65 U2a1 R66 K R67 M R68 U7 R69 H R72 T R73 U R74 U R76 JT R77 U aD t h it h d ARTICLE A haplogroup determination for 71 individuals oup multiplex PCR Mutational differences from rCRS (determined via multiplex PCR analysis) Whole-genome ID mt-DNA haplogroup (determined via whole-genome sequencing) Population label (determin via whole-genome sequenc 15043, 3384, 7094, 11215 I2868 M33d Roopkund_A 2706, 12705, 11719, 14766, 16223 I2869 H6b1 Roopkund_B 16294, 16223, 12633, 11251, 15452, 8701, 15607, 1888, 14905, 11215, 9540, 8697, 16126, 12633, 4216, 709 I2870 T1a Roopkund_B 15043, 482, 16294 I2871 M3c1a Roopkund_A 15043, 4216 .. .. .. 15043, 16126, 482 I2872 M3c2 Roopkund_A 11467, 8701 .. .. .. 15043, 16126, 482, 5783, 10727 I3342 M3a2 Roopkund_A 1888, 11467, 12308, 2706, 12705, 8701, 1811 .. .. .. 15043, 16126 I3343 M3 Roopkund_A 11467, 12308, 8701, 3714, 13188 I3344 U2c1 Roopkund_A 12007, 15043 .. .. .. 16223, 14766, 11719, 12705, 9540, 3010, 2706 I3345 H1 Roopkund_B 9540, 8701, 1598 .. .. .. .. I3346 M30c Roopkund_A 16223, 11719, 5301, 3434, 12705, 9540, 3010, 2706 I3348 H1c Roopkund_B 16223, 14766, 11719, 8701, 12705, 9540, 2706 I3349 M5a Roopkund_A 15043, 482, 4703 I3350 H60a Roopkund_B 9540, 12705, 8701, 11719, 14766, 16223 I3351 M4 Roopkund_A 9540, 12705, 8701, 11719, 14766, 16223 I3352 HV14 Roopkund_A 709, 16126, 207, 9540, 8701 .. .. .. 8701, 11719, 14766, 16223 I3401 W1 Roopkund_B 15043, 9540, 8701, 12361 .. .. .. 1888, 15043, 7094, 7859, 11215, 8701, 16172, 13104, 16223 .. .. .. 709, 11083, 15043, 8502, 16274, 12810 I3402 U1a1a Roopkund_A 709, 1888, 15043 .. Sample ID Skeletal codes No. libraries produced Population label Sex Mitochondrial DNA haplogroup (based on Sequenom genotyping) Mitochondrial DNA haplogroup (based on mt capture) Y-chromosome haplogroup Proportion of endogenous human DNA before capture (best library) 1240k coverage (average) No. of SNPs hit on autosomes C-to-T damage rate at terminal bases (average) X-chromosome contamination point estimate (for males with > 200 SNP ) Mitochondrial DNA match rate to consensus sequence Calibrated radiocarbon datesc δ13C (‰)d δ15N (‰)d .. .. 9540, 12705, 8701, 16223 .. .. .. 15043, 16126, 5301 .. .. .. 1888, 15043 .. .. .. 15043 .. .. .. 12007, 15043, 5301, 3714, 13104, 16223, 16294 .. .. .. 1888, 11467, 12308, 2706, 9540, 12705, 8701, 1811 .. .. .. 15043 .. .. .. 16126, 9540, 12705, 8701, 1811, 16223 .. .. .. 11467, 12308, 9540, 12705, 8701, 1811, 16223 .. .. .. 6221, 6371, 9540, 8701 .. .. .. 2706, 9540, 12705, 8701, 11719, 14766, 16223 I3403 X2d Roopkund_B 2706, 9540, 12705 I3404 H12 Roopkund_B 16223, 14766, 11719, 8701, 12705, 9540, 2706, 16126, 15043, 4491 .. .. .. 709, 1888, 4216, 12633, 16126, 8697, 9540, 14905, 15607, 8701, 15452, 11251, 12633, 16223 I3405 J1b Roopkund_B 4216, 16126, 3010, 9540, 16612, 12705, 8701, 12406, 15452, 16069, 11251, 16223 I3406 M30 Roopkund_A 12007, 15043 I3407 M3a1 Roopkund_A .. I6934 .. Roopkund_A 15043, 16126, 482, 4703 I6935 .. Roopkund_B 2706, 9540, 12705, 8701, 11719, 14766 .. .. .. 15670, 207, 4703 I6936 H1a Roopkund_B 2706, 9540, 12705, 16223 I6937 H12a Roopkund_B 11467, 12308 .. .. .. 6221, 9540, 8701 I6938 X2p Roopkund_A 15043, 5082, 5301 .. .. .. 2706, 3010, 9540, 12705, 8701, 11719, 14766, 16223 I6939 H1 Roopkund_B 15043, 13359, 15607 I6940 M24a Roopkund_C 4216, 12007, 16126, 3010, 9540, 12612, 12705, 8701, 15452, 16069, 16172, 11251, 16223 I6941 J1b1a1 Roopkund_A 15043 .. .. .. 12007, 15043 I6942 R30b2a Roopkund_A 15043, 3010, 5178, 8414 .. .. .. 12007, 15043 .. .. .. 15043, 16126, 482, 4703 I6943 M3a1 Roopkund_A 16223, 1811, 8701, 12705, 9540, 12308, 11467 I6944 U4d3 Roopkund_A 11467, 12308, 9540, 12705, 8701, 1811, 16223 .. .. .. 12007, 15043 I6945 M30 + 16234 Roopkund_A 11467, 12308, 9540, 12705, 8701, 10609, 1811, 16223 I6946 U8b1a1 Roopkund_A 11467, 12308, 8701, 1811, 16223 I6947 K1a Roopkund_B 15043 .. .. .. 11467, 12308, 9540, 12705, 8701, 14569, 1811, 16223 I7035 U7a2 Roopkund_A 709, 2706, 9540, 12705, 8701, 11719, 14766, 16223 I7036 H13a2a Roopkund_A 4216, 16126, 9540, 12705, 8701, 16223 .. .. .. 3741, 12308, 11467 .. .. .. 11467, 12308 .. .. .. 16126, 12308 .. .. .. 11467, 12308 .. .. .. Sample ID Skeletal codes No. libraries produced Population label Sex Mitochondrial DNA haplogroup (based on Sequenom genotyping) Mitochondrial DNA haplogroup (based on mt capture) Y-chromosome haplogroup Proportion of endogenous human DNA before capture (best library) 1240k coverage (average) No. of SNPs hit on autosomes C-to-T damage rate at terminal bases (average) X-chromosome contamination point estimate (for males with > 200 SNP ) Mitochondrial DNA match rate to consensus sequence Calibrated radiocarbon datesc δ13C (‰)d δ15N (‰)d logroup determination for 71 individuals x PCR Mutational differences from rCRS (determined via multiplex PCR analysis) Whole-genom 15043, 3384, 7094, 11215 I2868 2706, 12705, 11719, 14766, 16223 I2869 16294, 16223, 12633, 11251, 15452, 8701, 15607, 1888, 14905, 11215, 9540, 8697, 16126, 12633, 4216, 709 I2870 15043, 482, 16294 I2871 15043, 4216 .. 15043, 16126, 482 I2872 11467, 8701 .. 15043, 16126, 482, 5783, 10727 I3342 1888, 11467, 12308, 2706, 12705, 8701, 1811 .. 15043, 16126 I3343 11467, 12308, 8701, 3714, 13188 I3344 12007, 15043 .. 16223, 14766, 11719, 12705, 9540, 3010, 2706 I3345 9540, 8701, 1598 .. .. I3346 16223, 11719, 5301, 3434, 12705, 9540, 3010, 2706 I3348 16223, 14766, 11719, 8701, 12705, 9540, 2706 I3349 15043, 482, 4703 I3350 9540, 12705, 8701, 11719, 14766, 16223 I3351 9540, 12705, 8701, 11719, 14766, 16223 I3352 709, 16126, 207, 9540, 8701 .. 8701, 11719, 14766, 16223 I3401 15043, 9540, 8701, 12361 .. 1888, 15043, 7094, 7859, 11215, 8701, 16172, 13104, 16223 .. 709, 11083, 15043, 8502, 16274, 12810 I3402 709, 1888, 15043 .. 9540, 12705, 8701, 16223 .. 15043, 16126, 5301 .. 1888, 15043 .. 15043 .. 12007, 15043, 5301, 3714, 13104, 16223, 16294 .. 1888, 11467, 12308, 2706, 9540, 12705, 8701, 1811 .. 15043 .. 16126, 9540, 12705, 8701, 1811, 16223 .. 11467, 12308, 9540, 12705, 8701, 1811, 16223 .. 6221, 6371, 9540, 8701 .. 2706, 9540, 12705, 8701, 11719, 14766, 16223 I3403 2706, 9540, 12705 I3404 16223, 14766, 11719, 8701, 12705, 9540, 2706, 16126, 15043, 4491 .. 709, 1888, 4216, 12633, 16126, 8697, 9540, 14905, 15607, 8701, 15452, 11251, 12633, 16223 I3405 4216, 16126, 3010, 9540, 16612, 12705, 8701, 12406, 15452, 16069, 11251, 16223 I3406 12007, 15043 I3407 .. I6934 15043, 16126, 482, 4703 I6935 2706, 9540, 12705, 8701, 11719, 14766 .. 15670, 207, 4703 I6936 2706, 9540, 12705, 16223 I6937 11467, 12308 .. 6221, 9540, 8701 I6938 15043, 5082, 5301 .. 2706, 3010, 9540, 12705, 8701, 11719, 14766, 16223 I6939 15043, 13359, 15607 I6940 4216, 12007, 16126, 3010, 9540, 12612, 12705, 8701, 15452, 16069, 16172, 11251, 16223 I6941 15043 .. 12007, 15043 I6942 15043, 3010, 5178, 8414 .. 12007, 15043 .. 15043, 16126, 482, 4703 I6943 16223, 1811, 8701, 12705, 9540, 12308, 11467 I6944 11467, 12308, 9540, 12705, 8701, 1811, 16223 .. 12007, 15043 I6945 11467, 12308, 9540, 12705, 8701, 10609, 1811, 16223 I6946 11467, 12308, 8701, 1811, 16223 I6947 15043 .. Sample ID Skeletal codes No. libraries produced Population label Sex Mitochondrial DNA haplogroup (based on Sequenom genotyping) Mitochondrial DNA haplogroup (based on mt capture) Y-chromosome haplogroup Proportion of endogenous human DNA before capture (best library) 1240k coverage (average) No. of SNPs hit on autosomes C-to-T damage rate at terminal bases (average) X-chromosome contamination point estimate (for males with > 200 SNP ) Mitochondrial DNA match rate to consensus sequence Calibrated radiocarbon datesc δ13C (‰)d δ15N (‰)d We also found no evidence that the individuals were infected with bacterial pathogens, providing no support for the suggestion that these individuals died in an epidemic, although we caution that failure to ﬁnd evidence for pathogen DNA in long bone powder may simply reﬂect the fact that it was present at too low a concentration to detect (Supplementary Note 3)11. Skeletons at Roopkund Lake were deposited in multiple events. The discovery of multiple, genetically distinct groups among the skeletons of Roopkund Lake raises the question of whether these individuals died simultaneously or during separate events. We used Accelerator Mass Spectrometry (AMS) radiocarbon dating to determine the age of the remains. We successfully generated radiocarbon dates from all but one of the individuals for which we have genetic data, using the same stocks of bone powder that we used for genetic analysis to ensure that the dates correspond directly to the genetic groupings. We ﬁnd that the Roopkund_A and Roopkund_B groups are separated in time by ~1000 years, with the calibrated dates for individuals assigned to the Roop- kund_A group ranging from the 7th–10th centuries CE, and the calibrated dates for individuals assigned to the Roopkund_B group ranging from the 17th–20th centuries CE (Table 1; Fig. 3a; Supplementary Data 4). The single individual assigned to Roop- kund_C also dates to this later period. These results demonstrate that the skeletons of Roopkund Lake perished in at least two separate events. For Roopkund_A, we detect non-overlapping 95% conﬁdence intervals (for example individual I6943 dates to 675–769 CE, while individual I6941 dates to 894–985 CE), sug- gesting that even these individuals may not have died simulta- neously (Fig. 3a). In contrast, the calibrated dates obtained for 13 Roopkund_B individuals and the single Roopkund_C individual all have mutually overlapping 95% conﬁdence intervals. Roopkund skeletons form three genetically distinct groups. Sample ID Skeletal codes No. libraries produced Population label Sex Mitochondrial DNA haplogroup (based on Sequenom genotyping) Mitochondrial DNA haplogroup (based on mt capture) Y-chromosome haplogroup Proportion of endogenous human DNA before capture (best library) 1240k coverage (average) No. of SNPs hit on autosomes C-to-T damage rate at terminal bases (average) X-chromosome contamination point estimate (for males with > 200 SNP ) Mitochondrial DNA match rate to consensus sequence Calibrated radiocarbon datesc δ13C (‰)d δ15N (‰)d We explored the genetic diversity of the 38 Roopkund individuals using a previously established Principal Component Analysis (PCA) that is effective at visualizing genetic variation of diverse present-day people from South Asia (a term we use to refer to the territories of the present day countries of India, Pakistan, Nepal, Bhutan, Bangladesh, and Bhutan) relative to West Eurasian-related groups (a term we use to refer to the cluster of ancestry types common in Europe, the Near East, and Iran) and East Asian-related groups (a term we apply to the cluster of ancestry types common in East Asia including China, Japan, Southeast Asia, and western Indonesia)12. We ﬁnd that the Roopkund individuals cluster into three distinct groups, which we will henceforth refer to as Roopkund_A, Roopkund_B, and Roopkund_C (Fig. 2a). Individuals in Roopkund_A (n = 23) fall along a genetic gradient that includes most present-day South Asians. However, they do not fall in a tight cluster along this gradient, suggesting that they do not comprise a single endoga- mous group, and instead derive from a diversity of groups. Individuals belonging to the Roopkund_B cluster (n = 14) do not fall along this gradient, and instead fall near present-day West Eurasians, suggesting that Roopkund_B individuals possess West Eurasian-related ancestry. A single individual, Roopkund_C, falls far from all other Roopkund individuals in the PCA, between the Onge (Andaman Islands) and Han Chinese, suggesting East Asian-related ancestry. Differences in diet correlate with genetic groupings. We carried out carbon and nitrogen isotope analysis of femur bone collagen for 45 individuals. Femur bone collagen is determined by diet in the last 10–20 years of life17, and therefore is not necessarily correlated with the genetic ancestry of a population, which reﬂects processes occurring over generations. Nevertheless, we ﬁnd evidence of dietary heterogeneity across the genetic ancestry groupings, providing additional support for the presence of multiple distinct groups at Roopkund Lake. We ﬁrst observed that the Roopkund individuals are characterized by a range of δ13C values indicating diets reliant on both C3 and C4 plant sources, as well as δ15N values indicating varying degrees of consumption of protein derived from terrestrial animals (Fig. 3b and Supplementary Note 4). The δ13C values are non-randomly associated with the genetic groupings for the 37 individuals for whom we had both measurements. Sample ID Skeletal codes No. libraries produced Population label Sex Mitochondrial DNA haplogroup (based on Sequenom genotyping) Mitochondrial DNA haplogroup (based on mt capture) Y-chromosome haplogroup Proportion of endogenous human DNA before capture (best library) 1240k coverage (average) No. of SNPs hit on autosomes C-to-T damage rate at terminal bases (average) X-chromosome contamination point estimate (for males with > 200 SNP ) Mitochondrial DNA match rate to consensus sequence Calibrated radiocarbon datesc δ13C (‰)d δ15N (‰)d R48 M2a1a 15670, 207, 4703 I6936 H1a Roopkund_B R49 H12 2706, 9540, 12705, 16223 I6937 H12a Roopkund_B R50 U4 11467, 12308 .. .. .. R51 X 6221, 9540, 8701 I6938 X2p Roopkund_A R52 M6 15043, 5082, 5301 .. .. .. R53 H1 2706, 3010, 9540, 12705, 8701, 11719, 14766, 16223 I6939 H1 Roopkund_B R54 M24 15043, 13359, 15607 I6940 M24a Roopkund_C R55 J1b1a1 4216, 12007, 16126, 3010, 9540, 12612, 12705, 8701, 15452, 16069, 16172, 11251, 16223 I6941 J1b1a1 Roopkund_A R56 M 15043 .. .. .. R57 P4b1 12007, 15043 I6942 R30b2a Roopkund_A R59 D4 15043, 3010, 5178, 8414 .. .. .. R60 M4″67 12007, 15043 .. .. .. R61 M3a1 15043, 16126, 482, 4703 I6943 M3a1 Roopkund_A R62 U2e3 16223, 1811, 8701, 12705, 9540, 12308, 11467 I6944 U4d3 Roopkund_A R63 U2e3 11467, 12308, 9540, 12705, 8701, 1811, 16223 .. .. .. R64 M4″67 12007, 15043 I6945 M30 + 16234 Roopkund_A R65 U2a1 11467, 12308, 9540, 12705, 8701, 10609, 1811, 16223 I6946 U8b1a1 Roopkund_A R66 K 11467, 12308, 8701, 1811, 16223 I6947 K1a Roopkund_B R67 M 15043 .. .. .. R68 U7 11467, 12308, 9540, 12705, 8701, 14569, 1811, 16223 I7035 U7a2 Roopkund_A Table 2 Mitochondrial DNA haplogroup determination for 71 individuals NATURE COMMUNICATIONS \| (2019) 10:3670 \| https://doi.org/10.1038/s41467-019-11357-9 \| www.nature.com/naturecommunicatio 4 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11357-9 ancestry from individuals of the main Roopkund_B cluster relative to diverse comparison populations (Supplementary Data 3), and so we lump all the Roopkund_B individuals together in what follows. robust and tall individuals (outside the range of almost all South Asians), and more gracile individuals, and hypothesizes based on this the presence of at least two distinct groups of individuals, consistent with our genetic ﬁndings (Supplementary Note 2). g g pp y Our analysis of the genome-wide data from 38 Roopkund individuals shows that they include both genetic males (n = 23) and females (n = 15)—consistent with the physical anthropology evidence for the presence of both males and females (Supple- mentary Note 2). The relatively similar proportions of males and females is difﬁcult to reconcile with the suggestion that these individuals might have been part of a military expedition. We detected no relative pairs (3rd degree or closer) among the sequenced individuals10, providing evidence against the idea that the Roopkund skeletons might represent the remains of groups of families. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11357-9 The plotted data are provided in a Source Data ﬁle d Roopkund_A –0.04 –0.02 0.00 0.02 0.04 0.06 0.08 –0.08 –0.06 –0.04 –0.02 0.00 0.02 0.04 PC1 PC2 a b –0.04 –0.02 0.00 0.02 0.04 –0.02 0.00 0.02 0.04 0.06 0.08 PC1 PC2 Roopkund_A Roopkund_B Roopkund_C Greek Cretan lranian Onge Han French Indian cline populations Roopkund_A Roopkund_A Roopkund_B Roopkund_B Roopkund_C Roopkund_C –0.04 –0.02 0.00 0.02 0.04 0.06 0.08 –0.08 –0.06 –0.04 –0.02 0.00 0.02 0.04 PC1 PC2 a Onge Han French Indian cline populations Roopkund_A Roopkund_A Roopkund_B Roopkund_B Roopkund_C Roopkund_C e p b –0.04 –0.02 0.00 0.02 0.04 –0.02 0.00 0.02 0.04 0.06 0.08 PC1 PC2 Roopkund_A Roopkund_B Roopkund_C Greek Cretan lranian Roopkund_B c Roopkund_B WHG EHG Anatolia_N Iran_N Greek Crete.DG Roopkund_B Roopkund_A Roopkund_C Brahmin_Tiwari GujaratiB GujaratiA GujaratiC GujaratiD Palliyar Mala Han Malay Vietnamese Fig. 2 Genetic Structure of the Skeletons of Roopkund Lake. a Principal component analysis (PCA) of 1,453 present day individuals from selected groups throughout mainland South Asia (highlighted in gray). French individuals (representing the location where West Eurasian populations are known to cluster) are shown in purple, Chinese individuals are shown (representing the location where East Asian populations are known to cluster) in orange, and Andamanese individuals are shown in teal; the 38 Roopkund individuals are projected. b PCA of 988 present day West Eurasians with the Roopkund individuals projected. The PCA plot is truncated to remove Sardinians and southern Levantine groups; Present-day Greeks are shown in blue, Cretans in pink, Iranians in green, and all other West Eurasian populations in gray. A gray polygon encloses all the individuals in each Roopkund group with > 100,000 SNPs. c ADMIXTURE analysis of 2344 present-day and 1877 ancient individuals with K = 4 ancestral components. Only a subset of individuals with ancestries relevant to the interpretation of the Roopkund individuals are shown. Consistent with the PCA, Roopkund_A has ancestry most closely matching Indian groups; Roopkund_B has ancestry most closely matching Greek and Cretan groups; and Roopkund_C has ancestry most closely matching Southeast Asian groups. Genetic differentiation (FST) between Roopkund_A (d) and diverse present-day populations, and Roopkund_B (e) and diverse present-day populations. We only plotted present-day populations for which we have latitudes and longitudes; deeper red coloration indicates less differentiation to the Roopkund genetic cluster being analyzed. Sample ID Skeletal codes No. libraries produced Population label Sex Mitochondrial DNA haplogroup (based on Sequenom genotyping) Mitochondrial DNA haplogroup (based on mt capture) Y-chromosome haplogroup Proportion of endogenous human DNA before capture (best library) 1240k coverage (average) No. of SNPs hit on autosomes C-to-T damage rate at terminal bases (average) X-chromosome contamination point estimate (for males with > 200 SNP ) Mitochondrial DNA match rate to consensus sequence Calibrated radiocarbon datesc δ13C (‰)d δ15N (‰)d We ﬁnd that all the Roop- kund_B individuals (with typically eastern Mediterranean ancestry), as well as the Roopkund_C individual, have δ13C values between −19.7‰ and −18.2‰ reﬂecting consumption of terrestrial C3 plants, such as wheat, barley, and rice (and/or animals foddered on such plants). In contrast, the Roopkund_A individuals (with typically South Asian ancestry) have much more varied δ13C values (−18.9‰ to −10.1‰), with some implying C3 plant reliance and others reﬂecting either a mixed C3 and C4 derived diet, or alternatively consumption of C3 plants along with animals foddered with millet, a C4 plant (a practice that has been documented ethnographically in South Asia17). The difference in the δ13C distribution between the Roopkund_A and Roopkund_B groupings is highly signiﬁcant (p = 0.00022 from a two-sided Mann-Whitney test). y To further understand the West Eurasian-related afﬁnity in the Roopkund_B cluster, we projected all the Roopkund individuals onto a second PCA designed to distinguish between sub- components of West Eurasian-related ancestry13,14 (Fig. 2b). Individuals assigned to the Roopkund_A and Roopkund_C groups cluster towards the top right of the PCA plot, close to present-day groups with Iranian ancestry, consistent with where populations with South Asian or East Asian ancestry cluster when projected onto such a plot13. Individuals belonging to the Roopkund_B group cluster toward the center of the plot, close to present-day people from mainland Greece and Crete15. We observe consistent patterns using the automated clustering software ADMIXTURE16 (Fig. 2c) and in pairwise FST statistics (Fig. 2d, e, Supplementary Data 2). The visual evidence from the PCA suggests that two individuals from the Roopkund_B group might represent genetic outliers (Fig. 2b). However, symmetry f4- statistics show that the two apparent outliers (one of which has relatively low coverage) are statistically indistinguishable in Genetic afﬁnities of the Roopkund subgroups. We used qpWave18,19 to test whether Roopkund_B is consistent with forming a genetic clade with any present-day population (that TURE COMMUNICATIONS \| (2019) 10:3670 \| https://doi.org/10.1038/s41467-019-11357-9 \| www.nature.com/naturecommunications 5 5 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11357-9 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11357-9 –0.04 –0.02 0.00 0.02 0.04 0.06 0.08 –0.08 –0.06 –0.04 –0.02 0.00 0.02 0.04 PC1 PC2 a b c e d –0.04 –0.02 0.00 0.02 0.04 –0.02 0.00 0.02 0.04 0.06 0.08 PC1 PC2 Roopkund_A Roopkund_B Roopkund_C Greek Cretan lranian Onge Han French Indian cline populations 10–1 10–2 10–3 Roopkund_A Roopkund_A Roopkund_A Roopkund_B Roopkund_B Roopkund_B Roopkund_C Roopkund_C WHG EHG Anatolia_N Iran_N Greek Crete.DG Roopkund_B Roopkund_A Roopkund_C Brahmin_Tiwari GujaratiB GujaratiA GujaratiC GujaratiD Palliyar Mala Han Malay Vietnamese Fig. 2 Genetic Structure of the Skeletons of Roopkund Lake. a Principal component analysis (PCA) of 1,453 present day individuals from selected groups throughout mainland South Asia (highlighted in gray). French individuals (representing the location where West Eurasian populations are known to cluster) are shown in purple, Chinese individuals are shown (representing the location where East Asian populations are known to cluster) in orange, and Andamanese individuals are shown in teal; the 38 Roopkund individuals are projected. b PCA of 988 present day West Eurasians with the Roopkund individuals projected. The PCA plot is truncated to remove Sardinians and southern Levantine groups; Present-day Greeks are shown in blue, Cretans in pink, Iranians in green, and all other West Eurasian populations in gray. A gray polygon encloses all the individuals in each Roopkund group with > 100,000 SNPs. c ADMIXTURE analysis of 2344 present-day and 1877 ancient individuals with K = 4 ancestral components. Only a subset of individuals with ancestries relevant to the interpretation of the Roopkund individuals are shown. Consistent with the PCA, Roopkund_A has ancestry most closely matching Indian groups; Roopkund_B has ancestry most closely matching Greek and Cretan groups; and Roopkund_C has ancestry most closely matching Southeast Asian groups. Genetic differentiation (FST) between Roopkund_A (d) and diverse present-day populations, and Roopkund_B (e) and diverse present-day populations. We only plotted present-day populations for which we have latitudes and longitudes; deeper red coloration indicates less differentiation to the Roopkund genetic cluster being analyzed. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11357-9 b We show normalized δ13C and δ15N values for samples with isotopic data: 37 for which genetic data were generated (circles with colors indicating their cluster), and eight for which no genetic data were generated (labeled Roopkund_U). In cases where multiple measurements were obtained, we plot the average of all measurements. The plotted data are provided as a Source Data ﬁle Asians, we observe a weakly signiﬁcant difference in the proportion of West Eurasian-related ancestry in males and females (p = 0.015 by a permutation test across individuals; Supplementary Note 8), with systematically lower proportions of West Eurasian-related ancestry in males than females. This suggests that the males and females were drawn from signiﬁcantly different mixtures of groups within South Asia. Asians, we observe a weakly signiﬁcant difference in the proportion of West Eurasian-related ancestry in males and females (p = 0.015 by a permutation test across individuals; Supplementary Note 8), with systematically lower proportions of West Eurasian-related ancestry in males than females. This suggests that the males and females were drawn from signiﬁcantly different mixtures of groups within South Asia. a group would be expected to have admixture with groups with more typical South Asian ancestry (as the Kalash do), or would be expected to be inbred and to have relatively low genetic diversity. However, the Roopkund_B individuals have evidence for neither pattern (Supplementary Note 9). Combining dif- ferent lines of evidence, the data suggest instead that what we have sampled is a group of unrelated men and women who were born in the eastern Mediterranean during the period of Ottoman political control. As suggested by their consump- tion of a predominantly terrestrial, rather than marine-based diet, they may have lived in an inland location, eventually traveling to and dying in the Himalayas. Whether they were participating in a pilgrimage, or were drawn to Roopkund Lake for other reasons, is a mystery. It would be surprising for a Hindu pilgrimage to be practiced by a large group of travelers from the eastern Mediterranean where Hindu prac- tices have not been common; Hindu practice in this time might be more plausible for a southeast Asian individual with an ancestry type like that seen in the Roopkund_C individual. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11357-9 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11357-9 500 700 Radiocarbon dates a b Stable isotopes Roopkund_A Roopkund_B Roopkund_C Roopkund (undefined) Individuals 900 1100 Calibrated date (calCE) 1300 1500 1700 1900 –22 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 –20 –18 –16 δ13 C (‰) δ15 N (‰) –14 –12 –10 –8 Fig. 3 Radiocarbon and Isotopic Evidence of Distinct Origins of Roopkund Genetic Groups. a We generated 37 accelerator mass spectrometry radiocarbon dates and calibrated them using OxCal v4.3.2. The dating reveals that the individuals were deposited in at least two events ~1000 years apart. In fact, the Roopkund_A individuals (shown in yellow) may have been deposited over an extended period themselves, as the 95% conﬁdence intervals for some of the radiocarbon dates (for example I6943 and I6941) do not overlap. Radiocarbon dates indicate that Roopkund_B (shown in red) and Roopkund_C (shown in white) individuals may have been deposited during a single event. Error bars indicate 95.4% conﬁdence intervals. Calibration curves are shown in Supplementary Fig. 1. b We show normalized δ13C and δ15N values for samples with isotopic data: 37 for which genetic data were generated (circles with colors indicating their cluster), and eight for which no genetic data were generated (labeled Roopkund_U). In cases where multiple measurements were obtained, we plot the average of all measurements. The plotted data are provided as a Source Data ﬁle b Stable isotopes Roopkund_A Roopkund_B Roopkund_C Roopkund (undefined) –22 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 –20 –18 –16 δ13 C (‰) δ15 N (‰) –14 –12 –10 –8 500 700 Radiocarbon dates a Individuals 900 1100 Calibrated date (calCE) 1300 1500 1700 1900 b a Fig. 3 Radiocarbon and Isotopic Evidence of Distinct Origins of Roopkund Genetic Groups. a We generated 37 accelerator mass spectrometry radiocarbon dates and calibrated them using OxCal v4.3.2. The dating reveals that the individuals were deposited in at least two events ~1000 years apart. In fact, the Roopkund_A individuals (shown in yellow) may have been deposited over an extended period themselves, as the 95% conﬁdence intervals for some of the radiocarbon dates (for example I6943 and I6941) do not overlap. Radiocarbon dates indicate that Roopkund_B (shown in red) and Roopkund_C (shown in white) individuals may have been deposited during a single event. Error bars indicate 95.4% conﬁdence intervals. Calibration curves are shown in Supplementary Fig. 1. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11357-9 The plotted data are provided in a Source Data ﬁle We performed a similar analysis on individuals belonging to the Roopkund_A group and ﬁnd that they cannot be modeled as deriving from a homogeneous group (Supplementary Note 6). Instead, Roopkund_A individuals vary signiﬁcantly in their relationship to a diverse set of present-day South Asians, consistent with the heterogeneity evident in PCA (Fig. 2a). We were unable to model the Roopkund_C individual as a genetic clade with any present-day populations, but we were able to model its ancestry as ~82% Malay-related and ~18% Vietnamese- related using qpAdm7, showing that this individual is consistent with being of Southeast Asian origin. We tested if any of the Roopkund groups show speciﬁc genetic afﬁnity to present-day groups from the Himalayan region, including four neighboring villages in the northern Ladakh region for which we report new genome-wide sequence data, but we ﬁnd no such evidence (Supplementary Note 7). Within the Roopkund_A group which has ancestry that falls within the variation of present-day South is, whether it is possible to model the two populations as des- cending entirely from the same ancestral population with no mixture with other groups since their split). We selected 26 present-day populations for comparison, with particular emphasis on West Eurasian-related groups (we analyzed the West Eurasian-related groups Basque, Crete, Cypriot, Egyptian, English, Estonian, Finnish, French, Georgian, German, Greek, Hungarian, Italian_North, Italian_South, Norwegian, Spanish, Syrian, Ukranian, and the non-West-Eurasian-related groups Brahmin_Tiwari, Chukchi, Han, Karitiana, Mala, Mbuti, Onge, and Papuan). We ﬁnd that Roopkund_B is consistent with forming a genetic clade only with individuals from present-day Crete. These results by no means imply that the Roopkund_B individuals originated in the island of Crete itself, although they suggest that their recent ancestors or they themselves came from a nearby region (Supplementary Note 5; Supplementary Data 5). NATURE COMMUNICATIONS \| (2019) 10:3670 \| https://doi.org/10.1038/s41467-019-11357-9 \| www.nature.com/naturecommunications 6 ARTICLE Discussion Th The genetically, temporally, and isotopically heterogeneous composition of the groups at Roopkund Lake was unanticipated from the context in which the skeletons were found. Radiocarbon dating reveals at least two key phases of deposition of human remains separated by around one thousand years and with sig- niﬁcant heterogeneity in the dates for the earlier individuals indicating that they could not all have died in a single catastrophic event. Combining multiple lines of evidence, we suggest a possible explanation for the origin of at least some of the Roopkund_A individuals. Roopkund Lake is not situated on any major trade route, but it is on a present-day pilgrimage route—the Nanda Devi Raj Jat pilgrimage which today occurs every 12 years (Fig. 1a). As part of the event, pilgrims gather for worship and celebration along the route. Reliable descriptions of the pilgrim- age ritual do not appear until the late-19th century, but inscrip- tions in nearby temples dating to between the 8th and 10th centuries suggest potential earlier origins20. We view the hypothesis of a mass death during a pilgrimage event as a plau- sible explanation for at least some of the individuals in the Roopkund_A cluster. Taken together, these results have produced meaningful insights about an enigmatic ancient site. More generally, this study highlights the power of biomolecular analyses to obtain rich information about the human story behind archaeological deposits that are so highly disturbed that traditional archae- ological methods are not as informative. p The Roopkund_B cluster is more puzzling. It is tempting to hypothesize that the Roopkund_B individuals descend from Indo-Greek populations established after the time of Alexander the Great, who may have contributed ancestry to some present- day groups like the Kalash21. However, this is unlikely, as such NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11357-9 Given that the Roopkund_B and Roopkund_C individuals died only in the last few centuries, an important direction for future investigation will be to carry out archival research to determine if there were reports of large foreign traveling parties dying in the region over the last few hundred years. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11357-9 We used smartpca69 to compute FST between the two major Roopkund groups (Roopkund_A and Roopkund_B) and all other groups composed of at least 2 individuals in the dataset, using default parameters, with the settings inbreed:YES and fstonly:YES. We successfully generated genome-wide DNA for 38 individuals (Supplementary Data 1). For each sample, approximately 75 mg of bone powder originally prepared at CCMB was further processed in dedicated ancient DNA clean rooms at Harvard Medical School using standard protocols, including DNA extraction optimized for ancient DNA recovery23, modiﬁed by replacing the Zymo extender/MinElute column assemblage with a preassembled spin column device24, followed by library preparation with partial UDG treatment25. The quality of authentic ancient DNA preservation in each sample was assessed by carrying out a preliminary screening of all libraries via targeted DNA enrichment, designed to capture mitochondrial DNA in addition to 50 nuclear targets26. We sequenced the enriched libraries on an Illumina NextSeq500 instrument for 2 × 76 cycles with an additional 2 × 7 cycles for identiﬁcation of indices. Based on this preliminary assessment, libraries that were deemed promising underwent a further enrichment using a reagent that targeted ~1.2 million SNPs6–9, and then were sequenced using an Illumina NextSeq500 instrument. We performed clustering using ADMIXTURE16. We carried out this analysis on all samples used for the PCA analyses, although we display only selected populations for the sake of clarity. Prior to analysis, SNPs in linkage disequilibrium with one another were pruned in PLINK using the parameters–indep-pairwise 200 25 0.4. We performed an ADMIXTURE analysis on the remaining 344,363 SNPs in the pruned dataset for values of k between 2 and 10, and carried out 20 replicates at each value of k. We retained the highest likelihood replicate at each k and displayed results for k (k = 4), which we chose because we observed that it is most visually helpful for discriminating the ancestry of the groups of interest. p g y g p We used qpWave18,19, with default parameters and allsnps:YES, to determine if any of the Roopkund populations was consistent with being a clade with any present-day populations. We included a base set of nine populations in each test, chosen to represent diverse ancestry from throughout the world. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11357-9 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11357-9 Ancient DNA laboratory Work. A total of 76 skeletal samples (72 long bones and four teeth) were sampled at the Anthropological Survey of India, Kolkata. Skeletal sampling was performed for all samples in dedicated ancient DNA facilities at the Centre for Cellular and Molecular Biology (CCMB) in Hyderabad, India. A subset of samples that underwent preliminary ancient DNA screening at CCMB, Ancient DNA laboratory Work. A total of 76 skeletal samples (72 long bones and four teeth) were sampled at the Anthropological Survey of India, Kolkata. Skeletal sampling was performed for all samples in dedicated ancient DNA facilities at the Centre for Cellular and Molecular Biology (CCMB) in Hyderabad, India. A subset of samples that underwent preliminary ancient DNA screening at CCMB, including three samples that did not yield sufﬁcient data to assign mitochondrial DNA haplogroups during preliminary screening (see Supplementary Note 1), were further processed at Harvard Medical School, Boston, USA, consistent with recommendations in the ancient DNA literature for repeating analyses in two independent laboratories to increase conﬁdence in results22. We used smartpca69 to perform principal component analysis (PCA) using default parameters, with the settings lsqproject:YES and numoutlier:0. We projected the Roopkund individuals onto two PCA plots designed either to reveal a cline of West Eurasian-related ancestry in South Asian populations18, or to reveal the genetic substructure in present-day West Eurasians13. The ﬁrst PCA (Fig. 2a) included 1453 present-day populations12 in addition to the Roopkund individuals, while the second PCA (Fig. 2b) included 986 present-day populations13, in addition to the Roopkund individuals and two individuals from present-day Crete (population label Crete.DG). The PCA plots show that the samples cluster into three distinct groups, which we label Roopkund_A, Roopkund_B and Roopkund_C, and treat separately for subsequent analyses. 69 including three samples that did not yield sufﬁcient data to assign mitochondrial DNA haplogroups during preliminary screening (see Supplementary Note 1), were further processed at Harvard Medical School, Boston, USA, consistent with recommendations in the ancient DNA literature for repeating analyses in two independent laboratories to increase conﬁdence in results22. p At CCMB, samples were prepared for processing by wiping with a bleach solution, followed by deionized water. The samples were then subjected to UV irradiation for 30 min on each side to minimize surface DNA contamination. Bone powder was then produced using a sterile dentistry drill. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11357-9 We searched for 1st, 2nd, and 3rd degree relative pairs in the dataset by analyzing patterns of allele sharing between pairs of individuals (we found none)10. To identify Y-chromosome haplogroups in genetically male individuals, we used a modiﬁed version of the procedure reported in Poznik, et al.31, which performs a breadth-ﬁrst search of the Y-chromosome tree. We made Y chromosome haplogroup calls using the ISOGG tree from 04.01.2016 [http://isogg. org], and recorded the derived and ancestral allele calls for each informative position on the tree. We counted the number of mismatches in the observed derived alleles on each branch of the tree and used this information to assign a score to each haplogroup, accounting for damage by down-weighting derived mutations that are the result of transitions to 1/3 of that of transversions. We assigned the closest matching Y-chromosome reference haplogroup to each male based on this score (Supplementary Data 6). We caution that males with fewer than 100,000 SNPs have too little data to conﬁdently assign a haplogroup. In some instances, collagen samples were too poorly preserved and were pre- treated at Penn State using a modiﬁed XAD process72 (Supplementary Data 4 shows that there were no systematic differences in the dates obtained based on the XAD and modiﬁed Longin pretreatment extraction methods.) Samples were demineralized in 0.5 N HCl for 2–3 days at 5 °C. The demineralized collagen pseudomorph was gelatinized at 60 °C in 1–2 mL 0.01 N HCl for 8–10 h. The gelatin was then lyophilized and percent gelatinization and yield determined by weight. The sample gelatin was then hydrolyzed in 2 mL 6 N HCl for 24 h at 110 °C. Supelco ENVI-Chrom® SPE (Solid Phase Extraction; Sigma-Aldrich) columns were prepped with 2 washes of methanol (2 mL) and rinsed with 10 mL DI H2O. Supelco ENVIChrom® SPE (Solid Phase Extraction; Sigma-Aldrich) columns with 0.45 µm Millex Durapore ﬁlters attached were equilibrated with 50 mL 6 N HCl and the washings discarded. 2 mL collagen hydrolyzate as HCl was pipetted onto the SPE column and driven with an additional 10 mL 6 N HCl dropwise with the syringe into a 20 mm culture tube. The hydrolyzate was ﬁnally dried into a viscous syrup by passing UHP N2 gas over the sample heated at 50 °C for ~12 h. Population genetic analyses. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11357-9 (3) We used ANGSD29 to determine the degree of heterogeneity on the X- chromosome in males (who should only have one X chromosome) and excluded from analysis individuals with contamination rates greater than 1.5%. AMS radiocarbon dating. We subjected bone powder from 37 samples to radiocarbon dating. We dated the remaining bone powder (360–750 mg) from the same samples that were processed for ancient DNA. We were unable to generate a radiocarbon date for individual I3401, as there was not enough remaining bone powder for analysis. AMS radiocarbon dating. We subjected bone powder from 37 samples to radiocarbon dating. We dated the remaining bone powder (360–750 mg) from the same samples that were processed for ancient DNA. We were unable to generate a radiocarbon date for individual I3401, as there was not enough remaining bone powder for analysis. chromosome in males (who should only have one X chromosome) and excluded from analysis individuals with contamination rates greater than 1.5%. y g We determined the mitochondrial haplogroup of each individual in two ways. For individuals with whole mitochondrial genome data, we determined the mitochondrial haplogroups using haplogrep230. We also determined mitochondrial haplogroups from mitochondrial DNA genotyping using multiplex PCR (see Supplementary Note 1). At the Pennsylvania State University AMS radiocarbon dating facility, bone collagen for 14C and stable isotope analyses was extracted and puriﬁed using a modiﬁed Longin method with ultraﬁltration70. Samples (200–400 mg) were demineralized for 24–36 h in 0.5 N HCl at 5 °C followed by a brief (<1 h) alkali bath in 0.1 N NaOH at room temperature to remove humates. The residue was rinsed to neutrality in multiple changes of Nanopure H2O, and then gelatinized for 12 h at 60 °C in 0.01 N HCl. The resulting gelatin was lyophilized and weighed to determine percent yield as a ﬁrst evaluation of the degree of bone collagen preservation. Rehydrated gelatin solution was pipetted into pre-cleaned Centriprep71 ultraﬁlters (retaining >30 kDa molecular weight gelatin) and centrifuged 3 times for 20 min, diluted with Nanopure H2O and centrifuged 3 more times for 20 min to desalt the solution. We determined the genetic sex of the individuals by computing the ratio of the number of sequences that align to the X chromosome versus the Y chromosome. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11357-9 We include an additional 5–15 populations of either South Asian, West Eurasian, or Southeast/ East Asian ancestry in tests involving Roopkund_A, Roopkund_B and Roopkund_C respectively, chosen to provide additional resolution for each group based on their position in the previous PCA. Based on the observed genetic heterogeneity in the Roopkund_A population, we modeled each individual separately (Supplementary Note 6). For each test, the Left population set included the Roopkund population or individual of interest in addition to one of the selected present-day analysis populations, while the remaining populations were included in the Right population set. In the case of individuals belonging to the Roopkund_A and Roopkund_C groups, we also used qpAdm7, with default parameters and allsnps: YES, to determine whether these populations could be considered to be the product of a two-way admixture between any of the selected present-day populations (Supplementary Note 6). In this case, the Left population set included the Roopkund individual of interest in addition to all possible combinations of two of the selected present-day analysis populations, while the remaining populations were included in the Right population set. Bioinformatic processing. We used SeqPrep to trim adapters and molecular barcodes, and then merged paired-end reads that overlapped by a minimum of 15 base pairs (with up to one mismatch allowed) and aligned to the mitochondrial rsrs genome27 (for the mitochondrial screening analysis) or hg19 (for whole-genome analysis) using samse in bwa (v0.6.1)28. We identiﬁed duplicate sequences based on having the same start position, end position, orientation, and library-speciﬁc barcode, and only retained the copy with the highest quality sequence. We restricted to sequences with a minimum mapping quality (MAPQ ≥10) and minimum base quality (≥20) after excluding two bases from each end of the sequence. We obtained pseudo-haploid SNP calls by using a single randomly chosen sequence at SNPs covered by at least one sequence. We subjected the resulting data to three tests of ancient DNA authenticity: (1) we analyzed the mitochondrial genome data to determine the rate of matching to the consensus sequence using contamMix, and excluded from analysis samples that exhibited a match rate less than 97%8. (2) We removed samples that exhibited a rate of C-to-T substitutions less than 3%: the minimum recommended threshold for authentic ancient DNA that has been subjected to partial UDG treatment25. Methods h The genetic analysis of Himalayan populations (described in Supplementary Note 7) was approved by the Institutional Ethical Committee of the Centre for Cellular and Molecular Biology in Hyderabad, India. 7 NATURE COMMUNICATIONS \| (2019) 10:3670 \| https://doi.org/10.1038/s41467-019-11357-9 \| www.nature.com/naturecommunications ARTICLE Collagen turnover in the adult femoral mid‐shaft: Modeled from anthropogenic radiocarbon tracer measurements. Am. J. Phys. Anthropol. 133, 808–816 (2007). Stable isotope measurements. The isotopic measurement procedure at Yale University for the 37 samples for which we performed direct radiocarbon dating are described in the previous section. Stable isotope measurements. The isotopic measurement procedure at Yale University for the 37 samples for which we performed direct radiocarbon dating are described in the previous section. 18. Moorjani, P. et al. Genetic evidence for recent population mixture in India. Am. J. Hum. Genet. 93, 422–438 (2013). We also obtained isotopic measurements for long bone samples from 19 individuals (including data from 11 of the same individuals that were also analyzed at Yale) at the Max Planck Institute for the Science of Human History. Bone samples of 1 g were subsequently cleaned using an air abrasive system with 5 μm aluminum oxide powder and then crushed into chunks. Collagen was extracted following standard procedures78. Approximately 1 g of pre-cleaned bone was demineralized in 10 mL aliquots of 0.5 M HCl at 4 °C, with changes of acid until CO2 stopped evolving. The residue was then rinsed three times in deionized water before being gelatinized in pH 3 HCl at 80 °C for 48 h. The resulting solution was ﬁltered, with the supernatant then freeze-dried over a period of 24 h. 19. Reich, D. et al. Reconstructing native American population history. Nature 488, 370–374 (2012). 20. Sax, W. 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Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. 72. Lohse, J. C., Culleton, B. J., Black, S. L. & Kennett, D. J. 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https://openalex.org/W2176839263	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0136577&type=printable	English	null	Two Dimensional Yau-Hausdorff Distance with Applications on Comparison of DNA and Protein Sequences	PloS one	2,015	cc-by	6,965	RESEARCH ARTICLE OPEN ACCESS Citation: Tian K, Yang X, Kong Q, Yin C, He RL, Yau SS-T (2015) Two Dimensional Yau-Hausdorff Distance with Applications on Comparison of DNA and Protein Sequences. PLoS ONE 10(9): e0136577. doi:10.1371/journal.pone.0136577 Editor: Yang Zhang, University of Michigan, UNITED STATES Received: March 18, 2015 Accepted: August 5, 2015 Published: September 18, 2015 Copyright: © 2015 Tian et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by National Nature Science Foundation of China (Grant No. 31271408), Tsinghua University startup fund and Tsinghua University Independent Research Project grant. Kun Tian1, Xiaoqian Yang1, Qin Kong1, Changchuan Yin2, Rong L. He3, Stephen S.- T. Yau1* 1 Department of Mathematical Sciences, Tsinghua University, Beijing 100084, China, 2 Department of Mathematics, Statistics and Computer Science, The University of Illinois at Chicago, Chicago, IL 60607- 7045, United States of America, 3 Department of Biological Sciences, Chicago State University, Chicago, IL 60628, United States of America * yau@uic.edu a1111 Abstract Comparing DNA or protein sequences plays an important role in the functional analysis of genomes. Despite many methods available for sequences comparison, few methods retain the information content of sequences. We propose a new approach, the Yau-Hausdorff method, which considers all translations and rotations when seeking the best match of graphical curves of DNA or protein sequences. The complexity of this method is lower than that of any other two dimensional minimum Hausdorff algorithm. The Yau-Hausdorff method can be used for measuring the similarity of DNA sequences based on two important tools: the Yau-Hausdorff distance and graphical representation of DNA sequences. The graphical representations of DNA sequences conserve all sequence information and the Yau-Haus- dorff distance is mathematically proved as a true metric. Therefore, the proposed distance can preciously measure the similarity of DNA sequences. The phylogenetic analyses of DNA sequences by the Yau-Hausdorff distance show the accuracy and stability of our approach in similarity comparison of DNA or protein sequences. This study demonstrates that Yau-Hausdorff distance is a natural metric for DNA and protein sequences with high level of stability. The approach can be also applied to similarity analysis of protein sequences by graphic representations, as well as general two dimensional shape matching. Two Dimensional Yau-Hausdorff Distance with Applications on Comparison of DNA and Protein Sequences Kun Tian1, Xiaoqian Yang1, Qin Kong1, Changchuan Yin2, Rong L. He3, Stephen S.- T. Yau1* Two Dimensional Yau-Hausdorff Distance sequence alignment uses dynamic programming techniques to identify the globally optimal alignment solution, and is the most popular method in sequence comparison. However, the sequence alignment problem is NP-hard, making it infeasible for studying large data-sets. The moment vector approach characterizes the DNA space by assigning each DNA sequence a vector consisting of moments obtained from its graphical curve. The distance between sequences is then defined to be the Euclidean distance between their corresponding vectors. This approach is effective and operates in linear time. There is no criterion yet to determine the dimension of the moment vector, and the method does not present the DNA or protein space accurately, as we will show in this paper. On the other hand, it is obvious that the corre- spondence between feature vectors and DNA sequences is not one-to-one. Thus, the feature vector method is not reliable due to loss of information about nucleotide. New methods on sequence comparisons are being continuously developed. For example, Liu et al developed the Python package for generating various modes of feature vectors for sequences [4]. This method depends on fifteen types of feature vectors of sequence, which can be extremely large for computing DNA sequences of long lengths. Zou et al proposed the centre star MSA strat- egy for sequence alignment [5]. It offers new tools to address large-scale data for multiple sequence alignment. In this article, we establish a new approach to measure the distance between DNA (or pro- tein) sequences: the Yau-Hausdorff method. This study arises from the graphical representa- tion of DNA or protein sequences proposed by Yau [2, 6], in which each DNA or protein sequence is represented by a curve in two-dimensional plane. The graphical representation method results in one-to-one mapping between DNA sequences and the graphical curves. However the question on how to measure the true distance between two DNA curves has not been addressed up to now. The main contribution of this study is to introduce a new distance between two dimensional curves defined by the DNA (or protein) sequences. Although many techniques for two dimensional distance are available, presently the most useful criterion to measure the similarity between two-dimensional point sets is the Hausdorff distance [7, 8]. This distance can be used to determine the degree of resemblance between two point sets that are superimposed. PLOS ONE \| DOI:10.1371/journal.pone.0136577 September 18, 2015 Introduction Comparison of DNA sequences or protein sequences is a problem that has been studied in biological sciences for years. It is an important mean to understand the nature of known pro- teins and predict the unknown functions of the sequences. Many approaches have been pro- posed for measuring the similarity between DNA sequences and protein sequences, including multiple sequence alignment [1], moment vectors [2] and feature vectors [3]. Multiple Competing Interests: The authors have declared that no competing interests exist. 1 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0136577 September 18, 2015 Graphical representation of DNA sequences Yau proposed a unique method to represent DNA sequences by a two dimensional graph [6]. Compared with previous methods, the graphical representation resolves sequence degeneracy and is proven to eliminate circuit formation. We use Yau’s method to generate graphical repre- sentations of DNA sequences. We chose following four vectors to represent the four nucleo- tides A, G, C and T respectively: (1,2/3) ! T, (1,1/3) ! A, (1, −1/3) ! C, (1, −2/3) ! G. An illustrative example is given in Fig 1 for the graphic representation of the first 500 bp human mtochondrial DNA sequence. However the general Hausdorff distance does not satisfy our requirements, since we wish to measure the minimum distance between two point sets under rigid motions including translation and rotation. The minimum Hausdorff distance under rigid motions is a well-defined metric and not only measure the distance of two point sets, but also the similarity of their shapes. Mathematicians have tried to find efficient algorithms to compute this distance, but none of the existing algorithms reaches the level of efficiency required for analyzing long DNA (or protein) sequences. In this article, we define the Yau-Hausdorff distance, a new metric which measures the simi- larity between two-dimensional point sets. This new metric possesses some advantages: it is a well-defined metric in mathematics; it is a natural generalization of the minimum one-dimen- sional Hausdorff distance; it takes translation and rotation into full consideration; and it is much more efficient to compute than the existing two-dimensional minimum Hausdorff dis- tance. These advantages enable it to be a powerful tool for comparing two-dimensional point sets, particularly for comparing DNA or protein sequences. In the first section, we introduce two important methods: the Yau-Hausdorff distance and the graphical representation of DNA (or protein) sequences. In the second section, results from applying the Yau-Hausdorff method to several biological examples are presented and compared with results achieved by previous approaches. In the third section we discuss the advantages of the Yau-Hausdorff method and its broader applications. 2 / 19 Two Dimensional Yau-Hausdorff Distance Two Dimensional Yau-Hausdorff Distance The construction procedure for the graphical representation is natural in that it only collects information based on the hydrophobicity scale of the 20 amino acids and the protein sequence, and the information is then transformed into an intuitive two-dimensional graph, which natu- rally reflects the biological characteristics of a protein. Now that we have the graphical representation of DNA(or protein) sequences, all we need is a criterion that measures the similarity between two curves to characterize similarity of sequences. Huang proposed an approach using the feature vector as the numerical characteri- zation of a DNA sequence [3]. The feature vector consists of a 10-dimensional vector formed from the tallest peak, the lowest point, and the central points of the graphical representation of a DNA sequence. However, the feature vector may not preserve the complete information in a graphical representation because the feature vector does not contain enough information to reconstruct the curve, and thus cannot fully represent the distribution of nucleotides in a DNA sequence. Furthermore, translations or rotations of the curves are not considered in the method, so the feature vector approach may be unreliable in DNA sequence comparison. Our approach differs from the feature vector method in that we compare DNA or protein sequences by measuring the similarity between graphical representations directly, so our approach does not lose information within DNA or protein sequences. In addition, we take translation and rotation into account when making comparisons. To accomplish this, we pro- pose a new criterion for two-dimensional point set comparison, the Yau-Hausdorff distance. PLOS ONE \| DOI:10.1371/journal.pone.0136577 September 18, 2015 Graphical representation of protein sequences We use the approach proposed by Yau in 2008 [2] to generate graphical representations of pro- tein sequences. Here is a brief description of the process. A protein sequence is a string composed of 20 fundamental amino acids. Fauchere and Pliska assigned a value to each of these 20 amino acids according to its hydrophobicity [9], and Yau [2] mapped each value to a number between -1 and 1, such that the 20 numbers are uni- formly distributed on the positive and the negative axes respectively. Having determined the correspondence between each amino acid i and a real number yi 2 [−1,1], a vector can be defined with the horizontal component 1 and the vertical component yi. Given a protein sequence, its graphical representation is the collection of the corresponding vectors of the amino acids in the protein sequence, i.e., a point set with the size l+1, where l is the sequence length. Fig 1. Graphical representation of human mitochondrial DNA (1–500 bp, GenBank:X93334). doi:10.1371/journal.pone.0136577.g001 Fig 1. Graphical representation of human mitochondrial DNA (1–500 bp, GenBank:X93334). doi:10.1371/journal.pone.0136577.g001 doi:10.1371/journal.pone.0136577.g001 3 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0136577 September 18, 2015 Minimum two-dimensional Hausdorff distance We first introduce Hausdorff distance, one of the most widely used criteria for point set com- parisons [7]. For two point sets A and B, the Hausdorff distance between point A and point B sets is defined by ð1Þ hðA; BÞ ¼ max f max a2A min b2B ja bj; max b2B min a2A jb ajg ð1Þ Intuitively, this can be considered as the minimum distance for which at least one point in set B is accessible from any point of set A, and vice versa. Intuitively, this can be considered as the minimum distance for which at least one point in set B is accessible from any point of set A, and vice versa. When comparing graphical representations of DNA or protein sequences, we emphasize the level of shape similarity. Thus, the ideal metric should consider the optimal fit under rigid motions including translation and rotation. Since the general Hausdorff distance measures the distance between two fixed sets, it shall not be a good candidate for an ideal metric although the general Hausdorff distance is a defined metric. The minimum two-dimensional Hausdorff distance as defined below is a well-defined met- ric that indeed meets this requirement. H2ðA; BÞ ¼ min y2½0;2p min t2R2 hðA þ t; ByÞ ð2Þ ð2Þ where h is Hausdorff distance deﬁned in Eq (1) and h(A+t,Bθ) stands for the Hausdorff dis- tance between A and B after shifting A rightward by t and rotating B counterclockwise by θ. The minimum two-dimensional Hausdorff distance is widely used in graph comparison, and several algorithms have been proposed for this central distance. Some of the algorithms rely on the assumption that there are only grid points in the point sets. These algorithms are mainly used in pixel image matching such as photo identification and MRI analysis. But as comparing graphical representations of sequences requires precise rotation of each point, clearly the grid point assumption is not satisfied. The best matching of two shapes under translation and rota- tion can be obtained by the minimum Hausdorff distance. For two point sets with size m and n, the time complexity of the minimum Hausdorff distance by the Huttenlocher algorithm is O((m PLOS ONE \| DOI:10.1371/journal.pone.0136577 September 18, 2015 4 / 19 Two Dimensional Yau-Hausdorff Distance +n)6log(mn)) [10]. That algorithm for minimum Hausdorff distance under Euclidean motion was improved later with the complexity as O((m+n)5log2 mn)) [8]. Minimum two-dimensional Hausdorff distance However, these algorithms are still not feasible for comparing graphic curves of long DNA (or protein) sequences of more than 10000 bp. These limitations highlight the need to improve the algorithm of minimum Hausdorff distance. In this study, we present a new metric, the Yau-Hausdorff distance, for matching two-dimensional curves under translation and rotation. Yau-Hausdorff distance We propose here the Yau-Hausdorff distance in terms of the minimum one-dimensional Hausdorff distance [11]. The minimum Hausdorff distance between two one-dimensional point sets A and B under translation is defined as H1ðA; BÞ ¼ min t2R hðA þ t; BÞ ð3Þ ð3Þ where h(A+t,B) is the Hausdorff distance between A and B after shifting A rightward by t. This equation can be rewritten as H1ðA; BÞ ¼ min t2R max f max a2Aþt min b2B ja bj; max b2B min a2Aþt jb ajg ð4Þ ð4Þ The Yau-Hausdorff distance is then defined in terms of H1(A,B): The Yau-Hausdorff distance is then defined in terms of H1(A,B): DðA; BÞ ¼ max f max y min φ H1ðPxðAyÞ; PxðBφÞÞ; max φ min y H1ðPxðAyÞ; PxðBφÞÞg ð5Þ ð5Þ where Px(Aθ) is an one-dimensional point set representing the projection of A on the x-axis after being rotated counterclockwise by θ. The Yau-Hausdorff distance D defined above possesses the following properties: The Yau-Hausdorff distance D defined above possesses the following properties: 1. D can be proven as an metric (the proof is available in the supplementary materials). 2. D is defined in terms of and inherits properties from the minimum one-dimensional Haus- dorff distance. It is so far the most accurate criterion for two-dimensional point set comparison. 3. D fully considers all translation and rotation in the two-dimensional space. 4. Using the projection of two-dimensional point sets, D successfully avoids calculation of the Hausdorff distance of two-dimensional sets, and can be computed efficiently. The Yau-Hausdorf distance is not equal to the two-dimensional minimum Hausdorff dis- tances. In fact, the Yau-Hausdorf distance is the lower bound of the minimum two-dimen- sional Hausdorff distances. The proof that H2(A,B) D(A,B) and an example showing this inequality are provided in the supplementary materials. Our algorithm to compute the Yau-Hausdorff distance D is as follows: 3 3 Let A = {a1, a2, . . ., an} ⊂R3, B = {b1, b2, . . ., bm} ⊂R3. Let A = {a1, a2, . . ., an} ⊂R3, B = {b1, b2, . . ., bm} ⊂R3. 1. Fix a1. For i = 2, 3, . . ., n rotate A such that a1 ai//x−axis. We get θ1, θ2, . . ., θn−1. 2. Fix a2. For i = 3, 4, . . ., n rotate A such that a2 ai//x−axis. We get θn, θn+1, . . ., θ2n−3. PLOS ONE \| DOI:10.1371/journal.pone.0136577 September 18, 2015 Similarity analysis of DNA or protein sequences by the Yau-Hausdorff distance Using the Yau-Hausdorff distance of graphic curves, similarity analysis of DNA or protein sequences is performed by clustering the sequences into phylogenetic trees. The dissimilarity matrix of given sequences is constructed from the Yau-Hausdorff distance of pairwise sequences. UPGMA (Unweighted Pair Group Method with Arithmetic Mean) hierarchical clustering method from a pairwise distance matrix is used to construct phylogenetic trees [13]. The resulting UPGMA tree reflects the structure and relationship of the sequences presented in the distance matrix. Yau-Hausdorff distance 3. Fix a3. For i = 4, 5, . . ., n rotate A such that a3 ai//x−axis. We get θ2n−2, θ2n−1, . . ., θ3n−6 4. Randomly rotate 1000 times by y0 1; y0 2; :::; y0 1000. 4. Randomly rotate 1000 times by y0 1; y0 2; :::; y0 1000. 5. Let the set of these rotations be M ¼ fy1; y2; :::; y3n6; y0 1; y0 2; :::; y0 1000g. PLOS ONE \| DOI:10.1371/journal.pone.0136577 September 18, 2015 5 / 19 Two Dimensional Yau-Hausdorff Distance 6. Similarly we get the set of rotations for B N ¼ fφ1; φ2; :::; φ3m6; φ0 1; φ0 2; :::; φ0 1000g 7. For each θ 2 M, we compute minφ 2 N H1(Px(Aθ),Px(Bφ)). 8. For all θ 2 M, we get D1 = maxθ 2 Mminφ 2 N H1(Px(Aθ),Px(Bφ)). 9. Similarly we get D2 = maxφ 2 N minθ 2 M H1(Px(Aθ),Px(Bφ)). 10. Take D(A,B) = max{D1,D2}. It shall be noted that the algorithms for calculating the one-dimensional minimum Hausdorff distance include the algorithm proposed by G.Rote in 1991 [11] and the improved algorithm proposed by Li [12]. Because of the improved efficiency in Li’s algorithm, we chose the Li’s algorithm for computing the Yau-Hausdorff distance. PLOS ONE \| DOI:10.1371/journal.pone.0136577 September 18, 2015 DNA sequence comparison We apply the Yau-Hausdorff method by comparing the DNA sequences of the COI genes, bar- coding, H1N1, and the Influenza virus neuraminidase (NA) genes to verify the accuracy of our method on its ability to cluster genomes. GenBank access numbers of DNA and protein sequences used in this study are listed in S2 File. COI dataset analysis. Paul D. N. Hebert [14] claimed that the mitochondrial gene cyto- chrome c oxidase I (COI) can serve as the core of a global bioidentification system for animals [14]. First we applied our method on the COI gene of nine species, including one spider and eight raptor. The average length of COI gene sequences is about 700 bp. These nine sequences are transferred to graphical representations using Yau’s method [6], then the Yau-Hausdorff distance between each two graphs is computed. We get the distance matrix and generated the corresponding hierarchical tree. We also use the natural vector method [15] to test the result. The distance provided by natural vector method is Euclidean distance of the vectors presented by DNA sequences in 12-dimensional space R12, while Yau-Hausdorff method is based on cal- culating the minimum Hausdorff distance of point sets coming from the graphical representa- tion of sequences. The clustering results of both the Yau-Hausdorff method and the natural vector method are compared as shown in Fig 2. Callobius bennetti is a spider which should be separated from those eight raptor. Fig 2 dem- onstrates that both methods successfully cluster Callobius bennetti outside the cluster of raptor. In addition, the Yau-Hausdorff method clusters Accipiter francesiae and Accipiter gularis closer than the natural vector method. According to biological classification, Accipiter france- siae and Accipiter gularis belong to the same genus Accipiter. Therefore, it is reasonable that these two species are closer to each other than other species. In this case, Yau-Hausdorff method is more reliable than the natural vector method. 6 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0136577 September 18, 2015 Two Dimensional Yau-Hausdorff Distance Fig 2. Hierarchical tree of COI sequences (Yau-Hausdorff method and natural vector method). doi:10.1371/journal.pone.0136577.g002 Fig 2. Hierarchical tree of COI sequences (Yau-Hausdorff method and natural vector method). doi:10.1371/journal.pone.0136577.g002 Barcoding DNA analysis. DNA sequence comparison To compare the clustering accuracy by the Yau-Hausdorff method and the feature vector method [3], we construct the UPGMA phylogentic trees of 18 species barcoding DNA data sets using the Yau-Hausdorff method and the feature vector method, the results are shown in Figs 3 and 4, respectively. Fig 3 manifests that the species with the same genus are grouped together accurately by Yau-Hausdorff method. The result is con- sistent with the known biological classification, by which the 18 species belong to 9 genera and 7 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0136577 September 18, 2015 Two Dimensional Yau-Hausdorff Distance Fig 3. Hierarchical tree of barcoding DNA sequences (Yau-Hausdorff method). d i 10 1371/j l 0136577 003 Fig 3. Hierarchical tree of barcoding DNA sequences (Yau-Hausdorff method). Fig 3. Hierarchical tree of barcoding DNA sequences (Yau-Hausdorff method). doi:10.1371/journal.pone.0136577.g003 Fig 3. Hierarchical tree of barcoding DNA sequences (Yau-Hausdorff method). d i 10 1371/j l 0136577 003 doi:10.1371/journal.pone.0136577.g003 doi:10.1371/journal.pone.0136577.g003 each genus contains two species. The hierarchical tree in Fig 4 shows that Eunicida sp BOL- DACJ5892 is gathered with genus Capitellida instead of Eunicida sp BOLDACJ9615 although they belong to the same genus, while our method clearly clusters the 18 species into 9 genera. each genus contains two species. The hierarchical tree in Fig 4 shows that Eunicida sp BOL- DACJ5892 is gathered with genus Capitellida instead of Eunicida sp BOLDACJ9615 although they belong to the same genus, while our method clearly clusters the 18 species into 9 genera. each genus contains two species. The hierarchical tree in Fig 4 shows that Eunicida sp BOL- DACJ5892 is gathered with genus Capitellida instead of Eunicida sp BOLDACJ9615 although they belong to the same genus, while our method clearly clusters the 18 species into 9 genera. The results acquired from Yau-Hausdorff method is better than that from feature vector method. The feature vector does not preserve the information contained in a graphical repre- sentation because the feature vector does not contain enough information to reconstruct the curve from the vector, and thus cannot fully represent the distribution of nucleotides in a DNA sequence. Furthermore, our method includes translations and rotations for the best match of graphical curves. Thus our method offers a natural and accurate comparison of DNA sequences through graphical representations. The results acquired from Yau-Hausdorff method is better than that from feature vector method. PLOS ONE \| DOI:10.1371/journal.pone.0136577 September 18, 2015 DNA sequence comparison The feature vector does not preserve the information contained in a graphical repre- sentation because the feature vector does not contain enough information to reconstruct the curve from the vector, and thus cannot fully represent the distribution of nucleotides in a DNA sequence. Furthermore, our method includes translations and rotations for the best match of graphical curves. Thus our method offers a natural and accurate comparison of DNA sequences through graphical representations. H1N1 virus analysis. We perform test on the Yau-Hausdorff method on H1N1 virus. The pandemic in 2009 was a new strain of swine-origin influenza virus(S-OIV). We analyze the polymerase PB2 segment of S-OIV (swine-origin influenza virus) 2009 as well as avian and tri- ple reassortment swine viruses. Many researchers have conducted comprehensive computa- tional searches to determine the origin of S-OIV. Previous study [16] indicated that the PB2 segment of S-OIV, avian, and triple reassortment swine viruses share a similar evolutionary history. We construct the phylogentic tree from the Yau-Hausdorff distance between the 63 virus genes (Fig 5). The hierarchical tree in Fig 5 contains four branches. The third and the fourth clusters are the nearest, and their union is juxtaposed with the second cluster, while the first cluster is the farthest from the other three. What we examine here is S-OIV 2009 PB2 genes, PLOS ONE \| DOI:10.1371/journal.pone.0136577 September 18, 2015 8 / 19 Two Dimensional Yau-Hausdorff Distance Fig 4. Hierarchical tree of barcoding DNA sequences (Feature vector method). doi:10 1371/journal pone 0136577 g004 Fig 4. Hierarchical tree of barcoding DNA sequences (Feature vector method). doi:10.1371/journal.pone.0136577.g004 which are all placed into the third cluster. On the other hand, the third group contains some Swine H3N2 and H1N2 triple reassortment viruses. These clustering results agree with previ- ous study by Kingsford [16]. The fourth group is the closest cluster to the third group. This group consists of human H1N1, triple reassortment H3N2, H2N1, chicken, and turkey virus. It was pointed out by Kingsford’s paper [16] that the PB2 gene of avian virus, reassortment H2N1, H3N2 and the S-OIV 2009 share evolutionary. The farthest group from S-OIV 2009 is the first group. This group is composed mainly of H1N1 that has been circulating in swine populations in Europe and Asia for decades. Again, these results coincide with Kingsford’s con- clusion [16] which claimed that the Eurasian H1N1 has a different phylogenetic origin than the 2009 outbreak strain sequences. PLOS ONE \| DOI:10.1371/journal.pone.0136577 September 18, 2015 PLOS ONE \| DOI:10.1371/journal.pone.0136577 September 18, 2015 PLOS ONE \| DOI:10.1371/journal.pone.0136577 September 18, 2015 Protein sequence comparison To assess our method on characterization of the protein space, we apply the Yau-Hausdorff method to classify the protein kinase C (PKC) family and the β-globin family. In addition to hierarchical tree, we employ the natural graph to represent the classifications of protein families [17]. In the natural graph, if there is a level-1 directed edge from protein A to protein B, it means B is the closest to A among the entire family, thus they are categorized into the same level-1 cluster. Similarly a level-2 directed edge means that one level-1 cluster is the closest to another level-1 cluster. The distance between two graphs is defined as the mini- mum distance between any protein in one graph and any element in the other. The length of an edge that connects two proteins is proportional to the Yau-Hausdorff distance between them. A shorter line indicates a higher level of similarity between two proteins. The natural graph allows us to view the relationship between proteins and subfamilies in an intuitive way. PKC family analysis. The protein kinase C family is a large group of enzymes regulating the Ca2+-dependent pathways in cells [18]. PKC is classified into six subfamilies: cPKC, nPKC, aPKC, PKCμ, PKC1 and PRK. We compute distance matrix from the Yau-Hausdorff distance of each pair of the 124 protein sequences in the PKC family. The natural graph constructed from the distance matrix is shown in Fig 7. The graph shows that Yau-Hausdorff method classifies the 124 PKC sequences into three level-3 clusters with one uni-directional edge and one bi-directional edge. The first cluster con- tains all two typical subfamilies: cPKC (conventional PKC) and nPKC (novel PKC). The sec- ond cluster contains all aPKC sequences. One bi-directional edge connects it to the first cluster, indicating that among all PKC subfamilies, aPKC has the highest similarity with nPKC and cPKC. This matches what would be expected from their biology. The third cluster contains all the controversial PKC subfamilies. PKCμ is considered to be PKD actually; PKC1 are found on fungi; PRK (PKC-related kinase)are like PKC1 but they are found on animals [17]. We observe from the graph that this cluster is far from the first two. Actually the graph tells us that the min- imum distance between the aPKC subfamily and the subfamilies cPKC and nPKC is 58.44, which is also the longest edge in the graph. DNA sequence comparison Influenza virus gene analysis. In the last example of DNA data set, we test the influenza virus neuraminidase (NA) gene since sequence alignment does not work well for the influenza virus NA genes. We applied our method to this data set which contains 52 sequences and got the phylogenetic tree in Fig 6. The result obtained here is mainly consistent with the known biological classification. The top of this figure includes some mallard, Zhejiang and winged-teal influenza as a group. In the middle part of this figure, the Illinois influenza are gathered here. The middle and lower parts are mainly composed of H7N9 viruses, and Hong Kong influenza are clustered at the bottom of this figure. Compared with the sequence alignment method which is time consuming and not effective for the influenza virus genes, this example shows that the Yau-Hausdorff distance method works better than sequence alignment method. PLOS ONE \| DOI:10.1371/journal.pone.0136577 September 18, 2015 9 / 19 Two Dimensional Yau-Hausdorff Distance Fig 5. Hierarchical tree of H1N1 virus sequences (Yau-Hausdorff method). doi:10.1371/journal.pone.0136577.g005 10 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0136577 September 18, 2015 Two Dimensional Yau-Hausdorff Distance Fig 6. Hierarchical tree of the influenza virus NA genes(Yau-Hausdorff method). doi:10.1371/journal.pone.0136577.g006 Fig 6. Hierarchical tree of the influenza virus NA genes(Yau-Hausdorff method). doi:10.1371/journal.pone.0136577.g006 11 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0136577 September 18, 2015 Two Dimensional Yau-Hausdorff Distance PLOS ONE \| DOI:10.1371/journal.pone.0136577 September 18, 2015 PLOS ONE \| DOI:10.1371/journal.pone.0136577 September 18, 2015 Protein sequence comparison The Yau-Hausdorff method classifies the PKC family accurately except that PKC No. 84, which is a cPKC, is clustered into the cluster containing all aPKCs. This observation coincides with the result given by the natural vector method on the same dataset [17]. That article ascribes this abnormality yet undiscovered cPKCs that should appear between PKC No.84 and the cPKC subfamily, making PKC No.77 the closest to No.84. Our result obtained from the Yau-Hausdorff method verifies this prediction. Examining the result more carefully we find that our clustering outcome is more accurate than that of the natural vector method. Firstly, in the results of the natural vector method, the nPKC subfamily is divided into two parts by two cPKC sequences, while the Yau-Hausdorff method clusters the PKC family into complete subfamilies. Second, the natural vector method cuts the PRK subfamily into two parts, while all pRKs are clustered into the same cluster, i.e., the same level-1 branch. That shows the Yau-Hausdorff method characterizes the distance between proteins in a way that is closer to the actual nature of the proteins than the natural vec- tor method does, resulting in a more accurate classification. β-globin analysis. We perform phylogenetic analysis of β-globin from 50 species by the Yau-Hausdorff method (Fig 8) and the moment vector method (Fig 9). [2]. The hierarchical tree in Fig 9 shows that Yau-Hausdorff method categorizes the 50 β-globin sequences better than moment vector method. Even though the moment vector method divides the data-set into three complete parts: mammals, birds and fish, it is not as accurate as the Yau- Hausdorff clustering in regard to the classification into subfamilies of mammals. The moment PLOS ONE \| DOI:10.1371/journal.pone.0136577 September 18, 2015 12 / 19 Two Dimensional Yau-Hausdorff Distance Fig 7. Natural graph the PKC family(Yau-Hausdorff method). doi:10.1371/journal.pone.0136577.g007 Fig 7 Natural graph the PKC family(Yau-Hausdorff method) Fig 7. Natural graph the PKC family(Yau-Hausdorff method). doi:10.1371/journal.pone.0136577.g007 PLOS ONE \| DOI:10.1371/journal.pone.0136577 September 18, 2015 13 / 19 Two Dimensional Yau-Hausdorff Distance Fig 8. Hierarchical tree of 50 β-globin sequences (Yau-Hausdorff method). doi:10.1371/journal.pone.0136577.g008 Fig 8. Hierarchical tree of 50 β-globin sequences (Yau-Hausdorff method). Fig 8. Hierarchical tree of 50 β-globin sequences (Yau-Hausdorff method). doi:10.1371/journal.pone.0136577.g008 doi:10.1371/journal.pone.0136577.g008 PLOS ONE \| DOI:10.1371/journal.pone.0136577 September 18, 2015 14 / 19 Two Dimensional Yau-Hausdorff Distance Fig 9. Hierarchical tree of 50 β-globin sequences (Moment vector method). doi:10.1371/journal.pone.0136577.g009 Fig 9. Hierarchical tree of 50 β-globin sequences (Moment vector method). Stability analysis To analyze the stability of the Yau-Hausdorff method, we apply a random perturbation within 10% to the y-coordinates of each nucleotide (amino acid) in the graphical representation of DNA (or protein) sequences. We repeat the tests on both DNA and protein data-sets several times with perturbation, and observe no structural change in the hierarchical tree or the natural graphical representation. This shows that Yau-Hausdorff distance is a natural metric with high level of stability. Protein sequence comparison doi:10.1371/journal.pone.0136577.g009 Fig 9. Hierarchical tree of 50 β-globin sequences (Moment vector method). doi:10.1371/journal.pone.0136577.g009 vector approach leaves the subfamilies bovidae, artiodactyla, perissodactyl, whale and canidae intermingled in the final results, while our method clearly clusters the 29 mammals into ele- phants, bovidae, rodents, artiodactyla, perissodactyl, whales, primates, bears and canidae from top to bottom. This result is in strong agreement with biological systematics, showing that our algorithm accurately clusters the 50 species. We conclude that Yau-Hausdorff method gives a more accurate result than the moment vector method. The only outlier in our result is that Shark is clustered to a single cluster instead of being group with other fish. Looking into the original data we find that the β-globin sequence of shark is the only one in the data-set with length 142, while all the 49 other sequences are of length 148. This is probably the cause of this outlier, since the algorithm generating the hierar- chical tree clusters the closest branches together in each iteration. The β-globin sequence of shark is far from all other 49 β-globin (verified by observing the distance matrix) because it is shorter, and consequently it becomes branch clustered in the last step of the iteration. To cor- rect this outlier, we represent the distance matrix with the natural graph clustering as shown in Fig 10. 15 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0136577 September 18, 2015 Two Dimensional Yau-Hausdorff Distance Fig 10. Natural graph of 50 β-globin sequences(Yau-Hausdorff method). doi:10.1371/journal.pone.0136577.g010 Fig 10. Natural graph of 50 β-globin sequences(Yau-Hausdorff method). doi:10.1371/journal.pone.0136577.g010 Fig 10. Natural graph of 50 β-globin sequences(Yau-Hausdorff method). doi:10.1371/journal.pone.0136577.g010 The natural graph in Fig 10 shows that although the edge from Shark to Catfish is the lon- gest in the directed graph, shark is clustered into fish by a level-1 uni-directional edge, which is what we expect. This example shows that the outlier can be resolved by the natural graph, dem- onstrating advantage of the natural graph and accuracy of the Yau-Hausdorff method. In sum- mary, our method provides a distance matrix that completely agrees with established biological clustering of all 50 species. Noise perturbation analysis We perform the noise perturbation analysis on the Yau-Hausdorff distance. We first construct 150 deletion mutations on random positions of an intron DNA sequence of length 350 bp. The deletion mutation DNA sequences have lengths from 349 bp to 200 bp. The Yau-Hausdorff dis- tances between each of the 150 mutation sequences and the original DNA sequence are shown in Fig 11. The result shows that the correlation of Yau-Hausdorff distance and deletion length is almost linear. This result indicates that the Yau-Hausdorff distance is robust when the complex- ity of the graphic representations increases or noise is introduced in the representations. 16 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0136577 September 18, 2015 Two Dimensional Yau-Hausdorff Distance Fig 11. The relationship between Yau-Hausdorff distance and deletion length of sequence. doi:10.1371/journal.pone.0136577.g011 Fig 11. The relationship between Yau-Hausdorff distance and deletion length of sequence. doi:10.1371/journal.pone.0136577.g011 doi:10.1371/journal.pone.0136577.g011 Complexity analysis Given two sequences with lengths m and n, the computational complexity of the Yau-Haus- dorff distance between these sequence curves is O(m+n) times the minimum one-dimensional Hausdorff distance. For two sets of m and n points, the complexity of their one-dimensional minimum Hausdorff distance is O((m+n) log(m+n)) based on Li’s algorithm [12]. Therefore, the complexity of our algorithm is O((m+n)2 log(m+n)). Existing algorithms which find the minimum Hausdorff distance for point sets under Euclidean motion are highly complex, with O((m+n)5 log2 mn)) for the fastest algorithm [8]. The Yau-Hausdorff distance method signifi- cantly decreases computational complexity of finding the minimum Hausdorff distance between two-dimensional shapes. PLOS ONE \| DOI:10.1371/journal.pone.0136577 September 18, 2015 Conclusion This article proposes a new approach for comparing DNA sequences and protein sequences. We introduce two fundamental tools of our method: the graphical representation of DNA(or protein) sequences and the Yau-Hausdorff distance. We then define the distance between two sequences using the Yau-Hausdorff distance between the two-dimensional graphical represen- tations of the sequences. Given a family of DNA(or protein), we use this approach to calculate the distance between each pair of sequences, getting a distance matrix that contains informa- tion about the family structure. In the tests on both DNA and protein data-sets, we use hierarchical tree and natural graphi- cal representation to analyze the distance matrix. Based on different kinds of datasets, our results show that the Yau-Hausdorff method gives the most accurate clustering compared with several other approaches including natural vector method, feature vector method, moment vector method and sequence alignment method on both DNA and protein families. In addi- tion, we perform our test repeatedly with perturbation. The perturbation does not affect our results. We conclude that the Yau-Hausdorff method is a natural, accurate and stable approach for comparing DNA sequences and protein sequences, and has general applications in shape and image matching. Two Dimensional Yau-Hausdorff Distance The minimum Hausdorff distance of shapes is an important measure for similarity compar- ison and shape matching and retrievals, but computation of the Hausdorff distance in two and higher dimensions is a challenging problem. We propose this novel solution to this problem and prove that Yau-Hausdorff distance in d-dimensional Euclidean space Rd is also a metric for d > 2 (proof available in S1 File). It has potential broad application prospects in measuring the similarity of 2 or 3-dimensional curves, such as shape matching, image retrieval, and com- parison of 3-dimensional protein structures. Although the Yau-Hausdorff method has these advantages, it also has some limitations. The phylogenetic tree constructed by this distance may contain uncertainties. For example, the phy- logenetic tree in Fig 8 constructed by this distance showed that shark is not grouped with other fish. Another problem is that the computation complexity is high if the sequences contain mil- lions of nucleotides. We will continue to solve the limitations and improve the complexity of the algorithm. Author Contributions Conceived and designed the experiments: SSTY. Performed the experiments: KT XY QK CY. Analyzed the data: KT CY XY QK RH SSTY. Contributed reagents/materials/analysis tools: KT XY QK CY. Wrote the paper: KT CY XY QK RH SSTY. Supporting Information S1 File. Proof that Yau-Hausdorff distance is a metric, and is always less than or equal to the minimum two-dimensional Hausdorff distance. S1 File. Proof that Yau-Hausdorff distance is a metric, and is always less than or equal to the minimum two-dimensional Hausdorff distance. (PDF) S2 File. GenBank access numbers of DNA and protein sequences used in this study. (XLSX) Discussion In this study, the Yau-Hausdorff method provides more accurate results compared with the existing methods. Unlike previous methods that attempt to numerically characterize DNA or protein sequence graphical curves, the Yau-Hausdorff method measures the distance between sequences by directly comparing the curves via the Yau-Hausdorff distance. 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W2052718654.txt	https://diglib.uibk.ac.at/download/pdf/3374062.pdf	de		Studien zur Geschichte Kapitalistischer Organisationsformen, Monopole, Kartelle und Aktiengesellschaften im Mittelalter und zu Beginn der Neuzeit, <i>by Jakob Strieder</i>	Political science quarterly	1,918	public-domain	205,667	Universitäts- und Landesbibliothek Tirol Studien zur Geschichte kapitalistischer Organisationsformen Strieder, Jakob München [u.a.], 1925 urn:nbn:at:at-ubi:2-14823 pj {i i- [a ÿ -fâ >* S£-i. K dPH'M U) 0hÂ t fc (M, DicA / paü >j,' ^ c£>< fc4£ Dcu fiai c tw »yt' ►> C' v H itt »^ 1 tr >1 Do \|/ Îô/cwi < vÿ >‘i h -. r ^ r •'' V u itu » ' .Vt v'- ■ Afe^ osstu owM^ S^ ytru ^ z Du . L>(?tl £ÂV<: 10 ^ 5 ^ 0. 3cv , UéVKÊlIt >f\0hi<Vh»ic Hm « A.C 1 HCh \ % LTÏ Studien zur Geschichte kapitalistischer Organisationsformen. Von demselben Verfasser erschienen früher die folgenden wirtschaftsgeschichtlichen Werke: Zur Genesis des modernen Kapitalismus . Forschungen zur Entstehung der großen bürgerlichen Kapitalvermögen zu Ausgang des Mittelalters und zu Beginn der Neuzeit zunächst in Augsburg . Leipzig (Duncker & Humblot) 1904. Die Inventur der Firma Fugger aus dem Jahre 1527. Ergänzungsheft XVII der Zeitschrift für die gesamte Staats¬ wissenschaft , herausgegeben von Karl Bücher . Tübingen (Verlag der Lauppschen Buchhandlung) 1905. Levantinisdie Handelsfahrten deutscher Kaufleute des 16. Jahrhunderts . Meereskunde , Heft 149 . Berlin (E. S. Mittler & Sohn) 1919. Studien zur Geschichte kapitalistischer Org anisationsformen Monopole, Kartelle und Aktiengesellschaften im Mittelalter und zu Beginn der Neuzeit Von Jakob Strieder o. Professor der Wirtschaftsgesdiidite Zweite , Termehrte an der Universität München Auflage / München und Leipzig Verlag von Duncker 1925 & Humblot / /n ?. .c a y Alle Rechte Vorbehalte ». Aloys Schulte in Verehrung und Dankbarkeit. V s Vît Vorwort zur zweiten Auflage. Seit längerer Zeit ist die erste Auflage des hier in zweiter, ver¬ mehrter Auflage vorliegenden Werkes vergriffen. Es war in der Zeit der schweren deutschen Wirtschaftsnot , während der ich an die Vorbereitung der zweiten Auflage ging, nicht denkbar, das ganze Werk neu zu setzen, so daß sich die Möglichkeit geboten hätte , all die größeren und kleineren Nachträge, Erweiterungen und Berich¬ tigungen aufzunehmen, die ich im Laufe der Zeit gesammelt hatte. Ich mußte schon froh sein, wenn sich der Verleger entschloß, von der ersten Auflage einen unveränderten (photo -chemischen) Neudruck herstellen zu lassen und mir für Nachträge bis zu zwei Bogen neuen Satzes konzedierte 1. Wer die Aufnahme verfolgt hat , die die erste Auflage meiner „Studien zur Geschichte kapitalistischer Organisationsformen, Mono¬ pole, Kartelle und Aktiengesellschaften im Mittelalter und zu Be¬ ginn der Neuzeit“ gefunden hat , wird vielleicht die Erwartung hegen, ich würde mich in den Nachträgen besonders mit der Frage der An¬ fänge der Aktiengesellschaften beschäftigen. Hier haben manche Kritiker mir insofern nicht folgen zu können erklärt , als sie die von mir als embryonale Aktiengesellschaften angesprochenen handels¬ gesellschaftlichen Bildungen des Mittelalters und des 16. Jahr¬ hunderts als solche nicht anerkennen wollten. Die naheliegende Frage freilich, unter welche Kategorie der Handelsgesellschafts¬ formen denn sonst die von mir herangezogenen Bildungen (etwa die Steyrer-Allgemeine-Eisenhandelskompagnie) eingereiht werden sollen, haben die Kritiker unbeantwortet gelassen. Es müßte denn sein, daß man sich mit der Erklärung Silberschmidts beruhigt, der die fraglichen Kompagnien als gemischt-wirtschaftliche Unterneh1Dagegen wird meine „ Genesis des modernen Kapitalismus, Forschungen zur Entstehung der großen bürgerlichen Kapitalver¬ mögen zu Ausgang des Mittelalters und zu Beginn der Neuzeit zu¬ nächst in Augsburg“, die ebenfalls seit langem vergriffen ist, in völlig umgearbeiteter Auflage (allerdings erst im Jahre 1926) erscheinen. VIII Vorwort zur zweiten Auflage. mungen charakterisiert und damit die Streitfrage zu lösen glaubt. Nun sind freilich mehrere dieser Gesellschaften auch nebenbei ge¬ mischt -wirtschaftliche Unternehmungen gewesen , so daß für die Geschichte dieser angeblich ganz modernen kapitalistischen Orga¬ nisationsform schönes Material von mir geliefert werden konnte, aber der Kernpunkt des Problems ist damit nicht getroffen , sondern umgangen . Eine gemischt -wirtschaftliche Unternehmung kann ver¬ schiedenen gesellschaftsrechtlichen Charakters sein. Sie kann sich der aktiengesellschaftlichen Form bedienen , braucht es aber nicht zu tun. So einfach liegen also die Dinge hier nicht . Ich denke im Ver¬ lauf der nächsten Jahre die Streitfrage in einem besonderen Werke, „Die Entstehung der Aktiengesellschaft “ betitelt , von neuem aufzurollen . Es gilt dabei besonders noch eine Anzahl von Kom¬ pagnien in mühsamer , archivalischer Arbeit zu untersuchen , die einen gewissen öffentlich -rechtlichen Einschlag haben , und die jenen Kom¬ pagnien ähnlich organisiert zu sein scheinen , die ich als embryonale Aktiengesellschaften angesprochen habe . (Vgl. unten S. 140 f.) Es gilt ferner auch eine Reihe englischer und anderer überseeischer Kompagnien zu untersuchen , die schon im 16. Jahrhundert ent¬ standen sind , also vor der holländisch -ostindischen Kompagnie , von der ab Karl Lehmann erst die Entwicklung der Aktiengesellschaft datieren will. In dem mir für diese zweite Auflage zur Verfügung stehenden Raum gedenke ich nicht auf das Problem der Entstehung der Aktien¬ gesellschaft einzugehen . Hier sollen vielmehr folgende Erweite¬ rungen zur Geschichte der Kartelle und Monopole des 16. Jahr¬ hunderts geboten werden : Ein erster Abschnitt handelt über das bisher unbekannte Kupfersyndikat der Firmen Fugger und Manlich, geschlossen im Jahre 1548 ; hierbei werden auch ein paar Ergän¬ zungen zum Kartell der Fugger und Höchstetter vom Jahre 1515 gebracht . In einem zweiten Abschnitt wird die Stellung der Fugger zu dem großen mitteleuropäischen Zinnmonopolisierungsprojekt in der Mitte des 16. Jahrhunderts eingehend dargestellt . Die führende Teilnahme Anton Fuggers an diesem Spekulationsunternehmen — einem der kapitalgewaltigsten des ganzen Zeitalters — konnte über die Vermutungen in der ersten Auflage hinaus genau festgelegt werden . In dem dritten Abschnitt der Nachträge endlich ist einiges Neue zur Geschichte des Idrianer Quecksilbermonopols geboten. Vorwort zur zweiten Auflage. IX So darf ich denn diese zweite Auflage mit dem Gefühl einer kleinen inneren Befriedigung hinausgehen lassen, daß sie — ob.wohl in der Hauptsache nur ein unveränderter photo-chemischer Neu¬ druck der ersten — doch über diese hinaus einen Fortschritt unseres wirtschaftsgeschichtlichen Wissens vom 16. Jahrhundert bedeutet. Auch ohne diese Ergänzungen freilich hätte sich vielleicht gerade in unserer Zeit eine Neuauflage meiner „ Studien zur Geschichte kapitalistischer Organisationsformen“ empfohlen. Ein guter Teil des Werkes ist der Geschichte des heute so aktuellen Monopol- und Kartellwesens gewidmet. Dabei stellte ich das Verhältnis von Staat und Gesellschaft zu diesen werdenden Kristallisationen moderner Wirtschaftsgesinnung in den Vordergrund meiner Betrachtungen und Untersuchungen. „ Staat und Wirtschaft “ ist ja auch heule wieder ein Problem, das alle wirtschaftspolitisch Denkenden inter¬ essiert. Ich meine für diejenigen, die heute ein Kartellgesetz for¬ dern, um damit dem Staat wiederum die Führung unserer wirt¬ schaftlichen Politik zu sichern, wird ebenso wie für diejenigen, die es ablehnen, die Erinnerung daran von Wichtigkeit sein, wie im 16. Jahrhundert staatliche Finanznot und finanzielle Abhängig¬ keit des Fürstentums vom Großkaufmann einer starken gesellschaft¬ lichen Antimonopolbewegung die Kraft des Sieges nahm. München, Ainmillerstraße 34, im August 1924. Jakob Strieder. * XI Vorwort und Einführung. Für die Geschichte des Frühkapitalismus , der — in Deutsch¬ land — in der zweiten Hälfte des 16. Jahrhunderts seinen Höhe¬ punkt erreicht, ist die Einzeluntersuchung gegenwärtig das Ge¬ gebene. Man darf sogar noch weiter gehen und behaupten : die auf neuem archivalischen Material aufgebaute Einzeluntersuchung ist das, was uns auf diesem geschichtlichen Gebiete zurzeit nottut l. Eine Zusammenfassung der bisherigen Literatur würde ebensowenig wie eine Verarbeitung des spärlich nur gedruckten Quellenmaterials ein den Kenner befriedigendes Bild der wirtschaftlichen Zustände und Kräfte jener Zeit geben. Namentlich die die Wirtschaft neu¬ organisierenden Bildungen der frühkapitalistischen Epoche, die neuen, aus dem Geiste des Kapitalismus geborenen Formen, ins¬ besondere der Groß Unternehmung, lassen sich nur mit Hilfe zeit¬ raubender und mühsamer archivalischer Untersuchung deutlich in ihrer Entstehung und Wirksamkeit erkennen. Eine solche Untersuchung ist im folgenden geboten. Ich bin darin den früheren Monopol- und Kartellbildungen sowie denjenigen Arten der Handelsvergesellschaftung nachgegangen, die über die älteren Formen der Handelsgesellschaft hinaus auf die moderne Aktiengesellschaft weisen. Mehr gelegentlich wurde auch, wo es sich zum Verständnis der Darstellung notwendig erwies, auf die Geschichte des Verlagssystems eingegangen. Selbstverständlich konnten — weil vorwiegend Archivmaterial als Grundlage der Arbeit in Frage kommt — nicht alle frühkapitalistischen Organi¬ sationsformen der genannten Art ausfindig gemacht und beschrieben werden. Hier muß die lokale Geschichtsforschungweiter arbeiten. Wohlverstanden die streng wissenschaftliche! Es würde mich freuen, 1 Noch vor einiger Zeit hat Gustav Schmoller in( seinem Jahrbuch Jahrg . 1912, S. 880) darauf aufmerksam gemacht, daß für viele unserer Nationalökonomen die Archivscheu das Haupt¬ hindernis fruchtbarer wissenschaftlicher Tätigkeit gewesen ist. XII Vorwort und Einlührung. wenn ich der gerade für die Wirtschaftsgeschichte so außerordentlich wichtigen lokalen Historik einen Erzgang gezeigt hätte , aus dem sich noch mancher wertvolle Fund erwarten läßt . Ich wüßte kaum ein Gebiet, das sich so gut für in sich abgeschlossene lokale Mono¬ graphien eignet, wie die Geschichte einzelner kapitalistischer Unter¬ nehmungen großen Stils, seien es nun Großgewerkschaften oder allgemeine Handelsgesellschaften auf aktiengesellschaftlicher Grund¬ lage, wie sie im folgenden für Steyr, Iglau, Amberg nachgewiesen werden, seien es Monopolunternehmungen oder Kartellbildungen etc. Dem eigentlichen Hauptteil der Arbeit gehen längere grund¬ legende Ausführungen voraus. Davon sind die ersten, in Buch I, Kapitel 1—3 untergebrachten , dem allgemeinen Nachweis der be¬ sonderen Bedeutung des Bergbaues und des Erzhandels für die Entstehung und Entwicklung des Kapitalismus gewidmet. Die anderen, in Buch II gegebenen, spüren dem Zusammenhang nach, der zwischen Staat , Kirche und den fortgeschrittenen frühkapita¬ listischen Organisationsformen besteht h Ich habe mich veranlaßt gefühlt, in einem Anhang, der etwas umfangreicher ausgefallen ist, als ich selbst wünschte, die wich¬ tigsten archivalischen Quellen, auf die sich die folgende Darstellung teilweise stützt , in extenso abzudrucken. So entstand eine Samm¬ lung von Dokumenten der frühkapitalistischen Wirtschaftsorgani¬ sation. Die Berechtigung zu dieser Aktenpublikation glaube ich in dem fast völligen Fehlen ähnlicher wirtschafts- und handels¬ rechtsgeschichtlicher Quellenveröffentlichungenzu sehen. Der Ver¬ fasser wenigstens hätte früher öfters gern für wirtschaftsgeschicht¬ liche Übungen zur Entstehung des Frühkapitalismus ähnliches Quellenmaterial bequem beieinander gehabt. Das allermeiste des Quellenanhanges ist bisher ungedruckt. Nur einige wenige Stücke finden sich bereits bei Franz Anton Schmidt, Chronologisch -systematische Sammlung der Berg¬ gesetze der österreichischen Monarchie. Doch war dort der Druck der betreffenden Akten so fehlerhaft, auch der Inhalt so entstellt wiedergegeben, daß sich ein Neudruck nötig machte . Bei der Technik der Quellenedition habe ich mich im ganzen an die Be1 Vgl . außer dem II . Buche hierfür auch die gelegentlichen Hinweise im ganzen Verlaufe des III . Buches und schließlich noch das Schlußwort. Vorwort und Einführung. XIII Stimmungen über Herausgabe von Urkunden und Akten der königl. sächsischen Kommission für Geschichte angelehnt. Für die vorliegende Arbeit ist das urkundliche Material ins¬ besondere den folgenden Archiven entnommen worden: Dem königlichen Hauptstaats -Archiv zu Dresden; dem gemeinschaftlichen Hauptarchiv des Sachsen-Ernestinischen Hauses zu Weimar; dem Stadtarchiv zu Leipzig; dem Königl. allgemeinen Reichsarchiv in München; dem Stadtarchiv zu Augsburg; dem k. und k. gemeinsamen Finanzarchiv (Hofkammer-Archiv) in Wien; dem k. und k. Haus-, Hof- und Staatsarchiv in Wien; dem ehemaligen Reichskammergerichtsarchiv in Wetzlar; Ferner mit Hilfe von Akten- bzw. Abschriftenübersendung den folgenden Archiven: Dem R. Archivio di Stato in Rom; dem R. Archivio di Stato in Neapel; dem k. und k. Statthalterei -Archiv zu Prag; dem Stadtarchiv zu Iglau in Mähren; dem Stadtarchiv zu Steyr; dem Stadtarchiv zu Leoben; dem Königl. Staatsarchiv zu Königsberg i. Pr . ; dem Königl. Staatsarchiv zu Danzig; der Stadtbibliothek zu Danzig; dem Stadtarchiv zu Elbing; dem Stadtarchiv in Goslar. dem Königl. bayrischen Kreisarchiv zu Amberg, O.-Pf.; dem Stadtarchiv zu Lindau i. B. Allen Herren Beamten der genannten Archive, die mich unter¬ stützt haben, möchte ich auch an dieser Stelle meinen herzlichsten Dank sagen für die freundliche Beihilfe zur Vollendung des vor¬ liegenden Buches. Dieser Dank gilt besonders Herrn Dr. Boden¬ stein vom k. u. k. Finanzarchiv in Wien und Herrn Dr. Oskar Frei¬ herrn von Mitis vom dortigen Haus-, Hof- und Staatsarchiv . Durch Kollationierung einer Abschrift aus dem römischen Archivio di Stato und einige Nachforschungen in den vatikanischen Archiven verpflichtete mich Herr Dr. Schäfer. XIV Inhaltsverzeichnis. Seile Vorwort zur zweiten Auflage . ................ Vorwort und Einführung .................... Inhaltsverzeichnis.................. VH—IX XI—XIII XIV -XXVII Verzeichnis der zitierten wichtigeren gedruckten Quellenwerke und der Literatur ................... XXVIII- XXXV Erstes Buch. Montanindustrie und Friihkapitalismus ........... Erstes Kapitel. Die quantitative Bedeutung des Bergbaues und Erzhandels für die Entwicklung der deutschen Volkswirtschaft am Ausgang des Mittel¬ alters und zu Beginn der Neuzeit ..... ... Bergbau und Erzhandel, die wichtigsten Zweige der Wirtschaft des hl. röm. Reiches deutscher Nation zu Anfang des 16. JahrhundertsS. 3. Schätjung der quantitativen Bedeutung dieser Erwerbszweige für die deutsche Volkswirtschaft jener Zeit in einer Urkunde Karls V. vom Jahre 1525. S. 3. Nachprüfung der dort ge¬ gebenen Schäljungswerte unmöglich. S. 4. Jedenfalls nahm bis Mitte des 16. Jahrhunderts das hl. röm. Reich im Bergbau und Erzhandel der Erde die führende Stelle ein. S. 5. Werterhöhung der deutschen Bergwerke durch Preissteigerung einzelner Metalle, besonders des Kupfers im 16. Jahrhundert S. 5. Deutsche im aus¬ wärtigen Bergbau als Unternehmer und Bergarbeiter tätig. In Spanien und dessen Kolonien, Schweden , England, Böhmen, Polen, Ungarn. S. 7 f. Die Thurzo von Krakau in Goslar S. 9. Verbin¬ dung der Thurzo und Fugger im ungarischen Kupfer- und Silber¬ bergbau. S. 10. Gewinne der Fugger dort. S. 11. Uinsülje deutscher Finnen im Erzhandel. S. 12. Zurücktreten des übrigen Warenhandels . Das Finanzgeschäft, vielfach aus dem Erzhandel erwachsen . S. 13. Zweites Kapitel. Bergbau und frühkapitalistische Vermögensbildung ........ Frage nach der Bedeutung des Bergbaues und Erzhandels für die ursprüngliche, erste Akkumulation bürgerlicher Geld¬ vermögen , auf denen sich dann der Frühkapitalismus aufrichtete. S. 14 f. Zunächst arme , glückliche Fundgrübner waren in der Regel von keiner direkten Bedeutung für die Entstehung des Früh¬ kapitalismus. S. 15. Ein größeres Kapitalvermögen war zu aller¬ meist die Voraussetjung, um im Bergbau und Erzhandel be- l—52 3 —13 13—38 Inh altsverzelchnis. deutende Gewinne machen zu können. S. 15. Beweise: Aus der Entwicklung in Italien und Flandern. Manche Bergwerke wurden nur im ganzen gegen hohe Summen verpachtet. S. 15f. Oft Aus¬ übung des Berg- und Hüttenbetriebes zunftmäßig organisiert (Er¬ haltung gleicher Kleinbetriebe). S. 16. Zychas Gegensalj zwischen Kaufleute-Gewerken und einheimischen Gewerken nicht möglich. S. 18 ff. Krokers Überschätjung der bergmännischen Gewinne für die Genesis des modernen Kapitalismus. S. 20. Entwicklung in Sadisen. S. 21f. Kosten des Bergbaus in Idria. S. 23. Das Ver¬ lagssystem blüht besonders im Bergbau. S. 24f. Mit seiner Hilfe Eindringen schon reicher kapitalistischer Kaufleute in die Montan¬ industrie. Ebenso durch Übernahme kostspieliger technischer An¬ lagen. S, 25. Schlackenwald. S. 25. Thurzo in Ungarn. S, 26. In Goslar. S. 27. Reiche Kaufleute werden aber besonders Erzhändler, Abnehmer der Erzprodukte. S. 27f. Viel Kapital dazu nötig für Verlag und Vorschüsse an die Regalherren usw. Gezeigt am Mansfelder Kupferhandel. S. 29f. Der sog. Wechsel, das Vorkaufs¬ recht der Regalherren. S. 30f. Die »Erzkäufe«. S. 32ff. Drittes Kapitel. XV Seite Bergbau und Entfaltung der frühkapitalistischen Organisationsformen 38- 52 Die innere Organisation der Wirtschaft wird im Bergbau und Erzhandel des endenden Mittelalters und der beginnenden Neuzeit einer tiefen Umgestaltung entgegengeführt. S. 38Î. Frühes Eindringen des Kapitalismus hier zu konstatieren. S. 39. Die herrschende Unternehmungsforin ist schon im Mittelalter fast über¬ all im Bergbau die kapitalistische Gewerkschaft. S. 39. Bedeutung des Bergbaues für die Entstehung des Proletariats in seiner modernen charakteristischenEigenart. S. 40. Im Bergbau Massen von Arbeitern konzentriert. Unsicherheit ihrer Existenz. Soziale Arbeitslosigkeit. S. 41. Nomadenhaftigkeit der Bergarbeiter. Deutsche Knappen in England, in Frankreich, in Spanien, in Amerika usw. Im Bergbau früh die Probleme der Wohnungsnot, der Kinder- und Frauenarbeit. S. 42. Trucksystem. Cottagesystem. S. 43. Streike 44. Antistreikverbände der Unternehmer. S. 44—45. In der Montanindustrie früh interessante wirtschafts- und betriebs¬ organisatorische Neubildungen: konzentrierte Großbetriebe. S. 46. Z. B. die Mansfelder Saigerhütten- und Saigerhandelsgesellschaften. S. 47. Tendenz der Erweiterung und Vergrößerung. S. 48. Größe der Tiroler und der ungarischen Montanunternehmungender Firma Haug, Langenauer, Link & Co. S. 48. Der Fugger besonders in Ungarn. S. 49. Fusionen. S. 49/50. Versachlichung, Objektivierung der Wirtschaftsbeziehungen im Bergbau und Erzhandel. An Stelle der einzelnen Gewerken tritt die Gewerkschaft, eine unpersönliche Gemeinschaft häufig wechselnder Kapitalisten. Die Kuxe werden das erste bedeutungsvolle Inhaberpapier. Frage : Liegen viel¬ leicht auch noch die Anfänge anderer Erscheinungen der hoch¬ kapitalistischen Wirtschaft im kleinen schon in der Montanindustrie des 16. Jahrhunderts verborgen ? Die Anfänge der Aktiengesell- XVI Inhaltsverzeichnis. Seite Schaft vielleicht , der Ursprung des Kartellwesens etwa oder die ersten Versuche großer internationaler Monopole ? Stand nicht die mittelalterliche Wirtschaftsethik einer solchen Betätigung kapita¬ listischen Geistes entgegen . S. 51/52. Zweites Buch. Kirche, Staat und Frühkapitalismus ............ Entstehung des kapitalistischen Geistes als wirtschaftlicher Individualismus im Italien der Renaissancezeit . S. 55. Verbreitung über ganz Westeuropa . S. 57. Wie konnte das geschehen trog der entgegenstehenden Wirtschaftsethik der mittelalterlichen Kirche und gegen die Staatsgewalt , die der Kirche ihren weltlichen Arm lieh ? S. 58. Die Wirtschaftsethik des Mittelalters . Ihre Licht- und Schattenseiten . S. 58—59. Das kanonische Zinsverbot zunächst in erster Linie gegen Konsumptionskredit erlassen , aber auch gegen Produktionskredit in der Judikatur der Kirche gehandhabt , S. 60—61. Volksprediger . Luther . S. 61. Lehre vom gerechten Preis . S. 61 f. Geiler von Kaisersberg , Luther darüber . S. 62—63. Dagegen Konrad Peutingers kapitalistische Auffassung der Preisbildung . S. 62. Erste Jahrzehnte des 16. Jahrhunderts : Höhepunkt der antikapitalistischen Tendenzen in Deutschland . S. 63. Kirche und Staat billigten theo¬ retisch diese Bestrebungen , in praxi hatten gerade diese zwei Lebensmächte die mittelalterliche Wirtschaftsethik längst über¬ wunden . Ihre Finanzpolitik zwang sie dazu . S. 64. Papsttum und kapitalistischer Kaufmann . S. 64—65. Staat und kapitalistischer Kaufmann . S. 65—67. Folgen des Widerspruchs zwischen Theorie und Praxis . S. 67. Bedeutung von Kirdie und Staat für die Ent¬ wicklung kapitalistischer Organisationsformen . S. 67. Von Konsor¬ tien und Aktienvereinen im mittelalterlichen Italien , der kapitalisti¬ schen Gewerkschaft . S. 68- 69. Der Kartelle und Monopole. S. 69—70. Antimonopolbewegung in Deutschland in den ersten Jahrzehnten des 16. Jahrhunderts auf den Reichstagen besonders zum Ausdruck gelangt . S. 71—73. Vorgehen des Fiskals (Reichs¬ anwalt ) gegen Augsburger Kaufleute . Monopolprozesse durch Karl V. niedergeschlagen . S. 73. Karl V . und die Handels¬ gesellschaften . S. 74—78. Die Habsburger und die Monopol¬ bewegung . S. 78 ff. »Majestätsbriefe * für die monopolistischen Kaufleute . S. 81. Gegeneinanderarbeiten der kaiserlichen Kanzlei und des Reichskammergerichts . S. 81. Das Toledaner Mandat Karls V. vom 13. Mai 1525 mit seiner Bestimmung , daß die Kontrakte , die den Erzgrosshandel in die Hand weniger Kaufleute auslieferten , nicht als monopolistisch im Sinne der Reichstags¬ verhandlungen angesehen werden sollten und dürften . S. 81. Interpretation dieses Geseßes und seiner Begründung . S. 82—85. Seine Wirkung . S. 85—86. Anklage der Welser wegen Monopol¬ vergehens . S. 87. Vorgehen gegen den Kapitalismus in Basel. S. 88. Stimmen fortschrittlicher Theologen für den Kapitalismus. S. 89- 92. 53—92 Inhaltsverzeichnis. Drittes Buch. XVU Seite Monopole, Kartelle und Aktiengesellschaftenim Mittelalter 93- 363 und zu Beginn der Neuzeit......... Erstes Kapitel. Deutsche Aktiengesellschaften , vornehmlich des 16. Jahrhunderts . . 95- 156 Erster Abschnitt: Die führende Form der Unternehmung 95- 110 im f rühkapitalistischen deutschen Handel ....... Im 16.Jahrhundert haben die aus Familien wirtschaften her¬ vorgegangenen offenenHandelsgesellscbaften die Führung im großen süddeutschen Handels-, Industrie- und Finanzgeschäft. S.95. Gegensatj zum hansischen Wirtschaftsgebiet. S. 96 ff. Die Loitj. S. 99—100. Festverzinsliche Einlagen in den Handelsgesellschaften des 16. Jahrhunderts. S. 101. Die rigorose Wirtschaftsethik da¬ gegen. Einlagen in die Höchstetter Kompagnie. S. 102. Frage, ob die Wechsel, die für Depositen ausgestellt wurden, jederzeit an Dritte weitergegeben werden konnten. S. 103. Kommanditistische Beteiligung an den Handelsgesellschaften. Nürnberger Privileg von 1464. Interpretation desselben gegen Keutgen. S. 104f. Gelegentliche Vergesellschaftungen im süd¬ deutschen Handelsleben. Konsortialbeteiligungender großen Firmen bei Finanzgeschäften. Gelegentliche Vergesellschaftungenzu Pfennwertshandelsgesellschaften. S. 105. Zu Gesellschaften des Bergund Schmelzwerkhandels S. 106. Die Fugger-Thurzosche Tochter¬ gesellschaft des «gemeinen ungarischen Handels». S. 106. Konsortien der süddeutschen Kaufleute im Spekulationsgeschäft mit indischen Gewürzen S. 107. Konrad Rott, seine Konsortien und Tochtergesellschaften.S.107— 108.DieMansfelderSaigerhandelsgesellschaftenS .109. Zweiter Abschnitt: Die Entstehung der Aktiengesellschaft: 110- 125 Frage : Wann treten Aktiengesellschaften zuerst auf? Allgemeine Meinung: Im mittelalterlichen Italien. S. 111. Diese Ansicht von Karl Lehmann bekämpft. S. 112. Von ihm die Entstehung der Aktiengesellschaft ins 17. Jahrhundert verlegt (Kolonialgesellschaften ). Ursprung des Wortes »Aktie« in Holland. Autochthone Ausdrücke dafür in Frankreich, Deutschland und England. S. 112f. Juden und Aktien S. 113 ff. Von Lehmann das Spekulationsmoment bei der Aktiengesellschaft zu sehr in den Vordergrund gedrängt. S. 115 ff. Spekulation in Wertpapieren, besonders Kuxen, viel älter. S. 117. Das wesentlich neue bei der Aktiengesellschaft ist die neue Art der Kapitalassoziation. S. 117f. In ihr »Demokratisierung« des kapitalistischen Erwerbs¬ triebes. Aktiengesellschaft und Wirtschaftsethik. Letztere der Entwicklung günstig. S. 119ff. Dr. Eck und der 5%-Streit Con¬ tractus trinus. S. 121. Die älteren Aktiengesellschaften tragen noch nicht alle Kennzeichen der modernen Aktiengesellschaft an sich. S. 123ff. Schmollers Charakteristik der Aktiengesellschaften des 17. und 18. Jahrhunderts trifft auch für die des 16. Jahrhunderts zu. S. 125. XVIII Inhaltsverzeichnis. Scite Dritter Abschnitt : Aktiengesellschaften im steiermärkischen und oberösterreichischen Eisenerzhandel des 15. und 16. Jahrhunderts ................. .... 125—141 Die Leobener allgemeine Eisenhandelsgesellschaft. Aktien¬ gesellschaft. Gegründet um 1415. S. 127 fi. Die Gründung der Steyrer allgemeinen Eisenhandelskompagnie-Aktiengesellschaft (1582). S. 129 ff. Frühere Organisation des Steyrer Eisenverlags und Handels. S. 129f. Ursachen der Änderung und Gründung einer allgemeinen Eisenhandelskompagnie. S. 130f. Gegner der Gründung. Gutachten Steinbergers S. 132f. Statuten und Organi¬ sation der Kompagnie. S. 133ÏÎ. Die »Gesellschaft des gestreckten Stahls« der Kompagnie einverleibt. S. 139. Die kleine Kom¬ pagnie. S. 140. Erwähnungen anderer ähnlich organisierter Kom¬ pagnien. S. 141. Vierter Abschnitt : Die Tglauer Tuchhandelskompagnie (Aktiengesellschaft) .................. .. . 142—145 Als Nachahmung der Steyrer Eisenhandels-Kompagnie entstanden und 1592 von Kaiser Rudolf II. bestätigt. S. 142. Statuten. S. 143. Monopolanklage. S. 144. Spätere Schicksale. S. 145. Fünfter Abschnitt : Die Gesellschaft des Amberger Zinn¬ blechhandels (Aktiengesellschaft) 145 . ........... - 156 Gründung aul Anregung der Regierung (1533/4 ). Der Landesherr und seine Räte bedeutende Anteilszeichner. S. 146. Anteil der Bürgerschaft. S. 147 ff. Monopolanklage. S. 149—150. Verhältnis zur öffentlichen Gewalt. S. 152f. Innere Organisation. S. 153. Vereinigung mit den Nürnberger Zinngroßhändlern. S. 154 ff. Zweites Kapitel. Kartelle des 14.—18. Jahrhunderts ................ 156—212 Frühe Kartellerscheinungen. Im römischen Altertum. S. 15/f. In Deutschland während des 15.—18. Jahrhunderts. Kar¬ tell der Naumburger- und Weißenfelser Bettfedernhändler (1743). S. 158. Kartelle in de- englischen Großindustrie des 18. Jahr¬ hunderts. S. 158. ln der Thüringer Glasindustrie des 18. Jahr¬ hunderts. S. 159. Kartelle der Mansfeldcr Saigerhttttengesellschaften des 16. Jahrhunderts. S. 159. Das Kupfersyndikat vom Jahre 1498. S. 159. Kartellbestrebungen Konrad Rotts. S. 160. Das Salzvertriebssyndikat der neapolitanischen und französischen Regierung bzw. ihrer Pächter aus dem Jahre 1301. S. 161 ff. Fis¬ kalismus und Kartelle. S. 162. Syndikat der großen italienischen Finanziers bei den Anleihen der Fürsten des Mittelalters. S. 166. Kartellbildungen im hansischen Wirtschaftsgebiet des Mittel¬ alters. S. 167. Das Aiaunkartell von 1470. S. 168 ff. Alaunhandel im Mittelalter bis ca. 1460 unter byzantinischer Herrschaft im Mittelmeer. S. 168. Unter türkischer Herrschaft. S. 169. Entdeckung der päpstlich-Tolfaer Alaungruben (1461). Ausgebeutet durch die societates Inhaltsverzeichnis. XIX Seite aluminum. Deren Organisation. S. 170. Die Medici übernehmen die Pacht der Tolfaer Gruben. Ihr Pachtkontrakt. S. 170f. Verbot für die ganze Christenheit, türkischen Alaun zu kaufen. Päpst¬ liches Monopol. Gewinne der Kurie. S. 171. Für die Zwecke der Cruciata verwendet. Kartell der Päpste mit dem König von Neapel. (Besiger der Alaungruben von Ischia.) 11. Juni 1470. S. 171. Genaue Wiedergabe des Inhalts ries Kartellstatuts. S. 172ff. Sonstige Maßnahmen der Kurie, um sich für ihr Tolfaer Alaun ein Monopol— wenigstens in der Christenheit —zu sichern. Verträge mit den großen Alaunhändlern und mit den einzelnen, besonders stark Alaun importierenden Staaten. S. 177 ff. Durch¬ brechung des päpstlichen Alaunmonopols. Starke Einfuhr türkischen Alauns. Eröffnung von Alaungruben in den meisten europäischen Staaten. S. 180. Sinken der päpstlichen Einnahmen aus der Ver¬ pachtung von Tolfa. S. 181. Um die Mitte des 16. Jahrhunderts erneutes Steigen der Tolfaer päpstlichen Pachteinkünfte. Versuche einer Erklärung hierfür. Die Antwerpener Firma Sehet; vereinigt in ihrer Hand die Pacht von Tolfa und der spanischen Alaun¬ gruben. S. 182. Kartellbestrebungen im süddeutschen Frühkapitalismus (be¬ sonders im 16. Jahrhundert). S. 183. Geseggebung und Kartelle im 16. Jahrhundert. S. 184 ff. Reichstagsverhandlungen darüber. Sächsische Landesgeseggebung. S. 185. Einkäuferkartelle in Sachsen, im böhmischen Bergbau, im bayrischen Salzhandel. S. 185 ff. Zünfte und Kartelle; Prinzipielles dazu. S. 187f. Sittenlehre und Kartelle im 16. Jahrhundert. S. 188f. Die Summistenliteratur. S. 188f. Geiler von Kaisersberg. S. 189ff. Analekten zur Kartellgeschichte des 17. und IS. Jahrhunderts. Salzkartelle zwischen der bayrischen und österreichischenRegie¬ rung als Monopolinhabem der Reichenhaller bzw. Haller (Inntal) Salzproduktion und des dortigen Salzhandels. S. 193 ff. Bestim¬ mungen des Statuts. S. 194 ff. Das Kartell von 1649 öfters erneuert und mit wichtigen Zusägen versehen. S. 196. Es liefert an das Ausland billiger, um die Konkurrenz des burgundischen Salzes abzuwehren. S. 197f. Kartell der österreichischen und bayrischen Regierung mit den burgundischen Fermiers généraux. S. 198. Monopolanklage. S. 199. Kartelle in der Calwer Zeugindustrie des 17. Jahrhunderts. S. 200. Kartelle in Brandenburg unter dem großen Kurfürsten. S. 201 f. Kartellbildungen in der böhmischen Glasindustrie des 18. Jahrhunderts. S. 202. Überblick über die Geschichte der böhmi¬ schen Glasindustrie und des böhmisenen Glashandels. S. 202 f. Konvention aller böhmischen Glasproduzenten (Verleger) und Glashändler, die nach Portugal exportieren (10. Oktober 1715). S. 205. Bestimmungen der Konvention. S. 205 ff. Syndikat der böhmischen Glasproduzenten (1739). S. 207. Ablehnende Stellung der österreichischen Regierung. Begründung dieser Haltung aus der Freiheit des commercium. Kartellfeindlichkeit der öster¬ reichischen Regierung bis heute erhalten. S. 208. XX Inhaltsverzeichnis. Seile Kartelle im schwedischen Eisenbergbau des 18. Jahrhunderts. S. 209. In Frankreich zu derselben Zeit. S. 210. Desgleichen in Sachsen (vogtländische Textilindustrie). S. 211. Drittes Kapitel. Monopole , Kartellbestrebungen und Aktiengesellschaften im sächsischen Zinnhandel des 15. und 16. Jahrhunderts ........... 212—257 Der »Zinnkauf* in Sachsen von 14S9—1497. Beteiligung Herzog Albrechts daran. S. 212f. Die privilegierte »Gesellschaft des Zinnkaufs« von 1498—1500. S. 214. Höhe des Gesellschafts¬ kapitals. Organisation. S. 215. Stellung im öffentlichen Recht. S. 216f. Geschäftstätigkeit, Verlagssystem. S. 218. Dr. Christoph Kuppener. S. 220. Die »Gesellschaft des Zinnhandels« vom Ende des Jahres 1500 bis zur Aufhebung ihres Monopols. S. 220. Seit dein Jahre 1500 genoß die Gesellschaft des Zinnhandels in Sachsen ein Monopol. S. 221. Veränderung in der Geschäftsführung. S. 222. Bestimmungen über Austritt. S. 222. Faktoren und Aufsichtsräte. S. 223. Hervorragende Teilhaber. S. 224f. Luthers Beurteilung des Kapitalismus. Herzog Georgs Bedeutung für die sächsische Wirtschaftsgeschichte. S. 225f. Aufhebung des Monopols. S. 226 f. Neue Versuche, eine große, mit Monopol ausgestattete Zinnhandels¬ kompagnie zu gründen (1518). S. 228. Die Denkschrift vom Jahre 1518. Monopol. Produktionsbeschränkung. S. 229. Morali¬ sche Begründung. S. 230. Kapitalbeschaffung. S. 230f. Im Not¬ fälle Säkularisation. S. 232. Teilhaber. S. 233. Ordnung der Ge¬ schäftsführung. S. 234. Produktionskartell mit den böhmischen Zinngewerken. S. 235. Die Zinnhandelsgesellschaft von 1521—1527. S. 236f. Das Projekt von 1518 nicht zur Ausführung gelangt. Ver¬ handlungen zwischen den Zinngewerken und den Leipziger Kauf¬ leuten. Neue Zinnhandelsgesellschaft, die von 1521—1524 privi¬ legiert wird. Hauptteilnehmer Michael Puffler, Ulrich Mord eisen und Hieronymus Walter, der Leipziger Faktor der Welser. Andreas Madtstedt, der Leipziger Faktor der Fugger, hatte sich gleichzeitig bemüht, den Zinnkauf in die Hände zu bekommen. Monopol. S. 238. Hans Alnpeck jun. aus Freiberg, Faktor der Gesellschaft. Sein Sig in Altenberg. Sein Kontrakt. S. 239. Zinnverlag der ärmeren Ge¬ werken von Geyer, Ehrenfriedersdorf und Thum durch Michael Puffler und Ulrich Mordeisen (1525). S. 240. Kontrakt der Alten¬ berger Zinngewerken mit den Genannten und Hieronymus Walter (1524). S. 240f. Freiverkaufskontingent für die Ge¬ werken. S. 241. Feste , billige Preise für die sächsischen Zinnverarbeiter(Kannengießer) festgeseijt. Merkantilistische Hand¬ werkerpolitik. S. 241. Das große Leipziger Monopolprojekt von 1527. S. 242ff. Mächtiger Aufschwung Leipzigs um die Wende des 15. und in den ersten Jahrzehnten des 16. Jahrhunderts. S. 242. Eifersucht der Nürnberger. Ihre Versuche, die Hansen, die am stärksten die Leip¬ ziger Messen besuchten, aus dem Handel mit Mansfelder Kupfer zu drängen. S. 243f. Monopolprojekt von 1527 als Gegenschlag Inhaltsverzeichnis. XXI Seite gedacht. Versuch, den gesamten mitteleuropäischen Metallhandel jn die Hand weniger Leipziger Kapitalisten und Kaufleute zu (bringen. S. 244 ff. Plan gescheitert. i Zeiten des freien Zinnverkaufs (bis 1538). Erneutes Monopol (1538 ff.) und dessen Ende (1541). S. 251 ff. Versuche Herzogs Georgs, wenigstens die sächsische Zinnproduktion in eine Hand zu bringen. S. 252f. Die Antwerpener Firma Scheg ins Auge dafür genommen. S. 253. Georg bringt das Altenberger Zinnmonopol in seine Hand und gibt es an Michael Puffler weiter. S. 254. Für Geyer, Ehrenfriedersdorf und Thum vermitteln die herzoglichen Räte den Zinnkauf mit Puffler. S. 254 f. Verlag. Höhe der Altenberger Zinnproduktion, Namen und Be¬ teiligungsziffern der einzelnen Gewerken der Jahre 1545/46 . S. 255. Formen des Verlags. S. 256f. Im Jahre 1540 Kündigung des Zinn¬ kaufs durch Puffler. S. 257. Viertes Kapitel. Monopol - und Kartellbestrebungenim böhmischen und sächsischen Zinngroßhandel seit der Mitte des 16. Jahrhunderts ...... 258—292 Erster Abschnitt : Die Monopolisierung der böhmischen Zinnproduktion in der Hand des Augsburgers Konrad Mayr ............................ 258—271 Kurz vor der Mitte des 16. Jahrhunderts war der Zinngroßhandel in Böhmen frei. S. 259f. Wohl besonders Nürnberger darin tätig. S. 260. Ende 1549 nimmt König Ferdinand I. den Zinn¬ kauf in Böhmen an sich. Verspricht den ärmeren Gewerken Ver¬ lag zu geben. S. 261. Welche Kapitalisten standen hinter Ferdi¬ nand I.? S. 262. Die Fugger , die Manlich und die Mayr werden genannt. Unterhandlungen noch mit anderen. S. 262f. Konrad Mayr. S. 264. Sein Vermögen. S. 265. Verbindung mit den Fuggern ? S. 265. Anlage des Hauptbuches für das böhmische Zinnmonopol Konrad Mayrs durch Mathäus Schwarz, Hauptbuch¬ halter der Fugger. S. 266. Einzelheiten des Kontraktes Mayrs mit König Ferdinand. S. 267f. Bevorzugung der Amberger Gesell¬ schaft des Zinnblechhandels. S. 268f. Konrad Mayr läßt sich gegen etwa vorkommende Belästigungen des Reichsanvvalts schüfen. S. 270. Ferdinand verspricht mit dem Kurfürsten von Sachsen in Unterhandlung einzutreten, die ein Kartell der Beherrscher der sächsischen und böhmischen Zinnproduktion herbeiführen sollten. S. 271. Zweiter Abschnitt : Vergebliche Versuche , den böhmischen und sächsischen Zinngroßhandel zu kartellieren (1549ff .) . Konrad Mayrs Fall .................. 272- 278 Kurfürst Morig verspricht Ferdinand, auch in Sachsen einen Zinnkauf einzuführen, der mit dem böhmischen korrespondieren solle. S. 272f. Moritjens dahingehende Versuche scheitern an dem Widerstand der großen sächsischen Zinngewerken. S. 273f. Damit im Grunde auch Konrad Mayrs Projekt erledigt. Zwar Versuch XXII Inhaltsverzeichnis. Seite einer Sperrung der Grenzen der Habsburger Lande gegen aus¬ wärtiges Zinn. S. 276. Zinnabsatj durch Mayr zurückgehalten. S. 277. Starke Kontrebande. S. 277. Sturz Mayrs. Preissturz des Zinns. S. 278. Dritter Abschnitt : Erneuter Plan einer Monopolisierung oder wenigstens Kartellierung der böhmischen und sächsischen Zinnproduktion (1569) ........... 279—292 Von Kaiser Maximilian II. aus Finanznot neue Kartell¬ pläne eingeleitet. S. 279f. Der Kaiser nimmt den böhmischen Zinnkauf in seine Hand. S. 281. Kein Kapitalist zu finden, der ihn abnehmen will. Vergebliche Bemühungen Georg Ilsungs in Augsburg usw. Alle Kaufleute dort fordern ein korrespondieren¬ des sächsisches Zinnmonopol. S. 281. In Sachsen die Lage für ein Zinnmonopol ungünstig. S. 282 ff. Selbst die ärmeren Altenberger Zinngewerken, mit einem Zinnkauf begabt , wie sie waren, ver¬ sagen sich. Die übrigen sächsischen Zinner erst redit . S. 284 f. Kurfürst August an sich für ein Monopol. S. 286f. Bemühungen Maximilians II. bei dem Kurfürsten. S. 287. Entweder sollte der böhmische und der sächsische Zinnkauf in ein und desselben Kapitalisten Hand gebracht werden oder an zwei verschiedene Gesellschaften (eine in Böhmen und eine in Sachsen), die dann ein Kartell zusammen abzuschließen hätten. & 288f. Kurfürst August versagt sich dem Plan. S. 289f. Seine Gründe: Die sächsischen Großgewerken sind dagegen. Handwerkspolitische Erwägungen. S. 290f. Besonders aber konnte August deshalb von dem gefährlichen Plan absehen, weil ihn nicht, wie Maximilian II., bittere Geldnot zu Finanzprojekten trieb. S. 291. Auch das böhmische Monopolprojekt fällt damit. Not der dortigen Gewerken und Arbeiter. S. 291 f. Fünftes Kapitel. Monopole und Kartelle im Idrianer Quecksilberhandeldes 16. Jahr¬ hunderts .......................... 292—359 Organisation des Idrianer Quecksilberbergwerks. Die ver¬ schiedenen Gewerkschaften. S. 292f. Kartell der Gewerkschaft des Fürstenbaues und der »Idrianer Gewerkschaft«. S. 294. Handels¬ monopolisierung des Idrianer Quecksilbers in der Hand der Höch¬ stetter (im Jahre 1525). S. 295ff. Einzelheiten des Kontraktes. S. 296 f. Schutj gegen den Reichsfiskal. S. 297f. Ferdinand I. Rücken¬ deckung bei Karl V. S. 298. Dessen Toledaner Mandat vom 13. Mai 1525 in erster Linie für Quecksilber- und Kupfermonopole gedacht Also neben den Fuggern (Jakob Fuggers Kupfermono¬ polisierungsbestrebungen. S. 299 ff.) für die Höchstetter erlassen. S. 299f. Die Höchstetter-Kompagnie, ihre Zusammense&ung und ihr Ruf. S. 301 ff. Kartell der Höchstetter mit Wigkell, dem Beherrscher der geringen böhmischen Quecksilberproduktion. S. 303. Plan der Höchstetter, durch Vereinigung des Idrianer und Almadener Mono¬ pols in ihrer Hand ein Weltmonopol für Quecksilber zu schaffen. Inhaltsverzeichnis . XXIII Seite S. 304 ff. Die Pacht von Alinaden. S. 304f. Die Fugger . S. 305. Ferdinand I. versucht die Pacht von Almaden für die Höchstetter zu erhalten. S. 305f. Mißglückt. S. 306f. Bankerott der Höch¬ stetter. S. 307f. Die Fugger in Almaden . S. 308 f. Die Idrianer Produktion geht seit 1529 erheblich zurück. S, 309. »Einzelne Quecksilberkäufe« mit den Baumgartnern und anderen abgeschlossen . Kontrakt mit dem Venezianer Nicola Venier. S. 310f. Not in Idria. Kartellverabredungen mit der Nürnberger Firma Steber (oder Stüber), die die böhmischen Queck¬ silbergruben beherrschte. S. 311. Versuche Ferdinands, bei Karl V. durchzusetjen, daß spanisches Quecksilber dem idrianischen in Venedig keine Konkurrenz machen dürfe. S. 312f. Hans Baumgartner übernimmt im Jahre 1539 einen fünfjährigen Idrianer Quecksilbermonopolkontrakt. S. 313 ff. Erneuert 1544. S. 317. Dann treten (im Jahre 1548) die Herwart in den Kontrakt. S. 317 ff. Brand in Almaden (1550). S. 318. Seit 1556,57 in Amerika starke Naclifrage nach Quecksilber für das Amalgamierungsverfahren in den dortigen Bergwerken . S. 319f. Almaden wieder aufgenommen , Fugger . S. 320. Ihre Pachtperioden und Gewinne. S. 321 f. Auch Idria hat enormen Vorteil von der erhöhten Nach¬ frage nach Quecksilber. S. 322. Die Herwart machen glänzende Geschäfte, erneuern ihren Kontrakt. S. 322 f. Ferdinand I. kennt ■die günstige Konjunktur genau . Ratschläge treten an ihn heran, den Idrianer Quecksilberhandel zu verstaatlichen. S. 323ff. Finanznot hindert ihn daran. S. 327. Im Jahre 1561 neuer Kontrakt mit den Herwart, von dem aber der Kaiser zurücktritt, als König Philipp II. von Spanien den Vorschlag eines festen Lieferungs¬ vertrags des idrianischen Quecksilbers an die Krone Spanien macht. S. 328 ff. Plan infolge Differenzen in der Preisansetjung gescheitert S. 331. Im Jahre 1562 neuer Kontrakt mit den Herwart. S. 331 ff. Enormer Verdienst des Kaisers. S. 332. Um die Hoch¬ konjunktur auszunußen , muß der kaiserliche Finanzagent Ilsung noch vor Ablauf des alten Kontraktes den Herwart einen neuen vorschlagen (mit Antizipationen der kaiserlichen Gewinne dabei natürlich). S. 333ff. Die Herwart lehnen ab und versuchen, sich über¬ haupt aus dem Monopol zu ziehen . S. 336 f. Unterhandlungen Ilsungs mit Melchior Manlich, der zunächst ein hohes Angebot ge¬ macht hatte, sich aber doch schließlich auch versagt . S. 337 ff. Gründe. S. 339 ff. Weitere vergebliche Versuche, das Idrianer Quecksilber¬ handelsmonopol unterzubringen. S. 341 ff. Die Fugger lehnen ab. S. 341. Ilsung beruhigt die Gewerken . S. 342 f. Herantreten an die spanische Regierung zwecks kontraktiver Übernahme der Idrianer Produktion. S. 343ff. Philipp II. lehnt ab. S. 345f. End¬ lich wird die Augsburger Firma David Haug , Hans Langenauer u. Co. bestimmt, das Monopol von Idria zu übernehmen . S. 346 ff. Einzelheiten des Kontrakts. S. 347 ff. Kontrakt erneuert. S. 349f. Zahlungsstockungen der Haug, Langenauer & Co. S. 350 if. Ver¬ legenheit der Habsburger als Idrianer Regalherren und der dortigen Gewerken. S. 351 f. XXIV Inhaltsverzeichnis. Seite Verstaatlichung der Idrianer Gruben (1574). S. 352. Liste der damaligen Kuxinhaber. S. 352f. Die Regierung übergibt von neuem den Haug, Langenauer & Co. das Monopol. Gründe dafür. S. 354 ff. Der Kontrakt darauf berechnet der Firma ein Moratorium zu geben. S. 356 f. Im Jahre 1595 tritt eine venetianische Firma Bart. Bontempelo dei Calice das Idrianer Monopol an. S. 357f. Andere Italiener folgen. S. 359. Sechstes Kapitel. Einige sonstige Monopole , besonders unter Ferdinand I. Schlußwort 359—363 Monopol der Danziger Firma Anton Schmidt& Co., Baiesalz in Schlesien einzuführen. S. 359f. Eibenholzungsmonopol in Tirol und im Lande ob der Enns. S. 360. Ehrenberg und die Monopolbewegung des 16. Jahrhunderts. S. 360f. Die Bedeutung der staatlichen Archive (gegenüber denen privater Kapitalisten) für die Erforschung der Geschichte kapitalistischer Organisations¬ formen. S. 361. Staat, Kirche und Kapitalismus. S. 362f. Anhang. Dokumente der frühkapitalistischen Wirtschaftsorganisation 365—475 I. Allgemeine Akten zur Geschichte des Monopolwesens im 16. Jahr¬ hundert ........................... 367—383 Nr. 1. Bernsteinappaltvertrag zwischen Albrecht von Bran¬ denburg und einer hansischen Handelsgesellschaft. Königsberg, 9. Januar 1518 ............ 367—368 Nr. 2. Kaiser Karl V. und Erzherzog Ferdinand verleihen dem Jobst Günther das Monopol des Eibenholz¬ schlages in dem Lande ob der Enns. Worms, 10. Februar 1521 ................. 368—370 Nr. 3. Kaiser Karl V. befiehlt dem Reichsfiskal das Ver¬ fahren wegen Monopolaufrichtung einzustellen, das derselbe gegen verschiedene Augsburger Kaufleute eingeleitet hatte. Burgos, 15. September 1523 . . . 370—371 Nr. 4. Kaiser KarlV. nimmt die Erben Jakob Fuggers gegen jede Belästigung seitens des Reichsfiskals in Monopol¬ angelegenheiten in Schu^. Granada, 19. Oktober 1526 371—375 Nr. 5. Kaiser Karl V. bestimmt, daß die Kontrakte, die den Erzgroßhandel in die Hände weniger reicher Kauf¬ leute ausliefem, nicht als monopolistisch im Sinne der Reichstagsverhandlungen über Monopole an¬ gesehen werden sollen und dürfen. Toledo (Castilien), 13. Mai 1525 ................ 375—381 Nr. 6. Der kaiserliche Fiskal beklagt Bartolome Welser u. Co. vor dem Kammergericht, Monopole errichtet und versucht zu haben. Speier, 9. März 1530 . . . 381—383 Inhal tsverzcich nis. XXV Seite II. Materialien zur Geschichte der Spekulation mit Wertpapieren im 16. Jahrhundert ....................... 384- 387 Nr. 7. Einige Bestimmungen über den Kuxhandel in Sachsen während des 16. Jahrhunderls ...... 384—387 III. Quellen zur Geschichte der Aktiengesellschaft im 16. Jahrhundert 388—404 Nr. 8. Statut der Allgemeinen Eisen-Handelskompagnie zu Steyr (Aktiengesellschaft), 14. September 1581 . . . 388—404 IV. Akten zur Kartellgeschichtedes 14.—17. Jahrhunderts ..... 405—413 Nr. 9. König Karl II. von Neapel befiehlt dem Seneschall der Provence , in des Königs Namen das Salz¬ vertriebssyndikat abzuschließen, das der Pächter der neapolitanischen Salinen in der Provence und der Pächter der königlich französischen Salinen in Aiguesmortes miteinander verabredet hatten. 6. Dezem¬ ber 1301 ..................... 405- 406 Nr. 10. Die bayrische und Tiroler (österreichische) Regierung als Monopolinhaber der Reichenhaller bzw. Haller (Inntal) Salinen und des dortigen Salzhandels schließen ein Kartell, in welchem sie die Preise und Konditionen festlegen, unter denen sie an die Groß¬ händler verkaufen dürfen. 5. August 1649. Mit späteren Zusagen ................. 406- 411 Nr. 11. Kartellvertrag der bayrischen und Tiroler (öster¬ reichischen) Regierung als Monopolinhabern der Reichenhaller bzw. Haller (Inntal) Salinen und des dortigen Salzhandels mit den Pächtern der burgundisdien Salinen. 3. November 1659 ...... 411—413 V. Dokumente der kapitalistischen Wirtschaftsorganisation im sächsi¬ schen und böhmischen Zinnhandel des 16. Jahrhunderts. . . . 414 —457 Nr. 12. Herzog Georg von Sachsen bestätigt die Gesellschaft des Zinnhandels 6. März 1498 ........... 414—415 Nr. 13. Aus dem Gutachten des Dr. Tileman Brander über die Gesellschaft des Zinnhandels um 1500 ..... 415—416 Nr. 14. Aus dem Gutachten des Dr. Christoph Kuppener über die Gesellschaft des Zinnhandels um 1500 ..... 416—418 Nr. 15. Das Monopolprivileg Herzog Georgs von Sachsen für die Gesellschaft des Zinnhandels. 14. September 1500 418- 420 Nr. 16. Projekt einer großen sächsischen Zinnkaufsgesell¬ schaft und eines Kartells mit den Beherrschern der böhmischen Zinnproduktion. 1518 ......... 420—424 Nr. 17. Privileg des Herzogs Georg von Sachsen für die Ge¬ sellschaft des Zinnkaufs. 20. Dezember 1520. . . . 424—427 Nr. 18. Konvention zwischen den sächsischen Fürsten, den Schlick von Joachimsthal und den Pflug v. SchlakkenWalde zur Regulierung des Angebots und der Nachfrage von Arbeitern auf den Bergwerken der Kontrahenten usw. 1521 ............. 427—430 XXVI Inhaltsverzeichnis. Seite Nr. 19. Herzogliche Bestätigung des Dienstvertrages, durch den die Gesellschafter des Zinnkaufs den Hans Alnpeck jun. zu ihrem Faktor annehmen. 4. März 1521 430—431 Nr. 20. Die Leipziger Kaufleute Michael Puffler und Ulrich Mordeisen verabreden mit den Gewerken von Geyer, Thum und Ehrenfriedersdorf einen Zinnkauf auf drei Jahre und eine Veriagsgewährung (zinslos) von 600 fl. pro Jahr. 24. April 1525 .......... 431—432 Nr. 21. Vorschläge eines Leipziger Bürgers (?), wie den Leipziger Kaufleuten die Herrschaft über den mittel¬ europäischen Metallhandel verschafft werden könne. Um Michaelis 1527 ................ 432—435 Nr. 22. Herzog Georg nimmt auf drei Jahre den Zinnkauf in Altenberg, Lauenstein und Bärenstein in seine Ver¬ fügungsgewalt. 27. Mai 1538 ........... 435—436 Nr. 23. Herzog Georg übergibt Michael Puffler den Zinnkauf auf dem Altenberg, Bärenstein und Lauenstein auf drei Jahre (nur die Alnpeck ausgenommen). 26. Mai 1538 ................... 436- 437 Nr. 24. Herzog Georg macht seinen Bergbeamten von der Verleihung des Zinnkaufs an Michael Puffler Mit¬ teilung, befiehlt den öffentlichen Anschlag der Ver¬ ordnung und den Schuß und die Unterstützung Pufflers bei der Ausübung seines Privilegs. 26. Mai 1538 ................... 437—438 Nr. 25. Herzog Georg vermittelt zwischen Michael Puffler und den Gewerken von Geyer , Ehrenfriedersdorf und Thum einen Zinnkauf auf drei Jahre. 29. Mai 1538 ................... 438 Nr. 26. Verlagskontrakt Michael Pufflers mit einigen Zinn¬ gewerken zu Altenberg. 1539 .......... 438—440 Nr. 27. Michael Puffler beklagt sich bei Herzog Heinrich von Sachsen, daß einige Altenberger Zinngewerken, die er verlegt hat, ihr Zinnprodukt ihm nicht übergeben wollen. 1541 ................... 440—441 Nr. 28. König Ferdinand überträgt dem Augsburger Bürger Konrad Mayr das böhmische Zinnhandelsmonopol auf drei Jahre. 6. Dezember 1549 ......... 441—446 Nr. 29. Errichtung eines Zinnkaufs zwischen den Gewerken des Altenberger Radschachts und den Leipziger Zinnhändlern. 6. Mai 1562 ............ 446—451 Nr. 30. Kurfürst August verspricht dem Hans Jenitz, Hans Harrer, Wolf Prager , Hans Biener und Hieronymus Krahwider nach Ablauf des Kontraktes mit den Leip¬ ziger Zinnhändlem den Zinnkauf aus dem Altenberger Radschacht auf acht Jahre unter der Bedingung, daß sie den Dr. Blasius Grunwald und Dominikus Breun aus Leipzig in die Zinnkaufsgesellschaftauf¬ nehmen. 24. August 1564 ............. 451—452 Inhaltsverzeichnis. Nr. 31. XXVII Seite Zinnkaufs, bestehend aus den in der vorigen Urkunde genannten Mit¬ gliedern, schließt mit den Gewerken des Altenberger Radschachts wiederum einen Zinnkauf auf zwei Jahre ab. 27. November 1566 ........... 452—457 Die Gesellschaft des Altenberger VI. Dokumente der kapitalistischen Wirtschaftsorganisation im Idrianer Quecksilberhandeldes 16. Jahrhunderts ........ 458- 475 Nr. 32. Ferdinand I. und die Gewerken von Idria schließen mit Ambrosius und Hans Höchstetter &Co. aus Augs¬ burg einen Quecksilber- und Zinnobermonopolkon¬ trakt. 1. Januar 1525 ............... 458—463 Nr. 33. Verabredung eines idrianiscben Quecksilberhandelsmonopols zwischen König Ferdinand, den Gewerken von Idria und Hans Baumgartner. 1. August 1539 463—474 Nr. 34. Hans Langenauer, der Chef der Firma David Haug, Hans Langenauer & Co. zu Augsburg ersucht seinen englischen Faktor Hans Loner, von der Königin Elisabeth eine größere Geldsumme zu verschaffen, da sonst die Firma über ihren englischen Bergwerksuntemehmungen fallieren müsse. 22. September 1570 474—475 Orts- und Personenregister .............. 476- 486 Nachträge bei Gelegenheit der zweiten Auflage des voran¬ stehenden Werkes .................... 487—521 1. Das Kupfersyndikat der Firmen Fugger und Manlich (ge¬ schlossen im Jahre 1548) mit Ergänzungen zum Kartell der Fugger-Höchstetter vom Jahre 1515 ........... 489—502 2. Die Fugger und das mitteleuropäische Zinnmonopolisierungs¬ projekt vom Jahre 1550 ff .................. 503—512 3. Einige Ergänzungen zum 5. Kapitel des 3. Buches, betitelt: Monopole und Kartelle im Idrianer Quecksilberhandel des 16. Jahrhunderts ....................... 513—521 Orts- und Personenregister der Nachträge bei Gelegenheit der zweiten Auflage .................. 522—523 XXVIII Verzeichnis der zitierten wichtigeren Literatur. I. Quellenwerke. Acta historica res gestas Poloniae illustrantia (1507—1795) Tom. VIII. Leges, privilegia et statuta civitatis Cracoviensis. I. 2. ed. Piekosiiiski, Franciscus. Acta Tirolensia III. 1. Quellen zur Geschichte des Bauernkrieges in Deutsch¬ tirol, 1525. I. Teil. Quellen zur Vorgeschichte des Bauernkrieges: Beschwerdeartikel aus den Jahren 1519—1525. Innsbruck 1908, ed. 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Schneid , J., Eck und das kirchliche Zinsverbot. In : Histor. polit. Blätter. Bd. 108 (1891). Schneider , Georg, Die finanziellen Beziehungen der florentinischen Bankiers zur Kirche von 1285 bis 1304. Leipzig 1899. Schulte, A ., Geschichte des mittelalterlichen Handels und Verkehrs zwischen Westdeutschland und Italien, mit Ausschluß von Venedig. 2 Bde. Leipzig 1900. Derselbe, Ein » wichtiger Fund zur Handelsgeschichte« in: Zeitschrift für Geschichte des Oberrheins. Neue Folge. Bd. 27 (1912), S. 33 ff. Scott, W . R., The Constitution and finance of an english copper mining Company in the sixteenth and seventeenth centuries: being an account of »the society of the mines royal« in der Vierteljahrsschrift für Sozialund Wirtschaftsgeschichte. 5 (1907), S. 525 ff. Sieveking, H ., Genueser Finanzwesen mit besonderer Berücksichtigung der Casa di S. Giorgio. I. Bd. Genueser Finanzwesen vom 12. bis 14. Jahrhundert. Freiburg i. B. 1898. II. Bd. Die Casa di S. Giorgio. Daselbst 1899. Soetbeer, Ad ., Edelmetallproduktion und Wertverhältnis zwischen Gold und Silber seit der Entdeckung Amerikas bis zur Gegenwart. 57. Er¬ gänzungsheft zu Petermanns Mitteilungen. Gotha 1879. Sombart , Werner, Der moderne Kapitalismus. 2 Bde. Leipzig 1902. Derselbe, Das Proletariat, Bilder und Studien. In : Die Gesellschaft Sammlung sozial-psychologischer Monographien, herausgegeben von Martin Buber. Derselbe, Die Juden und das Wirtschaftsleben. Leipzig 1911. Derselbe, Die deutsche Volkswirtschaft des 19. Jahrhunderts. Berlin 1903. Derselbe, Krieg und Kapitalismus. Leipzig 1913. Srbik , Heinr. v ., Der staatliche Exporthandel Österreichs von Leopold I. bis Maria Theresia. Wien und Leipzig 1907. Steinbeck , Aemilius. Geschichte des schlesischen Bergbaues. 2 Bde. Breslau 1857. Sternberg , Graf Kaspar, Umrisse einer Geschichte der böhmischen Bergwerke. 2 Bde. in 4 Abteilungen. Prag 1836—1838. Stieda, W ., Ältere deutsche Kartelle. In : Schmollers Jahrbuch XXXVII (1913), S. 725 ff. Straccha , Benv, Tractatus de mercatura seu mercatore, benütjt die Ausgabe Venedig 1575. Strieder , Jakob, Zur Genesis des modernen Kapitalismus. Forschungen zur Entstehung der großen bürgerlichen Kapitalvermögen am Ausgang des Mittelalters und zu Beginn der Neuzeit, zunächst in Augsburg. Leipzig 1904. Derselbe, Die sogenannte Füriegung, eine Institution des deutschen Ge¬ sellschaftsrechts im Zeitalter des Frühkapitalismus, in: Vierteljahrs¬ schrift für Sozial- und Wirtschaftsgeschichte X (1912), S. 521 ff. Derselbe, Ein Kartell deutscher Kaufleute aus dem Jahre 1743. Histo¬ risches Jahrbuch 1911, S. 49—62. Thorsch, O ., Materialien zu einer Geschichte der österreichischen Staatsschulden vor dem 18. Jahrhundert. Leipziger Disser¬ tation 1891. Verzeichnis der zitierten wichtigeren Literatur. Troeltsch, XXXV W ., Die Calwer Zeughandelskompagnie und ihre Arbeiter. Studien zur Gewerbe- und Sozialgeschichte Altwürttembergs. Jena 1897. Wackernagel, R ., Geschichte der Stadt Basel. Bd. 1—2, 1. Basel 1906—1910. Weber , Max, Zur Geschichte der Handelsgesellschaften im Mittelalter nach südeuropäischen Quellen. Stuttgart 1889. Werner , Karl, Urkundliche Geschichte der Iglauer Tuchmacherzunft. Leipzig 1861. Wiedenfeld , Kurt, Das Persönliche im modernen Unternehmertum. Schmollers Jahrbuch 34 (1910), S. 223 ff. Wygodzinski, W ., Wandlungen der deutschen Volkswirtschaft im 19. Jahrhundert. Köln 1907. Derselbe, Einführung in die Volkswirtschaftslehre. Leipzig 1912. Yver, G., Le commerce et les marchands dans l’Italie méridionale au XHIe et XlVe siècle. Paris 1903. Zippel, G., L’allume di Tolfa e il suo commercio. In: Archivio délia R. Société Romana di Storia Patria. Vol. XXX (1907). Z y ch a , A., Zur neuesten Literatur über die Wirtschafts- und Rechts¬ geschichte des deutschen Bergbaus. Vierteljahrsschrift für Soz. und Wirtschaftsgeschichte. Bd. 5 (1907), S. 238ff.; Bd. 6 (1908), S. 85ff. Derselbe, Das böhmische Bergrecht des Mittelalters auf Grundlage des Bergrechts von Iglau. 2 Bde. Berlin 1900. Erstes Buch. Montanindustrie und Frühkapitalismus. Strieder , Studien z. Gösch , kapitalist . Organisationsformen. 3 Erstes Kapitel. Die quantitative Bedeutung des Bergbaues und Erzhandels für die Entwicklung der deutschen Volkswirtschaft am Ausgang des Mittelalters und zu Beginn der Neuzeit. Wer sich mit der Geschichte des deutschen Frühkapitalismus beschäftigt , wie es der Verfasser seit einer Reihe von Jahren tut, der wird mit einer inneren Notwendigkeit dazu gezwungen , den wirtschaftlichen Verhältnissen im deutschen Bergbau eine inten¬ sive Beachtung zu widmen . Der Bergbau und der Erzhandel machten seit den letzten Jahrzehnten des 15. und in der ersten Hälfte des 16. Jahrhunderts durchaus den bedeutendsten Zweig der Wirtschaft des Heiligen Römischen Reichs Deutscher Nation aus . Es war nicht zuviel gesagt , wenn Karl V. in einem Mandat vom 13. Mai 1525 1die Bergwerke die größte Gabe und Nutzbar¬ keit nannte , „so der Almechtig teutschen Landen mitgetailt“ 2. Es war sicherlich auch nicht übertrieben , wenn der Kaiser den Wert der jährlichen Produktion aus den Gold-, Silber-, Kupfer -, Zinn-, Quecksilber-, Blei-, Eisen- usw. Bergwerken des Heiligen Römischen Reiches Deutscher Nation auf mindestens 2 000 000 Gold¬ florin schätzte . Und es war eher zu niedrig als zu hoch gegriffen, wenn Karl V. in dem genannten Mandat vom Jahre 1525 die in 1 Das Mandat ist (unvollständig) abgedruckt: M. Jans e n, Studien zur Fuggergeschichte. 3. Heft. Jakob Fugger der Reiche. Studien und Quellen I. Leipzig 1910, S. 400 ff. Vollständig, aber vielfach korrumpiert bei: F. A. Schmidt, Chronologisch-syste¬ matische Sammlung der Berggesetze d. österr. Monarchie. 3. Abtlg. Wien 1839, Nr. 52. Vgl. jetzt den Abdruck der neuaufgefnndenen Originalurkunde bei mir im Anhang. 2 Wir können hier darauf verzichten, die vielen Stimmen jener Zeit hören zu lassen, die sich in begeisterter Weise über die hohe Bedeutung des deutschen Bergbaues im 16. Jahrhundert vernehmen ließen. 1 4 Erstes Buch : Montanindustrie und Frühkapitalismus. der Bergbau- und der Hüttenindustrie beschäftigten Gewerbe¬ treibenden des Heiligen Römischen Reiches mit 100 000 bezifferte L Der Versuch, über die kaiserliche Schätzung hinaus die Höhe der Metallproduktion im Heiligen Römischen Reiche Deutscher Nation während des 15. und 16. Jahrhunderts statistisch einiger¬ maßen genau anzugeben, wird — mit untauglichen Mitteln nur zu unternehmen — unterbleiben müssen. Soetbeer hat wenigstens die deutsche Edelmetallproduktion jener Zeit zu schätzen versucht. Es muß hier genügen, auf seine Übersichten hinzuweisen 2. Wenn zwar neuerdings die Aufstellungen Soetbeers, für ihren spanischamerikanischen Teil der gewaltigsten Überschätzung überführt 3, 1 Zum Vergleich, obwohl selbstverständlich die Zahl in Karl V. Mandat nur einen Schätzungswert hat : Die deutsche Gewerbestatistik zählte nach dem ungeheuren Aufschwung in den siebziger Jahren (Gründerzeit ) 1882 430 134 im Bergbau, Hütten - und Salinenwesen Tätige. 2 Ad. Soetbeer, Edelmetallproduktion und Wertverhältnis zwischen Gold und Silber seit der Entdeckung Amerikas bis zur Gegenwart . 57. Ergänzungsheft zu Petermanns Mitteilungen . Gotha 1879, S. 107 f. 3 Durch F. de Laiglesia, Los caudales de Indias en la primera mitad dei Siglo XVI Madrid 1904. Der spanische Autor gelangt auf Grund ausgezeichneten Quellenmaterials (die Ab¬ rechnungen der spanischen Kroneinnahmen aus den amerikanischen Kolonien, im Indienarchiv zu Sevilla befindlich) zu folgenden Sum¬ men im gegenwärtigen Geldwerte: 1509—1514 ......... 995 925 Pesetas 1516—1523 ......... 634 210 1525 2 121 460 1526—1529 ......... 943 152 1530—1540 ......... 3 110 896 1541—1546 ......... 2 419 840 1547—1550 ......... 1 225 312 1551—1555 ......... 10 145 760 Summa : 21 596 555 Pesetas = 17 277 244 Mark. Nach Soetbeer dagegen betrug ungefähr in derselben Zeit die spanischamerikanische Edelmetallproduktion nach gegenwärtigem Geldwert gerechnet 1521—1544 ......... 13 645 000 Mark 1545—1560 ....... . . 59 549 000 „ Summa :73 194 000 Mark. Das ist nicht ein relativ geringes Abweichen, sondern Ergebnisse, die sich nicht vereinigen lassen. Erstes Kapitel: Bergbau, Erzhandclu. quantitative Wirtschaftsentwicklung . 5 auch in ihrer Gesamtheit an Wert bedeutend verloren haben , so dürfte doch die aus den genannten Tabellen ersichtliche Tatsache bestehen , daß bis um das Jahr 1545 etwa (d. h. bis zum Aufkommen der Silberminen von Potosi und der starken Steigerung der peru¬ anischen und mexikanischen Produktion ) die Silberproduktion im Heiligen Römischen Reiche die Gesamtproduktion der übrigen Erdteile übertraf . Noch erheblicher war das Übergewicht Deutsch¬ lands in anderen Metallen , besonders in Kupfer . Das bedeutete einen um so größeren Gewinn für die deutsche Volkswirtschaft des 16. Jahrhunderts , als zweifellos eine enorme Preissteigerung gerade dieses Metalles damals statthatte 1. Nach Dobels Forschungen 2 verkauften die Fugger ihr Tiroler Kupfer im Jahre 1527 den Zentner für 5 fl. 45 kr . bis 6 fl. 15 kr. „ „ 1537 ..... . 6 „ 50 „ „ 7 „ 45 „ ,, ,, 1556 ,, ,, ,, 10 ,, — ,, ,, 11 ,, 45 ,, », »» 1557 ,, ,, ,, 11 ,, - ,, ,, 12 ,, ,, Auch die Preise für das ungarische Kupfer der Fugger erlebten im Verlaufe der ersten Hälfte des 16. Jahrhunderts schon eine mächtige Steigerung . Dachkupfer z. B., das im Jahre 1536 pro Zentner 5 }Z ö - kostete , stieg bis zum Jahre 1547 auf 11 fl. Der Preis von Libether Kupfer erhöhte sich von 4 fl. auf 9— 11 fl. usw. 3. Ähnliche Preiserhöhungen für Kupfer lassen sich auch aus den un¬ veröffentlichten Handlungsbüchern anderer süddeutscher Firmen ablesen . Auf die Werterhöhung anderer Produkte der deutschen Bergwerke , besonders Zinn und Quecksilber , kommen wir noch in einem späteren Zusammenhänge zu sprechen . Hier genüge der flüchtige Hinweis , daß im Verlaufe des 16. Jahrhunderts die Er¬ zeugnisse des deutschen Bergbaues (mit Ausnahme des Silbers) an Wert gewannen , mithin dem deutschen Kapital ein bedeutender Anreiz gegeben war , diese Schätze so intensiv wie möglich auszunutzen. 1 Verursacht wohl besonders durch die erhöhte Nachfrage nach Kupfer für Kriegsmaterialien . Cfr . W . Sombart, Krieg und Kapitalismus . Leipzig 1913 , S. 104. ! Fr . Dobel, Über den Bergbau und Handel des Jakob und Anton Fugger in Kärnten und Tirol (1495— 1560 ). Zeitschr . d . histor. Vereins für Schwaben -Ncuburg , Bd . 9 (1882 ), S. 207. 3 E . Fink, Die Bergwerksunternehmungen der Fugger in Schlesien . Zeitschr . d. Vereins f. Gesch . u . Altertum Schlesiens. Bd . 28 (1894 ), S. 324. 6 Erstes Buch : Montanindustrie und Friihkapitalismus. Der glänzend entwickelte Bergbau des Heiligen Römischen Reiches, der der gesamten deutschen Volkswirtschaft einen ge¬ waltigen Vorsprung gegenüber anderen Ländern gab, kam nun manchen deutschen Landschaften, Städten und Personen besonders zugute. Die Blüte einzelner deutscher Landschaften und Fürsten¬ tümer beruhte durchaus auf dem Bergbau. Tirol blühte schon im 15. Jahrhundert besonders durch seinen Silber- und Kupfer¬ segen, desgleichen Sachsen und die Gegend am Harz. Steiermark machte der Eisenstein des steirischen Erzberges bei Leoben reich usw. usw. Die fortgeschrittene sächsische Volkswirtschaft des 16. Jahrhunderts , wie sie Georg der Bärtige anbahnte und August dann zur Blüte brachte, ist nicht denkbar ohne das Silbererz, ohne das Zinnerz usw., das in den sächsischen Bergen wuchs. Auf dieser wirtschaftlichen Blüte wiederum erwuchs Sachsens hervorragende politische Stellung unter den deutschen Staaten des 16. Jahr¬ hunderts. Ebenso können wir uns das Emporsteigen des Hauses Habsburg zur Weltmacht um die Wende des 15. und lb . Jahr¬ hunderts nur schwer denken ohne den Silberstrom, der aus Tirol fließend, die habsburgischen Söldnerscharen bezahlte, und ohne die gewaltigen Mengen edler und unedler Mineralien, die in den habs¬ burgischen Ländern gefunden wurden. Einzelne kleinere Herren, wie die Grafen von Mansfeld, die Grafen Schlick, die Pflug, die Herzoge von Münsterberg, erlangten auf Grund des Erzreichtums ihrer Ländchen und Herrschaften ein Ansehen und eine Macht, die in keinem Verhältnis zur Größe ihrer Gebiete stand. Hunderte deutscher Städte und von deutschen Bergleuten gegründete oder emporgebrachte außerdeutsche Städte verdanken ihr Entstehen und ihr Wachstum unmittelbar dem Bergsegen. Mittelbar aber nehmen alle deutschen Städte — die einen mehr, die anderen weniger — an der Ausbeute teil. Was die deutschen Kaufleute des 15. und 16. Jahrhunderts im Bergbau und im Handel mit Bergwerksprodukten verdient haben, geht in die Millionen, ja man darf sagen in die Milliarden. Dabei muß aber noch bei der Beurteilung der statistischen Angaben in dem obengenannten Er¬ lasse Karls V. das Folgende berücksichtigt werden: Weit über die Grenzen des Heiligen Römischen Reiches Deutscher Nation hinaus erstreckte sich die Betätigung deutschen Kapitals und deutscher Unternehmungslustim Bergbau und Erzhandel. Im spanischen Erstes Kapitel: Bergbau, Erzhandelu. quantitative wirtschaftsentwicklung. 7 Quecksilber- und Silberbergbau des 16. Jahrhunderts spielten die Fugger eine ganz hervorragende Rolle l. Die Welser griffen sogar in den Bergbau der spanischen Kolonien tätig ein 2. Wie im 14. und 15. Jahrhundert die hansischen Kaufleute, so wurden im 16. Jahrhundert die süddeutschen Großhändler für den schwe¬ dischen Kupferbergbau als Gewerken und Abnehmer von Be¬ deutung 3. Ebenso stand es in England. Auch dort hatte hansische Unternehmungslust schon seit dem 14. Jahrhundert große Gewinne, besonders aus dem hervorragenden Zinnbergbau, geschöpft4. Dann waren Metalle, besonders Blei und Zinn, immer bedeutende Artikel des hansischen Handels aus England gewesen. Im 16. Jahrhundert blickte die englische Regierung mit Vorliebe nach Süddeutschland, wenn cs galt, auswärtiges Kapital und fremde Unternehmungslust für die Ausdehnung des englischen Bergbaues und Erzhandels nutzbar zu machen 5. Hervorragend war auch 1 Vgl . besonders K. H ä b 1e r , Die Geschichte der Fuggerschen Handlung in Spanien . Weimar 1897. 2 K. Häbler, Die überseeischen Unternehmungen der Welser und ihrer Gesellschafter. Leipzig 1903, S. 50. 3 M . Jansen, Jakob Fugger der Reiche . S. 58, 123. 4 So überließ am 25. Juni 1347 Kronprinz Eduard dem Kölner (Dortmunder ) Kaufmann Tidemann von Limberg gegen ein Dar¬ lehn von 3000 Pfund Sterling auf drei Jahre das Monopol für den Zinnhandel in Cornwall und Devonshire, sowie den Schlagschatz der Zinnbergwerke in Cornwall. Cfr. Joseph Hansen, Der englische Staatskredit unter König Eduard III . (1327—1377) und die hansischen Kaufleute . Zugleich ein Beitrag zur Geschichte des kirchlichen Zinsverbotes und des rheinischen Geldgeschäftes im Mittelalter . In : Hansische Geschichtsblätter 16 (1910), S. 391. 5 Cfr . R . Ehrenberg, Das Zeitalter der Fugger. Geldkapital und Kreditverkehr im 16. Jahrhundert . 2 Bde. Jena 1896. Anastati¬ scher Neudruck Jena 1912. I, S. 217 f., S. 234. Derselbe, Ham¬ burg und England im Zeitalter der Königin Elisabeth . Jena 1896, S. 4 f. Dazu W. R . Scott, The Constitution and finance of an english copper mining company in the sixteenth and seventeenth cen¬ turies : being an account of “the society of the mines royal ’’ in Vierteljahrsselirift für Sozial- und Wirtschaftsgesch . 5 (1907), S. 525 ff. Zu den verfehlten Bergbauspekulationen der Augsburger Firma David I-Iaug, Hans Langenauer &Co. vgl. jetzt auch W. G. Collingw o o d , Elizabethan Keswick. Extracts from the original account books 1564—1577 of the german miners in the archives of Augsburg. Cumberland and Westmorland antiquarian and archaeological society, Tract series Nr. VIII , Kendal 1912. 2 5 8 Erstes Buch : Montanindustrie und Frühkapitalismus. seit altersher die Betätigung deutschen Kapitals im böhmischen, polnischen 1und ungarischen Bergbau und Erzhandel. Jn Ungarn, das erst 1527 zum Heiligen Römischen Reich kam, hatten im frühen Mittelalter deutsche Bergknappen den Bergbau recht eigentlich erst zu systematischem Betriebe erhoben. Dann waren hansische* und seit dem 15. Jahrhundert in wachsendem Maße süddeutsche Kaufleute 3 die bedeutendsten Abnehmer der Produkte des ungari¬ schen Bergbaues geworden. Eine neue glänzende Epoche des un¬ garischen Bergbaues — und damit auch der deutschen Arbeit dort — begann im letzten Viertel des 15. Jahrhunderts. Wahrscheinlich war es die in der zweiten Hälfte des 15. Jahrhunderts in Sachsen 4, in Tirol 8, im Mansfeldischen 6 und sonst in großem Stil anhebende bergbauliche Tätigkeit , die auch in den Karpathen einen neuen Aufschwung des Bergbaues veranlaßte. Diese Hebung des ungari¬ schen Bergbaues verknüpfte sich mit der Person eines Krakauer Bürgers und Ratmannes, Johann Thurzo mit Namen 7. Thurzo 1 Besonders für Blei cfr. M. Jansen, Jakob Fugger der Reiche , S. 141. Polen als Bleigewinnungsland auch genannt in „Die drei Flugschriften über den Münzstreit der sächs. Albertiner und Ernestiner um 1530" . Ed . W. L o t z , Leipzig 1893, S. 72/3. 2 E . R . Daenell, Die Blütezeit der deutschen Hanse . 2 Bde. Berlin 1906. I. Bd., S. 61 f., 89, 95. Die Hansen (bes. Kaufleute aus Thorn ) waren auch über Krakau hinaus direkt in den ungarischen Bergbau vorgedrungen , bis die polnischen Könige, Krakaus Stapelrecht aufrichtend , den Hansen das Vordringen über diese Stadt hinaus verwehrten. 3 Schon in der ersten Hälfte des 15. Jahrhunderts begegnen uns Nürnberger Firmen im ungarischen Bergbau . J o h. Falke, Die Geschichte des deutschen Handels . 2 Teile. Leipzig 1859—60. II, S. 49. Schon im 14. Jahrhundert erwähnt sie dort E. R . Daenell, Der Ostseeverkehr und die Hansestädte von der Mitte des 14. bis zur Mitte des 15. Jahrhunderts . Hansische Geschichtsblätter Jahr¬ gang 1902, S. 44 ff. 4 O . Hoppe, Der Silberbergbau zu Schneeberg bis zum Jahre 1500. Heidelberger Dissertation 1908, S. 8. 5 Archiv f. österr . Geschichte 53. Bd., S. 343. A. Z y c h a , Zur neuesten Literatur über die Wirtschafts - und Rechtsgeschichte des deutschen Bergbaues . Vierteljahrsschrift für Soz. u. Wirtschaftsgesch . Bd . 5 (1907), S. 238 ff. ; Bd . 6 (1908), S. 8.5 ff. 6 W. Möllenberg, Die Eroberung des Weltmarktes durch das Mansfeldische Kupfer . Gotha 1911, S. 10. 7 Jansen, Jakob Fugger der Reiche, S. 132. Auch Zycha a. a . O. Bd . 6 (1908), S. 114. Erstes Kapitel : Bergbau , Erzhandel u. quantitative Wirt Schaftsentwicklung . 9 scheint einer jener Bergsachverständigen gewesen zu sein, wie sie uns im 15. Jahrhundert öfters begegnen . Ein Mann , der es ver¬ stand , mit Hilfe einer Wasserkunst ersoffene Gruben wieder gang¬ bar zu machen , der aber zugleich auch die Kunst des Metallscheidens, des Saigerns 1 zu handhaben wußte . Thurzo begegnet uns ungefähr um dieselbe Zeit , als er im ungarischen Bergbau auftaucht , auch in Goslar. Das Goslarer Stadtarchiv besitzt eine Reihe Urkunden, die uns Johann Thurzo schon in den siebziger Jahren des 15. Jahr¬ hunderts als angesehenen Bergherrn zeigen . So beurkunden am 18. September 1478 vor Rat und Gemeinde zu Goslar Gewerken des Rammeisberges (mit Namen genannte Bürger der Stadt ), daß sie für die Zukunft jeder die Hälfte seines Teiles unter der Trostes¬ fahrt an Johann Thurzo , Bürger und Ratmann zu Krakau , an Johann Koler 2 aus Nürnberg und Johann Pedek aus Bautzen ab¬ getreten haben . Die Abtretung ist dafür geschehen , daß Thurzo ein Verfahren in Anwendung brachte , mit dessen Hilfe man die kupferhaltigen Erze des Berges zu Kupfer scheiden konnte , statt sie wie bisher in unrationeller Weise mit dem Bleierz zu ver¬ schmelzen 3. Koler und Pedek stellten bald ihre Zubußezahlungen 1 Die Kunst , mit Hilfe von Blei das Silber aus den silberhaltigen Kupfererzen zu ziehen (Saigerverfahren ), die nach der Mitte des 15. Jahrhunderts allgemeiner bekannt wurde , bedeutete das wichtigste technische Ereignis in der Geschichte des Kupferbergbaues , dessen Wert , Ertrag und Produktion damit eine gewaltige Steigerung er¬ fahren hat . W . M ück, Der Mansfelder Kupferschieferbergbau in seiner rechtsgeschichtlichen Entwicklung . Bd . 1. Geschichte des Mansfelder Bergregals ; Bd . 2 : Urkunden des Mansfelder Bergbaues. Eisleben 1910 , I . Bd . S. 57. 2 Ein Joh . Koler erscheint um 1489 im Dienste der Fugger ; vgl. M. Jansen, Studien zur Fuggergeschichte , herausgegeben von Max Jansen . I. Heft : Die Anfänge der Fugger (bis 1494), Leipzig 1907 , S. 53 und sonst . Es wäre sehr interessant zu wissen , ob das ein und dieselbe Person ist . Vielleicht läßt es sich mit Hilfe des Wappens feststellen . Der Mitgewerke Thurzos hat einen Kreis im Wappenschild, auf der Wappenzier einen Kreis , aus dem oben Strahlen ausgehen. Vgl . Siegel in einer Urk . vom 20. Sept . 1478 , Goslarer Stadtarchiv. 3 Stadtarchiv Goslar , Urkunde Nr . 863 . . . „ darum dat syn der Lydt de Ramelsbergk geweldiget wart de kopperertz de uth dem berge g'ekom (m )en syn (nicht eyn kleyne menige sunde vele ) nymands to nutte gekomen syn , sundern manck dem blygertz mit grotem schaden verschmulten . Welche kopperertz der ergute Johan Tursso mit synen medebenomten dorch hulpe godes unde ore kunst willen * 6 * 10 Erstes Buch: Montanindustrie und Frühkapitalismus. zu dem Unternehmen ein 1, Thurzo brachte eine neue Gewerk¬ schaft zusammen2, trat aber kurz darauf seine Rechte an Ulrich Schütz, den berühmten Chemnitzer Gewerken, Hans Leimbach, den Zehntner und Rentmeister der Herzoge von Sachsen, Merten Bauer aus Leipzig u. a. ab 3. Mit Johann Thurzo vereinigten sich in den neunziger Jahren des 15. Jahrhunderts die Fugger zur Pacht und Ausbeutung der Neusohler Silber- und Kupfergruben 4 in einer besonderen. Handels¬ gesellschaft, die in den Fuggerschen Handelspapieren „der gemeine ungarische Handel“ genannt wird 5. Über 30 Jahre blieben die Gesellschafter beisammen. Erst nach der Katastrophe, welche die nationale ungarische Eifersucht u. a. der Gesellschaft im Jahre 1525 bereitete 9, übernahmen die Fugger allein die Pacht der Neu¬ sohler Bergwerke7. Das ungarische Geschäft behielt auch jetzt noch als sog. ungarische Handlung neben der „gemeinen Handlung“ der Fugger eine selbständige Verwaltung. An Sorgen fehlte es dabei nicht. Die Türkengefahr, die Eifersucht der Ungarn auf die Deutschen, der Neid, häufige Streiks der Bergarbeiter usw. ließen die ungarische Unternehmung nicht zu ruhigem Fortgang kommen. Im Jahre 1546 hielt es Anton Fugger , der damalige Chef des Welt¬ handelshauses, für geraten, „bei diesen allersorglichsten und ge¬ fährlichsten Zeiten und Läufen“ König Ferdinand den Pacht¬ vertrag zu kündigen. Der Augsburger Handelsherr mochte erleichtert to gude und kopper daruth maken .“ Den ungarischen Berg¬ werken stellte Hans Thurzo schon 1475 seine technischen Kennt¬ nisse zur Verfügung . M. Jansen, Jakob Fugger der Reiche, S. 132. 1 Vgl. Urk . 891 des Goslarer Stadtarchivs. 2 Vgl. Urk . 890 daselbst. 3 Vgl. Urk . 903 und 951 daselbst. ■» Näheres darüber bei M. Jansen, Jakob Fugger der Reiche, S. 133 ff. 5 Vgl . auch das in Buch II Kapitel II , Abschnitt 1 von mir über diese Tochtergesellschaft der Fugger und Thurzo Gesagte. 6 M . Jansen, Jakob Fugger der Reiche , S. 160 ff ., besonders S. 179 ff. 7 Ein interessantes Privileg König Sigismund I. von Polen (10 . April 1527 ) mit dem den Fuggern die Abmachungen bestätigt werden, die sie mit der Stadt Krakau getroffen hatten , bei Franziscus Piekosinski, Acta historica res gestas Poloniae illustrantia (1507 — 1795) Tom . VIII Leges , privilegia et statuta civitatis Cracoviensis . I, 2. Nr . 750. Erstes Kapitel : Bergbau , Erzhandel u. quantitative Wirtschaftsentwicklung . 11 aufatmen , als er der Sorgen um seine Kapitalien in dem von inneren Zwistigkeiten und von äußeren Feinden bedrohten Lande ledig war . Darnach nahm die ungarische Regierung die .Neusohler Berg¬ werke auf einige Zeit in eigene Regie , aber sehr bald begegnen uns wieder große Augsburger Firmen als Pächter . So 1548 Mathias Manlich, dem 1560 die Gesellschafter Melchior Manlicll, Hans Langenauer , Melchior Link und Mitverwandte folgten . 1570 er¬ scheinen die Augsburger Wolfgang Paller , David Weiß & Co. als Pächter der Neusohler Gruben , 1603 Wolfgang Paller neben Bar¬ tholome Caste und Lazarus Henckel von Donnersmark 1. Es war offensichtlich : ohne das deutsche Kapital und ohne die deutsche Unternehmungslust waren die ungarischen wie die meisten anderen Bergwerke der Deutschland benachbarten Länder nicht mit dem möglichst höchsten Nutzen für die betreffenden Landesherren usw. instand zu setzen und instand zu erhalten. Die Geschichte des Eindringens der süddeutschen Kapitalisten in den ungarischen Bergbau ist nun deshalb so interessant und auch deshalb so vergleichsweise breit hier behandelt worden , weil wir wenigstens für die Fugger -Thurzo -Periode einen numerisch faß¬ baren Begriff für die Gewinne geben können , welche die deutschen Kaufleute im Bergbau und Erzhandel des 15. und 16. Jahrhunderts machten . Max Jansen hat auf Grund älterer Untersuchungen von Dobel und auf durchaus zuverlässiges Material gestützt , be¬ rechnet , daß die Fugger in jener Handelsgesellschaft , die sie mit der Familie Thurzo zwecks Ausbeutung der Neusohler Kupfer¬ gruben schlossen , in der Zeit von 1495 bis 1525 anderthalb Millionen Goldgulden verdienten 2. ln Wahrheit betrug der Verdienst der Fugger noch bedeutend mehr . Das ungarische Geschäft war nicht nur ein Kupfergeschäft . In den Saigerhütten in Ungarn , Kärnten und Thüringen gewann der „gemeine ungarische Handel “ auch aus 1 Nähere Literatur zusammengestellt bei H e i n r. v. S r b i k , Der staatl . Exporthandel Österreichs von Leopold I. bis Maria Theresia. Wien und Leipzig 1907, S. 35 Anin. 1. Mathias Manlich & Co. waren schon 1548 den Fuggern in der Pacht von Neusohl nachgefolgt . Vgl. für ihre und ihrer Nachfolger Tätigkeit in Ungarn und Polen auch F. PiekosiAski a. a. O. Nr . 757 (1548), Nr . 762 (1553), Nr. 763 (1553), Nr. 764 (1560); dazu S. 1052, Nr. 770 (1570). 9 M . Jansen, Jakob Fugger der Reiche , S. 152 ff. Auf die Gewinne der Firma im Almadener Quecksilberbergbau komme ich später zu sprechen. 12 Erstes Buch : Montanindustrie und Frühkapitalismus. den ungarischen Rolkupfererzen bedeutende Mengen Silber. Zweifel¬ los kauften nun die Fugger und ebenso die Thurzo, wie sie von ihrer Tochterfirma, dem „gemeinen ungarischen Handel“, Kupfer kauften, auch Silber. Der Verdienst an diesem Verkauf fiel nun wohl der Tochterfirma zu, aber durch den Weiterverkauf des Silbers gewannen auch die zwei Mutterfirmen bedeutende Summen. Leider wissen wir nicht, wie viel Silber (und zu welchem Preise) die Fugger von dem „gemeinen ungarischen Handel“ erwarben. Erst dann könnte der ganze Gewinn geschätzt werden, der den Fuggern unter Jakob dem Reichen aus ihrem Eindringen in den ungarischen Bergbau und aus ihrer Verbindung mit dem Thurzo erwuchs. Um welche Umsätze es sich bei dem Erzhandel der süddeutschen Großhandelsfirmenoft handelte, erkennt man deutlich aus einigen Inventuren Augsburger Kaufleute des 16. Jahrhunderts . Die Inventur der Firma Fugger aus dem Jahre 1527 berechnet ein Warenkonto von insgesamt 380 000 fl. in den einzelnen Faktoreien der Firma. „Die vorhandenen Waren bestanden größtenteils aus Kupfer, wovon in Antwerpen allein für über 200 000 fl. lagerte, sodann auch aus Silber, etwas Messing und ganz wenig Tuch, Damast und sonstigen Geweben1“. In der Bilanz von 1536 ist das Warenkonto auf 415 000 fl. berechnet. Davon fallen 289 000 fl. auf Kupfer, Silber und Messing2. Im Jahre 1546 erreichte das Fuggersche Warenlager einen Wert von 1 250 000 fl. Davon entfallen mehr als 1 000 000 fl. auf Kupfer. An Barchent, dem zweitwichtigsten damaligen Handelsartikel der Fugger war nur für 125 000 fl. vorrätig 3. In ähnlicher Weise wie in dem Fuggerschen Geschäft zeigt sich auch in anderen süddeutschen Großhandelsgesellschaftendes 16. Jahrhunderts bei wachsender Ausdehnung und Bedeutung der Firma ein Zurücktreten der übrigen Handelswaren gegenüber den Metallen4. Vielleicht wird man später einmal, wenn uns eine größere 1 R. Ehrenberg , Zeitalter der Fugger. I, S. 122. Näheres bei Jakob Strieder, Die Inventur der Firma Fugger aus dem Jahre 1527. Tübingen 1905. 2 R. Ehrenberg a. a. O., S. 133. 3 R. Ehrenberg a. a. O., S. 146. 4 Zum Beispiel bei der Firma Anton Haug, Hans Langenauer, Ulrich Link und Mitverwandte. Cfr. J. Hartung, Aus dem Geheim¬ buche eines deutschen Handelshauses im 16. Jahrhundert. Zeitschr. f. Soz. u. Wirtschaftsgesch. Bd. 6. (1898) S. 46. Zweites Kapitel : Bergbau und frühkapitalistische Vermögensentwicklung . 13 Anzahl frühkapitalistischer Handelsbücher im Druck vorliegt, diesen Verlauf als typisch bezeichnen dürfen. Die Ursache für das starke Hervortreten der Metalle als Handelsartikel der ganz großen Firmen liegt im folgenden. Je größer die einzelnen Handelshäuser wurden, um so mehr lenkten sich die Blicke der kaiserlichen, der fürstlichen usw. Finanzagenten, Anleihen suchend, auf sie. Als Äquivalent konnten die Regierungen, namentlich die stets geldbedürftigen Habsburger, unter anderem besonders den Erzreichtum ihrer Länder bieten, über den sie in verschiedenen Formen — wir kommen noch darauf zu sprechen — ein Verfügungs- oder doch Mitverfügungsrecht besaßen. Auf diese Weise gelangten die großen Handelsgesellschaften zugleich mit dem Anleihegeschäft in den Erzhandel, um häufig darin enorme Summen zu verdienen. Vom Erzhandel war dann nur ein Schritt zum Eigenerwerb von Berg¬ werksteilen und Hüttenwerken, also zum Eintritt in die Montan¬ industrie selbst. Zweites Kapitel. Bergbau und frühkapitalistische Vermögens¬ bildung. Die Erkenntnis der Bedeutung des Bergsegens für die gesamte deutsche Wirtschaft des ausgehenden Mittelalters und der be¬ ginnenden Neuzeit hat zu einer neuen These über die Genesis des modernen Kapitalismus Veranlassung gegeben. Bekanntlich sind in den letzten Jahren besonders folgende zwei geschichtliche Pro¬ bleme des modernen Kapitalismus behandelt worden. Einmal hat man sich gefragt: Wie sind die großen bürgerlichen Kapitalver¬ mögen entstanden, auf deren Grund sich in Italien und Flandern schon früh im Mittelalter, in den übrigen Kulturländern gegen Aus¬ gang jenes Zeitalters der Frühkapitalismus aufrichtete ? Und zweitens hat man die Frage aufgeworfen: Welches ist die Genesis des kapitalistischen Geistes, jenes außerordentlichstark entwickelten Erwerbstriebes, der die neue Wirtschaftsordnung schuf, die wir kurz die kapitalistische nennen? Werner Sombart hat die erste der ebengenannten Fragen mit seiner bekannten „ Grundrentenakkumulationstheorie“ beant- 14 Erstes Buch: Montanindustrie und Frühkapitaüsmus. wortet I. Ich habe diese Theorie auf zwei Wegen, auf einem mehr de¬ duktiven und einem mehr induktiven , zu widerlegen versucht 2. Auf de¬ duktivem durch einerechtsgeschichtlicheZurückf ührung der vermögen¬ bildenden Kraft der städtischen Grundrente im Mittelalter auf ihren relativ bescheidenen Stand . Auf induktivem mit Hilfe der kon¬ kreten Vermögensgeschichte der führenden Frühkapitalistenfamilien der handelsgeschichtlich wichtigsten Stadt Deutschlands zu Aus¬ gang des Mittelalters und zu Beginn der Neuzeit (Augsburg ). Als Resultat dieser induktiven Beweisführung ergab sich, daß die über¬ ragende Mehrzahl der großen Vermögen , die in der Hochburg des deutschen Kapitalismus in jener Zeit entstanden und die Grund¬ lage der dortigen frühkapitalistischen Entwicklung bildeten , nach¬ weislich in der Hand von handeltreibenden Emporkömmlingen ge¬ schaffen worden sind . Grundrentenakkumulation hat dabei keine Rolle gespielt . Für Venedig , Florenz und andere Zentren des Früh¬ kapitalismus ist Sombarts Theorie ebenfalls abgelehnt worden. Es dürfte demnach nichts anderes übrig bleiben , als den Handel als die Kraft anzusehen , mit deren Hilfe die großen bürgerlichen Vermögen entstanden , auf denen sich der Frühkapitalismus auf¬ richtete . Freilich nicht der Handel schlechtweg , sondern ein Groß¬ handel , wie er sich in einer Anzahl durch Verkehrslage usw. be¬ günstigter Städte vielfach in Verbindung mit einem Exportgewerbe schon im Mittelalter zu bilden vermochte . Die Schöpfer und Träger dieses Großhandels waren zumeist Emporkömmlinge aus den Kreisen der Handwerker und der Kaufleute älteren , kleineren Stils. In letzter Zeit hat , wie eben erwähnt , die Erkenntnis der Be¬ deutung des Bergsegens für die gesamte deutsche Volkswirtschaft des endenden Mittelalters und der beginnenden Neuzeit zu einer neuen These über die Entstehung der großen Vermögen geführt, auf denen sich der deutsche Frühkapitalismus aufrichtete . Man liât sich gefragt : Ja sind es denn nicht noch mehr als Handelsprofite die Schürfe glücklicher Gewerken gewesen, aus denen sich die Ver¬ mögen oder doch viele von den Vermögen bildeten , auf deren Grund 1 W . Sombart, Der moderne Kapitalismus . 2 Bde . Leipzig 1902 . I. Bd ., bes . S. 218 ff. 2 J a k obStried er, Zur Genesis des modernen Kapitalismus. Forschungen zur Entstehung der großen bürgerlichen Kapitalvermögen am Ausgang des Mittelalters und zu Beginn der Neuzeit , zunächst in Augsburg . Leipzig 1904 , S. 29 ff. 1 Zweites Kapitel: Bergbau und frülikapitalistische Verinügensentwicklung . 15 der deutsche Frühkapitalismus basiert werden konnte ? Die Frage muß meines Erachtens verneint werden. So bedeutsam die Berg¬ werksgewinne manchmal für die Vergrößerung schon vorhandener großer Vermögen wurden, so gering ist die Rolle der unmittelbaren (Edel-)Metallakkumulation bei der ursprünglichen ersten Ver¬ mögensakkumulationx. Und auf letztere kommt es doch in erster Linie an, wenn man der Genesis des Frühkapitalismus bis in die Keime nachforschen will. Schon duich einen Hinweis auf die Entwicklung in Flandern und Italien erhält unsere Verneinung einen hohen Grad von Wahr¬ scheinlichkeit. In Italien und Flandern, wo der europäische Früh¬ kapitalismus zuerst sich breit entfaltete, waren im Lande selbst Bergwerksgewinne in größerem Maßstabe nicht zu machen. Aus dem einfachen Grunde, weil keine bedeutenden Bergwerke dortzu¬ lande vorhanden waren. Und es versteht sich von selbst, daß zu auswärtigen Bergwerksunternehmungen der Flanderer und der Italiener (etwa in der Levante oder im Balkangebiete 2 usw .) bereits beträchtliche Kapitalien nötig waren, deren Ursprung eben zu erklären ist. So bliebe also die Entwicklung in den mit Bergwerken gesegneten Ländern, vor allem also im Heiligen Römischen Reiche Deutscher Nation, aber auch in Spanien (mit seinen bedeutenden Silber- und Quecksilbergruben), in England (besonders Blei- und Zinnbergwerke) usw. auf das genannte Problem zu untersuchen. Da scheiden nun vorerst als Quellen der Vermögensbildung für zunächst arme, aber glückliche Gewerken alle diejenigen Berg¬ werke aus, die als Ganzes von den betreffenden Eigentümern an einzelne große Kapitalisten gegen hohe, im voraus zu zahlende Geld¬ summen verpachtet wurden. Also z. B. die Neusohler Kupfer- und Silbergruben, welche den Fugger-Thurzo 1494 für eine Jahrespacht von 3000 Goldguldcn von dem Bischof von Fünfkirchen verliehen wurden 3. Oder die Almadcner Quecksilberbergwerke, die 1516 ff. 1 Auch Sombart, Moderner Kapitalismus , I, S. 280 L, ver¬ neint im ganzen die Frage der Vermögens b i 1 d u n g durch Berg¬ werksgewinn. 2 „Die serbischen Gold- und Silberbergwerke von Nowobrdo, Janowr und Kratowo waren um das Jahr 1433 für eine jährliche Zahlung von 200 000 Dukaten an die Venetianer verpachtet .“ S o el¬ be e r a. a. O. S. 37. 3 M . Jansen, Jakob Fugger der Reiche , S. 13'2 ff. 16 Erstes Buch : Montanindustrie und Frühkapitalismus. Alfonso Guttierez gepachtet hatte , bis 1525 die Fugger die Pacht auf Jahrzehnte hinaus übernahmen 1. Oder die Tolfaner Alaun¬ gruben , die von Anfang an — wir kommen noch hierauf zu sprechen — durch ihre Eigentümer , die Päpste , kapitalkräftigen Gesellschaftern, den sogenannten societates aluminum , verliehen wurden usw. usw. Die Beispiele ließen sich mehren , die angeführten dürften zum Be¬ lege der These genügen , daß in manchen Bergwerken schon auf Grund der Eigentumsverhältnisse an ein Reichwerden zunächst armer Ge¬ werken durch allmähliche Akkumulation besonders glücklicher Aus¬ beuten nicht zu denken war. In anderen Distrikten war ein Reichwerden der Bergbau enden in dem Bergbau selbst aus folgendem Grunde zumeist unmöglich . In vielen Gebieten war die Ausübung des Berg - und Hüttenbetriebes durchaus zunftmäßig organisiert . So hatten z. B. in dem wichtigsten Eisengewinnungsgebiete zwischen Donau und Main, im Sulzbachischen , die Hammerwerksbesitzer (Hammer¬ meister ) der Städtchen Sulzbach und Amberg bereits 1387 „eine zünftlerisch -kartellartige “ Vereinigung mit Aufteilung der Pro¬ duktion , Festsetzung der Arbeitslöhne usw. geschlossen 2. Auch die Ordnung der Produktion im steiermärkischen Eisenerz gab durchaus ein Seitenstück zur zünftigen Gewerbeordnung , wie sie allgemein aus den Städten des Mittelalters bekannt ist . „Betrieb mit eigenem Rücken , Erforderung des Bürgerrechts (1539), ins¬ besondere aber die Erhaltung der gleich en K leinbet riebe’ nach zünftlcrischem System , rücken die steiermärkischen Rad¬ meister den städtischen Handwerkern einigermaßen nahe . Gleiche 1 K . H übler, Geschichte der Fuggerschen Handlung in Spanien , S. 93 ff., S. 75. 2 Einiges über diese interessante , Jahrhunderte währende Organi¬ sation bietet Ludwig Beck, Die Geschichte des Eisens in lechnischer und kulturgeschichtlicher Beziehung . 5 Bde . Braunschweig 1884/1901 . I. Bd . S. 766 ff. ; IL Bd . S. 666 ff. Ich hatte die Absicht, die kurzen , dort nach Lori (Sammlung des bayrischen Bergrechts 1764) gegebenen Notizen zu einer Monographie der Hammerwerks¬ vereinigung erweitern zu lassen , höre aber , daß von anderer Seite diese hübsche Aufgabe schon übernommen ist. s Z y c h a a . a . O. Bd . 6 (1908 ), S. 88. Vgl . auch für das Schwarz¬ waldgebiet E . G o t h e i n , Wirtschaftsgeschichte des Schwarzwaldcs und der angrenzenden Landschaften . I . Bd . Straßburg 1892, S. 653 f. 1 Zweites Kapitel: Bergbau und frtihkapitalistlsche Vermögensentwicklung . 17 Leistungen unter gleichen Bedingungen , mit gleichem Gewinn, war auch da der Grundgedanke .” Mit Recht zieht Zycha aus dieser Ord¬ nung der Produktion den Schluß : „Darum ist aber auch die wirt¬ schaftliche Lage der Radmeister immer nur eine bescheidene ge¬ wesen. Unter den drei Gliedern des steiermärkischen Eisenwesens haben nur die Eisenhändler Reichtümer sam¬ meln können und nur als H ä n d 1e r zugleich haben es Radund Hammermeister zu Vermögen gebracht x“. Es lassen sich noch eine Anzahl anderer Gründe anführen , die es in manchen Bergbaudistrikten unmöglich machten , daß einzelne besonders glüokliche und tüchtige , zunächst arme Bergbauer i m Bergbau selbst Vermögen akkumulierten , groß genug , um darauf eine kapitalistische Unternehmung zu begründen . Aber in anderen Bergbaugebieten war diese Möglichkeit — durch die Wirt¬ schaftsverfassung wenigstens — theoretisch und praktisch nicht ausgeschlossen . Es soll nicht geleugnet werden , daß in manchen Bergbaudistrikten hie und da einem besonders vom Glück be¬ günstigten , zunächst armen Bergbauunternehmer durch eine Reihe glücklicher Schürfe in seinen Bergwerksteilen eine Reichtumsbildung möglich wurde . Vorausgesetzt , daß der betreffende Glückspilz dann den so erworbenen Reichtum kapitalistisch weiter nutzte — was von der größeren oder geringeren Ausbildung seines Ervyerbstriebes abhing —, so hätten wir hier eine der Quellen von Ver¬ mögen vor uns , auf deren Grund sich der Frühkapitalismus auf¬ richtete. Aber es fragt sich, ob diese Art von Vätern des modernen Kapitalismus häufig genug war , um von der Wissenschaft , die auf die Erkenntnis des typischen Verlaufs (d. h . der Regel und nicht der Ausnahme ) zielt , in Rechnung gestellt werden zu müssen 2. 1 Z y c h a a. a. O., 6. Bd. (1908), S. 88. — So-gelang es auch im Mansfeldischenmanchem Hüttenmeister vorwärts zu kommen, „sich aus dem Kreise der Standesgenossen herauszuheben.“ Sie fingen an, sich selber dem Saiger handel zuzuwenden. Möllenberg a. a. O. S. 31. 2 Für das 19. Jahrhundert werden wir uns die Möglichkeit denken können, daß einmal ein Glückspilz auf einem Lotteriegewinn das Fundament eines kapitalistischen Unternehmens aufrichtete. Trotz¬ dem wird man bei der Frage nach der Herkunft der großen Ver¬ mögen, auf denen sich der Hochkapitalismus des 19. Jahrhunderts aufrichtete, solche seltene Zufälle außer acht lassen dürfen. Strieder , Studien z. Geach . kapitnlist . Or, 'anisfttioneformen . 2 23 18 Erstes Buch : Montanindustrie und Frühkapi talis mus. Verschiedene Momente weisen darauf hin , daß dies nicht der Fall ist . Was wir in den alten Bergstadtchroniken und bei ihren späteren Ausschreibern von dem Reichwerden „glücklicher Fundgrübner“ lesen, muß mit der größten Vorsicht aufgenommen werden . Wir müssen bei jedem Fall uns fragen , ob auch wirklich der Bergbau als solcher und nicht etwa der Handel mit Bergwerksprodukten die Reichtumsgrundlage des Betreffenden — der daneben auch Bergwerksunternehmer gewesen sein kann — schuf . Ferner muß auch in jedem einzelnen Falle untersucht werden , ob der „glück¬ liche Fundgrübner “ nicht schon vermögend war , als er durch den Bergbau reich wurde . Ob also hier wohl eine Kapitalver¬ mehrung , eine Kapitalvervielfachung , aber nicht eine Kapital¬ bildung durch bergbauliche Produktion vorliegt . In dieser Beziehung hat neuerdings 0 . Hoppe darauf hingewiesen, daß Martin Römer und andere hervorragende sächsische Gewerken schon vermögend waren , als sie in der denkwürdigen Entwicklung des Schneeberger Silberbergbaues der siebziger Jahre des 15. Jahrhunderts Reich¬ tum auf Reichtum häuften 1. Und gerade Martin Römer galt den alten Bergchronisten als Prototyp des glücklichen Gewerken, der im Bergbau sein Vermögen gewann. Auch Zycha neigt der Ansicht zu, daß ein Teil der großen Kapitalvermögen , die wir im Bergbau um die Wende des 15. Jahr¬ hunderts sehen , im Bergbau selbst aus dem Nichts heraus sich ge¬ bildet hätten . Der genannte Forscher stellt in seinem mehrfach schon zitierten Aufsatz „Zur neuesten Literatur über die Wirt¬ schafts - und Rechtsgeschichte des deutschen Bergbaues “ die süd¬ deutschen Kapitalisten , die im Tiroler (Schwazer) Silberbergbau seit den letzten Jahrzehnten des 15. Jahrhunderts als Großge¬ werken vordrangen , als „Kaufleute “ in scharfen Gegensatz zu den einheimischen Tiroler Großgewerken , den Tänntzl , den Füger usw.. Für die auswärtigen Kaufleute gibt er ohne weiteres zu, daß sie ein bedeutendes „bergfremdes “ Kapital in den Bergbau mitbrachten, aber nicht für die einheimischen Großgewerken . Zycha will die Genesis der Vermögen , die diese in den Tiroler Bergen investiert hatten , aus „bergmäßigem Gewinn“ , also in der „bergmännischen Produktion “ entstanden , erklären ®. Ja , aber wer sagt denn Zycha, 1O. H o p p e , Der Silberbergbau zu Schneeberg, S. 8, 25 Anm. 27. 2 Z y c h a a. a. O., 5. Bd . (1907), S. 287, 279. 3 Kein „Handelskapital “ , S. 274. Zweites Kapitel : Bergbau und frlihkapitalistisclie Vermogeusentwicklung . 19 daß die Tänntzl, die Füger und andere keine Kaufleute waren, als sie in den Bergbau eindrangen, sich also nicht erst im Handel ein Vermögen erworben hatten , das sie dann in den Berg¬ bau investierend bedeutend vergrößerten? Der genannte Autor sagt auf den schlesischen Goldbergbau exemplifizierend selbst, daß es ganz verkehrt wäre, sich die Hauptmasse der Gewerken als nur Gewerken vorzustellen, „d. h. anzunehmen, daß ihnen ein eigener bürgerlicher Wirtschaftsstand fehlte. Es war nicht etwa ein Teil der Bürger der Bergstädte lediglich Bergbauunternehmer, ein anderer tätig im Gewerbe und Handel. Vielmehr gab es unter den bürgerlichen Berufen zahlreiche Gewerken und zwar gerade auch unter den städtischen Handwerkern. Gerade das bür¬ gerliche Einkommen bildete den Rückhalt der Gewerken. Die meisten hätten andernfalls nicht durchhalten können 1.“ Was insbesondere die Entwicklung in Tirol angeht, so gilt es zu beachten, daß die meisten erfolgreichen einheimischen Tiroler Großgewerken aus Städten stammten, die durchaus die Möglichkeit boten, im Handel ein Vermögen zu erwerben 2. So wird ein Jakob Tänntzl schon im Jahre 1370 unter den Bürgern Innsbrucks genannt, während erst seit den dreißiger Jahren des 15. Jahrhunderts Belehnungen der Familie mit Schwazer Grubenrechten bekannt sind 3. Der erfolgreiche Gewerke Hermann Ringsmaul, der spätestens seit den sechziger Jahren bis 1478 in Schwaz (Falkensteiner Berg¬ revier) baute, entstammte einer Familie, die 1420 nach Hall eingewandert war. Hermann Ringsmaul selbst wird schon 1459 im Haller Salzwerk als Unternehmer genannt 4. Ebenso stammten die beiden bedeutenden Schwazer Gewerken Hans Sigwein und Hans Füger (Fieger) aus alten vermögenden Haller Familien 6. Der Kaufherr Voglmair, der um 1580 eine große Rolle im Unterinntaler Kupferhandel spielte, „besaß zunächst ein Krämereigeschäft in Schwaz. 1575 übernahm er nebst dem Erzgießer Löffler den Handel mit dem im Unterinntal gewonnenen erzherzoglichen 1 Zycha a. a. O., 6. Bd. (1908), S. 122. 2 Besonders aus Innsbruck und Hall. Zycha (1907), S. 260 Anm. 4. 8 Daselbst S. 261. 4 Daselbst S. 262. 6 Daselbst S. 262; besonders auch Anm. 8. a . a. O., 5. Bd. 2* 20 Erstes Buch : Montanindustrie und Frühkapitalismus. Kupfer. Zu gleicher Zeit errichtete er eine Filiale seines Schwazer Geschäfts in Innsbruck und wurde bald der erste inländische Hof¬ lieferant für Seide, Wolle, Wachs, Fastenspeis und Spezereien1.“ Wie nötig es ist, hier die Fragen absolut eindeutig zu stellen, das sieht man an den Aufsätzen Krokers 2 über Leipziger Wirt¬ schaftsgeschichteim 16. Jahrhundert . Der genannte Autor meint einmal: „Bei den Untersuchungen über die Entstehung des Kapital¬ reichtums in den süddeutschen Städten sei die Tatsache der Be¬ teiligung der süddeutschen Kaufleute an dem Bergbau ihrer Zeit fast gar nicht berücksichtigt worden. Er glaube, auch in den älteren süddeutschen Städten stamme ein großer Teil des Kapitals, der sich dort aufgehäuft habe, aus den Bergwerken3.“ Daß beträchtliche Mittel- und Schlußstücke von den großen Vermögen, die uns in der Mitte etwa des 16. Jahrhunderts in Augs¬ burg, Nürnberg usw. begegnen, ihren Ursprung im Bergbau haben, ist so unzweifelhaft, daß es von niemandem geleugnet werden dürfte. Darauf kam es aber bei den Untersuchungen über die Genesis des modernen Kapitalismus, über die „Entstehung der großen bürgerlichen Kapitalvermögen am Ausgange des Mittel¬ alters und zu Beginn der Neuzeit“ gar nicht an. Dort fragte es sich nicht um die einzelnen Wirtschaftszweige, mit deren Hilfe die Groß¬ vermögen des 16. Jahrhunderts entstanden waren, dort handelte es sich darum, die wichtigste der subjektiven Voraussetzungender kapitalistischen Unternehmungen zu erkennen, die Frage nämlich: Auf welche Weise entstanden in der Hand einzelner Wirtschafts1 Jos . Hirn, Erzherzog Ferdinand II . von Tirol. Geschichte seiner Regierung und seiner Länder . 2 Bde. Innsbruck 1885/88. I, S. 414. 2 E . Kroker, Leipzig und die sächs. Bergwerke. In Schriften des Vereins für die Gesch. Leipzigs. IX (1909), S. 25 ff. Derselbe, Beiträge zur Geschichte der Stadt Leipzig im Rcformationszeitalter. In Neujahrsblätter der Bibliothek und des Archivs der Stadt Leipzig, 1908. (Nr. 3: Heinz Probst , ein Leipziger Wucherer ; Nr. 5 : Die sächs. Bergwerke und Leipzig; Martin Leubel, Heinz Scherl.) Derselbe, Leipzig und die alte Fundgrube in Schneeberg. In Leipziger Kalender 1909, S. 129 ff. Derselbe, Heinrich Kramer von Claußbrucb , ein Leipziger Handelsherr des 16. Jahrhunderts . In Quellen zur Ge¬ schichte der Stadt Leipzig II . Bd . (Leipzig 1895). S. 355 ff. 3 Kroker, Leipzig und die sächsischen Bergwerke, S. 34. Gemeint sein können nur meine Untersuchungen in dem Buche „ Zur Genesis des modernen Kapitalismus “ . Zweites Kapitel : Bergbau und frühkapitalistische Vermögensentwicklung , 21 Subjekte jene Urver mögen , auf welche gestützt eben diese Wirtschaftssubjekte an die Gründung kapitalistischer Unter¬ nehmungen gehen konnten ? Wenn Leipziger Bürger am Ende des 15. und zu Beginn des 16. Jahrhunderts Kuxe erwarben, Hütten¬ gesellschaften gründeten usw. und dadurch ihre Vermögen be¬ trächtlich vergrößerten, wenn Leipziger „K auf h erre n“, also Großhändler, sich am Bergbau zu beteiligen 1 anfingen , so wirt¬ schafteten sie bereits kapitalistisch. Es fragt sich aber für die Genesis des Kapitalismus, woher hatten sie die Vermögen, mit deren Hilfe sie Bergwerksteile kaufen und Hütten anlegen konnten ? Wenn wir diese Frage an die verschiedenen dankenswerten Skizzen halten, die uns Kroker aus dem Leipziger Wirtschaftsleben des 16. Jahr¬ hunderts geschenkt hat, so kommen wir zu demselben Resultat, zu dem wir auch für die Bewohner von Augsburg, der Metropole des deutschen Frühkapitalismus, gelangten. Es sind im Handel erworbene Vermögen, die die Basis zu den weiteren kapitalistischen Unternehmungen der Leipziger (in der Montanindustrie) abgeben. So war es bei Heinz Scherl, der von Nürnberg nach Leipzig einge¬ wandert, als armer Krämer seine kaufmännische Laufbahn begann, dann einen Handel in kostbaren italienischen Seidenstoffen führte, um erst später in den Montanbetrieb als Gesellschafter der Hütte Eisfeld und besonders der Hütte Luderstadt überzugehen2. Und genau so lag die Sache wohl bei Martin Leubel, der gleichfalls aus der Kramerinnung emporstieg3. Auch Georg Kreuziger hat sich erst mit Hilfe des Kramhandels das Vermögen erworben, das er dann in einer wachsenden Anzahl von Kuxen anlegen und in einer glücklich geführten Spekulation in Bergwerksteilen bedeutend ver¬ mehren konnte 4. Man mußte — es war nicht anders —, wollte man was Rechtes aus den Bergen ziehen, schon was Rechtes hineinzustecken haben. Der Bergbau, wenn er wirklich rentabel, also Vermögen in großem Stile bildend, betrieben werden sollte, erforderte je länger, je mehr, bedeutende Betriebskapitalien. Die ganz kleinen Gewerken, statt reich zu werden, büßten nicht selten ihre mühsam ersparten kleinen 1 Kroker a. a. O. S.-32, 33. 2 Kroker, Neujahrsblätter IV, S. 81 ff. 3 DaselbstS. 74. 4 Kroker, Schriften des Vereins für die Geschichte Leipzigs, IX, S. 55. 22 Erstes Buch : Montanindustrie und Frühkapitalismus. Vermögen durch Zubußzahlungen für komplizierte Stollenanlagen, für Wasserbehebungsvorrichtungen usw. wiederum ein. Gerade Kroker hat darauf aufmerksam gemacht und unter Benutzung sicherer Quellen (der Leipziger Stadtkassenrechnungen) den Nachweis geführt , daß selbst ein so kapitalkräftiger Gewerke wie die Stadt Leipzig in allen sächsischen Bergwerken schließlich mit Verlust spekulierte und nur im Schneeberger Bergbau einen — allerdings für die Höhe des Risikos — recht bescheidenen Gewinn davontrug . Von den 60 Schneeberger Zechen, an denen der Rat der Stadt Leipzig von 1472— 1535 Mitgewerke war , gaben überhaupt nur 15 Ausbeute ; 45 bedurften mehr oder weniger erheblicher , den Gewinn vernichtender Zubußen . Und unter den 15 guten Zechen waren nur 2 wirklich gute , die „ alte Fundgrube “ und der „Fürsten¬ stollen “ . Hätte man nicht in diesen beiden Gruben je vier Jahre lang ein erstaunliches Glück gehabt , so hätte die Rechnung mit einem sehr hohen Verlust abgeschlossen 1. Ähnlich ist es sicherlich einem großen Teile der Gewerken gegangen ; was sie in einer Zeche verdienten , davon setzten sie in anderen einen erheblichen Teil wieder zu. Oder was sie in guten Jahren in einer anfänglich reichen Zeche gewannen , das ging in den Jahren der Zubuße wieder darauf. Nur wenige ganz vorsichtige Gewerken zogen sich zur rechten Zeit zurück und hielten sich dann dauernd dem riskanten Bergbaubetrieb fern . Schon der Wunsch , ihr Kapital hochverzinslich anzulegen, drängte die meisten zu erneuter Investierung . Methodologisch ergibt sich daraus folgende Notwendigkeit für die Erforschung der Vermögensbildung im Bergbau . Man muß die Bergbauunterneh¬ mungen und zwar alle Bergbäuunternehmungen eines bestimmten Gewerken für eine möglichst lange Zeit untersuchen , um zu sicheren Resultaten und Schlüssen über die vermögenbildende Kraft des Bergbaues als solchen zu kommen . Ich lasse hier noch ein Ausbeuteund Unkosten - („ Samcost -“) Verzeichnis aus dem Idrianer Queck¬ silberbergwerk , dem bedeutendsten des 16. Jahrhunderts , folgen 2. Man ersieht daraus , daß der Idrianer Bergwerksgewinn bescheiden genannt werden muß . Währenddeß waren — wir kommen noch hierauf zu sprechen — die Gewinne, die die Großhändler mit Idrianer Quecksilber machten , ganz enorm. 1 K rok er , Leipzig und die sächsischen Bergwerke, a. a. O. S. 45. 2 Nach einem Manuskript des Finanzarchivs Wien. Inneröstr. Quecksilber 23, Nr. 18 321. Zweites Kapitel: Bergbau und frühkapitalistische Vermögenscntwicklung . 23 In Summa wurde vom 1. August 1539 bis zum April 1573 ver¬ kauft : Idrianer Quecksilber : 33 540 Centner 60 Pfund, Fein Zinnober : 6114 Centner 85 Pfund, Erlöst wurde daraus 1 100 611 fl. 2 kr. Davon geht ab für Fron und Wechsel (d. h. der zehnte und der achte Teil) 1233 879 fl. 50 kr . 2 Pfg. Bleibt den Gewerken 866 731 fl. 11 kr . 2 Pfg. An Samcost Jahr fl. kr. 1539 1540 1541 1542 1543 1544 1545 1546 1547 1548 1549 1550 1551 1552 1553 1554 1555 1556 7 519 11 130 10 056 12 546 9 711 10 080 22 067 7 956 8 903 15 363 20 214 16 004 10 743 14 821 14 251 8 214 15 331 14 140 26 9 34 10 55 56 19 1 22 13 12 50 45 27 39 6 19 33 wurde gebraucht: \| fl. Pf. Jahr 2 3 2 1557 1558 1559 1560 1561 1562 1563 1564 1565 1566 1567 1568 1569 1570 1571 1572 1573 2 — 2 3 2 3 3 3 1 2 1 1 2 — 1 3 11 280 14 616 13 677 16 290 576 17 337 13 274 16 519 28 268 23 997 19 995 10 363 15 966 21 464 14 488 16 068 4 953 kr. 26 1 25 47 40 33 24 12 52 10 35 30 24 58 44 16 1 Pf. _ — 2 3. 1 1 1 1 1 — 2 2 — 2 2 — — I Summa aller Samcost von 1539 bis 31. März 1573: 504 196 fl. 7 kr. 1 Pf. Zieht man diese Summe von obigen 866 731 fl. 11 kr . 2 Pfg. ab , so beträgt der Gewinn der Gewerken in der genannten Zeit 362 535 fl. 4 kr . 1 Pfg ., kommt Ausbeute auf ein Jahr : 10 358 fl. 8% kr . und auf einen Kux jährlich im Durchschnitt 80 fl. 55 kr. * * * Die Ausdehnung , die das sogenannte Verlagssystem in allen Bergwerksdistrikten nahm , ist der beste Beweis für das Gesagte. 1 Über diese Abgaben an den Regalherren später näheres. 2 Bis zum 31. März. 5 24 Erstes Buch: Montanindustrie und Frühkapitalismus. Überall mußten die meisten Gewerken Vorschüsse von Betriebs¬ mitteln zu bekommen suchen . Sie erhielten sie zumeist von den Kaufleuten der benachbarten oder entfernteren Städte , die durch den Erzhandel mit ihnen in Beziehungen standen . So war es in dem steirischen Eisenbergbau , wo die Rad - und Hammermeister durch die Kaufleute von Leoben und Steyr verlegt wurden 1. So im Mansfelder Kupferbergbau , wo die kleinen Bergbau - und Hütten¬ unternehmer , die sogenannten Hüttenmeister , je intensiver der Bergbau betrieben wurde , um so weniger fähig waren , ohne Hilfe von auswärtigem Kapital auszukommen 2'. Fast durchgängig bildete sich auch im Mansfeldischen ein Verlagssystem heraus : Geldgeber war der Kupferhändler , der dem Hüttenmeister zur Bestreitung von dessen Betriebsunkosten eine bestimmte Summe (den soge¬ nannten Verlag) — meist in Raten — vorschoß , wofür sich der Hüttenmeister verpflichtete , dem Kaufmann (Verleger) die von ihm innerhalb einer bestimmten Zeit produzierten Rohkupfer zu einem vertragsmäßigen Preise zu liefern . Dieselbe Sache im fchlesischen Gold- und Zinnbergbau 3, im sächsischen Zinn-, Silber¬ und Kupferbergbau 4, im böhmischen Zinn- und Silberbergbau 6U3W. Durch das Verlagssystem sind sehr häufig kapitalistische Kaufleute dann zu Gewerken geworden . Sie ließen sich nicht selten 1 L. Bittner, Das Eisenwesen in Innerberg-Eisenerz bis zur Gründung der Innerberger Hauptgewerkschaftim Jahre 1625. Archiv f. Österr. Gesch. Bd. 89, S. 514 ff. 2 W . Möllenberg a. a. O. S. 14 f. 3 C . Faulhaber, Die ehemalige schlesische Goldproduktion. Breslauer Dissertation 1896, S. 17 f. und E. F i n k a. a. O. S. 309. Aemilius Steinbeck, Geschichte des schlesischen Berg¬ baues. 2 Bde. Breslau 1857. II, S. 10. Zycha a. a. O., 6. Bd. (1908), S. 126. 4 Auf die Verhältnisse im sächsischen Zinnbergbau kommen wir im Verlaufe dieses Werkes noch ausführlich zu sprechen. Für Verlag sächs. Silbergewerken in Geyer um 1470 vgl. die interessanten Bitt¬ schriften, abgedruckt in den Mitteilungen des sächsischen Altertums¬ vereins 15. Heft, S. 26 f. und sonst: Für Schnceberg daselbst S. 113. Für Kupfer vgl. Dresden H. St. A. Loc. 7249 Bleyhandel 1524—32 Bl. 60. Die Besitzer von Saigerhütten verlegten oft die kupferbauenden Gewerken. In der angeführten Urkunde sagt ein gewisser Hans Flick: „Diese nachfolgenden 26 habe ich auf Kupfer verlegt." 5 A . von Sclieurl, Christoph von Scheurl. Auch über den böhmischen Zinnbergbau mit seinem Verlagswesen wird noch öfter zu sprechen sein. Zweites Kapitel: Bergbau und früh kapitalistische Vermögensentwicklung . 25 ihre Vorschüsse durch „Verhypothezierung “ der Bergwerksanteile der Verlagnehmer sicherstellen . Bei Zahlungsunfähigkeit des Ge¬ werken gingen seine verpfändeten „Kuxe “ dann an den kapita¬ listischen Kaufmann über 1. Vielfach vollzog sich das Eindringen des kapitalistischen Kauf¬ mannes in den Bergbaubetrieb als solchen auf folgende Weise. Man brauchte sein Geld zur Bewältigung größerer technischer Aufgaben, die sich überall in den Gruben mit der Zeit ergaben . Zunächst war das Abbauen des Erzes nicht allzu schwierig gewesen. Der Tagbau hatte vorgeherrscht . Aber je mehr der Bau in die Tiefe ging, um so größer wurden die Baukosten 2. Es mußten Stollen , d. h. Tunnel angelegt werden , die dem Bergwerk das Wasser nehmen und frische Luft bringen sollten usw. Weder der Grundherr noch die Gewerken konnten und wollten in den meisten Fällen ihr Geld an diese kost¬ spieligen und langwierigen Bauarbeiten wagen . Da überließen sie gern kapitalkräftigen auswärtigen Kaufleuten die Kosten und Ge¬ fahren der Stollenbauten usw . und gaben ihnen dafür die Mehrzahl der neuen Kuxe , gewisse Bergwerksgerechtigkeiten usw. So über¬ ließen Grundherr und Gewerken der böhmischen Bergstadt Schlacken¬ wald einem Konsortium süddeutscher Geldmänner , bestehend aus den Firmen Hans Schnöd, Welser und Scheuerl , gegen die Übernahme von umfangreichen Stollenbauten folgende Gerechtig¬ keiten : 1 O. O p e t , Das Gewerkschaftsrecht nach den deutschen Berg¬ rechtsquellen des Mittelalters in Zeitschr . f. Bergrecht 34 (1893 ), S. 309 . Z y c h a a. a. O. 5. Bd . (1907 ), S. 277 . Ein Beispiel , wie ver¬ schiedene süddeutsche Kaufleute (Jakob und Anton Welser , die Vöhlin - Gesellschaft , Peter Imhof & Gebr., die Humpiß - Gesellschaft, Lienhart Hirschfogel und Brüder , Hans Baumgartner , die Herwart, die Bimmel u. a.) in den Pfandbesitz von Bergteilen im schlesischen Goldbergbau gelangten , siehe K . W u 11 k e , Schlesiens Bergbau und Hüttenwesen (Codex dipl . Silesiae XX u . XXI ) 1900/01 , Nr . 329 bis 331 ; auch Z y c h a a. a. O. 6 . Bd . (1908 ), S. 116 f. Für die Fugger W u 11 k e a. a. O. Nr . 333 , Z y c h a , 6 . Bd . (1908 ), S. 117. Weitere Beispiele Z y c h a , 6 . Bd . S. 127. 2 Vgl . für das folgende E . Reyer, Städtisches Leben im 16. Jahrhundert . Kulturbilder aus der freien Bergstadt Schlacken¬ wald . Leipzig 1904 . Dazu K . Th . von Inama - Sternegg, Deutsche Wirtschaftsgeschichte , III . Bd . 2. Teil . Leipzig 1901. S. 163. 26 Erstes Buch : Montanindustrie und Frühkapitalismus. 1. Alle Erzvorkommnisse, welche der zu bauende Stollen an¬ fährt, sollen den Stollenbesitzern gehören bis auf 17 Lachter im Umkreis. 2. Alle Bergwerke, die durch den tiefen Stollen vom Wasser befreit und mit gutem „Wetter “ (d. h. Luft) versorgt werden, sollen den Gewerken des tiefen Erbstollens den Neunten der ge¬ samten Erzgewinnung abgeben. Im Jahre 1539 begannen die Arbeiten, elf Jahre später, so be¬ richtet Reyer, wurde das erzreiche Gebiet in der Hub’ angefahren und nun schüttete ein Kux, welcher vordem um ein paar Goldgulden erkauft werden konnte, in einem Jahre 30—40 fl. Ausbeute. Zu Ende des 16. Jahrhunderts war der Stollen mit seinen Strecken nahezu 4 Kilometer(eine Marschstunde) lang; 24 Schächte mündeten in denselben ein. In ähnlicher Weise wie hier die genannten drei Firmen wurde in Ungarn ein Krakauer Bürger und Ratsherr Großgewerke im Karpathengebict 1. Die dortigen ungarischen Bergwerke hatten bereits im früheren Mittelalter, von eingewanderten deutschen Bergleuten gefördert, eine gewisse Bedeutung gewonnen. Seit Ende des 14. Jahrhunderts aber war Wasser in die Gruben eingedrungen, das die damalige Technik nicht bewältigen konnte. Erst gegen Ende des 15. Jahrhunderts begann eine neue Epoche des ungarischen Silber- und Kupferbergbaues. Damals gewannen die sieben un¬ garischen Bergstädte Kremnitz, Neusohl, Schemnitz, Königsberg, Pukancz, Dilln und Libethen einen Bürger und Ratsherrn von Krakau, Job. Thurzo mit Namen, zu einem Versuch, das Wasser aus den Gruben mittels Hebemaschinen zu entfernen. In dem Ver¬ trag (vom 24. April 1475) wurde dem Thurzo ein ungarischer Gold¬ gulden als Wochenlohn versprochen, besonders aber sollte er 1/6 des mit Hilfe seiner Wasserkunst gewonnenen Erzes erhalten. In der Bestätigung, die König Mathias (am 15. Mai 1475) dem Vertrag zuteil werden ließ, gab er Johann Thurzo das Recht, „aus allen verlassenen Gruben das Wasser zu heben und dann nach Silber zu schürfen.” Für jede Gewichtsmark Silber wurden ihm 4 Goldgulden 1Vgl . für das Folgende Jansen, Jakob Fugger der Reiche, S. 132 f. Dort auch in Anm. 1 die Literatur. Dazu meine Besprechung in der Historischen VierteljahrsschriftJahrg. 1912. S. 98 ff. Endlich Z y c h a a. a. O. 6. Bd. (1908), S. 114 f. 1 Zweites Kapitel : Bergbau und frühkapitalistische Vermögensentwicklung . 27 als Entschädigung versprochen. Außerdem durfte er die für ihn und die Arbeiter notwendigen Lebensmittel abgabenfrei einführen. Um dieselbe Zeit , als die Tliurzo mit Hilfe des Besitzes einer fortgeschrittenen Technik und mit Hilfe von eigenem oder von ihnen aufgebrachtem Kapital als Gewerken in den ungarischen Bergbau eindrangen, diente ihnen dasselbe Mittel auch dazu, um sich in der Goslarer Erzproduktion einzunisten L* Auf den geschilderten Wegen der Kapitalbeschaffung für die Bergwerksproduzenten sind die vermögenden Kaufleute der näheren und entfernteren Städte in wachsendem Maße in den Bergbau selbst als Gewerken eingedrungen. Lange vorher hatten sie aber schon als E r z h ä n d 1e r an dem Bergsegen teilgenommen. Seit alters erwarben Kaufleute von den Gewerken oder von den Landesfürsten oder von sonstwie Bezugsberechtigten Erze und verkauften sie in alle Welt weiter. Verdienten die Kaufleute dabei Geld — und zu¬ meist taten sie es in hohem Grade —, so war es ein Handel mit Bergbauprodukten und nicht der Bergbaubetrieb selbst, der für die bürgerliche Reichtumsentwicklung von Bedeutung wurde. So sind die Fugger erst ziemlich spät in den Tiroler Bergbau einge¬ drungen. Sie waren bis zum Jahre 1522 nicht als Gewerken im Tiroler Bergbau tätig 2. Bis zum Jahre 1522, in welchem Jahre Jakob Fugger bedeutende Bergwerksanteile bei Scliwaz, Ratten¬ berg und Lienz aus der Konkursmasse des Martin Baumgartner erwarb, war der Anteil der Fugger an den bedeutendsten Tiroler Bergwerken, denen um Schwaz, von einer anderen Art. Von jener mehr kaufmännischen Art , wie sie sich auch nach 1522 noch neben der neuen erhielt. Seit 1488 traten die Fugger in verschiedenen, gleich noch zu besprechenden Formen, als Großkäufer von Tiroler Bergwerksprodukten, besonders Silber und Kupfer, bei den ver¬ schiedenen Bezugsberechtigten auf. In ähnlicher Weise wissen wir 1 Siehe oben i>. y. Wie mir Herr Archivdirektor Professor Dr. Hölscher freundlichst mitteilt, gibt es im Goslarer Stadtarchiv außer den von mir benutzten noch eine größere Anzahl Urkunden über Tliurzo, so daß dieser „deutlich im historischen Lichte“ dasteht. Ich behalte mir vor, auf diesen interessanten Krakauer Kapitalisten und Techniker, der sich so international im Bergbau betätigte, näher zurückzukommen. 2 Vgl . für das Folgende Strieder, Die Inventur der Firma Fugger aus dem Jahre 1527, S. 41 ff. Jetzt auch Z y c h a a. a. O. 5. Bd. (1907), S. 274 f. 13 5 28 Erstes Buch : Montanindustrie und FrühkapitaUsmus. aus den Handlung'sbüchern der Firma Ulrich Link, Anton Haug und Mitverwandten \ daß die Gesellschaft erst um 1553 Bergwerks¬ anteile zu Schwaz, Sterzing und Gossensass aus dem Besitz des Mathias Manlich 2 an sich brachte s. Bis dahin hatte die Firma keine 4 Kuxe besessen und nur einen bedeutenden Handel mit Berg¬ werksprodukten betrieben. Solche Beispiele relativ späten Eindringens der großen süd¬ deutschen Kapitalisten in den eigentlichen Bergbaubetrieb selbst legen die Vermutung nahe, daß es den großen Kaufleuten gar nicht so sehr darauf ankam, als Gewerken in den Anfang der bergbaulichen Produktion zu kommen. Wenn nicht eine zwingende Notwendigkeit vorlag, blieben sie lieber am Ende des Prozesses als Abnehmer stehen. Mochten andere die Gefahren tragen, die das Gewerkesein in Zu¬ bußen usw. mit sich brachte, der kluge Kaufmann begnügte sich mit dem größeren und sicheren Gewinne aus dem Absatz bergbau¬ licher Produkte 6. 1 Die Gesellschaft war unter der Firma „Anton Haug , Hans Langenauer , Ulrich Link & Co.“ 1531 auf der Grundlage des alten Bimmelschen Geschäftes (vgl. Strieder, Zur Genesis des modernen Kapitalismus , S. 146) errichtet worden und bestand als eine führende Augsburger Großunternehmung unter wechselnden Führern aus dem Verwandtenkreise der Haug und Link bis um das Jahr 1574. Von 1533 bis 1562 sind die Bilanzen der Handelsunternehmung in zwei starken Foliobänden im Augsburger Stadtarchiv erhalten (Geheim¬ buch I und II im folgenden zitiert ). Näheres über das Geschäft bei Ehrenberg, Zeitalter der Fugger I, S. 227 ff. Ferner bei Har¬ tung, Aus dem Geheimbuche eines deutschen Handelshauses, a. a. O. S. 36 ff. 2 Mathias Manlich war ein bedeutender Schuldner der Firma; vgl . Geheimbuch II , Bl. 41 a. 3 Geheimbuch II , Bl. 42. 4 Nur in Joachimstal hatten die Link noch aus der Zeit der Bimmel (Geheimbuch II , Bl. 35 a) einige Bergwerksteile mit Hans Rosenberg und Erasmus Herwart zusammen . Aber die Kuxe brachten keine Ausbeute. 5 Vgl . auch Artur Salz, Geschichte der böhmischen Industrie in der Neuzeit . München und Leipzig 1913, S. 70. Einmal bemerkt Kroker sehr richtig : „Andre Männer wurden in Schneeberg und in den übrigen Bergstädten reich, besonders solche, die mit dem Erz nur handelten , oder die in ihren Saigerhütten das Silber vom Kupfer scheiden ließen ; das war in den Händen eines guten Geschäfts¬ mannes ein gutes und sicheres Geschäft. Leipzig und die sächsischen Bergwerke, a. a. O. S. 47. 1 Zweites Kapitel : Bergbau und frühkapitalistisehe Vermögensentwicklung . 29 Es ist nun für die hier behandelte Frage von Bedeutung, daß im Verlaufe des endenden 15. und beginnenden 16. Jahrhunderts die Kapitalbasis, die dazu nötig war, um in größerem, bedeutenden Gewinn abwerfenden Stile den Handel mit Bergwerksprodukten zu treiben, sich immer mehr vergrößerte. Da mußten große Hütten¬ werke angelegt werden, um die Roherze, wie man sie von den Ge¬ werken erwarb, verkaufsfähig (zu Kaufmannsgut) zu machen, da mußte man den ärmeren Gewerken, auf deren Produkte man re¬ flektierte, oft Vorschüsse(Verlag) geben, usw. Wir werden an einer anderen Stelle darauf zu sprechen kommen, welch große Kapitalien für die Anlage solcher Hüttenwerke oft nötig waren. Aber auch die Verlagssummen nahmen oft riesige Dimensionen an. Wir kennen jetzt durch die Veröffentlichungvon M ü c k die Summen, um die es sich bei dem Verlag der Mansfelder Hüttenmeister (der Gewerken des vereinigten Mansfeldischen Berg- und Hüttenbetriebs) durch die kapitalistischen Kupferhändler oft handelte. Je mehr die Wert¬ schätzung des Kupfers und sein Preis im Verlaufe des 16. Jahr¬ hunderts stieg x, um so höher wurde der Verlag. Hatte man früher etwa 500 fl. auf die Jahresproduktion eines Feuers vorgestreckt, so betrug schon 1523 die „Leibung“ erheblich mehr. So mußte der Nürnberger Kupferhändler Sigmund Pfinzing, als er 1523 mit dem Hüttenmeister Dietrich Pockler einen Verlagskontrakt schloß, für die Hergabe der Jahresproduktion von dessen zwei Feuern (zum Preise von 11% fl. pro Zentner) 1400 fl. „zu guter Förderung und Verlegung der Kupfer“ vorschießen 2. Seitdem stieg die Höhe des Verlags rasch. In vier Kupferkaufverträgen, die verschiedene, be¬ sonders von Nürnberger und Leipziger Kapitalisten gebildete Saiger¬ handelsgesellschaften im Jahre 1524 mit Mansfelder Hüttenmeistern abschlossen3, wurden auf 4 gräfliche (Drachstedtsche) Feuer 8000, auf 2 Feuer des Hans Stelle 3000, auf drei Feuer des Wilh. Rincke und Merten Knebbel 3000, auf 3 Feuer des Jakob Luder und Hans Stellwagen 3000 fl. Verlag gewährt. In den beiden Kupferkaufkontrakten 4, welche die Grafen Ernst, Hoyer, Gebhart und Albrecht von Mansfeld am 14. August 1528 für 8 gräfliche (Drachstedtsche) Feuer mit der Arnstädter bzw. der 1 Siehe oben S. 5. 2 Über die Art der Rückzahlung Miick a . a. O. I , S. 103. 3 Vgl . Mü c k I, S. 103, und II, Nr. 275—278. •Jlück a . a . O. II , Nr . 280 , 281. 1 30 Erstes Buch : Montanindustrie und FrühkapitaUsmus. Leutenberger Saigerhandelsgesellschaft schlossen , wurden 26 000 fl. Verlag ausbedungen . Auf 2 Feuer des Hans Brugkner 3000 fl. . Um das Jahr 1530 wurden im Durchschnitt 1500 fl. auf die Jahreskupfer¬ produktion vorgeschossen 2. Vergrößert wurde das Kapitalbedürfnis von Einzelkapitalisten oder Gesellschaften , die Kupfer von den Hütten¬ meistern kauften , über die Verlagsforderungen hinaus noch dadurch, daß die Vcrlagssummen oft nicht rechtzeitig zurückgezahlt werden konnten und bei Ablauf des Kontrakts noch teilweise ausstanden 3. Die liier angeführten Zustände sind nun nicht etwa für den Mansfelder Kupferbergbau und Kupferhandel charakteristisch , sic sind vielmehr wirtschaftliche Organisationsformen , die im Bergbau bzw. Erzhandel des 16. Jahrhunderts allgemein üblich waren . Es braucht nur angedeutet zu werden , wie stark das auf die Ausbildung von konzentrierten Großbetrieben im frühkapitalistischen Erz - und Metallhandel wirken mußte . Von entscheidenderer Bedeutung für diese Entwicklung aber wurde fast noch das folgende : Die meisten Metalle waren dem freien Handel dadurch entzogen 4, daß öffent¬ liche Gewalten , der Landesfürst usw. alle gewonnenen Erze für sich forderten . Natürlich gegen Bezahlung , die aber weit niedriger war als der jeweilige Marktpreis der betreffenden Ware . Dieses Vorkaufs¬ recht der öffentlichen Gewalt nannte man den Wechsel (cambium, Lösung) s. Oft wird auch der Gewinn, der „Wechselgewinn “, den der Landesherr dabei machte , kurz „Wechsel “ genannt. Der Wechsel, das eben skizzierte Vorkaufsrecht , „ erscheint im Salzburgischen durch eine Urkunde König Heinrichs von 1195 auf Grund eines Reichsspruches für die dortigen Erzbischöfe sicher¬ gestellt ." Und noch in der „allgemeinen Freiheit “ von 1477 war 1 Mü c k a. a. O. II, Nr. 287. 2 Mü c k a. a. O. I, S. 103. 3 Vgl . darüber das Schreiben des Grafen Albrecht von Mansfeld an den Kurfürsten von Sachsen vom 2. Juli 1539. M ü c k a. a. O. I, S. 103. 4 Aber es gab auch Metalle und Gegenden, wo nicht ein Vorkaufs¬ recht der öffentlichen Gewalt den freien Verkehr mit Bergwerks¬ produkten hemmte. Vgl. z. B. E. G o t h e i n , Geschichte des Berg¬ baues im Schwarzwald. Zeitschr. f. Gesch. d. Oberrheins XLI (1887), S. 629 ff. 3 Z y c h a a. a. O. 5. Bd. (1907), S. 266 ff. Dort nähere Literatur. Vgl. auch Acta Tirolensia. Urkundl. Quellen zur Geschichte Tirols. III. Bd. I. Teil. Innsbruck 1908. S. 32 Anm. 3. Zweites Kapitel : Bergbau und t'rühkapitalistische Vermögensentwlcklun «. 31 vorgesehen, daß Kupfer und Blei dem Erzbischof „ um ein ziemlich landläufigen Kauf“ anzubieten sei. In Tirol begegnet uns das Vorkaufsrecht der Herzoge auf Silber zu Anfang des 15. Jahrhunderts deutlich ausgebildet In Sachsen wohl noch früher KIn Schlesien besaßen die Herzoge von Münsterberg das Vorkaufsrecht für das in ihren Bergen ge¬ wonnene Gold 2. In Goslar hatte der Rat der Stadt im Verlaufe des 14. und 15. Jahrhunderts die Verpflichtung durchzusetzen ver¬ standen, daß alles aus den Rammeisberger Bergwerken gewonnene Erz (es handelte sich besonders um Blei) nach seiner Verhüttung an ihn verkauft werden mußte 3. Im 16. Jahrhundert wußten dann die Herzoge von Braunschweig dieses Regal an sich zu bringen. Im preußischen Ordenslande war aller gefundene Bernstein an die Wirtschaftsbeamten des Ordens abzuliefern usw. usw. Es versteht sich leicht, daß das Erzvorkaufsrecht der öffent¬ lichen Gewalten und damit die Konzentration bedeutender Erz¬ quantitäten in einer Hand einen Großbetrieb im Erzhandel erzeugen mußte. Die öffentlichen Gewalten hatten nicht Lust und auch keinen genügenden Beämtenapparat, um die an sie abgelieferten Erzmengen im einzelnen zu verkaufen. Sie suchten womöglich einen einzigen und womöglich einen Abnehmer auf längere Zeit zu bekommen. Besonders auch aus dem folgenden Grunde! Während des 15. und 16. Jahrhunderts , in der Zeit also der Blüte des deutschen Bergbaues, war das Geldbedürfnis der Fürsten usw. aus den verschiedensten Gründen (Kriege, neue Verwaltungsauf¬ gaben, wachsende Kultur und damit Luxus usw.) außerordentlich gestiegen. Da bot sich in Abschlüssen langjähriger Erzlieferungen mit kapitalkräftigen Kaufleuten ein bequemes Mittel, um große Gelddarlehen zu erhalten. Wie überhaupt die vergleichsweise primi¬ tive Finanzwirtschaft des Mittelalters „ teilweise oder gar vornehmlich nicht mit den Einkünften selbst, sondern mit dem Kredit der Ein¬ künfte arbeitete 4, wurden sofort Kreditgeschäfte auf das Erz1 H. Ermisch, Das sächsische Bergrecht des Mittelalters. Leipzig 1887, S. CXXXVIII. 2 Zy cha a. a. O., 6. Bd. (1908), S. 118. 3 Vgl . darüber C. Neuburg, Goslars Bergbau bis 1552. Ein Beitrag zur Wirtschafts- und Verfassungsgeschichte des Mittelalters, Hannover 1892, an verschiedenen Stellen. 4 Vgl. die noch heute analogen Verhältnisse bei chinesischen und anderen Anleihen vonseiten europäischer großer Bankgruppen, 32 Erstes Buch : Montanindustrie und Frülikapitallsmus. Vorkaufsrecht der Krone fundiert 1.“ So entstanden als Kontrakte alleiniger Lieferung die sogenannten Erzkäufe (Silber-, Kupfer -, Blei-Käufe usw.), die eine so bedeutende Rolle in der Wirtschafts¬ organisation besonders des 16. Jahrhunderts spielen. Der erste Tiroler „ Silberkauf “ von großem Umfange 2 wurde im Jahre 1456 von dem Herzog von Tirol mit einem Augsburger Kaufmann und seiner Gesellschaft abgeschlossen . Ludwig Meuting 3 u, Co. liehen dem Herzog Sigmund 20 000 und dazu noch 15 000 fl. für den Ankauf des Silbers von den Gewerken bzw. Schmelzern. Dafür sollte der Gesellschaft und niemandem sonst bis zur völligen Rückzahlung der Schuldsumme alles Silber , das in den Berg¬ werken zu Schwaz und Gossensass und allenthalben in der Graf¬ schaft Tirol „gevellet und gemacht wirdt, “ zum Preise von 7% fl. rh . für die Mark Wiener Gewichts „kaufswéise “ überantwortet werden . Der Silberbedarf des Herzogs für seine Münze mußte gegen bare Bezahlung zu demselben Preise zurückgekauft werden 4. Nach den Meuting scheinen die Baumgartner von Kufstein einige Silber¬ käufe mit Sigismund abgeschlossen zu haben , bis seit 1488 die Fugger auf lange Zeit hinaus die führende Macht für die Silber¬ käufe der Herzoge von Tirol wurden s. Für sächsisches Silber war Nürnberg — neben Frankfurt a. M. und Venedig — der Hauptverkaufsplatz . Nürnberger Kaufleutc haben denn auch spätestens seit den achtziger Jahren neben dem die auf zukünftige Zoll- usw. Einkünfte basiert sind. Vgl. für das Folgende auch die Analogien, die sich noch heute z. B. im Kaffeeund Baumwollhandel finden . Die Großen von Turkestan und Siam verkaufen die Baumwolle ihrer Plantagen schon in Le Havre (zu¬ meist an die dortige Liquidationskasse ), wenn sie noch ungeerntet auf den zcntralasiatischen Feldern steht . So gewinnen sie bequem Kapital für neue Anpflanzungen . Ebenso die Kaffeepflanzer Bra¬ siliens. Köln. Zeitung 18. April 1913. 1 Z y c h a a. a. O., 5. Bd. (1907), S. 270. 2 Kleinere waren schon in der ersten Hälfte des 15. Jahrhunderts vorausgegangen . Vgl. Zycha, S . 270. 3 Für dieses Handelshaus vgl. Strieder, Zur Genesis des modernen Kapitalismus , besonders S. 102 ff. 4 Zycha S . 271. 8 Näheres darüber bei Zycha S . 271 ff. Ferner jetzt bei Jansen, Die Anfänge der Fugger, S. 44, 54 ff. und (Urkunden) S. 114 ff. und 150 ff. Derselbe, Jakob Fugger der Reiche, S. '10 ff., 79 ff. 1 Zweites Kapitel : Bergbau und frühkapitulistlsche Vermögensentwicklung . 33 Verkaufe sächsischen Silbers für Rechnung des Herzogs auch schon den „ Silberkauf “ im oben gekennzeichneten weiteren Sinne inne¬ gehabt \ Die Organisation des Goslarer Bleikaufs wird Maximilian Schmid demnächst in einer Monographie behandeln . Hier genüge zu sagen, daß der Großhandel mit Goslarer Blei während der ersten Hälfte des 16. Jahrhunderts stets durch sogenannte Bleikontrakte der Stadt Goslar (bzw. dann der Herzoge von Braunschweig ) als Monopol in die Hände von Leipziger Kapitalisten 2 gelegt wurde. Selbstverständlich gegen bedeutende Vorschüsse der letzteren ! Die Leipziger Kaufleute wurden bald von der sächsischen Regierung, die ebensowenig wie die sächsischen bergbauenden Gewerken das Goslarer Blei zur Silbergewinnung entbehren konnte , monopolistisch herangezogen . Die Kaufleute durften das Goslarer Blei nur in die sächsischen Silberbergwerke liefern , wenigstens solange als dort Bedarf dafür vorhanden war . Kurfürst August nahm dann den Blei¬ handel nach Jahren als zum Regal des Bergbaues gehörig überhaupt direkt an sich und „versorgte aus seinen oft sehr bedeutenden Vor¬ räten zu Freiberg und Dresden die bergbauenden Gewerken seines Landes “. Auch seine Hauptbezugsquelle war der Rammeisberg bei Goslar , wo er sich durch stets erneute Verträge mit den Her¬ zogen von Braunschweig den ungestörten Vorkauf von allem ge¬ wonnenen Blei zu sichern suchte . Am 31. Mai 1556 schloß er einen solchen Vertrag auf drei Jahre mit dem Herzog Heinrich , demzu¬ folge der Kurfürst alles im Rammeisberge gewonnene Blei, den Zent¬ ner zu 45 Schneeberger Silbergroschen , erhielt . Im Jahre 1558 wurde dieser Betrag unter Erhöhung des Bezugspreises auf 47 Groschen erneuert usw. 3. Für die sächsischen Schmelzer bestand dann natürlich die Verpflichtung , das Blei für den Schmelzprozeß von der herzoglichen Kammer zu beziehen . Dieselbe Pflicht war für Reichenstein . (Schlesien) von den Herzogen von Münsterberg schon zu Anfang des 16. Jahrhunderts (spätestens ) aufgerichtet . 4 1 Vgl . A. P u f f , Die Finanzen Albrechts des Beherzten. Leipzig 1911, S. 86 ff. 2 Z. B. Ulrich Lintacher, Wolfgang Wiedemann, Lucas Straub, Ulrich Rauscher usw. 3 Job. Falke, Die Geschichte des Kurfürsten August in volkswirtschaftlicher Beziehung. Leipzig 1868, S. 295 ff. 1 Z y c h a a. a. O., G. Bd., S. 118. Stfiv «liT , Stu <1ifi » l . Gesch . kapitalist . Organi sationaformen . .{ 1 5 34 Erstes Buch : Montanindustrie und Erühkapttulismus. Uber die Art der Bernstein kontrakte gibt ein Ver¬ tragsentwurf Aufschluß (Königsberg, 9. Januar 1518) demzufolge sich Albrecht von Brandenburg den Handelsgesellschaftern Nicolaus Pflaume und Georg Kramer zu Königsberg, Ebert Roge zu Danzig und Claus Lang zu Lübeck gegenüber 2zur alleinigen Lieferung des Bernsteins zu festbestimmtem Preise verpflichtete. Dafür hatten die genannten Kaufleute dem Herzog Albrecht 10 000 Mk. (geringer preußischer Münze) zinslos zu leihen. Großartig, was die Höhe der dabei von den Kapitalisten ge¬ zahlten Vorschüsse angeht, waren auch die Kupferlieferungskon¬ trakte , die Kupferkäufe, die die Mansfelder Grafen seit Übernahme des Regalbetriebes der Hütten und Bergwerke (1536) 3 schlossen 4. In dem Kupferkaufvertrage der vorderortischen Grafen mit der Firma Manlich vom 11. April 1557 verpflichtete sich das Augsburger Handelshaus zur Gewährung eines Darlehns in der Höhe von 300 000 fl. an die Grafen gegen Verpfändung ihrer drei Bergwerks¬ fünfteile s. Die Fürer von Nürnberg liehen 1561 bei Gelegenheit eines Kupferkaufs auf 1)4 vorderortische Bergwerksfünfteile 140 000 fl. 6; Wolfgang von Lindenau und Martin Mertens aus Leipzig 1561 auf ein hinterortisches 150 000 fl. 7,1562 auf ein vorderortisches Bergwerksfünf teil 50 000 fl. 8 usw . usw. Man nannte die Darlehen der Kapitalisten, die unter Pfandsetzung der betreffenden Berg- und Hüttenwerke gegeben waren, „Hauptgüter“. Da die Grafen zu allermeist nicht imstande waren, bei Endigung des Kupferkaufs die Hauptgüter zurückzuzahlen, so wurde entweder unter Weiterbelassung des „Hauptgutes“ der Kupferkauf mit dem bis1 Im Staatsarchiv zu Königsberg. Ich habe den interessanten Kontrakt im Anhang abgedruckt. 2 Die Genannten hatten schon vorher Bernsteinkontrakte mit dem Herzoge von Preußen abgeschlossen; siehe den Text des oben¬ genannten Vertragsentwurfes. 3 Im Jahre 1536 teilten die fünf verschiedenen Linien der Mans¬ felder Grafen ihren gemeinschaftlichen Besitz an den Berg- und Hüttenwerken; vgl. Mück a. a. O., I, S. 61 f. 1 Mücli a. a. O., I, S. 107 ff., besonders S. 111 die wichtigsten aufgeführt. 5 Mü c k a. a. O., I, S. 104, und II. Bei. Urk. Nr. 306. 6 Daselbst II. Bd. Urk.-Nr. 307. TDaselbst II. Bd. Nr. 308. Daselbst II. Bd. Nr. 309. 1 Zweites Kapitel : Bergbau und frühkapitalistische Vermögensentwicklung. 35 herigen Kontrahenten verlängert \ oder der neue Kupferkaufs¬ kontrahent mußte seinerseits das „Hauptgut “ seines Vorläufers, womöglich noch unter neuen Darlehensgewährungen, über¬ nehmen. Aber mit der Darleihung der sogenannten Hauptgüter an die Mansfelder Grafen waren die Anforderungen an die Kapitalisten, die Mansfelder Kupferkäufe abschließen wollten, noch nicht er¬ ledigt. „Neben den Hauptgütern hatten die Kupferhändler den Verlag auch weiterhin noch zu gewähren.“ Denn die Grafen waren trotz der Betriebsübernahme der Berg- und Hüttenwerke dazu nicht imstande. So enthielt jeder Kupferkauf neben der An¬ leihegewährung an die Grafen auch noch die Verpflichtung, den Hüttenleuten die Betriebsmittel vorzustrecken (Vorlegung, Ver¬ lag) 2. Auch das erforderte zumeist ganz enorme Summen. So be¬ trugen z. B. die mit den drei vorderortischen Bergwerksfünf teilen versicherten Forderungen der Kupferhändler im Jahre 1570 rund 650 000 fl. 3. Kein Wunder , wenn dann öfters selbst kapitalkräftige Händler bzw. Handelsgesellschaftenmit dem weiteren Verlage in Zahlungsschwierigkeiten gerieten 4. Weit über das Gebiet des Erzgroßhandels hinaus haben sich die sogenannten Käufe oder Kontrakte ausgedehnt. So schloß Sebastian Neidhart am 8. Juli 1530 einen Perlenkontrakt ab. Der Augsburger Großhändler lieh Karl V. 40 000 fl. Der Kaiser sollte die Schuldsumme binnen 3(4 Jahren zurückzahlen und zwar „durch allerhand Perlen“, die von Amerika in der Sevillaner Casa de con¬ tra tacion während der genannten Zeit eingingen. Würde, durch die Perleneingänge die geschuldete Summe in 3(4 Jahren nicht ge¬ deckt, so solle. Neidhart zwischen der sofortigen Bezahlung der Schuld oder einer Verlängerung des Perlenkontraktes wählen 5. Bekannt sind ferner die. Pfefferkontrakte, die die portugiesische Regierung mit den größten Kaufleuten Europas abschloß. In einem anderen Zusammenhang wird noch hierauf zu sprechen zu kommen 1 Mü c k a. a. O., I, S. 104. 2 Über die Art, wie das Verlagssystem ausgebildet wurde vgl. M lick a. a. O., I, S. 104 ff. 3 M ü c k a. a. O., I, S. 107. 4 Beispiele Mück a . a. ()., I, S. 107 Anin. 1. 5 Zeitschrift des historischen Vereins für Schwaben und Neuhurg 1911, S. 139. 3 36 Erstes Buch : Montanindustrie und Frühknpitalisinus. sein. Zunächst mag hier noch der Tatsache Erwähnung geschehen, daß auch in denjenigen Teilen des Bergbaues sogenannte Käufe vorkamen, in welchen den Fürsten nicht der Vorkauf des Berg¬ werksproduktes rechtlich zustand. Es wurde zum fürstlichen Bergwerksregal gerechnet, daß die Gewerken eines bestimmten Erzproduktionsgebietes von seiten der Regierung gezwungen werden konnten, auf eine bestimmte Anzahl Jahre einen alleinigen Erzlieferungskontrakt mit einer bestimmten Firma zu schließen. Sehr oft haben die Regierungen von diesem Teile ihres ßergwerksregals Gebrauch gemacht. Wir werden in späteren Abschnitten dieses Buches von „Zinnkäufen“ zu reden haben, die die sächsischen und auch die böhmischen Zinngewerken durch Vermittlung der betreffenden Regierungen mit Großkauf¬ leuten abschlossen. Ebenso von „ Quecksilberkäufen“, die die Idrianer Gewerken mit verschiedenen Augsburger Handelsgesell¬ schaften eingingen. Hier sei noch auf einige solche„Käufe“ im Erzliandel hingewiesen. Im Jahre 1586 schloß Wilhelm von Rosenberg durch Vermittlung Kaiser Rudolphs mit den Gewerken zu Tabor, Ratiborzicz und Przbram einen Erzkauf auf 30 Jahre ab 1. Interessant sind die Kobaltkontrakte, die die sächsischen Gewerken unter Zustimmung der Regierung mit — zumeist auswärtigen — Kaufleuten im 16., 17. und 18. Jahrhundert eingingen2. Zumeist drang die Regierung deshalb auf den Abschluß solcher Käufe, weil sie dann von dem betreffenden Kapitalisten eine Anleihe gewährt erhielt. Aber oft tat sie es auch aus weniger eigennützigen Gründen, um den Ge¬ werken und der Bergbevölkerung überhaupt mit einem gesicherten Verlag bzw. Absatz ihrer Erze die Grundlage einer gesicherten Existenz zu verschaffen. Aus letzteren Gründen verlieh Kurfürst August von Sachsen in den sechziger Jahren des 16. Jahrhunderts 1 A. Voigt, Beschreibung der böhmischen Münzen, III, S . 253. 2 Vgl . Christ . Melzer, Bergkläuftige Beschreibung der . . . Stadt Schneebergk . . . Schneeberg 1684, S. 484 ff. Dazu der Auf¬ satz „Kurzer Abriß des Schneeberger Silber- und Kobaltbergbaues von 1471 bis 1719“ in Bergmännisches Journal, I. Bd. (1793), S. 160 ff., und Meitzer, Stadt - und Bergchronik von Schneeberg 1716. Besonders aber W. Bruchmüller, Der Kobaltbergbau und die Blaufarbenwerkein Sachsen bis zum Jahre 1653. Leipziger Disser¬ tation 1897. Dort sind auch im Anhang verschiedene interessante Kobaltkontrakte abgedruckt. Zweites Kapitel : Bergbau und frühkapitallstischeVermögeusentwicklung . 37 dem Annaberger Kaufmann Hieronymus Kettwig mehrmals einen Eisensteinkauf in den „ oberen Bergstädten Annaberg , Marienberg, Schneeberg und den einverleibten Bergwerken, “ wo es bis dahin vielfach an Eisensteinkäufern gefehlt hatte . Auf Grund des „ Stein¬ kaufs “ waren die Gewerken gehalten , allen erbeuteten Eisenstein an Kettwig zu verkaufen 1. Auch die sächsischen Zinnkäufe , auf die wir noch in einem anderen Zusammenhang des längeren zu sprechen kommen werden , sind offenbar von der Regierung in un¬ eigennütziger Weise geschaffen worden. In manchen Bergwerksdistrikten scheint es oft nicht einmal des Eingreifens der staatlichen Gewalt bedurft zu haben , um die Gesamtheit der Gewerken 2 zum Abschluß von Erzkäufen mit reichen Kapitalisten zu veranlassen . So hören wir davon nichts bei dem Goldkauf , den im Jahre 1493 der reiche Breslauer Kauf¬ mann Franz Bottner mit der Gesamtheit der Reichensteiner Ge¬ werken abschloß . Dem Vertrage zufolge übernahm der Breslauer Kapitalist den Verlag der ärmeren Gewerken im dortigen Gold¬ bergbau unter der Bedingung , daß ihm alles gewonnene Gold „auf Abrechnung vom Vorschuß zu jenem Preise geliefert werde, der dem gemeinen Kauf zu Breslau entspräche 3.“ Unbeeinflußt von der Regierung scheint auch der „Vitriol¬ kauf “ zustande gekommen zu sein, den am 6. Mai 1523 die in Goslar zum Vitriolsicden Berechtigten mit einigen Braunschweigern schlossen. Dem Lieferungsvertrage entsprechend mußten die Vitriolsieder drei Jahre hindurch ihr sämtliches Produkt (nur etwas Weniges durften sie nach Frankfurt abgeben ) an die Braunschweiger Kapitalisten zum Preise von 2 Mk. pro Zentner liefern . Die jährliche Mindestlicferung sollte 100 Faß , a 12 Zentner betragen 4. 1 F a 1k e , Geschichte des Kurfürsten August in volkswirtschaft¬ licher Beziehung, S. 168, nennt einen Eisensteinkauf Kettwigs vom Jahre 1564 auf drei Jahre. Der Vertrag ist 1568 erneuert (wiederum auf drei Jahre). Hauptstaatsarchiv Dresden, Loc. 4491 Allerhand Privilegien, Bl. 27. Vgl. auch F a 1k e a. a. O., S. 188. 2 Daß einzelne Gewerken „Käufe“ abschlossen, d. h. gegen Vorschüsse sich zur alleinigen Lieferung ihres Produktes an einen Händler verpflichteten, war selbstverständlich etwas Altes. 3 Z y c h a a. a. O., 6. Bd. (1908), S. 126 nach K. Wuttke, Schlesiens Bergbau und Hüttenwesen (= Codex diplomaticus Silesiae XX und XXI , 1900) Nr. 265. 4 Neuburg, Goslars Bergbau, S. 275. 38 Erstes Buch : Montanindustrie und Frühkapitalismus. Leicht ließe sich die Aufreihung solcher „Käufe“ im Erzhandel des 16. Jahrhunderts noch um Dutzende von Beispielen vermehren 1. Was wir erkennen wollen, erkennen wir mit Hilfe der angeführten schon deutlich genug: Es unterliegt keinem Zweifel, daß die Aus¬ bildung des Systems der Erzkäufe wie nichts anderes die Entstehung von Großbetrieben im Erzhandel förderte und damit dem mittelmäßig Begüterten, der etwa Erzhandel aus erster Hand treiben wollte, den Weg verlegte. Es galt im allgemeinen erst in anderen Handelszweigen ein größeres Vermögen zu verdienen, ehe man Erzhandel aus erster Hand treiben konnte. Hier konnten je länger je mehr nur erste Firmen wirken. Der Erzgroßhandel gab, wie wir noch sehen werden, das wichtigste Feld von bedeutsamen Monopolisierungs¬ versuchen der ganz großen süddeutschen Handelsgesellschaftenab. Als Gesamtresultat der Untersuchungen dieses Kapitels mag dem¬ nach vielleicht das folgende angeführt werden: Auch der Bergbau, die bergmännische Produktion als solche, dürfte den Handel nicht aus seiner Stellung am Anfang, au der Schwelle des europäischen Frühkapitalismus abdrängen. Im Anfang war der Handel, heißt es für die Geschichte des modernen Kapitalismus nach wie vor. Drittes Kapitel. Bergbau und Entfaltung der frühkapitalistisdien Organisationsformen. Das erste Kapitel dieses Buches versuchte einen Begriff zu geben von der quantitativen Bedeutung des Bergbaues und des Erzhandels für die deutsche Wirtschaft des 15. und 16. Jahrhunderts. Wir sahen, daß diese Bedeutung nicht leicht überschätzt werden kann. Aber nicht nur die extensive, die numerische Wichtigkeit für die deutsche Volkswirtschaft im Zeitalter des Frühkapitalismus ist es, was die Wirtschaftshistoriker zu einem näheren Eingehen auf die Verhältnisse im Bergbau und Erzhandel der damaligen Zeit 1 Z. B. aus B eck a. a. O., II, S. 1062, S. 790, 1208 (17. Jahr¬ hundert). Drittes Kapitel: Bergbauu. Entfaltung frtthknpftallsl. Orgunisationsforim n. 39 veranlassen sollte ; wichtiger ist ein Zweites : Nirgends mehr als im Bergbau und Erzhandel wurde im Verlaufe des endenden Mittel¬ alters und der beginnenden Neuzeit auch das Quale der Wirtschaft einer tiefen Umgestaltung entgegengeführt . In keinem anderen Wirtschaftszweige ist das Eindringen des Frühkapitalismus so rasch und so tiefgreifend vor sich gegangen wie gerade in den genannten Produktionszweigen . Schon im Verlaufe des Mittelalters hatte sich aus der älteren Produktivgenossenschaft , in der alle Teilnehmer auch selbst am Berge mitarbeiteten , die spezifisch kapitalistische Gewerkschaft entwickelt . Als der Bergbau im Heiligen Römischen Reiche Deutscher Nation und den von deutschen Kaufleuten wirt¬ schaftlich beherrschten Ländern im 15, Jahrhundert seinen großen Aufschwung nahm , war überall die Unternehmungsform der kapi¬ talistischen Gewerkschaft schon völlig ausgebildet und durchaus die vorherrschende Wirtschaftsform im Bergbau . Mit Recht hat noch neuerdings ein so guter Kenner der geschichtlichen Verhältnisse des europäischen Bergbaues wie Zycha die Vorstellung zurück¬ gewiesen, als ob sich der spezifisch kapitalistische Betrieb während des 15. Jahrhunderts noch in einem Übergangsstadium aus der Periode der Arbeitsgenossenschaft befunden hätte Die Umwandlung der handwerksmäßigen Organisation des Ge¬ werbes in eine kapitalistische , die sich im Bergbau und Hüttenbetriebe früher durchsetzte als irgendwo anders in der Sphäre deutscher gewerb¬ licher Produktion , hat beizeiten in den Zentren der Montanindustrie die Begleiterscheinungen des modernen Kapitalismus geweckt. Eine große Anzahl der wirtschaftstheoretischen und sozialen Pro¬ bleme des viel später erst entstehenden Hochkapitalismus sind im deutschen Montangewerbe des 15. und 16. Jahrhunderts gleichsam in nuce schon vorgebildet . Die Zustände in der Berg- und Hütten¬ industrie des 16. Jahrhunderts lassen dem Beobachter diese Welt oft wie einen Mikrokosmos erscheinen , in welchem das meiste des¬ jenigen bereits im kleinen lebend erscheint , was dann später größten¬ teils erst in dem hochkapitalistischen Makrokosmos des 19. Jahr¬ hunderts seine völlige Ausbildung erfährt. So entstand in den Mittelpunkten des Montangewerbes früh 1 Zycha a. a. O., 5. Bd. (1907), S. 249, und sonst in dessen schon vielgenannten Artikeln „Zur neuesten Literatur über die Wirtschafts- und Rechtsgeschichte des deutschen Bergbaues“. 40 Erstes Buch: Montanindustrie und Frühkapltalismus. ein Proletariat . Der Ausdruck „Arbeiter “ in seinem spezifischen engeren Sinne taucht in den Quellen zur Bergwerksgeschichte des Mittelalters zuerst auf 1, um seitdem als Klassenbezeichnung für die Lohnarbeiter der kapitalistischen Industrie beibehalten zu werden . Und mit dem Namen ward damals die Sache geboren: eine neue soziale Schicht von ihrem Wochenlohn lebender , besitz¬ loser Lohnarbeiter , wie sie in der Menge und charakte¬ ristischen Eigenart noch nicht vorhanden gewesen war, taucht vor unseren Blicken auf . Wir hören von vergleichsweise bedeutenden Arbeitermassen , die sich in den Bergwerksdistrikt .en zusammenballten . Die Fugger -Thurzo hatten in ihren ungarischen Bergwerken um 1525 mehr als 500 Häuer im Dienst 2. Dazu kamen noch die übrigen Arbeiter , die Hüttenleute usw. Allein am Falken¬ stein , dem bedeutendsten Tiroler Bergwerk , war der Knappen¬ stand laut zuverlässiger Forschungen folgender 3: 1526 : 4596 Mann 1554 : 7460 „ 1589 : 4490 „ (4166). Mitte des 16. Jahrhunderts dürfte der Knappenstand aller Scljwazer Baue mit 12 000 Mann nicht zu hoch bemessen sein 4. Auch hierzu sind — um die Gesamtmenge der dortigen Arbeiterschaft fcstzustellen — die „ungelernten Arbeiter “ zu addieren . So arbeiteten nach dem sog. Ettenhardtschen Bergbuche im Jahre 1532 beim Falkensteiner Tiefbau täglich 500 bis 600 Wasserheber (jährlich zirka 20 000 fl. Arbeitslohn) B. Und diese Bergarbeitermassen, die uns hier begegnen , haben schon früh wichtige Eigenarten des modernen Proletariats ausgebildet . Sie haben schon die Mehrzahl der entscheidenden Charakteristika des modernen Proletariertums an sich, wie sie uns Sombart mit seiner künstlerisch -feinen Be¬ gabung für sozial - psychologische Nachempfindung geschildert 1 Auch schon der Ausdruck Lohnarbeiter kommt früh vor ; vgl. Gasteiner Goldrecht (1300—1350 ), S. 198 : „Welcher lonarbeiter seiner samchost und verdienten Ion von den gruebmeistern nit bezalet wirdet . . ." K . Th . v . Inama - Stemegg, Deutsche Wirtschaftsgeschichte III , 2, S. 162 Anm . 2. 2 Jansen, Jakob Fugger der Reiche , S. 170. 3 Z y c h a a. a. O., 5. Bd . (1907), S. 256. 4 Ebenda S . 256 Anm . 3. * Ebenda S. 256 Anm . 1. * * Drittes Kapitel: Bergbauu. Entfaltung frühkapltalisL Organisationsformen . 41 hat 1. Da ist die Unsicherheit der Existenz bedingt durch nicht selten auftretende „ soziale Arbeitslosigkeit“ . Eine Grube ersäuft oder Feuer zerstört das mühsam geschaffene Werk der Schächte2. Ein Bergwerk erscheint nicht mehr lohnend. Hunderte verlieren Arbeit und Brot. Da ist schon die Nomadenhaftigkeit. Wenn das Proletariat des 19. Jahrhunderts in besonderer Weise dadurch mit charak¬ terisiert wird, daß ihm die Seßhaftigkeit fehlt, daß es „von Stadt zu Stadt , von Land zu Land zieht, wohin es gerade die Konjunktur, der Arbeiter heischende Kapitalismus“ ruft , so hat auch hier das 19. Jahrhundert nur etwas vergrößert und intensiviert, was wir im Keime schon im Leben des Bergarbeiters des 15. und 16. Jahr¬ hunderts finden. Es gibt kaum ein europäisches Land im 15. und 16.Jahrhundert , das nicht deutscheBergarbeiter—gerufen zumeist von deutschen kapitalistischen Unternehmern -— in größerer Anzahl sah. So ließen sich, um nur einige Beispiele zu nennen, deutsche Bergknappen zur Aufbesserung des Betriebes in die Bergwerke der spanischen Provinz Galicien anwerben s. Im Jahre 1528 konnte der Augsburger Kapitalist Joachim Höchstetter, den der König von England zum „principal surveyor and master of all mines in England and Ireland“ ernannt hatte , sich erbieten, die von ihm entdeckten Bergwerke mit sechs anderen deutschen Unternehmern und 1000 Arbeitern zu bearbeiten 4. In den französischen Minen sind in erster Linie Deutsche als Bergarbeiter beschäftigt 6. Ja, als man in den Bergen der amerikanischen Kolonien Spaniens Edelmetalle fand und Krone und Kapitalismus dadurch zum Berg1 W. So mb art, Das Proletariat , Bilder und Studien . In: Die Gesellschaft. Sammlung sozial-psychologischer Monographien, herausgegeben von Martin Buber. 2 „Ein schwerer Schlag traf 1585 die Knappen am Röhrerbühel in Tirol . In einem Hauptschachte dieses Bergwerks brach Feuer aus, welches mehrere Tage nicht unterdrückt werden konnte . 11 Ar¬ beiter verloren dabei das Leben, mehr als 1000 andere mußten längere Zeit die Arbeit einstellen.“ Jos . Hirn, Erzherzog Ferdinand II. von Tirol. Bd . I, S. 559. 8 K . Häbler, Die überseeischen Unternehmungen der Welser, S. 61. 4 R . Ehrenberg, Hamburg und England im Zeitalter der Königin Elisabeth , S. 5. P . ImbartdelaTour, Les origines de la Réforme. 2 Bde. Paris 1905/09. Bd. I, S. 232 Anm. 1: „En 1483, dans le Conserans, le personnel des mines est encore presque entièrement allemand .“ 42 Erstes Ruch : Montanindustrie und Frühkapitulismus. bau angereizt wurden , da fanden sich in Joachimstal und Schwaz Bergleute , die dem Rufe des Kapitalismus auch über das Welt¬ meer willig folgten . Mehr als 50 Bergknappen warben allein die Welser für die spanischen Kolonien an 1. Mit der Massenhaftigkeit und der „Unseßhaftigkeit “ der prole¬ tarischen Bergwerksbevölkerung des 15. und 16. Jahrhunderts tritt auch das Problem der Wohnungsnot bereits in den Kreis der sozial¬ psychologischen Geschichtsbetrachtung . Erst eine eingehende Er¬ forschung der sozialen Lage der Bergarbeiterschaft im Zeitalter des Frühkapitalismus , die vielfach auf noch ungedrucktem Quellen¬ material zu beruhen hat , wird uns die vielerlei Fragen nach den Wohnungsverhältnissen des damaligen Industrieproletariats be¬ antworten können . Auch die Frage , inwieweit das Elend der Frauenund Kinderarbeit schon in der Montanindustrie und den damit zusammenhängenden Industrien um sich gegriffen hatte , wird dabei näher zu erforschen sein 2. Zweifellos waren beide Übel schon vor¬ handen . So wissen wir , daß Anfang des 17. Jahrhunderts im Steyrer Eisenhandwerk die Zahl der weiblichen Hilfskräfte überwog . Für 300 Messerermeister arbeiteten nur 150 Gesellen, aber 1500 Mägde 3. Was aber die Kinderarbeit im Bergbau angeht , so wird man an die schlimmsten Zustände etwa in der englischen Industrie zu Anfang des 19. Jahrhunderts erinnert , wenn man Schilderungen des 16. Jahr¬ hunderts wie folgt liest : „ Knaben im zarten Alter von 10 bis 12 Jahren müssen als „Kläuberbuben “ schon zur Tagarbeit heran , um „Bruch und Zagei“ zu scheiden und zu säubern . Waren sie soweit erstarkt, um mit beladener Bergtruhe laufen zu können , dann traten sie in den Dienst als .Truhenläufer oder Hundzieher “. Erst nach Ab¬ lauf dieser Bubendienste wurden sie .Hauer mit Schlägel und Eisen “. In der Hüttenindustrie begann mancher Junge zuerst als Kaener (Kohlenbrenner ) und Holzknecht seine harte Laufbahn , um dann bei günstigem Geschick an einem Plähhaus oder Schmelzwerk Plähknecht (Schmelzer) oder Wäscher zu werden “ 4. ‘Häblera . a. 0 „ S. 61 ff. 2 Ebenso die Frage der Sonntagsruhe. Vgl. hierzu Oberbair. Archiv XIX S. 127: Ludwig dem Reichen wird von Rom aus er¬ laubt, Sonntags in den Salinen von Reichenhall arbeiten zu lassen. 3 Bittner a . a. 0 „ S. 549, 551. 1 H. P e e t z , Volkswissenschaftliche Studien. München 1880, S. 17 ff. Z y c h a a. a. O., 5. Bd. (1907), S. 255 Anm. 5. 2 6 Drittes Kapitel: Bergbauu. Entfaltung frühkapltallst. Organisationsformen . 43 Die Beispiele würden sich aus der berggeschichtlichen Literatur und besonders aus Archivalien leicht mehren lassen , hier muß ich dazu übergehen , auf einige andere , besonders augenfällige Er¬ scheinungen in der Sozialgeschichte der Bergarbeiterschaft des ausgehenden Mittelalters und der beginnenden Neuzeit aufmerksam zu machen , die deutliche Analogien zur späteren hochkapitalistischen Entwicklung bieten . Da gilt es zunächst auf das Trucksystem hinzuweisen ! Ein echtes Kind der frühkapitalistischen Montan¬ industrie , ist es zuerst als eine Wohltat für die Arbeiterschaft ent¬ standen . Es war für die oft sehr zahlreiche Bergarbeiterschaft unmöglich , genügende Nahrung und Kleidung usw. in der näheren Umgebung ihrer Arbeitsstätte zu bekommen . Oft lagen ja auch die Gruben fern ab jeder sonstigen Ansiedelung 1. Da war es gut, wenn die Bergarbeiter ihren Lohn nicht lediglich in Bargeld , sondern teilweise in „Pfriend “ erhielten , d. h. in Korn oder Mehl, Schmalz, Tuch oder sonstigen unentbehrlichen Bedürfnissen , Pfennigwirtschaft oder Pfennwertshandlung genannt 3. Aber bald wußten manche Unternehmer die „Pfennwertshandlung “ dazu zu benutzen , um sich zu bereichern 3. Dagegen erhob sich vielerorts ein Kampf der Bergarbeiter , der, unterstützt von den Regierungen und oft von den Bürgern der den Bergwerken benachbarten Städte 4, zu¬ meist von Erfolg gekrönt war B. Vielfach gingen mit den Be1 Im Jahre 1558 bewilligte Kurfürst August von Sachsen dem Schneeberger Andreas Sonnebrunn u. a. für ein neu anzulegendes Zinnbergwerk ein größeres Terrain , damit er, „w eil die Gegend unbewohn t “ , seine Bergleute und Arbeiter auf dem verliehenen Boden „bauen , wohnen und denselben zu irem underhalt erblich brauchen und geniessen lassen möge .“ Dresden , H . St . A . Loc . 4491 Allerhand Privilegien usw . Bl . 13 a. 2 Vgl. besonders Zycha a. a. O., 5. Bd . (1907 ), S. 256 , wo wichtiges berggeschichtliches Material über das Trucksystem zusammen¬ gestellt ist . Dazu Salz a. a. 0 ., S. 96. 3 Gute Beispiele bei Hirn a. a. O., Bd . I, S. 557 f. 4 Die sich natürlich durch das Trucksystem um einen Teil ihrer Kundschaft bedroht sahen . Vgl . die Bewegung der ungarischen Bergstädte , besonders Neusohl , gegen die Pfennwertshandlung der Fugger -Thurzo dort . Jansen, Jakob Fugger der Reiche , S. 165 ff. 6 Auch das sog . Cottagesystem (der Unternehmer baut Wohnhäuschen und zwingt die Arbeiter , sie ihm äbzumieten ) ist wohl schon im 16 . Jahrhundert bekannt . Aus dem 17. Jahrhundert sei folgendes Beispiel aus den Akten des Hauptstaatsarchives zu Dresden (Loc . 4491 Allerhand Privilegien ) angeführt : Im Jahre 1680 wird * * 44 Erstes Buch : Montanindustrie und Frühkai >italismus, schwerden über die Auswüchse des Trucksystems Klagen über allzu lange Arbeitszeit 1und besonders Lohn Streitigkeiten Hand in Hand. Oft kamen diese aber auch gesondert vor und waren vielfach mit heftigen Streiken verbunden. Eine ausführliche inter¬ essante Schilderung eines solchen Bergarbeiterstreikes hat uns neuerdings Jansen in seinem „Jakob Fugger“ gegeben 2. Andere Beispiele mit Bedrohung der Streikbrecher usw. waren in größerer Anzahl schon vorher durch die Literatur bekannt 3. Bekannt ist es auch, wie vielfach in den Bauernaufständen des 16. Jahrhunderts sich das Bergarbeiterproletariat mit dem bäuerlichen Proletariat verband 4. Dagegen dürfte es noch neu sein, daß auch schon Unter¬ nehmer des beginnenden 16. Jahrhunderts eine Art Antistrikeverbände schlossen. Im Jahre 1520 vereinigten sich die Herzoge von Sachsen, die Schlick als Herren von Joachimstal und die Pflug als Herrschaftsbesitzer in dem böhmischen Schlackenwald 6 zu einem Abkommen, das einen interessanten Beitrag zur Wirtschafts¬ und Sozialgeschichte jener Zeit darstellt 6. Das Abkommen sollte einer Firma , die neue Eisenhämmer in menschenleerer Gegend er¬ bauen will, bewilligt „etliche Wohnhäuserlein vor die Arbeiter , Bergund Handwerksleute . . . aufzubauen und sie mit Leuten zu besetzen.“ 1 Die Unternehmer suchten die normale Arbeitszeit dadurch zu umgehen , daß sie dem Arbeiter die Zeit des Einfahrens in die tiefen Stollen nicht anrechneten . Hirn a. a. O., I, S. 560. 2 a. a. O. S. 175 ff. 3 Streik der Schneeberger Bergleute im Jahre 1496. Vgl. Chr. Heizer, Bergkläufftige Beschreibung der Bergk-Stadt Schneebergk . 2. Aufl. S. 924. Der damalige deutsche Ausdruck für Streik ist „Aufstehen , Uflauf“ üsw. Streik der Joachimstaler Bergleute bei P . A 1b i n u s , Meißnische Bergchronik , S. 76. Der Mansfeldischen in Verbindung mit dem Bauernkrieg : Albinus a. a. O., S. 109. Vgl. weitere Meldungen von Bergarbeiterstreiken bei Salz, S . 31 und Anmerkungen ; Mück a. a. O., I, S. 115. 4 Vgl . auch Sitzungsberichte der Wiener Akademie der Wissen¬ schaften , hist .-pliil. Klasse X (1853), S. 403. 8 Alle drei Bergherren hatten bedeutende eigene Bergwerke und Bergwerksteile. 6 H. St . A. Dresden, Loc. 4486. - Bergwerkssachen de anno 1487 bis 1599. Bl. 72—74. Bei mir im Anhang abgedruckt ; für die Vorverhandlungen vgl. auch Loc. 9848 , Handlung durch der Herzoge zu Sachsen Räte in Altenburg , usw. Bl. 1 ff. Die Ver¬ abredung ist 1540 erneuert worden. Loc. 7215. Schlicksche Sachen usw. 1520—1540. Bl. 68 ff. Vgl. auch Ernestinisches Gesamtarchiv zu Weimar . Reg.T. Abt . 2. Bl. 258. Drittes Kapitel: Bergbauu. Entfaltung fl'Ubkapitalisl. Oiganisatiimsforinen . 45 die Abwanderung der Bergleute von einem Bergwerksgebiet in das benachbarte andere verhindern. Zu dem Zwecke verabredeten die genannten Kontrahenten, in ihren Bergwerken auf keinen Fall eine Lohnerhöhung eintreten zu lassen; vielmehr sollte auf allen Bergwerken ein gleicher Lohn (berechnet nach dem Werte der Münze des betreffenden Landes) gezahlt werden (§ 1). Ferner sollte es keinem Häuer in den Bergwerken der genannten Herren gestattet werden, zwei Schichten einzufahren (§ 2). Wenn ein Schichtmeister, ein Steiger oder ein anderer Bergmann Schulden bei den Gewerken oder den Bergarbeitern oder auch der Berg¬ herrschaft (aus dem Zehnten) mache und nun auf das Bergwerks¬ gebiet eines der Kontrahenten' entfliehen würde, so soll der bös¬ willige Schuldner dort „keine Freiheit noch Sicherung genießen“ (§ 4). Ebensowenig soll ein Agitator, der „bei den Bergleuten Un¬ willen, Aufstehen und Aufruhr“, d. h. Streik, anstifte und deshalb ausgewiesen würde, auf einem Bergwerk der Kontrahenten Arbeit bekommen (§ 5). Schwarzes Buch! Einen tiefen Einblick in die sozialen Verhältnisse des 16. Jahr¬ hunderts gestattet auch § 7. Dort verabreden die genannten Berg¬ herren, daß sie streikende Bergarbeiter, die in hellen Haufen aus den Gebieten des einen Kontrahenten in die Gebiete der anderen ziehen, nicht aufnehmen, sondern „die Entwichenen zu der Ge¬ rechtigkeit halten und ihres Mutwillens strafen“ wollen. Man sieht, die Verabredungen der Bergherren sind im Grunde wirtschaftlicher Natur ; sie sollen dazu dienen, Angebot und Nach¬ frage von Arbeitskräften in einer den Kontrahenten günstigen Weise zu regeln 1. Aber nicht nur soziale Probleme des modernen Kapitalismus begegnen uns in großer Anzahl bereits in der Geschichte der Montan¬ industrie des ausgehenden Mittelalters und der beginnenden Neu¬ zeit. Interessant ist es auch, zu beobachten, wie sich hier viele von jenen wirtschafts- und betriebsorganisatorischenUmbildungen schon zeigen, die später, besonders im 19. Jahrhundert , ihre bisher höchste Ausbildung erfahren haben. 1Uber heutige Verbände der Arbeitgeber gegen die Arbeiter (Antistreikverbände ) vgl . R . Liefmann, Die Untemehmerverbände (Konventionen , Kartelle ), ihr Wesen und ihre Bedeutung. Freiburg i. Breisgau 1807 , S. 72 f. 46 Erstes Buch : Montanindustrie und Frühkapitalisinus. Ich denke dabei zunächst an die Tatsache , daß in Deutschland erst innerhalb der Montanindustrie des 16. Jahrhunderts wirkliche konzentrierte kapitalistische Großbetriebe in größerer An¬ zahl zu entstehen begannen 1. Hatten bis dahin die kapitalistischen Kaufleute die gewerbliche Produktion in manchen Gewerbszweigen mit Hilfe des Verlagssystems schon mittelbar dirigiert , so schufen sie sich jetzt selbst eigene industrielle Großbetriebe und wurden außer Kaufleuten auch unmittelbar gewerbliche Produzenten . Das ungarische Geschäft der Fugger -Thurzo und ihrer Nachfolger , das Unternehmen der Fugger in den spanischen Quecksilbergruben von Almaden , die vielen Hüttenwerke , die vereinigten Hütten - und Bergwerksbetriebe einzelner und vergesellschafteter süddeutscher Kaufleute in Tirol und sonst („ Gesellschaften des Berg- und Schmelz¬ handels “ genannt) 2, die großen Saigerhandelsgesellschaften im Mansfeldischen , die uns neuerdings Möllenberg beschrieben hat, das alles waren konzentrierte Großbetriebe , in denen sich Pro¬ duktion und Handel zu modernen , bis dahin nicht gekannten kapitalistischen Unternehmungsformen vereinigten. Zweifellos hatte der Fortschritt in der Technik des Berg - und Hüttenwesens zu der gekennzeichneten Entwicklung sein gut Teil beigetragen , wie umgekehrt gerade die Ausbildung von Groß¬ betrieben in der Sphäre des Berg- und Hüttenwesens (wir könnten auch sagen das Eindringen des Kapitalismus ) wie nichts anderes den Fortschritt der Technik dort begünstigte und ermöglichte. Vorzüglich kann man den Zusammenhang von technischem Fort¬ schritt und der Bildung von Montangroßbetrieben in der Geschichte der Thüringer Saigerhandelsgesellschaften beobachten . Seit die Technik des Kupfersaigerns 3 in den sechziger und siebziger Jahren des 15. Jahrhunderts allgemeiner bekannt wurde , schossen die 1 Wenn wir von einigen Buchdruckereigroßbetriebenabsehen. Sombart, Der moderne Kapitalismus I, S. 405. 2 Beispiele bei Zycliaa. a. O., 5. Bd. (1907), S. 285 ff. und sonst aus der Literatur zusammengestellt. „Verbindungen der berg¬ männischen Produktion mit dem Fernhandel in Metallen, insofern nämlich die hervorragendsten Händler auch die ersten Gewerken und Schmelzer wurden.“ 8 D . h. die Kunst, das Silber mit Hilfe von Blei aus den silber¬ haltigen Kupfererzen zu ziehen. Näheres über das Verfahren bei M ö 11e n b e r g a. a. O., S. 5. 3 Drittes Kapitel: Bergbauu. Entfaltung frühkapitulist. Orge nisationsformen . 47 Saigerhütten und Saigerhandelsgesellschaftenwie Pilze aus dem Boden 1. Hauptsächlich Nürnberger, aber auch Augsburger und bald Leipziger Kapitalisten gründeten Saigerhandelsgesellschaften und bauten Saigerhütten in großer Anzahl. Solche Hütten aber waren sehr kostspielige Anlagen und deshalb dem Kleinbetrieb entzogen. „Die Einrichtung einer auf etwa 7000 Zentner Kupfer jährlich berechneten Saigerhütte bestand aus 8 Schmelzöfen, 10 Saigeröfen, 3 Garherden, 3 Treibherden und 2 Dörröfen“ 2. Der Bau der Hütte Leutenberg dauerte mehrere Jahre und be¬ anspruchte ein Kapital von weit mehr als 10 000 fl. a. Es versteht sich von selbst, daß bei solchen Anforderungen nur das Großkapital auf dem von der Technik neu gewonnenen Gebiete arbeiten konnte. Und auch das Großkapital mußte sich oft noch gesellschaftlich zusammentun, um die neuen Aufgaben erfüllen zu können. Uni so mehr als die jungen Großbetriebe nun ihrerseits wieder eine starke Tendenz der Ausweitung in sich trugen. Gerade wie ihre Nach¬ kömmlinge im 19. Jahrhundert. Auch schon aus dem geringen Material, das uns bis jetzt über diese Dinge vorliegt, können wir diese Tendenz der montanistischen Großindustrie erkennen. Als die Fugger-Thurzo im Jahre 1494 bei Neusohl eine Saigerhütte zur Verarbeitung der dortigen Kupfer¬ erze bauten, da warfen sie 1000 fl. für den Bau aus und berechneten die Bauzeit auf etwa ein halbes Jahr 4. Die Leutenberger Saiger¬ handelsgesellschaft, die Mansfelder Kupfererze verarbeitete, brauchte, wie gesagt, schon mehrere Jahre und ein Kapital von weit über 10 000 fl. für die Herstellung ihrer Hütte . Gerade die Geschichte der Mansfeldischen Saigerhandelsgesellschaften zeigt die Schnellig1 Mö 11 e n b e r g a. a. O., S. 7. 2 Mö 11e n b e r g a. a. O., S. 5. 3 Möllenberg a. a. O., S. 25. — Eine Geschichte der tech¬ nischen Fortschritte im Berg- und Hüttenwesen des endenden 15. und 16. Jahrhunderts tut uns bitter not ! Im Bergbau dieser Zeit liegen auch die Anfänge eines Erfinderschutzes. Man kannte dort schon im 16. Jahrhundert Patente. Vgl. z. B. Falke, Geschichte Kurfürst Augusts von Sachsen in volkswirtschaftlicher Beziehung, S. 202. 'Jansen, Jakob Fugger der Reiche, S. 135. Außer dem Neusohler besaßen die Fugger noch ein Saigerhüttenwerk in Kärnten und eines in Thüringen (Hütte Hochkirch). 23 5 48 Erstes Buch : Montanindustrie und Frühkapitullsmus. keit , mit der sich Kapitalien in solchen neuen , industriellen Groß¬ betrieben zu konzentrieren pflegten . Die Gesellschaft der Hütten Schwarza und Mansfeld , im Jahre 1472 gegründet , hatte noch mit einem Stammkapital von 6000 fl. auskommen zu können gemeint; in die 1502 gegründete Arnstädter Hüttengesellschaft hatten die Inhaber bereits 31 500 fl. eingeschossen. Die höchste Entwicklung zeigte dann die Gesellschaft der Hütte unter Leutenberg . Die Gesellschaft hatte mit dem gewiß stattlichen Stammkapital von 70 000 fl. ihre Geschäftsfähigkeit im Jahre 1524 eröffnet , 1526 waren bereits 91 000 fl., 1527: 108 380 fl., 1532: 120 710 fl. in¬ vestiert 1. Auf ähnlicher Höhe standen die Stammkapitalien der 1536 resp . 1537 neugegründeten Hütten Luderstadt (80 000 fl.) und Steinach (93 000 fl. eingezahlt ). Zeitweise hatte ein einziger Gesellschafter der Hütte Luderstadt , der Leipziger Kapitalist Heinrich Scherl, 40 000 fl. investiert a. Das Betriebskapital der Steinadler stieg bis 1554 auf über 236 000 fl. ®, später , wie es scheint, noch über diese enorme Summe hinaus 4. Nicht minder groß als die in den führenden Mansfeldischen Saigerhandelsgesellschaften eingeschossenen Kapitalien waren die Kapitalien , die sich in anderen montanindustriellen Großbetrieben des 16. Jahrhunderts konzentrierten . Die Aktiva , welche die Firma Haug , Langenauer , Link & Co . in ihren Schwazer Montanunter¬ nehmungen angelegt hatten , stiegen von 60 262 fl. im Jahre 1533 auf 194 416 fl. im Jahre 1555 6. In den Faktoreien des ungarischen Erzhandels stiegen die Aktiva der genannten Firma von 13 873 fl. im Jahre 1560 auf 87,016 fl. im Jahre 1562 6. Die Fugger und Thurzo schossen bei der Gründung ihres ungarischen BergwerksUnternehmens im Jahre 1494 kein bestimmtes Kapital ein, die Fugger legten vielmehr dem Unternehmen das nötige Betriebs¬ kapital nach Bedarf allmählich vor . Von Ende 1494 bis Mai 1504 1 Möllenberg a. a. O., S. 25. 2 I.aut seinem Testament. Kroker, Neujahrsblätter IV, S. 82. 3 Mö 1l e n b e r g a. a. O., S. 109. 4 Fr i e d r. B o t h e . Frankfurter Patriziervermögen im 16. Jahr¬ hundert. Berlin 1908, S. 61. 5 Vgl. die Tabelle I bei Hartung. Aus dem Geheimbuch eines deutschen Handelshauses a. a. O., S. 39. 6 Vgl . die Tabelle I bei Hartung a. a. O., S. 39. Drittes Kapitel: Bergbauu. Entfaltung fvühkapitalist.Organisationsformcn . 49 hatten die Fugger für „offizielle Erfordernisse , Pachtgelder , Wege¬ bau u. a . und offizielle Schmiergelder , um bei den Herren den Handel zu bekommen und zu behalten , 1 064 499 ungarische Gold¬ gulden 65 Pfennige ausgegeben “. Dieser Summe standen an Bar¬ einnahmen nur 1 001 837 fl. (ungarische ) 64 Pfennige gegenüber. Der „gemeine ungarische Handel “ blieb daher den Fuggern 82 662 fl. (ungarisch ) 1 Pfennig (= 83 589 fl. rh . 35 Pfennig ) schuldig . An Materialvorrat war für 241 913 fl. ungarisch vorhanden , so daß die Aktiva der Fugger -Thurzoschen Montanunternehmung (abzüglich des Fuggerschen Guthabens von 62 662 fl. ungarisch ) noch 179 251 fl. ungarisch betrugen 1. Groß waren auch die Kapitalien , die sich in dem „ Schwazer Berg-, Schmelz- und Pfennwerthandel “ konzentrierten , den 1526 die Fugger mit Benedikt Burkhart , Christof Herwart aus Augsburg, Anton und Hans Bimmel aus Augsburg — alles Firmen , die längst im Tiroler Berg- und Schrnelzhandel tätig waren — schlossen. „Das zusammengeschossene Kapital betrug 84 000 fl., wovon Burkhart mit 12 000 fl., die anderen drei Parteien mit je 24 000 fl. partizipierten . Mit Schluß des Jahres 1527 verkaufte Burkhart zwei Drittel seines Anteiles an die Gebrüder Bimmel, ein Drittel an die Fugger “ 2. Noch bedeutender war die Gründung des „ Jenbaclier Berg- und Schmelzwerkhandels “, an dem neben den Fuggern zunächst auch die Haug & Co. und Michael Katzbeck erheblichen Anteil hatten . Die Fugger investierten in die neue Gesellschaft „ihre auf 114 422 fl. veranschlagten Bergteile im Inn -, Eisack - und Etschtal mit den dazugehörigen Hütten - und Schmelzwerken .“ 1575 brachten die Fugger den Haugschen , 1578 den Katzbeckschen Anteil des „ Jenbacher Handels “ an sich 3. Mit vollständigen Fusionen scheinen wir cs bei der Jenbacher und der Schwazer Gründung nicht zu tun zu haben. Wenigstens reservierten sich die Fugger nach der Gründung des Jenbacher Berg- und Schmelzwerkhandcls ihre Anteile in Kärnten und am Röhrerbühel mit dem Hüttenwerk Litzlfeldcn als „Tiroler und Kärtner Propriohandel“ oder auch „ Schwazer Proprio1 Jansen , Jakob Fugger der Reiche, S. 152 ff. 2 Dobel, Bergbau und Handel d. Jak. und Ant. Fugger in Kärnten und Tirol, a. a. O., S. 202. 3 DaselbstS. 213. Strieder , Studien x. Gesch . kapit &list . Organisations rönnen . 4 1 50 Erstes Buch: Montanindustrie und Frühkapitalismus. handel “. Vielleicht haben aber die anderen Firmen sich keinen „Propriohandel “ zurüekbehalten , sondex-n alle ihre Tiroler Bergwerks¬ unternehmungen und ihren gesamten dortigen Erzhandel in die Jenbacher Gesellschaft fusioniert . Hier werden uns monographische Untersuchungen über die Organisationsformen solcher einzelner Großunternehmungen , die ein Bedürfnis vertiefter wirtschafts¬ geschichtlicher Forschung sind , später erst ganz klar blicken lassen. Die Fusionierung einzelner montanindustrieller Unternehmungen in großen Gesellschaften des „Berg- und Schmelzwerkhandels “ be¬ deutet einen Schritt weiter auf dem Wege der Versachlichung , der Objektivierung der Wirtschaftsbeziehungen in dem Montangroß¬ gewerbe, auf die wir als Ausdruck fortgeschrittener kapitalistischer Wirtschaftsorganisation nunmehr einen Blick werfen wollen Im eigentlichen Bergbau hatte die Versachlichung der Wirtschafts¬ beziehungen bereits früher bedeutende Fortschritte gemacht . Zu¬ nächst hatte z. B. der einzelne Gewerke (durch die Hand des Bergbeamten ) und nicht etwa die Gewerkschaft als Ganzes den Berg¬ arbeitern den Lohn gezahlt 2. Dieses persönliche Verhältnis ver¬ wandelte sich allmählich in ein. sachliches . Der einzelne Gewerke entrückt dem Gesichtskreise des Arbeiters , die Lohnzahlung über¬ nimmt die Gewerkschaft , eine unpersönliche Gemeinschaft häufig wechselnder Kapitalisten. In den Kuxen über Bergwerksanteile war ferner das erste versachlichte Kreditverhältnis im ( weitesten Sinne des Wortes Kredit ), das erste bedeutungsvolle Inhaberpapier ge¬ schaffen worden . Was wir über den Kuxhandei und die Kux¬ spekulation schon aus dem 16. Jahrhundert wissen, läßt keinen Zweifel darüber , daß viele Inhaber von Kuxen persönlich dem 1 In seinem Buche „ Die Juden und das Wirtschaftsleben “ (Leipzig 1911) hat Werner Sombart auf die verschiedenen Arten der Ver¬ sachlichung der wirtschaftlichen Beziehungen als eine „ für alles hochkapitalistische Wesen mehr denn irgendein anderer Vorgang kennzeichnende Erscheinung “ hingewiesen. Man wird die sub¬ tilen Untersuchungen auch dann mit Genuß lesen, wenn man den Anteil der Juden bei dieser Entwicklung nicht so hoch wie Sombart einschiilzt und wenn man dieAn länge der „Kommerzialisierung" des europäischen Wirtschaftslebens etwas früher ansetzt als es der genannte Autor tut. 2 O . O p e t , Das Gewerkschaftsrecht , a. a. O., S. 312 f. ; Z y c h a a. a. O., 5. Bd. (1907), S. 256 f.; S a 1z a. a. O., S. 26. Drittes Kapitel : Bergbau u. Entfaltung Irühkapitalist , Organisationsformen . 51 Unternehmen , dessen Mitbesitzer sie durch ihre Anteilscheine waren, durchaus fremd gegenüberstanden l . Zeigt sich in all den genannten Organisationsformen die Montan¬ industrie des 16. Jahrhunderts als ein Vorbild des späteren Hoch¬ kapitalismus im kleinen , so erhebt sich für uns die Frage : Liegen vielleicht auch noch die Anfänge anderer Erscheinungen der hoch¬ kapitalistischen Wirtschaft im kleinen schon in der Montanindustrie des 16. Jahrhunderts verborgen ? Die Anfänge der Aktiengesellschaft vielleicht , der Ursprung des Kartellwesens etwa oder die eisten Versuche großer internationaler Monopole ? Da sind nun bei solcher Fragestellung unsere Theoretiker schnell bei der Hand , eine innere Unmöglichkeit zu konstatieren , der zufolge Gebilde wie Aktien¬ gesellschaften nicht im „Mittelalter “ entstanden sein können . So schreibt F . Wernicke in seinem Buche „Der Kampf um den wirt¬ schaftlichen Fortschritt “ 2, nachdem er hervorgehoben hat , wie die kanonische Wucherlehre der Bildung von Kapitalassoziationen widerstrebte : „ So suchen wir denn auch die Form einer Gesellschaft, die lediglich durch den Zusammenfluß von Kapitalbeträgen ohne. Arbeitstätigkeit irgendeines der Zuschießenden entsteht , im Mittelalter vergebens , kein einziger kanonistischer Schriftsteller hat den Begriff einer reinen Kapitalassoziation konstruiert , weil eine solche Gesellschaft handgreiflich ein Widerspruch gegen die Unfruchtbar¬ keit des Geldes gewesen wäre , für den sich wohl keine Rechtfertigung hätte finden lassen 3“ . Und nach Endemann fügt dann Wernicke in Sperrdruck hinzu : „Es ist sonach kein Zufall, daß wir reine Kapitalvereine in den großen Handelskompagnien zuerst in Eng¬ land und den Niederlanden , also in protestantischen Ländern und zu einer Zeit , da es mit der Wucherlehre dort reißend bergab ging, entstehen sehen“ 4. Die methodische Geschichtsforschung hat ge¬ lehrt , daß man mit Urteilen aus Gesetzesvorschriften aller Art 1 Vgl. Anhang, auch unten den Abschnitt: Entstehung der Aktiengesellschaft. Georg Kreuziger, ein Leipziger Kaufmann, spekulierte in der ersten Hälfte des 16. Jahrhunderts mit mehr als einem halben Tausend von Kuxen. K r o k e r , Schriften des Vereins für die Geschichte Leipzigs IX, S. 38 ff., besonders S. 58. 2 .Jena 1910, S. 40 ff. 3 We r n i c k e a. a. O., S. 40. 4 W . Ende mann, Studien in der romanisch-kanonistischen Wirtschafts- und Rechtslehre bis gegen Ende des 17. Jahrhunderts. 2 Bde. Berlin 1874, 1883. I. ßd ., S. 371 ff. 4* 52 Erstes Buch: Montanindustrie und Frühkapitalismus. auf die Realität der Dinge sehr vorsichtig sein muß. Wir wissen heute , daß die Wirtschaftsethik der mittelalterlichen Kirche die Ausbreitung kapitalistischen Geistes und kapitalistischer Wirtschaft wohl hat hemmen, aber nicht — auch schon im Mittelalter nicht — hat aufhalten können. Ein besonderer Teil „ Staat ,Kirche und Frühkapitalismus “ soll zunächst hierüber näheren Aufschluß geben, che wir uns im dritten Buche den neuen, aus dem kapitalistischen Geiste geborenen wirt¬ schaftlichen Organisationsformen selbst zuwenden. Zweites Buch. Kirche, Staat und Frühkapitalismus. 55 Kirche, Staat und Frühkapitalismus. „Not kennt kein Gebot .“ Die Geschichte der Entfaltung des kapitalistischen Geistes in Europa ist die Geschichte der Entfaltung des Individuums , der einzelnen kräftigen Persönlichkeit auf dem besonderen Gebiete der materiellen Kultur , auf dem Gebiete . des Wirtschaftslebens. Wie der Gesamtindividualismus in jener Kulturbewegung , die wir Renaissance nennen , so tritt auch der wirtschaftliche Individualismus zuerst in 11 a 1i e n in einem größeren Maßstabe in die Erscheinung als eine Teilmanifestation des gewaltigen Geistes, der an die Arbeit geht , die moderne Welt zu begründen . Wie auf politischen , auf religiösen, auf künstlerischen Gebieten , so hoben sich in Italien schon seit dem 10. Jahrhundert auch auf dem Gebiete des Wirt¬ schaftslebens zuerst einzelne, dann im Verlaufe des 12. und 13. Jahr¬ hunderts immer mehr besonders kräftige Individualitäten aus der großen Masse heraus . Menschen, die über ihre Kreise , über ihre Handels - und Gewerbsgenossen hinauszukommen suchten und hinauszukommen wußten . Männer , die sich nicht mit der im all¬ gemeinen üblichen Wirtschaftsweise begnügten , mit der Wirtschafts¬ form, die wohl eine gewisse standesgemäße Nahrung verbürgte, aber nicht viel darüber hinaus . Persönlichkeiten , die neue Erwerbs¬ möglichkeiten kühnen Geistes erfaßten , die neue Methoden der Betätigung eines gesteigerten Erwerbstriebes sich zu eigen machten. Es fragt sich, welches waren die Motive, die diese Naturen zu einer so intensiven Betätigung des Erwerbstriebes vermochten? Der Wunsch , in die sozial höhere Klasse hinaufzusteigen , Ehrgeiz, Ruhmsucht , Sorge um die Familie ? Das und manches andere noch hat zweifellos mitgespielt , aber im Grunde , und gerade bei den schöpferischsten dieser Naturen am meisten , war es noch etwas anderes . Im Untergründe , oft hinter der Bewußtseinsschwelle, trieb sie das Betätigungsbedürfnis der eigenen kräftigen Persönlich¬ keit . Jener unaufhörliche Schaffensdrang , der sich betätigen muß auf dem Gebiete , auf das sein Träger — man möchte sagen zufällig — 56 Zweites Buch : Kirche , Staat und Frühkapitalismus. gestellt ist . Diese stete Schaffensbereitschaft , die immerfort nach Befriedigung drängt , läßt sich nicht erklären , läßt sich nicht rest¬ los in einzelne Komponenten auflösen . Sie ist mehr als Freude am Gelingen und an der Macht , mehr als Gewohnheit , obgleich dies alles und vieles andere mitspricht . Sie muß in ihrer tiefsten Wesen¬ heit schlechterdings als etwas allen schöpferischen Naturen Eigenes angesehen und hingenommen werden . In klassischer Reinheit kommt die geschilderte Seelenstimmung in einem vielzitierten Worte Jakob Fuggers , des bedeutendsten deutschen Frühkapitalisten, zum Ausdruck . Als dem einst sein Schwager riet , er solle sich vom Geschäft zurückziehen und den so mühsam erworbenen Reichtum in einem ruhigen Lebensherbst genießen , da gab ihm der Fugger zur Antwort : „Er hätte viel einen anderen Sinn, er wolle gewinnen, dieweil er könnte .“ Warum , ist nicht gesagt , konnte auch von Jakob Fugger nicht gesagt werden . Der Erwerb von Geld und immer wieder von Geld ist in seinem Ausspruch als eine Art Selbstzweck gedacht. Die äußeren Umstände , die wirtschaftliche Weltlage , wenn man sich so ausdrücken darf , war seit dem 11. Jahrhundert günstig für die Entfaltung und Verbreitung des kapitalistischen Geistes in Italien . Auf die schlechten Zeiten des 9. und 10. Jahrhunderts mit ihren Sarazenen - und Magyarenstürmen und anderen Schädi¬ gungen des Wirtschaftslebens waren bessere Zeiten gefolgt . Der nie ganz unterbrochene Handel mit Byzanz und der Levante über¬ haupt konnte in größerem Stil aufgenommen werden . Dann brachten die Kreuzzüge die italienischen Kaufleute direkt in die griechisch¬ arabische Welt hinein , .die sich ihnen bis dahin fast nur auf dem Markt von Byzanz durch Vermittlung der Byzantiner dargeboten hatte . Eine Fülle von Betätigungsmöglichkeiten ergab sich in¬ folge dieser Vorgänge kühnen , aufwärtsstrebenden Naturen unter den italienischen Kaufleuten . Aber nicht genug , daß sich lohnendere Einkaufsgebiete eröffneten , auch Käufer von orientalischen Waren traten in wachsendem Maße seit dem 11. Jahrhundert an Italien heran . Die steigende Kultur in Flandern , in Deutschland , in Frank¬ reich , England usw., später auch in den skandinavischen und slawischen Ländern forderte orientalische Waren . In erster Linie waren es naturgemäß die italienischen Städte , bei denen man die steigende Nachfrage zu decken suchte. In und mit den genannten und vielen anderen Betätigungs¬ möglichkeiten , sie teilweise verursachend und von ihnen wiederum Zweites Buch ; Kirche , Staat und Frühkapitalismus. 57 angeregt und beeinflußt, hat sich der kapitalistische Geist, der Geist des ökonomischen Individualismus in Italien seit dem 12. Jahr¬ hundert besonders in großem Stil und in glänzender Weise ent¬ wickelt. Als seine Träger kommen kräftige, unermüdliche Per¬ sönlichkeiten im Wirtschaftsleben aller Städte empor. Ihre Ver¬ mögen wachsen in diesem Auf steigen. Kredit und Einlagen von Verwandten und Fremden vergrößern noch deren kapitalbildende Wirkung. Man glaubt an diese rastlos tätigen Menschen, man gibt ihnen Glauben, d. h. Kredit. Ein Großhändlerstand bildet sich und ein Bankierstand von bald internationaler Bedeutung. Ein großer Teil der orientalischen Industrien wird nach Italien verpflanzt. Im Mittelmeer entsteht eine Kolonialwirtschaft der italienischen Stadtstaaten , die in ihren Organisationsformen für die Spanier und Portugiesen des 15. und 16. Jahrhunderts , ja selbst für die Holländer des 17. Jahrhunderts noch vorbildlich werden konnte. Wie der Geist der italienischen Renaissance rationelle, das heißt vernunftgemäß ausgesonnene Methoden der Staatsregierung, der Verwaltung, der Diplomatie, der Kriegskunst usw. ersann, so schuf er auf wirtschaftlichem Gebiete den ökonomischen Ratio¬ nalismus. In großartigen, fein durchdachten Formen der Handelstechnik, des Kredits, des Gesellschaftswesens usw., besonders aber in der doppelten Buchführung schuf sich der Renaissancegeist des italienischen Unternehmertums eine durch und durch rationelle Grundlage seiner Wirksamkeit. Der größten Schöpfung des Gesamt¬ geistes der italienischen Renaissance, dem Staate als Kunstwerk, stellt sich eine aus demselben Geiste geborene Schöpfung der auf sich selbst gestellten Persönlichkeit an die Seite: Die Wirtschaft als Kunstwerk, das moderne Geschäft, die kapitalistische Unter¬ nehmung. Von Italien aus hat sich dann der kapitalistische Geist über ganz Westeuropa verbreitet. Zum Teil in direkter Übernahme, dadurch, daß die Kaufleute jener Länder bei den Italienern in die Lehre, gingen. Oft im wahren Sinne des Wortes. Mehr aber noch, weil sich auch in den übrigen westeuropäischen Ländern eine individualistische Geistesrichtungangebahnt hatte , die naturgemäß auch auf das wirtschaftliche Gebiet Übergriff und sich hier, weil die äußeren Bedingungen günstig waren, in einzelnen kräftigen Persönlichkeiten entwickeln, auswirken und von ihnen aus ver¬ breiten konnte. 58 Zweites Huch: Kirche, Staat, und Frühkapitalismus. Am Ende des 15. und zu Anfang des 16. Jahrhunderts ist der kapitalistische Geist über eine ansehnliche Oberschicht der wirt¬ schaftlich Tätigen in ganz Europa verbreitet. Für den, der diese Entwicklung genauer verfolgt, erhebt sich dabei immer und immer wieder die Frage: Wie konnte sich der kapitalistische Geist durch¬ setzen gegen die enlgegenstehende Wirtschaftsethik der mittelalter¬ lichen Kirche und gegen einen Staat, der der Kirche seinen welt¬ lichen Arm lieh? Es kann hier nicht meine Aufgabe sein, im einzelnen zu schildern, wie die Wirtschaftsethik der mittelalterlichen Menschheit aussah. Ich kann nur ganz flüchtig darauf eingehen, um zu zeigen, welche wirtschaftsmoralischen Anschauungen der aufkommende und umsichgreifende kapitalistische Geist zu zersetzen sich anschickte. Die mittelalterliche Wirtschaftsmoral, wie sic beispielsweise bei Thomas von Aquino zusammengefaßt niedergelegt ist, beruht durchaus auf der Idee der gerade auskömmlichen Nahrung, auf der Forderung, der einzelne solle danach streben, sein standes¬ gemäßes Auskommen zu haben, aber auch nicht viel darüber hinaus l. In der Zunftverfassung liât die mittelalterliche Stadt, hat der mittelalterliche Staat versucht, diese wirtschaftsethische Forderung zu verwirklichen. Es wird meines Erachtens für die bekannte Streitfrage nach der Entstehung der Zünfte im Mittelalter viel zu wenig oder vielmehr gar nicht beachtet, daß das wichtigste Motiv für die bischöflichen Stadtherrn 2, die Zunftbildung zuzulassen, in dem Wunsche bestand, durch Innungen das christliche Wirtschafts¬ ideal zu verwirklichen. Das christliche Wirtschaftsideal mit seinem Ausschluß der heftigen Konkurrenz, mit seiner Garan¬ tierung eines standesgemäßen Auskommens für möglichst alle Bewohner der Stadt. Man hat mit Recht betont 3, daß dieses Ideal nicht zu wirtschaftlichen Großtaten hinzureißen imstande ist, daß es vielmehr leicht zu „quietistischer Behaglichkeit und Ruhe¬ seligkeit“ verführe. Aber derselbe Beurteiler ist weitherzig genug, um diesem Verdikt die folgende Würdigung hinzuzufügen: Solange 1 Vgl. etwa Max Maurenbrecher, Thomas von Aquinos Stellung zum Wirtschaftsleben seiner Zeit . 1. Teil . Leipzig 1898. 2 Die meisten und wichtigsten ersten Städte des früheren Mittel¬ alters waren Bischofsstädte. 2 W . Wygodzinski, Wandlungen der deutschen Volks¬ wirtschaft im 19. Jahrhundert . Köln 1907 , S. 14. Zweites Buch : Kirche , Staat und Frühkapitalismus. 59 das Wirtschaftsideal des Mittelalters nicht „Ausartungen der Re¬ pression“ erzeugte, basierte es doch auf einer „Weltanschauung, der niemand seine Achtung versagen kann. Es setzt nicht den höchsten Wert des Lebens in den Geldgewinn, sondern es betrachtet um¬ gekehrt nur den Erwerb als notwendige Grundlage für ein Leben, das anderen und höheren Zielen zu dienen bestimmt ist. Es will den Kampf ums Dasein auf wirtschaftlichemGebiete, das rücksichts¬ lose Niederkämpfen des Schwächeren nach Möglichkeit ausschalten, indem es allen eine zwar bescheidene, aber sichere Existenz gewähr¬ leistet.“ Wygodzinski hat auch bereits erkannt, daß das Wirtschafts¬ ideal des Mittelalters „in voller Reinheit nur durchzuführen ist, so¬ lange die Bevölkerung stationär bleibt.“ Also in der geschlossenen Stadtwirtschaft, die ja notwendigerweise zum mittelalterlichen Wirtschaftsideal gehört und die auch von Thomas von Aquino als Ideal proklamiert wird. Es ist kein Zufall, wenn ein neuzeit¬ licher katholischer Sozialethiker, Kempel mit Namen, der aller¬ dings von den berufenen Organen der katholischen Kirche ab¬ gelehnt worden ist, die Beseitigung des Kapitalismus etwa folgender¬ maßen fordert: „Man beschränke jede einzelne Stadtgemeinde, große und kleine, auf möglichst allen Gütererzeugungsgebieten wieder auf sich selber und ordne in ihr selber die Erwerbs- und übrigen Gesellschaftsverhältnisse . . . Die tolle, ungebundene Provinzial-, Volks- und Weltwirtschaft hat dann alsbald von selbst ein Ende; die der Freiwirtschaft eigene, grenzenlose Verwicklung aller Geschäfte, das dadurch bedingte Emporkommen der Stärkeren über die Schwächeren, die Ansammlung der materiellen Güter¬ erzeugungsmittel in immer wenigen Händen, die Dienstbarmachung breiter Arbeitermassen durch wenige Schlotbarone, das Gewerk¬ schaftswesen, Kartellwesen und alle anderen, den Gesellschafts¬ körper wild durchwühlenden Interessenbestrebungen, kurz der ganze neuzeitliche industrielle Kapitalismus hat sein Ende ge¬ funden“ 1. So toll die Forderung ist, logisch ist die Erkenntnis, daß ein unkapitalistisches Wirtschaftsleben nur in einer ge¬ schlossenen Stadtwirtschaft mit Zünften usw. existieren kann. Der Versuch, das gesamte Wirtschaftsleben der mittelalter¬ lichen Städte in das Zunftsystem zu bringen, ist aber doch nur sehr teilweise geglückt. Es ist zweifellos, daß schon zu Thomas 1F . Kempel, Göttliches Sittengesetz und neuzeitliches Er¬ werbsleben. Mainz 1902. 60 Zweites Buch: Kirche, Staat und Frühkapitalismus. von Aquinos Zeiten, in Italien besonders, sein wirtschaftliches Idealbild mit der Wirklichkeit in den bedeutenden Handelsstädten in krassem Widerspruche stand. Die großen Kaufleute von Pisa und Florenz, von Venedig, Genua usw. hatten „ viel einen anderen Sinn“ als den, sich mit einem knappen, standesgemäßen Auskommen zu begnügen. In ihnen hatte der kapitalistische Geist die zünftige Beschränkung der Betätigung des Erwerbssinnes längst zunichte gemacht. Älinlich lagen die Dinge in dem wirtschaftlich so fort¬ geschrittenen Flandern schon im 13. Jahrhundert . Auch in Deutsch¬ land gab es in Köln, Mainz, Regensburg usw. schon im 13. Jahr¬ hundert Städte, in denen sich ein wachsender Stand von Groß¬ händlern über die handwerksmäßige Auffassung des Erwerbslebens erhob. Im 14. und 15. Jahrhundert vermehrte sich dann überall die Zahl der Städte mit kapitalistisch fühlenden und kapitalistisch handelnden Kaufleuten bedeutend. Es fragt sich, wie hatte sich der kapitalistische Geist, der kapitalistische Kaufmann durchzusetzen vermocht gegen die ent¬ gegenstehende Wirtschaftsethik der Kirche und des Staates? Sprechen wir zunächst von der Kirche. Wie hatte sich der kapitalistische Geist ausbreiten können gegenüber einer kirch¬ lichen Leime, die im kanonischen Zinsverbote einen Riegel vor die Ausbreitung jeder Kreditwirtschaft zu schieben sich mühte ? Bei der Macht, die die mittelalterliche Kirche über die Gemüter aus¬ übte, ist diese Frage besonders wichtig. Man hat hier und da den Umfang des kanonischen Zinsverbotes abzuschwgchen versucht. Man hat behauptet, das kanonische Zinsverbot habe sich nur auf den Konsumtionskredit bezogen, nicht aber auf den Produktivkredit, es sei nur gegen die Ausbeutung der Notlage des Kreditsuchenden erlassen worden. Neuerdings hat Franz Keller in einer interessanten Schrift 1Unter¬ „ nehmung und Meinwert" die genannte Ansicht wieder vertreten. So angenehm seine Anschauung für eine Erklärung des Eindringens des kapitalistischen Geistes in die Wirtschaftsordnung des Mittel¬ alters wäre, so wenig läßt sie sich mit den liistorischen Tatsachen in Einklang bringen. Wohl ist das Zinsverbot von der Kirche in einer •FranzKeller , Unternehmung und Mehrwert. Eine sozial¬ ethische Studie zur Geschäftsmoral. Köln 1912. (Vereinsschrift der Görresgesellschaft.) Zweites Buch : Kirche , Staut und Frühkapitalismus. 61 Zeit vorwiegend naturaler Wirtschaft aufgestellt worden , zu einer Zeit, als Geldkapital noch eine geringe Rolle spielte , als die Kapital¬ eigenschaft des Geldes noch kaum erkannt war und die zinsbare Verwertung des Geldes dem allgemeinen Stande der Wirtschaft zuwiderlief 1. Zu einer Zeit also, wo man fast allein Geld zu dem Zwecke lieh , um sich Konsumtionsmittel damit zu kaufen , und nicht zu produktiven Zwecken , d. h. um mit Hilfe des geliehenen Geldes Geschäfte zu treiben . Das alles ist richtig und für die Be¬ urteilung der Entstehung des Zinsverbotes von Bedeutung . Aber es muß für unsere Frage ''hervorgehoben werden , daß die Kirche theoretisch und in der Judikatur das kanonische Zinsverbot noch aufrecht erhalten hat , als es durch die veränderten wirtschaftlichen Zustände unhaltbar geworden war . Das ganze Mittelalter hindurch haben besonders die einflußreichen Volksprediger und Volksbeichtiger der Dominikaner und Franziskaner sich gegen das Zinsnehmen gewendet . Noch Luther verwarf es ganz generell . Er steht hier völlig auf dem Standpunkte der extremsten Scholastik. Das Zinsverbot war nun aber nur ein Teil der wirtschafts¬ ethischen Anschauungen der christlichen Kirche , die einer schnellen und ungehinderten Entfaltung des kapitalistischen Geistes hindernd im Wege standen 2. Andere Anschauungen wirkten in derselben, die Ausbreitung des Kapitalismus aufhaltenden Richtung . Ich hebe hier nur noch die Forderung des „gerechten Preises “ hervor. Die Frage nach dem justum pretium , nach dem turpe lucrum , nach der moralisch erlaubten Höhe auch des Warengewinnes , hat das ganze Mittelalter , mit Einschluß des 16. Jahrhunderts , lebhaft be¬ schäftigt . Die Kanonisten unterschieden drei Stufen des gerechten Preises einer Ware ; einen niedrigsten oder gnädigen Preis (pretium 1 Handwörterbuch der Staatswissenschaften. 2. Aufl. 7. Bd. S. 961. 2 Der Begriff Wucher umfaßte im Mittelalter nicht nur Wucher im Darlehen, sondern auch im Kauf. Wucherer war jeder, der den gerechten Preis überschritt. Wucher schließlich jeder übermäßige Gebrauch des Kapitals im alltäglichen Verkehr. M. Neumann. Geschichte des Wuchers in Deutschland bis zur Begründung der heutigen Zinsengesetze (1654). Halle 1865, S. 91. Noch am Ende des 17. Jahrhunderts wird von einem Monopolpächtergesagt: „Die Einwohner werden eines Privati Kontribuenten und seines Wuchers Sklaven.“ Vgl. von S r b i k , Der staatliche Exporthandel Öster¬ reichs, S. XXXII Anm. 3. 62 Zweites Buch : Kirche , Staat und Frühkapitalismus. infimum seu pium), unter welchem man gerechterweise nichts kaufen dürfe, einen mittleren oder mäßigen Preis (pretium medium seu moderatum) und einen höchsten oder harten Preis (pretium supre¬ mum seu rigorosum) , über welchen hinaus man gerechterweise nichts verkaufen dürfe. Jenseits dieser letzten Stufe beginnt der ungerechte Preis, das turpe lucrum 1. Dementsprechend lehrte noch Geiler von Kaisersberg (1445—1510) : „Der kaufen will als wolfail er immer mag und einer verkaufet als thüer er verkaufen immer mag, denen beiden soi man daz heilig Sacrament nicht geben . . . das ist wider brüderliche liebe . . . Du soit deinen zimlichen gewin daruff setzen, dein müe und arbeit magst du wol darin schlahen, aber als thüer kaufen und verkaufen als du immer magst , daz ist falsch (confessores debent esse occulatissimi!) 2. Und fast wörtlich schrieb dann auch Luther in seiner bekannten Schrift „Vom Kaufshandel und Wucher“ : „Es sollt nicht so heißen, ich mag meine Waar so theur geben als ich kann oder will; sondern also: Ich mag meine Waar so theur geben als ich soll , oder , als recht und billig ist .“ Man beachte, wie anders etwa Konrad Peutinger, der juristische Berater der Augsburger Hochfinanz des beginnenden 16. Jahr¬ hunderts die Dinge auffaßte, wenn er schrieb: „Unusquisque mer¬ cator merces suas prout potest et in facultate eius est de iure vendit et in hoc contra ius nihil admittit nec illicita pacta facit, sicut etiam saepe contingit quod species viliori pretio quam emerunt merca¬ tores vendere solent et sic potius damnum quam lucrum procurant et sentiunt 3. Das Ideal des christlichen Kaufmanns, wie es noch durchaus in mittelalterlicher Art und Weise auch den einflußreichen Volks¬ predigern des 15. und 16. Jahrhunderts vorschwebte, hat Geiler von Kaisersberg wie folgt gezeichnet: „Ein kaufman soi ein rechte meinung hon. Sein meinung nit daruff setz und sein sach uff vil 1 Vgl . noch L u d o v i c u s Molinaim 2 . Bde. seines Werkes de jure et justicia, der „ de contractibus" betitelt ist. Disputatio 347. „De pretio justo.“ Hier wird die Meinung der Doktoren, d. h. der alten Kanonisten, wiedergegeben. 2 Kaiserbergs Brösamlein. S. XLVIII. 3 Aus einem lateinischen Gutachten Dr. Konrad Peutingers über Monopole und Handelsgesellschaften de anno 1530. Manuskript in der Augsburger Stadtbibliothek Cod. 2 ° Aug. 380 fol. 180v. Zweites Buch : Kirche , Staat und Frühkapitalismus . 63 gewins und uff gross guet zu heuffcn ; aber darum , dass er sein kind und weib erner als recht und billig ist und dass er armen lüten zu hilff kum und gotes dinst fürdern mög.“ Und an einer anderen Stelle : ..... Ist auch böss, da einer kaufmanschatz treibt uss gitickeit (propter inexplebilem avaritiam ). An seinem gewin ist kein endt, er kan nitt uffhören und der sack mag nit fol werden . Er hat keinen boden . Er ist bodenlos . Er facht hübschlich an und gat stetz für sich. Zu dem ersten , so treit er seinen krom in einem wenlin hin und her , streel und Spiegel. Wan er etwas überkumpt , so will er darnach ein gedemli haben und würt darnach ein kaufman und haltet hüss . Er hört nit uff, er sei denn in einer geselschaft . Noch hört er nit auf, als für und für . Er wil ein Galeen uff dem mer haben. Und also huglet er uff sein gütt und gedencket nit weiter me an seinen anfang und vergisst seins ends got des allemechtigen und seins tods . Er lügt allein das er vil güts gewönne “ 1. In ähnlicher Weise wendet sich dann später auch Luther und andere protestan¬ tische Theologen gegen ein übermäßiges Gewinnstreben und gegen die Erhebung aus dem sozialen Stande , in den der einzelne hinein¬ geboren ist. * * * Zu Anfang des 16. Jahrhunderts ist in Deutschland von den Bußpredigern die Forderung einer strengen Wirtschaftsethik am schärfsten betont worden , wie überhaupt die antikapitalistische Tendenz im 16. Jahrhundert in Deutschland ihren Höhepunkt er¬ reicht . Man muß die Reichstagsverhandlungen über Monopole lesen, wie sie besonders seit 1512 einsetzten , um den ganzen In¬ grimm der kleineren öffentlichen Gewalten gegen den kapitalistischen Geist zu erkennen . Was im Mittelalter unter „Fürkauf “ bekämpft worden war , der Versuch , durch einen teilweisen Aufkauf der Waren sich einen gewissen Monopolpreis zu verschaffen , das bekämpfte man jetzt unter dem neuen Schlagworte der Monopolia. Jede Ver¬ abredung kartellartiger oder monopolistischer Natur , die einen Ausschluß der Konkurrenz und damit eine Hebung der Preise be¬ zweckte , wird als „wucherischer Kontrakt “ gebrandmarkt und 1 Aus der Predigt. „Von dem Wannenkremer und der Kaufleute Handtierung.“ In den Brösamlein S. XCII. 64 Zweites Buch : Kirche, Staat und Frühkapitalismus. streng in verschiedenen Reichstagsabschieden verboten 1. Ich werde gleich noch ausführlicher auf diese Dinge zu sprechen kommen, hiev müssen wir zunächst zu der Frage zurückkehren , die ich vorhin stellte , und die wir nach dem inzwischen Gesagten noch tiefer ver¬ stehen gelernt haben werden . Ich frage von neuem : Wie kam es, daß sich der kapitalistische Geist, daß sich der kapitalistische Kauf¬ mann gegen die Unsumme von Hemmungen durchzusetzen ver¬ mochten , die sich aus den wirtschaftsethischen Anschauungen der mittelalterlichen Kirche und des Staates heraus ihnen entgegen¬ stemmten ? Die Antwort auf diese Frage ist nicht schwer zu geben . Die Kirche und der Staat konnten den kapitalistischen Kaufmann für ihre eigenen stattlichen Aufgaben , für die sich mehrenden Aus¬ gaben der großen Politik , des Kriegswesens , der Verwaltung usw., schon im Mittelalter nicht mehr entbehren . Sie waren durch ihre damit zusammenhängenden Geldbedürfnisse und ihre zunehmenden Geldgeschäfte gezwungen , den kapitalistischen Kaufmann zu Hilfe zu rufen , ihn zu gebrauchen und so seine Bedeutung und Wirksam¬ keit zu vergrößern . Was sie theoretisch so streng verurteilten , die beiden Mächte Kirche und Staat , das konnten sie in praxi nicht entbehren 2. Es ist ein eigenes Geschick um Ideen in der Welt¬ geschichte . Als eines der sonderbarsten ist mir immer die Tatsache erschienen , daß vielleicht nichts die Verbreitung der Kreditwirtschaft und der kapitalistischen Wirtschaft praktisch mehr gefördert hat als das Papsttum , also als die Spitze jener Institution , die theoretisch sich am heftigsten gegen den kapitalistischen Geist gewendet hatte und sich trotz ihrer entgegenstehenden Praxis im Verlaufe des Mittelalters immer wieder wandte. Im großen Zuge gesehen , verlief die praktische Förderung des kapitalistischen Kaufmanns durch die Kurie etwa folgendermaßen: Die römische Kurie sah sich, besonders seit dem Anfänge des 13. Jahr¬ hunderts , infolge ihrer wachsenden Verwaltungsaufgaben und in1 Auch in den Niederlanden wurden unter Karl V. scharfe Monopol¬ verbote erlassen. A. Henne, Histoire du règne de Charles V. en Belgique. V. Bd. S, 354 f. Die englische Entwicklung siehe bei L e v y a. a. O. 2 Vgl. Beispiele bei R. Pöhlmann, Die Wirtschaftspolitik der Florentiner Renaissance und das Prinzip der Verkehrsfreiheit. Leipzig 1878, S. 85 ff. Zweites Buch : Kirche, Staat und Frulikapitalismus . 65 folge ihrer zunehmenden Weltmachtspolitik gezwungen, ein dichter und dichter werdendes System von Steuern und Abgaben über die Christenheit, namentlich über den Klerus zu legen 1. Für die Über¬ weisung der Abgaben aus den verschiedenen Ländern nach Rom, bald auch für Vorschüsse auf diese Abgaben und auf anderes hin, konnten die Päpste, ebensowenig wie die Abgaben leistenden Kleriker, je länger je weniger den kapitalistischen Kaufmann der italienischen Städte entbehren. Oft brauchte die Kurie die Kaufleute auch, um größere Geldsummen bei ihnen als Depositen niederzulegen und zur rentablen Anlage zu bringen. Es ist bekannt, wie gerade die vielfachen Aufgaben, die das päpstliche Finanzsystem stellte, dazu beitrugen, aus der italienischen Kaufmannswelt als Oberschicht ein mächtiges, internationales Bankiertum erwachsen zu lassen. Dabei kann es nach den grundlegenden Forschungen Gottlobs und anderer nicht, mehr zweifelhaft sein, daß die Päpste schon im 13. Jahrhundert bewußt und mit Absicht in verschiedenen Formen ihren kauf¬ männischen Helfern und Gläubigern Zins gezahlt haben. Selbst die religiösesten Päpste mußten sich, „ wie alle anderen Menschen dem Machtgebot der Umstände beugen“ 2. Ohne Zinsenzahlung war kein Geld zu beschaffen für die wachsenden Aufgaben einer Weltorganisation, wie sie die Kirche geworden war. So blieb nichts anderes übrig, als im Widerspruch zum kanonischen Ideal in praxi Zinsen zu zahlen und damit den sich verbreitenden kapitalistischen Geist moralisch und praktisch zu fördern. Und wie es der Kirche gegangen war, so ging es auch dem Staate. Auch der Staat, der zunächst durchaus auf dem Stand¬ punkte der mittelalterlichen Wirtschaftsethik gestanden hatte, konnte bald für seine Zwecke, in erster Linie für seine Kriegszwecke3, den kapitalistischen Kaufmann nicht mehr entbehren. Das zeigte sich schon deutlich in den italienischen Renaissancestaaten seit 1 A. Gottlob, Päpstliche Darlehnsschulden des 13. Jahr¬ hunderts . Historisches Jahrbuch 20 (1899 ), S. 665 ff. 2 A. Schulte, Geschichte des mittelalterlichen Handels und Verkehrs zwischen Westdeutschland und Italien , mit Ausschluß von Venedig . 2 Bde . Leipzig 1900 . I. Bd ., S. 268. 3 Neuerdings hat W . S o m b a r t die Bedeutung des Kriegs¬ wesens für die Entwicklung des Kapitalismus ausführlich und ein¬ dringend klargelegt . Krieg und Kapitalismus . München und Leipzig 1913. Strieder , Studien z. Gescl «. kapitatist . Or ^anisationsformen . b 66 Zweites Buch : Kirche , Staat und Frühkapitalismus. dem 13. Jahrhundert . Hier wurde der Satz „pecunia nervus belli“ zuerst ausgesprochen. Aber auch in jedem anderen christlichen Staate schon des 14. und 15. Jahrhunderts scheiterte jeglicher Wunsch, gegen den vordringenden kapitalistischen Geist Stellung zu nehmen, an den realen Geldbedürfnissen des Alltags. Solange dabei in den außeritalienischen christlichen Staaten die Juden und die landesfremden italienischen Geldwechsler, die sogenannten Lom¬ barden oder Kawertschen, das Geldbedürfnis der Fürsten befriedigten, war der Widerspruch zwischen Theorie und Praxis noch nicht so auffällig. Für die Juden galt nach einer verbreiteten Anschauung das kanonische Zinsverbot nicht, und den landesfremden Lom¬ barden zählte man auch kaum unter die Christenmenschen. Anders wurde die Sache, als die Kaufleute des eigenen Landes die Juden und die Kawertschen immer mehr aus dem reichen Ge¬ winn abwerfenden Geldgeschäft verdrängten und die staatlichen Gewalten nun mit diesen heimischen Kapitalisten arbeiten mußten. Erst jetzt klaffte zwischen der kanonischen Zinstheorie des christ¬ lichen Staates und der Praxis ein unüberbrückbarer Widerspruch. Und doch konnten selbst die gewissenhaftesten Fürsten daran nichts ändern. Auch sie kamen ohne den kapitalistischen Kaufmann nicht mehr aus. Die gewissenlosen Fürsten aber haben nicht selten den Widerstreit zwischen dem christlichen Wirtschaftsideal, das noch immer die öffentliche Meinung beherrschte, und der Praxis be¬ nutzt, um reiche Gläubiger zu vernichten oder doch um ihre her¬ geliehenen Kapitalien zu bringen. Zumeist stand selbst in dem Europa des 16. Jahrhunderts noch die öffentliche Meinung auf der Seite des Fürsten , der sich bankrott erklärte und den Kapita¬ listen, die ihm aus schweren Finanznöten geholfen hatten , die Zahlung verweigerte. Für die große Menge waren die Kaufleute, die sich mit Geldgeschäften befaßten, eben alle mehr oder weniger Wucherer. Selbst Matheus Schwarz, der Hauptbuchhalter der Fugger, schreibt zu Anfang des 16. Jahrhunderts noch in einem Lehrbuche über Buchhaltung an der Stelle, wo er die im kauf¬ männischen Leben üblichen Fremdworte verdeutschte : „ Interesse, das ist höflich gewuchert; Finanzen [d. h. Finanzgeschäfte treiben] \ ist gleich höflich gestohlen.“ 1„ Sono sempre intrigati e obligati con l’usure, che loro chiamano finanze“. A 1b è r i , Relazioni degli ambasciatori veneti al senato I. 2. S. 204. Zweites Buch: Kirche, Staat und Frühkapitalismus. 67 Der vielgenannte Zwiespalt zwischen Theorie und Praxis wirkte natürlich hemmend auf das Wirtschaftsleben und korrum¬ pierend auf die öffentliche Moral. Die kleinste staatliche Gewalt und nicht wenige Privatleute glaubten sich bei Zahlungsunfähigkeit oder bei Zahlungsunlust hinter den Wucherparagraphen und das kanonische Zinsverbot zurückziehen zu können. Und selbst das Raubrittertum , das die Kaufleute plünderte, suchte sich gern aus der antikapitalistischen Tendenz der mittelalterlichen Wirtschafts¬ ethik ein Mäntelchen für sein gewalttätiges Gewerbe zu machen. * * * Man kann noch weiter gehen als zu sagen, das Geldbedürfnis von Staat und Kirche für Verwaltungs-, Kriegs- und weltmachts¬ politische Zwecke hat das Aufkommen des Kapitalismus, aller mittelalterlichen Wirtschaftsethik zum Trotz, zugelassen und ge¬ fördert. Man darf ohne Übertreibung sagen, das kirchliche und das staatliche Geldbedürfnis hat nicht selten bedeutungsvolle kapita¬ listische Organisationsformen unmittelbar selbst mit geschaffen. Ich denke dabei z. B. an Konsortien von Kaufleuten, die sich — oft durchaus schon international — bildeten, um besonders große oder besonders gefährliche Anleihen der Kurie oder der Fürsten unterzubringen 1. Bei manchen fürstlichen Finanzgeschäften, z. B. den sogenannten Silber- und Kupferkäufen mit den Habsburgern als den Landesherren von Tirol, mußten auch aus dem Grunde 1 Beispiele bei Gottlob, Päpstliche Darlehensschulden, a. a. O., S. 697 . Ferner bei O. M e 11 z i n g , Das Bankhaus der Medici und seine Vorläufer . Jena 1906 , S. 49 ff ., S. 58 ff ., 77 usw . ; Georg Schneider, Die finanziellen Beziehungen der florentinischen Bankiers zur Kirche von 1285 bis 1304 . Leipzig 1899 ; auch in dem zusammenfassenden Aufsatz von Josef Kulischer, Warenhändler und Geldausieiher im Mittelalter . Zeitschrift für Volkswirtschaft , Sozialpolitik und Verwaltung . Bd . XVII (1908 ), S. 29 ff . Für die Konsortien hansischer Kaufleute in England im 14. Jahrhundert vgl . Josef Hansen, Der englische Staatskredit unter König Eduard III . und die hansischen Kaufleute . Hansische Geschichtsblätter 1910 , S. 323 ff . Auch G. Grosch, Geldgeschäfte hansischer Kaufleute mit englischen Königen im 13 . und 14 . Jahr¬ hundert , im Archiv für Kulturgeschichte , Bd . II (1904 ). In Frank¬ reich mußte sich zu Anfang des 15. Jahrhunderts ein Konsortium von 20 Kapitalisten bilden , um Karl VII . Geld zu verschaffen . Cfr. 5* * 68 Zweites Buch : Kirche, Staat und Frühhapitalismus. oft mehrere große Handelsgesellschaftenzu Konsortien zusammen¬ treten, weil die Habsburger mehreren Firmen gegenüber Geld¬ verpflichtungen hatten , die sie durch Gewährung von Kontrakten der genannten Art auf einmal befriedigen wollten 1. In ähnlicher Weise veranlaßte der Wunsch der Fürsten, auf einen Schlag mehrere Gläubiger zu befriedigen und deren gegenseitige Eifersucht zu dämpfen, die Entstehung von Konsortien im Gewürzhandel der portugiesischen Krone usw. Obwohl natürlich auch hier zugleich die Größe der Gefahr die Kaufleute zu gemeinsamem Vorgehen in Konsortialbeteiligungen bewog. Ich denke ferner bei der oben genannten These über den Zu¬ sammenhang obrigkeitlicher Finanznot und der Entstehung wichtiger kapitalistischer Organisationsformen an die frühen italienischen Aktienvereine, die ins Leben traten , um öffentliche Anleihen auf¬ zubringen 2, oder auch an die älteren Bankgründungen, die notorisch oft aus dem Grunde geschahen, um dem Staate damit neue Ein¬ nahmequellen zu eröffnen3. Es mag in diesem Zusammenhänge auch nicht unerwähnt bleiben, daß die rasche Verbreitung der kapitalistischen Gewerkschaft und die damit zusammenhängende Verdrängung der älteren Arbeitsgenossenschaftaus dem Bergbau — seit dem 13. Jahrhundert etwa — wesentlich durch die Finanz¬ bedürfnisse der Regalherren gefördert wurde. Das Interesse der Bergherren, das zunächst in einer möglichst hohen Quote des Zehnten und der übrigen obrigkeitlichen Anteile an der Ausbeute H . P r utz, Jacques Coeur von Bourges . Geschichte eines patrio¬ tischen Kaufmannes aus dem 15. Jahrhundert . Berlin 1911 , S. 51. Für die großen Konsortien , besonders italienischer und süddeutscher Kaufleute des 16. Jahrhunderts , ist natürlich vor allem Ehren¬ berg, Zeitalter der Fugger , heranzuziehen . Vgl . dort besonders auch I. Bd . S. 399 f. 1 Für solcher Art Konsortialbeteiligungen der Fugger vgl . M. Jansen, Jakob Fugger der Reiche , S. 80 f, 83, 85, 88, 96, 115 und sonst . Für Frankreich vgl . P . Imbart de la Tour, Les origines de la Réforme . Bd . I, S. 424. 2 L. Goldschmidt, Universalgeschichte des Handelsrechts. Stuttgart 1891 , S. 291 . Trotz des Widerspruchs von Karl Leh¬ mann Die ( geschichtliche Entwicklung des Aktienrechts bis zum Code de commerce . Berlin 1895) halte ich daran fest , daß die Wiege der modernen Aktiengesellschaft in Italien stand . Näheres darüber unten im dritten Buch Kapitel I Abschnitt 2. 3 Schindlers Jahrbuch 1913 , S. 1168. 8 Zweites Buch : Kirche, Staat und Frühkapitalismus . 69 gesehen wurde, „führte von selbst darauf, daß eine rasche und intensive Ausbeutung der Erzlager viel eher von größeren Betrieben, die mit einer zahlreichen Belegschaft arbeiten konnten, zu erwarten sei, als von den alten kleinen Arbeitsgenossenschaften, die in selbst¬ genügsamer Beschränkung nur einen Schacht nach dem anderen anschlugen“ 1. Besonders gut aber läßt sich der Zusammenhang zwischen „Fiskalismus“ und „Kapitalismus“ an der Entstehung der kapita¬ listischen Organisationsformen der Monopole und Kartelle ver¬ folgen2. Die frühesten Kartelle, die mir bisher bekannt geworden sind, erscheinen nicht so sehr als Schöpfungen von Kaufleuten, sondern ebensoviel als Erzeugnisse einer staatlichen Finanzpolitik, die für sich die Berechtigung in Anspruch nahm, ein Regal, das ihr zustand, durch vertragsmäßigen Ausschluß einer lästigen Konkurrenz vor der Wertminderung zu bewahren. Das gilt z. B. von dem Salz¬ vertriebssyndikat, das im Jahre 1301 König Karl II . von Neapel als Besitzer von provenealischen Salinen und Philipp der Schöne von Frankreich als Regalherr der Salinen von Aiguesmortes ihre Bankiers und Salinenpächter, die Florentiner Kaufleute Franzesi und Bardi, abschließen ließen 3. Das gilt auch von dem Kartell, 1 Inama - Sternegg , Deutsche Wirtschaftsgeschichte . III. Band . 2. Teil, S. 165 f. 2 Auch für die ältere Geschichte des Seeversicherungswesens ist die fiskalische Wirtschaft von Bedeutung geworden . Gegen eine Prämie von 6 % übernahm im 16. Jahrhundert der König von Por¬ tugal , selbst während der Kriege Karls V. gegen Frankreich , die Ver¬ sicherung der Schiffe, die indisches Gewürz von Portugal nach Ant¬ werpen usw. brachten . Nach dem Kriege wurde die Prämie sehr ermäßigt . Cfr. das Gutachten Peutingers de anno 1530, Msc. Augs¬ burger Stadtbibliothek . Cod. 2 ° Aug. 386 fol. 189r f. . . mani¬ festum est, quod regia celsitudo Portugallensis negotiatores nationum omnium qui solent cum ea contrahere ac in Germaniam inferiorem transnavigantes etiam bellorum tempore , quae inter sacram caes. maiestatem et Francorum regem sunt gesta salvos et cum omni securitate mercium suarum acceptis sex pro centum et etiam extra illa bella pro minori summa hactenus conservavit . . Fol . 200 r nennt Peutinger diese Versicherung direkt „Securitates “ . In Portugal hatte übrigens 1367—1387 schon eine „ Zwangssozietät zum Zwecke der Seeversicherung“ existiert . Handwörterbuch der Staatswissen¬ schaften , 3. Aufl., VII , 1250. 8 Der Syndikatsvertrag ist in den Regesten Davidsohns For¬ ( schungen zur Geschichte von Florenz . III. Berlin 1901, Nr . 382) erwähnt. 2 70 Zweites Buch: Kirche, Staat und Frühkapitaiismus. das im Jahre 1470 Papst Paul II . mit König Ferrante von Neapel einging, um die gegenseitige Konkurrenz ihrer Alaungruben von Tolfa und Ischia auszuschalten 1. , Auch die bedeutendsten Monopole des Mittelalters und der beginnenden Neuzeit sind im wesentlichen Schöpfungen der staat¬ lichen Finanzpolitik. Dieser Satz gilt von den Monopolen im sizilischen Königreich und sonst seit dem 12. und 13. Jahrhundert , ja noch früher. Er gilt von dem päpstlichen Alaunmonopol des 15. Jahrhunderts , a«r das wir noch zu sprechen kommen werden. Er gilt auch besonders von den Monopolen im Gewürzhandel3 und im Erzgroßhandel des 16. Jahrhunderts , also jenen Formen einer kapitalistischen Wirtschaft, die besonders stark von den Sozialethikern der Zeit bekämpft wurden. Gerade hier haben wir es ganz offenbar mit Schöpfungen fürstlicher Finanzpolitik zu tun . Um in außergewöhnlich schwierigen Finanznöten besonders hohe Darlehns¬ summen von den Kaufleuten zu erhalten, bewilligten ihnen die Fürsten das alleinige Großhandelsrecht mit einer Ware, über die den Landesherren auf Grund von Regalien ein gewisses Verfügungs¬ recht oder Mitverfügungsrechtzustand. Die kapitalistischen Kauf¬ leute griffen bei einem solchen Angebot zumeist gern zu. Oft gaben sie auch entsprechende Anregungen4. Vielfach waren eben ihre 1 Vgl. A. Gottlob, Aus der Camera apostolica des 15. Jahr¬ hunderts. Innsbruck 1889, S. 296; ferner G. Zippel, L’allume dl Tolfa e il suo commercio. In Archivio délia R. Società Romana di Storia Patria. Vol. XXX (1907), S. 34 ff. Das hochinteressanteKartell¬ instrument ist in extenso abgedruckt bei A. Theiner, Codex dipl. dominii temporalis sanctae sedis. 3. Bd. Rom 1862, S. 463 ff. Wir müssen in einem späteren Kapitel dieses Werkes noch einmal auf diese frühen Kartelle zurückkommen. Dann werden auch „fiskalische Kartelle" der späteren Jahrhunderte genannt werden. 2 Ad. Schaube, Handelsgeschichte der romanischen Völker des Mittelmeergebiets bis zum Ende der Kreuzzüge. München und Berlin 1906, S. 509 f., und sonst, vgl. Register unter Monopol. 3 R . Ehrenberg, Das Zeitalter der Fugger. II. Bd. S. 14, I. Bd. S. 398f. 4 Selbstverständlichhatten die großen Kaufleute ihre eigenen Gedanken über die ethische Beurteilung von Monopolbestrebungen. Conrad Peutinger, ihr beredter Anwalt, dürfte die communis opinio wenigstens der Augsburger Großfinanz treffen, wenn er einmal schrieb: „Praeterea etiam in praetensa consultatione et in eius articulis pluri¬ bus haec verba ,aigennutzig handtierungen' usurpantur, quae studium * Zweites Buch : Kirche , Staat und Frühkapitalismus. 71 Darlehen an die Fürsten so hoch angelaufen , daß sie überhaupt nur noch mit Hilfe von so außerordentlichen Maßregeln wie Mono¬ polen getilgt werden konnten . Nichts ist verkehrter , als die Monopol¬ bewegung des 16. Jahrhunderts nur aus einer exorbitanten Gewinn¬ begierde der Kaufleute erklären zu wollen. Das lebendige Interesse, das z. B . die deutsche königliche und kaiserliche Finanzpolitik hier hatte , zeigt sich deutlich an dem Eifer , mit dem sie die monopol¬ inhabenden Kaufleute gegen eine Belästigung durch das Reichs¬ regiment schützte 1. Während auf den deutschen Reichstagen seit Beginn des 16. Jahrhunderts die kleinen staatlichen und stän¬ dischen Gewalten gegen die Monopole wetterten und ihre strenge Bestrafung beschlossen, verpflichteten sich der deutsche König Fer¬ dinand und sein kaiserlicher Bruder Karl V. in den Monopolkontrakten , die sic mit den Kaufleuten abschlossen , heimlich , die Monopolisten gegen jedes Eingreifen der Reichsgewalt zu ver¬ teidigen . Nur so waren vielfach die Kaufleute zur Annahme der Monopole zu bewegen. Wir müssen , um hier deutlich zu sehen , einen Blick auf die deutsche Antimonopolbewegung des 16. Jahrhunderts werfen , wie sie besonders auf den Reichstagen sich abspielte . Der Reichstags¬ abschied von Trier -Köln (1512) brachte in den Paragraphen 16 und folgende des 4. Stückes eine scharfe Absage an die Monopolisten. Da heißt es : „Und nachdem etwa viel grosse Gesellschafft in Kauffmannsschafften in kurtzen Jahren im Reich aufgestanden, auch etliche sondere Personen sind , die allerley Waar tind Kauffmanns -Giiter , als Specerey , Ertz , Wöllen -Tuch und dergleichen proprii commodi in illis negotiationibus interpretari solent, et licet indebite societatibus ad iniuriam referuntur , cum tamen proprium commodum quaerere prout de jure non solum in negotiationibus sed etiam aliis in actionibus omnibus permittitur , nulli prohibetur , sic etiam omnibus mercatoribus et eorundem societatibus , qui non solum bona et res, sed et corpora laboribus et periculis exponunt , dum contra ius non contrabunt , proprium commodum sicut et aliis qui otiosi acquirunt fovere et illi studere convenit “, 1Unser vorliegendes Werk dürfte die völlig falschen Auffassungen korrigieren , die H. L e v y (Monopole, Kartelle und Trusts in ihren Beziehungen zur Organisation der kapitalistischen Industrie . Dar¬ gestellt an der Entwicklung in Großbritannien . Jena 1909, S. 68) über die Verbreitung des Monopolwesens in Deutschland im frühkapitalis¬ tischen Zeitalter vertreten hat. 72 Zweites Buch: Kirche, Staat und Fxühkapitalismus. in ihre Händ und Gewalt allein zu bringen unterstehen , Fürkauff damit zu treiben , setzen und machen ihnen zum Vortheil solcher Güter den Wehrt ihres Gefallens , fügen damit dem hl. Reich und allen Ständen desselbigen mercklichen Schaden zu, wider gemein beschriebene Kayserliche Recht und alle Erbarkeit : Haben Wir, zur Fürderung gemeines Nutz und der Nothdurfft nach , geordnet und gesetzt und thun das hiemit ernstlich und wollen, dass solche schädliche Handthierung hinführo verboten und ab sey und sie niemands treiben oder üben soll. Welche aber wider solches thun würden , deren Haab und Güther sollen confiscirt und der Obrig¬ keit jeglichen Orts verfallen sevn . Auch dieselbe Gesellschafft und Kaufficut hinführo durch kein Obrigkeit im Reich geleitet werden , sie auch desselben nicht fähig seyn , mit was Worten , Meynungen oder Clausuln solche Geleit gegeben werden. § 17. Doch soll hiedurch niemands verboten seyn , sich mit jemand in Gesellschafft zu thun , Waar , wo ihnen gefällt , zu kaufl’en und zu verliandthicrcn ; dann allein , dass er die Waar nicht unter¬ stehe in eine Hand zu bringen und derselben Waar einen Wehrt nach seinem Willen und Gefallen zu setzen , oder dem Kauffer oder Verkauffer andinge solche Waar niemands dann ihm zu kauften zu geben oder zu behalten ; oder dass er sie nicht näher geben wolle, dann wie er mit ihm überkommen hat. § 18. Wo aber die, denen liierinn Kauffmannssehafft zu treiben, wie obsteht , unerlaubt ist , unziemliche Theueruug in ihren Waaren zu machen unterstehen würden , darin soll eine jede Obrigkeit mit Fleiss und Ernst sehen , solche Theueruug abzuschaffen und einen redlichen , ziemlichen Kauff verfügen . Wo aber einige Obrigkeit in solchem lässig oder säumig seyn und das an unsern kayserlichen Fiscal gelangen wird , so soll unser Fiscal solches der Obrigkeit, da solche Kauffleut oder Handthierer gesessen oder wohnend seyn , zu erkennen geben und sie ermahnen , solche beschwerliche Handlung abzuschaffen und zu straffen in Monats -Frist . Dann wo die Obrigkeit solches in bestimmter Zeit nicht thät , so wolt und müsst er auss seinem Amte in solchem procediren und fürnehmen, wie sich gebührt ; alsdann er auch solches zu thun , Macht und Recht haben , auch unverzüglich thun soll.“ 1 1 Neue und vollständigere Sammlung der Reichs-Abschiede, 4 Teile. Frankfurt 1747. II . Teil, S. 144. Zweites Buch : Kirche, Staat und Frühkapitalismus. 73 Der Reichstagsabscliied von Trier-Köhl (1512) brachte die Reichsverhandlungen über die Monopole nicht zum Stillstand. Auch in den nächsten Jahrzehnten sollten sich das Regiment, Reichs¬ tag und Reichskammergericht mit der Frage der Monopole lebhaft beschäftigen. Besonders eindringlich setzten die Verhandlungen im Regiment 1522/23 ein, nachdem man dort schon vorher über die reichsrechtliche Behandlung von Monopolien verhandelt hatte 1. Soviel ich sehe, hat denn auch zuerst im Jahre 1523 der Fiskal yon der Gewalt Gebrauch gemacht, die ihm der Reichstag im Jahre 1512 in die Hand gegeben hatte . Eine ganze Anzahl Augs¬ burger Handelsherren, Jakob Fugger, Andreas Grander, Christoff Herwart, Ambrosius Höchstetter, Bartholome Welser, Andreas Rem und ihre Mitgesellschafter wurden von Caspar Marth, dem damaligen Anwalt des Reiches, vor Gericht geladen. Mit fieber¬ hafter Eile suchten die bedrohten Augsbui'ger Schutz vor dem nahenden Unheil. Sie zogen von allen Seiten hochmögende Bundes¬ genossen heran. Jakob Fugger wandte sich an Herzog Georg von Sachsen, mit dem er in enger geschäftlicher Verbindung stand 2, ebenso an Erzherzog Ferdinand. Besonders aber baten die Augs¬ burger Kaufleute Karl V. um Hilfe. Nicht umsonst! Von Burgos in Kastilien aus richtete der Kaiser schon am 15. September 1523 ein energisches Schreiben an den Fiskal und befahl ihm, sofort das Verfahren gegen die Augsburger Kaufleute einzustellen3. Gleichzeitig forderte der Kaiser auch seinen Bruder, den Erzherzog Ferdinand, auf, den Prozeß des Fiskals gegen die Augsburger Handelsherren niederzuschlagen4. Ferdinand möge dem Fiskal befehlen, „daß er all information und unterricht des handeis, so er wider die öbgemelt kaufleut, hat “, dem Kaiser „unter seinem betschaff zuschick“. Karl werde die Akten „übersehen und weiter darin nach des heiligen reichs Ordnung, gemainen rechten, der 1 Die Verhandlungenvon 1522/23 sind ausführlich wiedergegeben in „Deutsche Reichstagsakten jüng. Reihe“. 3. Bd. (1901). Bearbeitet von Adolf Wrede. S. 554 ff. Die Wormser von 1521 daselbst 2. Bd. S. 351. 2 Jansen, Jacob Fugger der Reiche, S. 261. 3 Urkunde im Anhang abgedruckt. 4 Burgos in Castilien, 15. September 1523. K. und K. Haus-, Hof- und Staatsarchiv in Wien. Reichsregistratur Bd. 3 Fol. 235/236. Am 16. Dezember 1524 wiederholte der Kaiser noch energischer die Mahnung an Caspar Marth. Reichsregistratur 5. Bd. Bl. 11 ff. 1 74 Zweites Buch : Kirche, Staat und Frühkapitalismus. pilligkait und wie des hl. reichs notturft und aufnemung erfordern wirdet, handeln lassen und solchs alles alsdan“ dem Regiment und dem Reichskammergericht mitteilen. Sollte schon etwas gegen die angeklagten Augsburger Kaufleute „gehandelt, aus¬ gesprochen oder furgenomen“ worden sein, so möge Ferdinand das „von stund an genzlich bis auf weitern bevelh abthun und nichtigen und in vorigs wesen sezen und stellen.“ Im Ernst dachte natürlich Karl V„ finanziell völlig abhängig von den süddeutschen Geldmännern wie er war, gar nicht daran, den Prozeß des Reichsfiskals gegen die vorhin genannten Augs¬ burger Kaufleute wieder aufleben zu lassen. Das ergibt sich deut¬ lich genug schon aus der Tatsache, daß die Augsburger keine Mühen und Kosten scheuten, um es auf dem Nürnberger Reichstage (1523/24) durchzusetzen, daß die endgültige Regelung der Monopolienfrage dem Kaiser übertragen würde L Tatsächlich erreichte man diese Übertragung. Wenn auch mit der Beschränkung, daß der Kaiser verpflichtet sein sollte, bis zur Frankfurter Fastenmesse des nächsten Jahres eine Entscheidung über die Monopolangelegen¬ heit zu treffen. Geschah das nicht, so sollten die Bestimmungen des Kölner Abschieds von 1512 in Kraft treten 2. Erich König hat aus dem Peutinger-Nachlaß, der in der Augs¬ burger Stadtbibliothek aufbewahrt wird, den Nachweis geliefert, daß die Augsburger Kaufmannschaft alle Hebel in Bewegung setzte, um den Kaiser zum schnellen Erlaß eines Handelsgesetzes zu be¬ wegen, in welchem die Monopolfrage erledigt wurde. Namentlich darauf kam es den Augsburgern an, gegen ein willkürliches, instanzen¬ überspringendes Eingreifen des Reichsfiskals einen stärkeren gesetz¬ lichen Schutz zu haben als die Formulierung des Trier-Kölner Abschieds (1512) bot. Zwar wurde dort die gerichtliche Zuständig¬ keit der ordentlichen Obrigkeit (also des städtischen Rats für Augs¬ burg usw.) auch für Monopolprozesseaufrechterhalten, aber sie wurde doch nicht entschieden genug betont und gegen Übergriffe 1 Laut einer Denkschrift Dr. Konrad Peutingers, vgl. Erich König, Peutingcrstudien. In Studien und Darstellungen aus dem Gebiete der Geschichte. IX. 1. und 2. München 1914, S, 119. 2 Deutsche Reichstagsakten jüngere Reihe. 4. Bd. (1905). Heraus¬ gegeben von Adolf Wrede. Nr. 110, Art. 4 und Nr. 149 § 27, IV. (Endgültige Beschlußfassung und Abschied.) Zweites Buch: Kirche , Staat und Friilikapitalismus . 75 # anderer Behörden geschützt 1. Das sollte in Zukunft anders werden. Höchstwahrscheinlich hat Konrad Peutinger selbst das Handels¬ gesetz entworfen, das nach längeren Bemühungen der Augsburger am spanischen Hofe von Karl V. am 10. März 1525 zu Madrid Rechtskraft erhielt. Das Gesetz bestimmt im wesentlichen das folgende bezüglich der Gerichtszuständigkeit in Monopolprozessen2. Als Ankläger wegen Monopolvergehens darf nur die Obrigkeit des¬ jenigen Ortes auftreten, in welchem der Führer der betreffenden Firma seinen Wohnsitz hat (nicht etwa die Obrigkeit des Ortes, nach dem die monopolisiertenWaren verbracht worden sind oder die Obrigkeit der das Geschäft vermittelnden Faktoren). Nur dann, wenn die zuständige Obrigkeit bei offenkundigen Über¬ tretungen der Monopolverbote nicht einschreitet, oder wenn sie verdächtige Fälle nicht untersucht, bzw. in einem eröffneten Ver¬ fahren willkürlich Stillstand eintreten läßt, nur dann hat der kaiser¬ liche Kammergerichtsfiskal ein Recht, seinerseits vorzugehen; er muß aber zunächst die erste Instanz mahnen und erst, wenn sie daraufhin einen Monat untätig verstreichen läßt , hat er die Be¬ fugnis, den Fall vor das Kammergericht zu ziehen. Wurde so die Gerichtshoheit der Städte usw. bei Monopol¬ vergehen einem willkürlichen Eingreifen des Fiskals gegenüber sichergestellt, so blieb die Ächtung und Strafbedrohung bei Mono¬ polen und Monopolversuchen, die auf gleiche Stufe mit Fälschungen und Betrug gestellt wurden, selbst bestehen. Ebenso das Verbot aller Syndikate, die den Zweck der Preissteigerung verfolgten. Wichtig ist es, daß nur diejenigen Monopole mit Strafe bedroht wurden, deren das gemeine Recht Erwähnung tut . Erwähnens¬ wert erscheint mir auch die Tatsache, daß die schweren Straf¬ bestimmungen des Kölner Reichstages in Karls V. Gesetz vom 10. März 1525 einigermaßen gemildert auftreten . So sollte in Zukunft nicht mehr das ganze Vermögen des Monopolisten der Obrigkeit verfallen sein, sondern nur derjenige Teil, mit dem das Monopolvergehen ausgeübt worden war. Ferner sollte jetzt der Käufer monopolistischer Waren ungestraft bleiben, gleichviel ob 1 Kö n i g a. a. O. S. 115 ff. 2 Die folgende Inhaltswiedergabe des Gesetzes vom 10. März 1525 lehnt sich größtenteils frei an König an, der auch im Anhang seines Werkes die wichtige Quelle in extenso abgedruckt hat. S. 169 ff. 1 76 Zweites Buch ; Kirche , Staat und Frühkapitalismus. er von dem Monopol gewußt hat oder nicht ; der Verkäufer wird mit der Konfiskation des erzielten Kaufpreises bestraft. Man wird der Bedeutung des Gesetzes vom 10. März 1525 am meisten gerecht werden, wenn man es nicht so sehr in der Monopolienfrage, als vielmehr in der Frage der obrigkeitlichen Be¬ schränkung einer freien Entwicklung der Handelsgesellschaften epochebildend ansieht. Heftiger nämlich fast noch als die Bemühungen gewisser Kreise, im Reiche die Monopolien zu vernichten, waren die Versuche dieser Idealisten, die Ausdehnung der Kapitalkraft derHandelsgesellschaften zu verhindern. Seit Generationen waren bereits Angriffe auf das Sozietätswesen erfolgt. Schon in den Zunftaufständen von 1425—1429 wurde in Konstanz die Abschaffung der Handelsgesellschaftenge¬ fordert h In dasselbe Horn stieß die Reformation Kaiser Sigis¬ munds im 7. Kapitel, „von den geselschaften in den stetten “ über¬ schrieben. Da heißt es: „ Item es sind groß geselschaften aufgestanden, die zusamen spannent und treibent groß kaufmannschatz, es ge in wol oder übel. Sy schybent es ye darnach, das sy nit verliern. Das kompt auch aller gemain in den stetten und auf dem land übel. Man soi dawider sein, das solich puntnuss abgestellet werd und nyndert mer gefunden, weder von edlen noch von bürgern. Wer aber darüber in dhain geselschaft punde, ist er ain burger in ainer reichstatt, so soi sein koufmanschatz der statt ledig und gar vervalen sein und soi inn darinne nichtzit schiermen. Ist er edel, so soi er sein in des reichs Un¬ gnaden und sein koufmanschatz menigklich erlaubet sein. Man soi vor allen aufsätzen in allen kauffen vesteklich verhüten , wenn laider yetz die aufsätz allen lannden we tond, es schaidet trüw und gemainsame gar“ 2. 1 Schulte, Geschichte des mittelalterlichen Handels . I . Bd. S. 608. 2 Zitiert nach der Ausgabe von Heinrich Werner . III . Ergänzungshe £t des Archivs für Kulturgeschichte . (1908 .) S. 73. Übrigens wendete sich der Autor der Reformation des Kaiser Sigismund auch gegen die Versuche , Monopole zu bilden . In Kapitel 18 („ Wie man das furkouffen furkomen sol “ überschrieben ) liest man : „ Man soi auch wissen , das notturftig ist der gmainen cristenhait zu versorgen umb alles verkauften , es sey wein , körn , fleisch , smaltz , allerlay gemuss, was man messen sol . In ainem land gerätt das denn , in dem andern da vindet man mangen , der darauf sicht und furkauft ; so es im fug- 1 Zweites Buch : Kirche, Staat und Frühkapitalismus. 77 Aber eine rechte Hetze gegen die großen Handelsgesellschaften hebt doch erst in der volkstümlichen Literatur und in der Gesetz¬ gebung des beginnenden 16. Jahrhunderts an \ Man kann dabei von jenen Fanatikern absehen, die eine völlige Abschaffung der Handelsgesellschaften forderten. Ihre extremen Wünsche hatten keine Aussicht, Gesetzeskraft zu erlangen. Anders stand es mit den Vorschlägen der besonnenen Feinde der kapitalistischen Ent¬ wicklung, wie sie sich in der mächtigen und schnellen Ausdehnung der Handelsgesellschaftenoffenbarte. Die Forderungen einer Ein¬ dämmung der Handelsgesellschaften durch Beschränkung der in¬ vestierten Kapitalien, der zugeiassenen Mitglieder (nur Vater, Sohn und Schwiegersohn sollen sich in Gesellschaft zusammen¬ tun dürfen), der Anzahl der Faktoreien usw., diese Forderungen durften schon eher auf Zustimmung der maßgebenden Kreise hoffen. Standen doch selbst Städte wie Nürnberg, Ulm, Frank¬ furt usw. solchen Prohibitivmaßregeln nicht unfreundlich gegen¬ über. Die genannten Städte wurden dabei vielfach von Neid gegen Augsburg, das eigentliche Herz des deutschen Frühkapitalismus, getrieben 2. Durch das Handelsgesetz Karls V. vom 10. März 1525 wurden nun mit einem Schlage die Hoffnungen der mehr oder weniger weitgehenden Gegner einer freien Entfaltung der Handelsgesell¬ schaften zerstört. Der Kaiser bestimmte in dem Erlaß, es dürfe weder jetzt noch künftig ehrbarem Kaufmannshandel und Wandel — dem großen, dem mittleren und dem kleinen — durch irgendwelche einengende, gesetzlich nicht begründete Verfügungen Behinderung und Schmälerung widerfahren. Vielmehr sollte es lieh ist , so schlecht er ungewonlich gwinn darauf und dringet arm leut . . .“ In Werners Ausgabe , S. 88 . Ich möchte gegen Werner (und Böhms ältere Ausgabe ) lesen . . . „ da vindet man mangel . Der darauf sicht und furkauft , so es im fuglicli ist , so schlecht er . . .“ 1 Die meisten Literaturangaben bei Ang. Kluckhohn, Zur Geschichte der Handelsgesellschaften und Monopole im Zeitalter der Reformation . In Historische Aufsätze , dem Andenken an Georg Waitz gewidmet . Hannover 188S , S. 666 ff . Dazu vgl . Deutsche Reichstagsakten jüngere Reihe . III . Bd ., S. 554 ff. ; IV . Bd ., S. 467 ff. Was sich auf den Reichstagen im großen abspielte , das wiederholte sich im kleinen auf den Landtagen usw. 2 Kö n i g a. a . O., S. 121 und dic Rcichstagsakten IV , S. 257, 260 , 641 , 673. 1 78 Zweites Buch: Kirche, Staat und Frühkapitalismus. einem jeden erlaubt sein, ganz frei und uneingeschränkt Kauf¬ mannschaft zu treiben, wie, wann, an welchen und an wieviel Orten innerhalb und außerhalb des Reiches, durch welche Personen, in welcher Art und mit welchen Gütern und Waren er wolle, sei es allein oder in Gesellschaft mit anderen und mit jedem beliebigen Kapital , gleichviel, ob dies sein, oder seiner Gesellschafter oder anderer Leute eigen sei L Man sieht, offenbar ging die Tendenz des Gesetzes auf eine freie Entwicklung der Handelsgesellschaften hinaus. Das Reichsregiment hat denn auch den Erlaß, als den Wünschen des Großhandels allzusehr entgegenkommend, nicht publiziert, sondern zu den Akten gelegt 2. Bedeutete das Madrider Handelsgesetz Karls V. vom 10. März 1525 für die Kämpfe um die Ausdehnung der Handelsgesellschaften einen gewissen— für die Gegner des Großhandels sehr ungünstigen— Abschluß, so sollte in der nächsten Zeit auch die Frage der Monopolien eine den Augsburger Großkaufleuten recht annehmbare Wandlung bringen. Die Habsburger, Karl V. und Ferdinand I., wie die meisten damaligen Fürsten überhaupt 3, mußten die Antimonopolbe¬ wegung, wie sie sich in der Literatur ihrer Zeit und auf den Reichs- und Landtagen ihrer Länder abspielte, mit recht gemischten Gefühlen betrachten. Ihrer Finanzpolitik war die Bewegung eines¬ teils günstig. Das drohende Unheil, das in der Gestalt eines Reichs¬ kammergerichtsprozessesgleich einem Damoklesschwert über den Häuptern der damaligen deutschen haute finance hing, mußte die reichen Kapitalisten den kaiserlich-königlichen Anleihewünschen gegenüber gefügig machen. Bei der obersten Reichsgewalt lag ja der einzige Schutz gegen Eingriffe des Fiskals. Ferner bedeuteten auch die Schutzbriefe des Kaisers für die monopolistischen Kauf1 Nach Königs Interpretation des Gesetzes. 2 Kö nig a. a. O., S. 119. 3 Peutinger nennt folgende Monopole , die zu seiner Zeit besonders bekannt waren . Außer dem Pfeffer seien monopolisiert gewesen: Korallen in Hippo und anderen Orten Afrikas , Alaun in Sizilien und Toskana , Rosinen in Achaia , Erdharz , sog . Asphalt (nach seinem Vorkommen im Asphaltsee in Judäa , sog . Totes Meer) in Palästina. Ob Peutingers Behauptung des etymologischen Herkommens des Wortes Apalto (in Italien für Monopol gebraucht ) richtig ist , lasse ich dahin gestellt. Zweites Buch : Kirche, Staat und Frühkapitalismus . 79 leute eine nicht geringe Einnahmequelle für die kaiserliche Kammer¬ kasse 1. So konnte also die Antimonopolbewegung der Finanzpolitik der Habsburger manchen Vorteil bringen. Aber die Sache hatte nun auch ihre Kehrseite. Zum mindesten war es doch eine Mit¬ schuld, die die Habsburger bei der Entstehung der meisten mono¬ polistischen Organisatiçnsformen traf , die damals im heiligen römischen Reiche entstanden. In einem Gutachten Augsburger Bürger über Monopole aus jener Zeit 2 ist der Kern der Sache ge¬ troffen, wenn gesagt wird: „Nit allain durch die gesellschaften monopolia, als man’s nenen thut und furkeff gesehechen. Dan so man’s woll ermessen will, wirt man finden, das durch kaufen, verkaufen, klains und großes, durch gemain vom hösten bis auf den geringesten stand, der menschen aigner und laider wenig gemainer nucz, brüderliche lieb gesucht wurdt (will fälschen des guts, betrug etc. geschweigen). Ja , torf te man ’s sagen , monopolia oder furkeff durch die großen heren gestuirt 3 und verkauft , wie kondpar am tag liegt mit alon 4, specerei , ge wand, merlo 5, metall und and er m etc.“ Tatsächlich läßt sich nachweisen, daß das Kupfersyndikat vom Jahre 1498 — wir kommen noch auf dieses Kartell zu reden — „auf k u n i g 1. Majestät Will und Befehl“ zwischen 1 Für solche „Briefe“ wurden 40—100 fl. Taxe gezahlt. Auch die Schutzbriefe des Kaisers, die verhindern sollten, daß die Kauf¬ leute unter dem Vorwand, sie seien der „Lutherei“ verdächtig, von ihren Feinden belästigt und geplündert wurden, boten dem Reich eine gute Einnahmequelle. Den Schutzbrief für die Fugger hat Jansen Jakob ( Fugger der Reiche, S. 394 ff.jabgcdruckt. Andere für die Firma Adler-Augsburg, Prechter-Straßburg, Welser-Augsburg usw. sind in den ReichsregistraturbüchernBd. V BI. 151 ff., 178 ff., 35 ff. usw. zu finden. Oft ist in ihnen auch Schutz gegen Anklage wegen Monopolvergehen noch mit erwähnt. 2 Deutsche Reichstagsakten jüngere Reihe. III . Bd. S. -559 f. Ich möchte annehmen, daß das Gutachten von K. Peutinger be¬ einflußt ist. 3 stürn= stacheln, antreiben. 4 Alaun. 5 Nach dem Herausgeber Ausdruck für Kabeljau. 1 80 Zweites Buch : Kirche, Staat und Frühkapitalismus. einigen Augsburger Großkaufleuten abgeschlossen wurde ' . Un¬ zweifelhaft lief das Syndikat darauf hinaus, den Augsburger Firmen eine Monopolstellung auf dem bedeutenden venezianischen Kupfer¬ markte zu sichern. Und was waren denn die sogenannten Silber- und Kupferkaufe, die Kaiser Maximilian in Tirol mit den Augsburger Kauf¬ leuten, vor allem mit den Fuggern in großer Anzahl abgeschlossen hatte , anderes, als Monopolisierungengroßen Stils. Dann hatte König Ferdinand seine Hand im Spiele gehabt als zwischen der Idriancr großen Gewerkschaft und der des Fürstenbaues ein Queck¬ silberkartell zustande kam 2, und derselbe Herrscher war es gewesen, der — wie wir im einzelnen noch sehen werden — durch seine immer gesteigerten fiskalischen Profitwünsche das von ihm geschaffene Idrianer Quecksilberhandelsmonopolimmer drückender für die Konsumenten gestaltete. König Ferdinand hat es auch einmal direkt ausgesprochen, daß er die Idrianer Quecksilbermonopol¬ kontrakte zu dem Zwecke abgeschlossen habe, damit das Queck¬ silber „um so viel höher gesteigert werden möge“3. Die Beispiele einer führenden Anteilnahme der deutschen Könige und Kaiser bei der Entstehung von Monopolbildungen des endenden 15. und des beginnenden 16. Jahrhunderts ließen sich mehren. Schon das Gesagte dürfte genügen, um zu erkennen, daß die oberste Reichsgewalt nicht wohl anders konnte, als die monopolinhabenden Kaufleute gegen eine Verfolgung durch den Fiskal in Schutz zu nehmen. Im gegenteiligen Falle hätte sich schwerlich bald noch ein Kapitalist gefunden, der mit Sr. Majestät den Monopolgewinn zu teilen bereit war. Die meisten großen Kaufleute verließen sich dabei übrigens bald nicht mehr auf den guten Wißen der Herrscher, falls der Fiskal gegen sie wegen Handels¬ vergehen vorging. Sie ließen sich vielmehr sofort bei der Eingehung des Monopolvertrags von dem König bzw. Kaiser bestätigen, daß er sie bedingungslos beschützen würde, falls man von Reichs wegen 1 Vgl . das Consilium in causa societatis cupri von K. Pcutinger. Manuskript in der Augsburger Stadtbibliothek. Cod. 2 ° Aug. 398. Fol. 193r. König a. a. O. S. 109. Dazu Ehrenberg n. a. O. I. 396ff. 2 Siehe weiter unten das Kapitel „Monopole und Kartelle im Idrianer Quecksilberhandel des 16. Jahrhunderts.“ 3 Näheres siehe im Kapitel „Monopole und Kartelle im Idrianer Quecksilberhandeldes 16. Jahrhunderts.“ 81 Zweites Buch: Kirche, Staat und Frühkapitalismus. ihre Abmachungen als unerlaubte Wirtschaftsorganisationen an-sehen und gegen sie als Monopolisten einschreiten würde. Ein entsprechender Paragraph wird uns in den Monopolkontrakten im böhmischen Zinnerzhandel des 16. Jahrhunderts , besonders aber im Idrianer Quecksilberhandelderselben Zeit begegnen. Ein Schritt weiter war es, wenn einzelne Kaufleute auch ohne die bestimmte Veranlassung des Abschlusses eines Monopolkon¬ traktes sich nachträglich für frühere Übertretungen der strengen Wirtschaftsethik durch „Majestätsbriefe“ Sicherung verschafften. So ließen sich die Erben Jakob Fuggers kurz nach dessen Tod von Kaiser Karl V., in einer aus Granada vom 19. Oktober 1526 datierten Urkunde 1garantieren , daß niemand sie wegen ver¬ gangener oder zukünftiger Monopole gerichtlich belangen oder belästigen dürfe. Die Urkunde enthält das äußerste, was mir aus jenen Jahrzehnten an Diskrepanz, an Gegeneinanderarbeiten, zwischen der kaiserlichen Kanzlei und dem obersten kaiserlichen Gerichte begegnet ist 2. Welche Handelsgeschäfte übrigens den Fuggern besonders schwer auf dem Gewissen lagen, das erkennt man aus einer Urkunde Karls V. vom 26. Oktober 1525 (gegeben zu Toledo) 3. Es waren die Tiroler Kupfer- und Silberkäufe, be¬ sonders jener „Kauf“, den Jakob Fugger am 7. November 1514 für sich allein und jener, den er am 30. Oktober 1515 mit Ambrosius und Hans Höchstetter und Compagnie abgeschlossen hatte. Von den Fuggerschen Salvierungspatenten vom 26. Oktober und vom 19. Oktober, besonders von letzterem aus, war es nur noch ein Schritt, wenn der Kaiser auch offen für die völlige reichs¬ gesetzliche Beseitigung des Monopolverbotes, wenigstens auf dem wichtigsten Gebiete des damaligen deutschen Großhandels, eintrat. Das geschah durch das Toledaner Mandat Karls V. vom 13. Mai 15254. Der Gesetzeserlaß bestimmte, daß die Kontrakte, die den Erzgroßhandel in die Hände weniger Kaufleute auslieferten, 1 Abgedruckt im Anhang. An demselben Tage wurde eine gleiche Urkunde für den Welser ausgestellt. RcichsregistraturKarlsV. 5. Bd. Bl, 278 ff. 2 Man bedenke, daß sich Karl V. in seiner Wahlkapitulation ver¬ pflichtet hatte, gegen den Mißbrauch der Monopole vorzugehen. Vgl. Reichstagsakten jüng. Reihe, I, Nr. 387, Art. 19. 3 Bei Jansen, Jakob Fugger der Reiche, S. 401 ff., leider nur teilweise abgedruckt. 4 Siehe Anhang. Strieder , Studien z, Gesell , kapitalist . OrganiHalioiißforiiMin . (> 82 Zweites Buch : Kirche , Staat und Frühkapltalismus. nicht als monopolistisch im Sinne der Reichstagsverhandlungenan¬ gesehen werden sollten und dürften. Meines Erachtens gehört das genannte Mandat Karls V. zu den interessantesten Dokumenten der Wirtschaftsgeschichte. Zum ersten Male ist hier öffentlich von seiten der höchsten staatlichen Gewalt der Christenheit der Grundsatz durchbrochen, der die mittelalter¬ liche Wirtschaftsethik beherrscht hatte : Die Forderung des ge¬ rechten Preises, des alten pretium justum, ist vom Kaiser — wenn auch nur für eine bestimmte Produktionssphäre — fallen gelassen worden. Den Monopolinhabern für Bergbauprodukte 1 wird aus¬ drücklich das Recht zugestanden, ihre Erze und Metalle zu dem höchsten Preise zu verkaufen, den sie erhalten können. „Zum höchsten und nach den besten wirden, wie sie des statfinden künden nach irem gefallen“ heißt es wörtlich in dem kaiserlichen Erlasse. Wie anders hatte die Forderung der Scholastiker gelautet und wie anderes hatte noch Luther gefordert, als er schrieb: „Es sollt nicht so heißen, ich mag meine Waar so theur geben als ich kann oder will, sondern also, ich mag meine Waar so theur geben als ich soll oder als recht und billig ist.“ Im höchsten Grade interessant und für den Geist der neuen Zeit bezeichnend ist die Begründung der genannten kaiserlichen 1Auch in dem Gutachten Peutingers de anno 1530 werden für den Erzgroßhandel besondere Freiheiten bzgl. Bildung kapi¬ talistischer Organisationsformen gefordert. Dort heißt es : „Mani¬ festum est quod minerae et metalla et etiam eorum negotiationes habent speciales proprietates et condiciones ante et praeter omnes caeteras mercaturas, adeo quod, si possibile esset, uti non est, omnes minerae et omnia metalla in manum unam pervenerint, nullum esset hoc monopolium saltem contra rempublicam et eius utilitatem, verum, tanto altius et carius venderentur et in magno pretio continerentur, eo melius minerae et fodinae tamquam magnum et unicum donum Dei interteneri possent . . . Sed ex hoc satis et plane constat, quod isti suggestores, qui statibus, imperii et eorum deputatis haec exposuerunt, libenter illas mineras et fodinas tamquam magnum et utile Dei donum, ex quo mirum in modum utilitas reipublicae promoveri solet, supplantare vellent et impedire, sicut etiam in minoribus causis et negotiis ex quibus reipublicae utilitas communi nationi Germanicae resultat libenter fecissent et de praesenti facerent ubi hoc non provideretur et praecavebitur.“ Manuskript in der Augs¬ burger Stadtbibliothek. Cod. 2 ° Aug, 386 fol. 184v. 83 Zweites Buch : Kirche, Staat und Frühkapitalismus. Ausnahmemaßregel mit dem allgemeinen Wohl 1 und mit den Lebensnotwendigkeiten einer deutschen Volkswirtschaft , d. h. mit der überragenden Bedeutung des Bergbaues und des Erzhandels für die gesamte Wirtschaft des heiligen römischen Reiches und mit der Unmöglichkeit , die Blüte dieses ökonomisch wichtigsten Zweiges der deutschen Volkswirtschaft anders als durch Monopole aufrecht zu erhalten . Nur wenn den Gewerken , die ihr Kapital in den ohne¬ hin schon riskanten Bergbau steckten , eine entsprechend hohe, möglichst gleichmäßige Gewinnquote , wie sie durch Monopole garantiert werde , gesichert sei, könne die Bergbaulust nachhalten und sich zum Nutzen der gesamten deutschen Volkswirtschaft noch vermehren . Im besonderen sind dann noch die Schäden , die Preis¬ schwankungen der Bergwerksprodukte und das Ablassen des Kapitals von unrentablen Gruben für die dortige Arbeiterbevölkerung haben mußten , in glücklicher Weise für die Monopolverteidigung ins Treffen geführt. Interessant ist es auch , daß Karl V. die Erlaubnis zu Mono¬ polen im Erz -, besonders im Kupfer - und Quecksilbergroßhandel damit begründet , daß die genannten Metalle keine Bedarfsartikel im strengsten Sinne des Wortes seien und nur zum kleinen Teil „in deutschen Landen “ verkauft , zum größten ins Ausland ex¬ portiert würden . Die Handwerker , die in Deutschland Metalle verarbeiteten , aber dürften sich über die Preissteigerung z. B . des Kupfers — wie sie sich aus Monopolen ergäbe — nicht beschwert fühlen 2. In demselben Maße wie die Kupferpreise , stiegen ja auch die Preise ihrer Fertigwaren . Auch gälte es zu bedenken , daß 1 Der Kaiser betont ausdrücklich , es käme nicht darauf an, ob die aus den Monopolen resultierende Preiserhöhung einzelnen zum Nachteil gereiche. Der allgemeine Nutzen der Monopole für die gesamte deutsche Volkswirtschaft sei ausschlaggebend : „Obgleich das alles in ctlich weg etwa sondern perso neu für nachtailig geacht oder verstanden werden soit, dieweil doch solchs sunst in vil mehr weg und dem gemeinen nutz dienstlich , fruchtbar und gut ist .“ 2 „Item die kupfersmid beswarn sich, dass die gesellschaften das eisen und kupier verdeurn , pitend solhes abzustellen , auch daran und darob zu sein, dass uns Taufrer lcupfer von gewercken zu kaufen geben werde und nit von den geselschaften .“ Aus den Beschwerde¬ artikeln der Tiroler Bauern 1519—1525. Vgl. H. Woptncr, Quellen zur Geschichte des Bauernkrieges in Deutschtirol , 1525. I. Teil. Quellen zur Vorgeschichte des Bauernkrieges : Besch Werdeartikel aus den Jahren 1519—1525. ActaTirolensia III. 1. Innsbruck 1908, S. 120. 6 84 Zweites Buch : Kirche , Staat und Frühkapitalismus. ohne die Monopolerlaubnis für den Erzgroßhandel die Bergwerke nicht in dem starken Maße wie m i t derselben abgebaut , also Mangel an dem betreffenden Metall entstehen und die Preise erst recht in die Höhe getrieben würden. Richtige und falsche ökonomische Erkenntnis war in diesen Ausführungen Karls V. gemischt . Auf den Kernpunkt der Sache aber war nicht cingegangen . In Wahrheit zwang die bitterste Finanznot den Kaiser , die oberste staatliche Macht der Christenheit, von den Prinzipien der alten christlichen Wirtschaftsethik ab¬ zugehen . Es bahnt sich dabei , aus der Not geboren , eine weit¬ herzigere Beurteilung des Kapitalismus und des kapitalistischen Unternehmers an . Man begann langsam zu verstehen , daß der Kapitalismus nicht notwendigerweise , wie heute noch die Sozial¬ demokratie meint , etwas Unsittliches sein muß . Man fing an , den Kapitalismus als die historisch gegebene Begleiterscheinung einer Volkswirtschaft zu begreifen , eines Wirtschaftslebens also, das sich über die ältere Stadtwirtschaft und die ihr eigenen Wirtschaftsprinzipien erhob. Die Begründung der Monopole mit den nationalen Inter¬ essen und dem allgemeinen Wohl spielt in den Argumenten der Monopolfreunde , namentlich auch bei Peutinger eine wichtige Rolle. Es sind dieselben Beweisgründe , die um dieselbe Zeit (1524) zur Begründung der „Erbarkeit “ eines Silberringes geltend ge¬ macht wurden , den die an der Silberproduktion interessierten deutschen Fürsten auf Vorschlag des Nürnberger Kaufmanns Christoph Führer schließen sollten , um den Silberpreis hinauf¬ zutreiben 1. Da heißt es in einer Denkschrift 2:Dieweil „ denn alle fremde Nationen , die Gott mit etwas begnadet , dess man zu mensch¬ licher Nothdurft und Gemeinschaft bedürftig , dasselbe , so hoch sie wissen und vermögen verlassen und anwerden , der Allmächtige aber in deutscher Nation uns und unser Land mit Bergwerken 1 Die drei Flugschriften über den Münzstreit der sächsischen Albertiner und Ernestiner um 1530, hcrausgegeben von Walther L o t z, Leipzig 1893, S. V, imd besonders Willi. Pückert, Das Münzwesen Sachsens 1518—1545. I. Abt . (Die Zeit von 1518 bis 1525 umfassend ). Leipzig 1862, S. 80 f. 2 Die Denkschrift atmet durchaus kapitalistischen Geist, wenn sie sagt : „eö sei sonder zweifei einem jeden zugelassen, wird auch also gehalten , das Seine so hoch er vermag auszu¬ bringen,“ d . h. zu verkaufen . P ü c k e r t a . a. O., S. 80. Zweites Bach : Kirche, Staat und Frühkapitalismus. 85 begnadet : verhoffen wir, so wir solcher Gottes Gabe und Waare uns zu Gut , auch zu stattlichem Schutz unsrer Land und Leut, weiter denn bisher genössen , wir seien dessen auch nicht zu verdenken .“ Und ein anderes Mal ist betont, der Plan hänge eng zusammen mit dem „gemeinen Nutzen, den wir am höchsten und am meisten zu fördern und zu betrachten geneigt sind “. Denn wenn durch das Syndikat „ das Silber am Kaufe gesteigert , würden sonder Zweifel die Bergwerke baß denn jetzt gefördert und wie zu hoffen neue erregt , damit dann unsre Land und Leute gereichert und also gemeiner Nutz gestärket “ 1. Die Anklänge an das Edikt von Toledo sind unverkennbar. Ich kann nicht mit Bestimmtheit sagen , ob das Toledaner Mandat vom 13. Mai 1525 das Schicksal des von König aufgefundenen Handelsgesetzes Karl V. vom 10. März 1525 insofern teilt , als es vom Reichsregiment nicht publiziert wurde . Der Reichstagsabschied von Speier (1526) jedenfalls 2 dekretierte ganz generell : „Nachdem die Monopolien und große Gesellschaften ein eigennützige unleid¬ liche Handlung , die. in gemeinen kayserlichen Rechten bey hoher Pön und Straff verboten ist , so soll der kayserliche Fiscal gegen denselbigen , wie sich im^Rechten gebührt , ernstlich procediren und handeln , damit dieselbige abgethan und der gemeine Nutz ge¬ fördert werde “ 3. Und wörtlich wiederholte der nächste Abschied (Speyer 1529) dasselbe Verbot 4. Dagegen stellte sich der Augs¬ burger Reichstagsabschied von 1530 und die Reichspolizeiordnung von 1548 wieder auf den Standpunkt des Trier -Kölner Beschlusses von 1512, den sie nahezu wörtlich wiederholen 5. Das heißt sie bestimmen , daß der Fiskal erst dann gegen die Monopolisten Vor¬ gehen dürfe , wenn die ordentliche Obrigkeit sich versage . Immerhin 1 Püclcert a. a. O., S. 80. Vgl. auch König a. a. O., S. 124, Anm. 3. 2 Laut einer Eingabe Augsburgs an den Kaiser (verfaßt von Peutinger ) wurde der scharfe Abschied von Speier angenommen „ als schon viele Fürsten abgereist gewesen wären und ohne Anhörung der interessierten Stände .“ König a. a, O., S. 125. 3 Neue und vollständigere Sammlung der Reichsabschiede. II . Teil, S. 278. 4 a. a. O. S. 300. 6 a. a. O., S. 327 bezw. 597 f. Für die Verhandlungen über die Monopolienfrage auf dem Augsburger Reichstag (1530) vgl. auch K. E . Förstemann, Urkundenbuch zur Geschichte des Reichs¬ tags zu Augsburg im Jahre 1530. 2Bde . Halle 1835. IL Bd., S. 191 ff., * 86 Zweites Buch : Kirche , Staat und Frühkapitalismus. nahm der Abschied des Augsburger Reichstags das Erz aus der Reihe der Waren, mit denen Monopole zu treiben verboten sei, aus. In Wirklichkeit hat weder das strengere noch das lässigere Monopolverbot etwas geholfen. Klagend mußte die Reichspolizeiordnung von 1548 betonen 1: „Wiewol die Monopolia, betrügliche, gefährliche und ungebührliche Fürkäuff, nicht allein in gemeinen, beschriebenen Recht, sondern auch in gemachten und publicierten Reichsabschieden bey großen Pönen und Straffen, als Verlust aller Haab und Güter und Verweisung des Lands verbotten, so ist doch solchen Satzungen , Abschieden und Verbott biß an¬ her mit gebührlicher und schuldiger VolIn¬ ziehung gar nicht nachkotnmen noch gelebt worden, sondern seynd in kurtzen Jahren etwa viel große Gesellschaft in Kauffmanns-Geschäfften, auch etliche sonderbare Personen 2, Handtierer und Kauffleut im Reich auffgestanden, die allerley Waaren und Kauffmanns-Güter, auch Wein, Korn und anders dergleichen von den höchsten biß auff die geringsten (in welchem sie dann in den Landen hin und wieder gute Kundschafft und Verwarnung haben, sonderlich wann die Waaren verderben, oder sonst in Aufschlag kommen und ehe die andern Kauffleut solches gewahr werden) in ihre Pland und Gewalt allein zu bringen unterstehen, Fürkäuff damit zu treiben und denselben Waaren einen Wehrt nach ihrem Willen und Gefallen zu setzen, oder dem Käuffer oder Verkaufter anzudingen, solche Waaren nimands dann ihnen zu kauffen zu geben oder zu behalten, oder daß er, der Ver¬ kaufter, sie nicht näher 3oder anders geben woll, dann wie mit ihme überkommen, fügen damit dem heiligen Reich und allen Ständen desselben mercklichen Schaden, wider obvermelte gemeine, geschriebene Recht und alle Erbarkeit, zu“ 4. dazu S. 806; vgl. auch das lateinische Gutachten Peutingers von 1530. Manuskript in der Augsburger Stadtbibliothek . Cod. 2 ° Aug. 386. Bl. 176r ff. Die jüngere Reihe der deutschen Reichstagsakten, die hoffentlich bald wieder fortschreitet , wird wohl manchen Beitrag zur Frage der Monopole und der großen Handelsgesellschaften bringen. 1 Neue und vollständigere Sammlung der Reichsabschiede . 11, 597. 2 D. h. einzelne Kaufleute , nicht Gesellschaften. 3 D. h. billiger. 4 Dieselbe Klage wiederholt auch die Reichspolizeiordnung von 1577, a. a. O., III . Teil, S. 388. Zweites Buch : Kirche , Staat und Frühkapitalismus. 87 Auch die Belohnung , die die Polizeiordnung von 1548 dem Angeber von Monopolisten in Aussicht stellte (% des verwirkten Gutes) dürfte nicht allzu viel gefruchtet haben , waren doch die¬ jenigen , die wirklich etwas Genaues auszusagen imstande waren — die Monopolisten selbst —, bei gegenseitiger Anzeige von der Belohnung ausgeschlossen 1. Unter den Prozeßakten des Fiskals, die in den Repertorien des ehemaligen Reichskammergerichts ver¬ zeichnet sind , habe ich nur eine einzige Monopolklage,ausfindig machen können . Es ist die Klage , die 1529 der kaiserliche Fiskal Caspar Marth bei dem Reichskammergericht gegen die Firma Bartolome Welser & Co. vorbrachte . Der Reichsanwalt beschuldigte die Augs¬ burger Firma , sie habe Monopole aufgerichtet . Besonders ver¬ dammungswürdig erschien dem Ankläger eine Verabredung der Welser mit dem König von Portugal , laut welcher sich der letztere kontraktlich verpflichtete , niemandem Spezereien zu demselben niedrigen Preise zu verkaufen wie er sie Bartolome Welser & Co. verkaufte 2. Auf Grund der Klage des Fiskals lud Karl V. für den 7. März 1530 die Firma Welser & Co. vor das Reichskammergericht. Es mag kein Tag der Freude für das alte , aristokratische Geschäfts¬ haus gewesen sein, als am 7. Februar 1530 Ihrer Römischen Kaiser¬ lichen Majestät Kammergerichtsbote in der Schreibstube erschien und dem Seniorchef des Hauses , dem alten Bartolome Welser , die Ladung feierlich überreichte , als gleichzeitig die Klage Wegen Monopolvergehens der Firma am Rathause von Augsburg öffentlich angeschlagen wurde 3. Über den Verlauf des Prozesses ist nicht viel bekannt geworden. Ganz bestimmt half der Kaiser auf Grund des oben erwähnten Privilegs unserer Firma die Angelegenheit niederschlagen , trotz der reichsrechtlichen Bestimmung , daß alle Privilegien , Begnadigungen und Verjährungseinreden usw. bei Monopolvergehen kraftlos seien und im Gegenteil „den schädlichen Monopolisten und Handtierern, so sich damit beschirmen wollten , viel mehr nachteilig , sträflich und derselbigen Strafe gravieren und häufen sollten .“ Stärker als das Recht war die Finanznot des Kaisers . Es wird in diesem Welserschen Falle wie in so vielen anderen gegangen sein, wie in der ganzen 1 a. a. O., II , S. 598. 2 Die Anklage ist im Anhang wiedergegeben. 3 Nach den Akten des Reichskammergerichts , jetzt in München. Vgl. auch König a. a. O., S. 126 ff. 88 Zweites Buch : Kirche, Staat und Frühkapitalismus. Monopolistenverfolgungüberhaupt : Der Kraftaufwand, mit dem Reichsstände und öffentliche Meinung, mit dem auch die Buß¬ prediger katholischen und lutherischen Bekenntnisses gegen den Drachen der Monopolia wie gegen alle „wucherischen Kontrakte“ kämpften, stand im schreiendsten Mißverhältnis zu dem geringen Erfolg. Die vorangehenden Auseinandersetzungendürften gezeigt haben, wo die Wurzeln dieser Mißerfolge lagen. Nur an wenigen Stellen, besonders dort, wohin die Macht des deutschen Kaisers nicht reichen konnte oder wollte, gelang es dem Zunftgeiste, die Oberhand zu bekommen über den aufblühenden Frühkapitalismus. In Basel war im 15. Jahrhundert ein schon älterer Großhandelsstand an Zahl und Geldkraft mächtig gewachsen. Ein durchaus modernes Gewinnstreben beseelte diese Kaufleute, besonders jene, die seit dem 6. und 7. Jahrzehnt des 15. Jahr¬ hunderts daran gingen, sich zu Gesellschaften mit monopolistischem Charakter zusammenzusehlicßen. Seitdem begann ein harter Kampf des Kleinbürgertums gegen das Kapital, ein Kampf, der im Verlauf der ersten Jahrzehnte des 16. Jahrhunderts unter dem Eindruck der allgemeinen Kämpfe gegen Großhandelsgesellschaften und Monopole zuungunsten des Kapitals entschieden wurde. Der Baseler Rat löste die „ gemeinschädlichen Gesellschaften auf, schützte die. schwer gefährdeten Detaillisten gegen die Konkurrenz der Groß¬ kaufleute. Als dann gar die Zünfte zum Regiment in Basel ge¬ langten, da setzten sie 1526 eine Gewerbeordnung durch, die die unbeschränkte Herrschaft des Handwerks auf dem städtischen Markte begründete. Dem Handel blieb nur der notdürftigste Spielraum, er wurde zum Kleinbetrieb verurteilt. Es ist ein voller Triumph des Mittelalters über die andrängende neue Zeit, eine Reaktion des Zunftgeistes gegen die Freiheit des Handels“, was wir in Basel in dieser Zeit sich vollziehen sehen1. Es wäre nun sehr verkehrt, zu glauben, dass sich nicht schon in der mittelalterlichen Welt führende Theologen gefunden hätten, 'KurtKaser , Deutsche Geschichte im Ausgange des Mittel¬ alters (1438—1529). 2. Bd. Deutsche Geschichte zur Zeit Maxi¬ milians I. (1486—1519). Stuttgart und Berlin 1912. S. 444 ff. ; nach R. Wackernagel, Geschichte der Stadt Basel. Bd. 1—2, 1. Basel 1906—1910. II, 1. S. 525 ff. Zweites Buch : Kirche, Staat und Frühkapitalismus . 89 die einer gerechteren Beurteilung des kapitalistischen Geistes die Wege ebnen halfen. Es fehlte dort durchaus nicht an bedeutungs¬ vollen Stimmen, die darauf hinwiesen, daß es durchaus kein Verstoß gegen die Forderung des justum pretium sei, wenn gewisse Unter¬ nehmer einen erhöhten Unternehmergewinn anstrebten und auf Grund einer besonders intensiven organisatorischen, weit über die einfache wirtschaftliche Arbeit hinausgehenden Unternehmertätig¬ keit auch erzielten. Franz Keller ist in dem historischen Teile seiner oben schon genannten sozialethischen Studie zur Geschäfts¬ moral neuerdings diesen Auslassungen bedeutender Theologen und Philosophen des Mittelalters nachgegangen. Will man die Bedeutung dieser Stimmen für die hier behandelte Frage abwägen, für die Frage nach den Verumständungen, die dem kapitalistischen Geiste zum Durchbruch durch die entgegenstehende Wirtschaftsethik des Mittelalters verhalten, so darf man meines Erachtens ihre Wirkung nicht allzuhoch einschätzen. Einmal stand der gemäßigten Richtung, die sie vertraten , eine andere, der Ent¬ wicklung des kapitalistischen Geistes weniger günstige gegenüber. Und gerade diese beherrschte mehr oder weniger die öffentliche Meinung. Zweitens aber liegen die den kapitalistischen Geist in etwa verteidigenden Stimmen hinter einem ersten starken Hervor¬ treten des Kapitalismus in Italien, so daß sie nicht wohl als Ursache dafür geltend gemacht werden können, daß der kapitalistische Geist die mittelalterlichen ethischen Hemmungen durchbrechen konnte. Vielmehr müssen die von Keller verzeichneten Auslassungen als Rechtfertigungen des schon geschehenen Durchbruches angesehen werden. Es war nicht anders; bei der ersten Ausbreitung des kapitalistischen Geistes und des Kapitalismus mußten schon die genannten vitalen Finanzinteressen der Kirche und des Staates helfend eintreten, sonst wäre die anfänglich schwache Pflanze in den Hemmungen, die sich aus der mittelalterlichen Wirtschafts¬ ethik ergaben, erstickt. Wenn Karl V. in dem vorhin erwähnten Toledaner Mandat vom 13. Mai 1525 die Monopolbildungen im Erzhandel durch einen Hinweis auf das allgemeine Wohl zu rechtfertigen ver¬ suchte, wenn das auch, wie wir sahen und noch sehen werden, 90 Zweites Buch: Kirche , Staat und Frühkapitalismus. von anderer Seite ebenfalls geschah, so war das nichts Neues. Schon die großen Moralisten des Mittelalters hatten in mancher Beziehung die Wege hierfür gebahnt. Duns Scotus(um 1300 lehrend) und nach ihm Bernhardin von Siena, der große soziale Prediger des beginnenden 15. Jahrhunderts , hatte alle geschäftlichen Unter¬ nehmen verurteilt, wenn sie dem Gemeinwesen schadeten 1. Ab¬ gesehen davon, daß die „wucherischen“ Geschäfte an sich schlecht seien, wurden sie von dem zuletzt genannten Theologen besonders auch deshalb zurückgewiesen, „weil sie unsozial wirken und die Blüte des Vaterlandes untergraben“ 2Bernhardin ( v. Siena, Opera II, S. 750 ff.). Von hier bis zur Umkehrung, daß alle dem allgemeinen Wohl dienenden Geschäfte sittlich erlaubt seien, war kein weiter Schritt und tatsächlich kam es z. B. vor, daß die mittel¬ alterliche Zinsdoktrin bei Anleihen der öffentlichen Gewalten, der Fürsten und Städte, eine Ausnahme von ihrem Verdammungs¬ urteile machte, indem sie erklärte, diese Anleihen dienten dem öffentlichen Wohle 3. Und ebenso wurde auch in der Frage der Monopole von den mittelalterlichen Moraltheologen empfunden. Es genüge hier auf Ludovicus Molina (1535—1600) hinzuweisen, der die Anschauungen des Mittelalters über die Frage, wann Mono¬ pole erlaubt seien und wann nicht, im 2. Bande (de contractibus) seines Hauptwerkes (De iure et iustitia) zusammenfaßt. Da heißt es nach einer Definition der fraglichen' Erscheinungen4: Monopole sind in der Regel unrecht. Nur dann sind sie es nicht, wenn sie durch den Staat selbst aus Erfordernis des all¬ gemeinen Wohles auf gerichtet werden 5. Wenn nämlich ein Staat gewisse Waren nicht hat und auch niemand da ist, der sie (wegen allzu großer Gefahr oder Arbeit) ohne Monopolprivileg importieren will, dann ist es erlaubt, ein Monopolprivilegzu ge¬ währen und mit der nötigen Vorsicht einem oder mehreren Kapita¬ le e 11 e r a . a . O., S. 34. 2 Keller a . a . O., S. 34. 3 Ehrenberg, Zeitalter der Fugger . I, S. 33. 4 „Monopolium est quasi unius duntaxat in provincia aliqua, civitate , aut oppido , venditio ." 5 „Dixi monopolia regulariter esse iniqua et reipublicae iniuriosa; quoniam aliquando iniqua non sunt , quando videlicet pub¬ lica auctoritate fiunt , bono communi id efflagi¬ tant e." Zweites Buch : Kirche , Staat und Frühkapitalismus . 91 listen zu übertragen 1. Molina führt als Beispiel neben dein Drucker¬ privileg das indische Gewürzhandelsmonopol des Königs von Portugal an 3und beruft sich dabei auf die Autorität eines anderen berühmten spanischen Moraltheologen Johannes de Medina3gest ( . 1546). 1 „Si enim respublica mercibus aliquibus indigeat , nec sit qui illas asportare velit (quia forte cum labore maximo, et periculo amit¬ tendi ea negotiatio fieret) nisi privilegium concederetur , quod nullus alius eas merces asportare aut vendere posset , sane tunc licitum erit concedere eiusmodi privilegium , taxato moderato pretio , attentis circumstantiis omnibus concurrentibus , ne, qui eo privilegio usuri sunt , plus iusto rempublicam grauent . Si autem desit qui officium aliquod reipublicae expediens suscipere velit , ut vendendi merces aliquas , aut asportandi peregrinos, poterit tunc concedi alicui, vel aliquibus , ut ipsi soli tale munus excerceant , constituto eis moderato pretio , si timor sit ne excedant pretium iustum .“ 2 Quia Lusitaniae rex suis expensis navigationem in Indiam aperuit , locaque illarum regionum subegit , statuere in suum potuit commodum (quod in reipublicae etiam commodum redundat , dum id occasio est ut , unde eam defendat , habeat , neque novis tributis illam gravet ) ut nullus praeter ipsum aut praeter eos quibus certo pretio eam negotiationem concesserit, asportare ex India certas merces possit, aut illas ex Lusitania in Indiam deferre, aut inibi eas vendere .“ 3 Johannes de Medina, De restitutione et contractibus in titulum poenitentiae quaestio 30 § penult . addit damnandum non esse si rex, in publica aliqua necessitate constitutus pro certa aliqua pecuniae summa concedat aliquibus mercatoribus , vel opificibus ut ipsi soli vendere possint aliquid in suo regno, constituto eis moderato pretio, ne rempublicam plus iusto gravent . Regem quippe tunc a peccato excusat publica necessitas , in qua est, et mercatores privilegium iusta illa de causa a rege ipsis concessum. Sicut enim rex exigere a subditis poterat ut contribuerent ad publicae illi necessitati sub¬ veniendum , ita subiicere illos potest illi gravamini ; modo tamen mode¬ ratum sit et cum minori ipsorum molestia ac detrimento . Quo loco obserua , hac de re commodiorem aliam tradi non posse regulam, quam tunc licere eiusmodi privilegia concedere, quando id postulat recta ratio ac commune bonum , attentis atque expensis circumstantiis omnibus tam ex parte subditorum quam ex parte regis atque illorum quibus conceduntur : simulque attento , ut subveniendo communi bono, quoad fieri possit , non magis graventur , quaedam reipublicae partes , quam aliae, quando ad omnes aequaliter subvenire illi spectat. Quando autem irrationabiliter atque in subditorum praeiudicium , con¬ cederentur eiusmodi privilegia et peccaret lethaliter rex aut respublica, quae illa concederet et simul qui ea impetrarent aut illis uterentur, tenerenturque homines ad restitutionem subditis damnorum omnium, quae contra ipsorum voluntatem inde ipsis sequerentur .“ 92 Zweites Buch : Kirche , Staut und Frühkapitalisraus. Johannes de Medina weist besonders auch auf die Not hin, die publica necessitas, die den Herrscher rechtfertige, der den Kauf¬ leuten gegen bestimmte Darlehenssummen Monopole bewillige. Verschaffe sich der König oder die anderen Vertreter des Staats¬ wesens nicht durch Gewährung von Monopolprivilegien Gelder, so müsse er aus den Untertanen auf andere, vielleicht drückendere Weise die Mittel zur Inganghaltung der Staatsmaschine heraus¬ pressen. Deutlich ist auf die Necessitas als Rechtfertigungsgrund der Monopole auch in dem Werke De republica libri sex et viginti 1 des Petrus Gregorius Tholzanus hingewiesen, wenn es heißt : „Potest nocessitas reipublicae, hoc genus conquirendarum pecuniarum inter negotiationem excusare et aliquando principem laudabilem reddere, qui ita parcit tributis et oppressionibus vectigalium, ut eosdem tanquam filios propriis laboribus relevat. Sic refert Rhodericus Dubraius 2( lib . 19 constitutionum Bohemiae), Rodolphum, Bohemiae regem, Alberti Caesaris filium negotiationem aut in argenti fodinis Cuthuensibus exercuisse: vinum, sal, hordeum, triticum ad panes et cerevisiam coquendam ex Austria, ex horreis suis convehendo, pretiaque rebus promercalibus ex arbitrio suo constituendo atque in eo genere mercaturae monopolium sibi vindicando. Et se hac necessitate compulsum huc se descendisse, dixisse: ut hoc modo summam aerarii inopiam sublevaret et debita per utrumque Wenceslaum regem contracta, dissolveret. Atque ideo duo millia aureorum illis ipsis montibus hebdomatim aeri alieno dissolvendo tribuisse.“ Selbstverständlich sprachen sich dann die Staatsrechtslehrer des 17. und des 18. Jahrhunderts noch unbedingter für das Recht des Staates, Monopole zu erlauben, aus. 1 2 Bde. Pontimusani 1596. 1. Bd. S. 97 f. 2 Über ihn sagt Jöchcr (Gelehrtenlexikon ) : „Rodericus de Dubrawa, ein böhmischer Rechtsgelehrter, in welchem Saeculo aber ist unbekannt, hat iura et constitutiones regni bohemici geschrieben, die noch nicht gedruckt sind.“ Drittes Buch. Monopole , Kartelle und Aktiengesell¬ schaften im Mittelalter und zu Beginn der Neuzeit. V 1 95 Erstes Kapitel. Deutsche Aktiengesellschaften vornehmlich des 16. Jahrhunderts. Erster Abschnitt. Die führende Form der Unternehmung im frühkapitalistischen deutschen Handel. Wenn man die führenden Großunternehmungen, die größten Firmen des süddeutschen Frühkapitalismus im 16. Jahrhundert und des deutschen Hochkapitalismus um die Wende des 19. Jahr¬ hunderts miteinander vergleicht, so ist namentlich der folgende Unterschied deutlich erkennbar. Im 19. Jahrhundert liegt die Führung im Wirtschaftsleben durchaus bei den „unpersönlichen“ Aktiengesellschaftenx. Die Großbanken, die größten Schiffahrts¬ gesellschaften, die bedeutendsten Industrie- und Handelsunter¬ nehmungen usw., die meisten haben die Form der Aktiengesell¬ schaft 2. Im 16. Jahrhundert dagegen sind aus Familienwirtschaften hervorgegangene offene Handelsgesellschaften die Führer im großen Handels-, Industrie- und im Finanzgeschäft. In Augsburg, dem Zentrum des deutschen Frühkapitalismus des 16. Jahrhunderts , sind die Fugger, die Welser, die Herwart, die Neidhart, die Manlich, die Baumgartner und viele andere 1 So konnte R . Lief mann in seinem Buche : Beteiligungs¬ und Finanzierungsgesellschaften (Eine Studie über den modernen Kapitalismus und das Eff.ektenwesen. Jena 1909, S. 11 ff. 2. Aufl ., 1913) die Periode des Hochkapitalismus im 19. Jahrhundert , nach der Art der neu hinzugekonmienen Kapitalsform , als „ Effektenkapilalismus " dem Frühkapitalismus gegenüberstellen. 2 Vgl . auch Kurt Wiedenfeld, Das Persönliche im modernen Unternehmertum . Schmollers Jahrbuch 34 (1910), S. 223 ff. 96 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. Firmen solche Familiengesellschaften1. Besonders gut läßt sich quellenmäßig die Angliederung der Verwandtschaft und Schwäger¬ schaft an eine vom „ Stammvater“ gegründete Firma bei dem Geschäft der Familie Bimmel erkennen, das schon von den Söhnen des Gründers zu einer Finanzmacht erhoben, dann von den an¬ geheirateten Familien Haug, Link, Langenauer usw. fortgesetzt wurde und zu europäischem Ruf gelangte 2. In ähnlicher Weise kann ein genealogisch interessierter Historiker die bedeutendsten Augsburger Steuerzahler leicht um ein paar große Familienfirmen gruppieren. Dieselbe Erscheinung der Führerschaft von Familiengesell¬ schaften im Wirtschaftsleben gewahren wir in Nürnberg, in Ulm und in den übrigen Metropolen des süddeutschen Frühkapitalismus des 15. und 16. Jahrhunderts 3. Besonders erwähnt möge wenigstens noch die große Ravensburger Gesellschaft werden, der Zahl der Mit¬ glieder nach wohl die größte Familiengesellschaft jener Zeit eines machtvoll sich entwickelnden süddeutschen Frühkapitalismus 4.. Die Tatsache des Uberragens der aus der Familiengemeinschaft hervorgegangenen offenen Handelsgesellschaft im süddeutschen Früh¬ kapitalismus des 15. und 16. Jahrhunderts bildet einen bemerkens¬ werten Gegensatz zum Wirtschaftsgebiet der hansischen Seestädte 5. 1 Vgl. R. Ehrenberg, Das Zeitalter der Fugger. I. Bd. 2. Kapitel ; J. Strieder, Zur Genesis des modernen Kapitalismus. III . Abschnitt. 2 Strieder a. a. O., S. 146 ff., 151 ; Ehrenberg a. a. O. I. Bd., S, 227 ff. und J. Hartung, Aus dem Geheimbuche eines deutschen Handelshauses, a. a. O., S. 36 ff. 3 Vgl . unter anderem A. Schulte, Geschichte des mittelalter¬ lichen Handels und Verkehrs. Kapitel 53 ff. 4 Außer Schulte a. a. O. besonders W. H e y d , Die große Ravensburger Gesellschaft. Stuttgart 1890. Für die nächste Zeit sind interessante Veröffentlichungen über die genannte Gesellschaft zu erwarten. Schulte wird ihre jüngst im Kloster Salem auf¬ gefundenen Handelspapiere aus dem endenden 15. und beginnenden 16. Jahrhundert in mehreren Bänden herausgeben. Cfr. A. Schul¬ tes Artikel „Ein wichtiger Fund zur Handelsgeschichte“ in Zeit¬ schrift für die Geschichte des Oberrheins. Neue Folge. Bd. 27 (1912), S. 33 ff. 5 Dieser Gegensatz ist auch angedeutet bei Hugo Rachel, Die Handelsverfassung der norddeutschen Städte im 15. bis 18. Jahr¬ hundert. Sehmollers Jahrbuch 34. Bd. (1910), S. 1032/33. Erstes Kapitel : Deutsche Aktiengesellschaften vornehmlich des 16. Jahrh . 97 Herrscht in Süddeutschland auf die geschilderte Weise die Kon¬ zentration des Kapitals, so in den hansischen Seestädten dessen Zersplitterung. Eine Zersplitterung in der Art, daß der hansische Kaufmann sein Kapital in einzelnen, voneinander unabhängigen Unternehmungen auf dem Wege der Vergesellschaftung unter¬ bringt 1. Der hansische Kaufmann trieb Geschäfte, der süddeutsche Kaufmann hatte — in der Regel wenigstens — ein Geschäft, das er Zeit seines Lebens allein oder mit seinen Kompagnons inne¬ behielt und ausbaute. Damit soll nun nicht gesagt sein, daß der oder die Inhaber solcher süddeutscher Firmen nicht auch manchmal Teile ihrer Kapitalien zu besonderen, zu gelegentlichen Gesell¬ schaften mit anderen Firmen zusammenlegten. Das kam häufig vor und im Verlaufe des 16. Jahrhunderts , wie wir sehen werden, immer mehr. Aber die Hauptsache blieb ihnen doch ihr „eigenes“ Geschäft. Dieses zu führen und zu vergrößern, war ihr Beruf und ihr Stolz. Auf solche Weise bildeten sich in Süddeutschland besonders seit dem 15. und 16. Jahrhundert viele ganz große Familienfirmen heraus. Geschäfte, in denen Brüder und Vettern, Oheime und Neffen, Schwäger und andere Anverwandte das kleinere Unter¬ nehmen der ersten Generation zusammenhielten und zu dauernden, großen, sich gleich oder doch ähnlich bleibenden, jedenfalls organisch aus dem älteren Betriebe herauswachsenden Handelsoperationen weiterführten 2. 1 Vgl. die Aufsätze F. Keutgens über hansische Handels¬ gesellschaften vornehmlich des 14. Jahrhunderts in der Vierteljahrs¬ schrift für Sozial- und Wirtschaftsgeschichte. 4. Bd. (1906) S. 278 ff., 461 ff. und 567 ff., besonders S. 502 ff. Seit dem 14. Jahrhundert spätestens wurden die hansischen' Gesellschaften zumeist nicht mehr auf ein „individualisiertes Unternehmen“, sondern auf bestimmte Zeitdauer (oder auch auf unbestimmte) abgeschlossen. 2 Daß man oft Faktoren, Handlungsdiener, auch wenn sie nicht zur Verwandtschaft gehörten, zur Beteiligung zuließ, geschah, um die Betreffenden zu Eifer und treuer Pflichterfüllung für die Firma an¬ zuregen. Über das Institut der „Fürlegung“, das die Möglichkeit schuf, auch unbemittelte Faktoren zu Gewinn und Verlust zu be¬ teiligen, siehe meinen Aufsatz „Die sogenannte Fürlegung, eine In¬ stitution des deutschen Gesellschaftsrechts im Zeitalter des Früh¬ kapitalismus“ in Vierteljahrsschrift für Sozial- und Wirtschafts¬ geschichte X (1912), S. 521 ff. Strieder , Studien z. Gesch kapitalist . Orgamsationeformen . 7 98 Drittes Buch: Monopole , Kartelle und Aktiengesellschatten im Mittelalter. Ganz anders, im allgemeinen, im Handels- und Wirtschafts¬ betrieb der hansischen Seestädte. Wohl ist die früher vielfach verbreitete Vorstellung falsch, die Annahme, als ob der hansische Handel im wesentlichen von kapitalarmen Händlern getragen worden sei, denen Rentner Teile ihres Vermögens in vielfacher Zer¬ splitterung zu Handelszwecken auf die eine oder die andere Art vergesellschafteten. Davon kann nicht die Rede sein. Nein, auch die Eigentümer des Kapitals, mit dem der hansische Handel ge¬ trieben wurde, waren in der Regel Kaufleute. Aber — und hierin liegt der grundlegende Unterschied zu Süddeutschland — der Unternehmungen, in die sich solch ein hansischer Kaufmann einließ, waren viele und verschiedenartige. Bald schloß er sich mit diesem, bald mit jenem einheimischen oder fremden Kaufmann zusammen. Mögen die Gesellschaften auch zumeist in einem be¬ grenzten Bekanntenkreise eingegangen und oft erneuert worden sein, so daß sie häufig sehr lange dauerten ; die Tatsache besteht, daß die Kapitalien der einzelnen reichen hansischen Kaufleute in verschiedenen Unternehmungen investiert, zersplittert wurden. So wissen wir aus Eintragungen in das Lübecker Niederstadtbuch l, daß der Kaufmann Hermann Mornewech von 1323—1335, also im Verlaufe von nur 13 Jahren , sich zu 18 verschiedenen Malen mit Berufsgenossen zu geschäftlichen Unternehmungen verband, 18mal Gesellschaftsverträge abschloß und erneuerte. Höchstwahrschein¬ lich aber hat Hermann Mornewech in der genannten Zeit noch viel mehr als 18 Gesellschaftsverträge abgeschlossen. Es steht nach neueren Forschungen fest, daß durchaus nicht alle Gesellschafts¬ verträge der Kaufleute in das Lübecker Niederstadtbuch eingetragen werden mußten. Wir haben es in den Eintragungen nicht, wie man leicht glauben könnte, mit einer Art Handelsregister mit Eintrags¬ pflicht zu tun . Die Veröffentlichung des Wittenborgischen Hand¬ lungsbuches2aus der Mitte des 14. Jahrhunderts beweist das voll¬ kommen deutlich. Der Kaufmann Wittenborg „hat sich nämlich 1 C , W . Pauli, Lübeckische Zustände im Mittelalter . 3 Bde. Lübeck (resp . Leipzig ) 1847— 1878 . I . Bd . S. 140 . Uber das Lübecker Niederstadtbuch vgl . auch Paul Rehme, Die Lübecker Handels¬ gesellschaften in der ersten Hälfte des 14. Jahrhunderts . Zeitschrift für das gesamte Handelsrecht 42 (1894 ). 2 Das Handlungsbuch von Hermann und Johann Wittenborg, herausgegeben von Carl Mollwo . Leipzig 1901. Erstes Kapitel: Deutsche Aktiengesellschaften vornehmlich des 16. Jahrh. 99 der Rubrik .societates1im Stadtbuch nur ein einziges Mal bedient, als er einmal einen Gesellschaftsvertragmit einem Manne abschloß, mit dem er sonst nicht in Verbindung stand 1. Dagegen liât er seinen regelmäßigen Ge¬ sellschaftern stets nur in sein privates, jetzt veröffentlichtes Geschäftsbuch eingetragen“. So wird man die vorhin durchgeführte Unterscheidung der süddeutschen führenden Handelsgesellschaften des 15. und 16. Jahr¬ hunderts und der hansischen gelten lassen müssen. Die Gegenüber¬ stellung in Gesellschaften, die aus familienrechtlichen Gemeinschaften hervorgehend, das gesamte verfügbare Kapital einer Reihe von ver¬ wandten und verschwägerten Kaufleuten zusammenfassen und andererseits (auf hansischer Seite) in solche, die „von vornherein auf freier Vereinbarung beruhend“ 2, nur einen Bruchteil der Handelskapitalien der Vertragsschließenden enthalten. Selbstverständlich hat es in Süddeutschland auch Handels¬ gesellschaften mit dem Charakter von letzterer Art gegeben. In Menge sogar. Und umgekehrt fehlen im Norden die großen Familienfirmen nicht ganz. So wissen wir von dem bedeutenden Geschäft, das die vier Söhne(Michael, Simon, Stephan und Hans jr.) des reichen Stettiner Kaufmanns Hans Loitz nach dem Tode ihres Vaters (1539) gemeinsam weiterführten 3. Die Firma trieb Flandel im ganzen Umkreise des hansischen Gebietes von Narwa im Osten4 die zahlreichen Verträge mit 1 Keutgen a . a . O., S. 473/4. 2 Max Weber, Zur Geschichte der Handelsgesellschaften im Mittelalter nach südeuropäischen Quellen . Stuttgart 1889 . Ab¬ schnitt II . Die seehandelsrechtlichen Sozietäten , S. 15 ff . Zitiert bei Keutgen a . a . O. 3 Vgl. Hering, Die Loytzen , in Baltische Studien , heraus¬ gegeben von der Gesellschaft für pommersche Geschichte und Alter¬ tumskunde . 11. Jahrgang . (1845 ), S. 80 ff Einiges über den Geschäftsverkehr des sächsischen Kaufmanns und Kammermeisters Hans Harrer (unter Kurfürst August ) mit den Loitz aus Danzig und Stettin cfr . : Neues Archiv für sächsische Ge¬ schichte Bd . 15 (1894 ), S. 95. 4 Vor dem Reichskammergericht spielte seit 1566 ein Prozeß unes Lübeckers (v . Dicke oder vom Dyke ) contra Hans , Bernhard und Stephan Loitz in Lübeck und Stettin . „ Arrestanlage auf zwei Schiffe zu Lübeck , welche Kläger angeblich durch seinen Diener in Dänemark mit Salz beladen und nach der Narva segeln ließ, ivo aber dieser Diener die Schiffe an die Verklagten (Loitz ) verkaufte .“ 7* * 100 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. bis nach England 1im Westen und bis tief landeinwärts die Oder und Weichsel hinauf. Hans Loitz leitete vom Stettiner Stammsitz aus die Firma. Zwei der Brüder, Michael und Simon, führten die Geschäfte zu Danzig, Stephan zu Lüneburg. Faktoreien besaß die Gesellschaft ferner in Leipzig, Frankfurt, Breslau, Prag usw. Außer dem Warengeschäft trieben die Loitz auch sehr bald das große Anleihegeschäft mit den großen Herren des Nordens und Ostens. Besonders König Sigmund August von Polen scheint ihnen stark verpflichtet gewesen zu sein. Die Loitz mußten zum Zwecke dieser fürstlichen Geldgeschäfte bedeutende Depositen aufnehmen. Der Ruf der Firma sorgte dafür, daß nicht nur pommersche (besonders aus dem Landadel), sondern Kapital¬ besitzer aus dem ganzen deutschen Norden und Nordosten gern Depositengläubiger der Firma waren. Die Gesellschaft galt viele Jahr¬ zehnte hindurch für absolut mündelsicher, so daß selbst milde Stiftungen und öffentliche Institute ihr Geld in bedeutender Höhe anvertrauten . Um so schlimmer gestaltete sich 1572 der Bankrott der Firma, der nicht nur die Familie Loitz, sondern auch eine ganze Anzahl ihrer Depositengläubigerin ganz Norddeutschland ins Ver¬ derben riß. * * * Die süddeutschen Familiengesellschaften sind nun häufig nicht mit ihrem eigenen Kapital ausgekommen, um der Ausweitung ihrer Die Akten dieses Prozesses sind 1851 an das Staatsarchiv in Lübeck abgegeben worden, wo sie sich jetzt noch befinden. 1 Zu ersehen aus folgenden Urkunden des Königlichen HauptStaatsarchives zu Dresden Loc. 7250, Bleyhandel anno 1543—46. Bl. 5, 8, 87 f. Herzog Philipp von Pommern an Herzog Moritz von Sachsen. Wolgast 26. Dezember 1543. Seine Untertanen Michael, Simon, Stephan und Hans Loitz (zu Alt-Stettin und Danzig wohnhaft) hätten ihm erklärt, daß sie geneigt seien, in die sächsischen Bergwerke und Hütten die Zufuhr (und den Verlag) des Bleis zu besorgen. Sie würden das Blei aus England beziehen (das besser sei als das Goslarer), wohin sie viel handelten. Er (Herzog Philipp) sei den Loitzen sehr zu Dank verpflichtet und habe sie stets als treue Geschäftsleute kennen gelernt. Tatsächlich brachten die Loitz Mitte 1546 2000 Zentner englisches Blei über Hamburg in die Joachims¬ taler Bergwerke. Weimar, Staatsarchiv Reg. T. Fol. 501, S. 11, 3 (pag. 56). Erstes Kapitel: Deutsche Aktiengesellschaften vornehmlich des 16. Jahrh. 101 Handels- und Gewerbsgeschäfte, besonders aber, um den erhöhten Ansprüchen des fürstlichen Geldbedarfes zu genügen. Immer mehr verbreitete sich in .Süddeutschland um die Wende des 15. Jahr¬ hunderts die schon ältere Sitte, in die Handelsgesellschaftenfest verzinsliche Einlagen aufzunehmen1stillliegendes („ Geld“ in der Terminologie des 16. Jahrhunderts genannt). Auch Firmen, die miteinander in Kontokorrent standen oder die das Kontokorrent¬ geschäft trieben, ließen sich als Sicherheit festverzinsliche Einlagen machen. Zu allermeist war ein Satz von 5 % als Entgelt für De¬ positen üblich, in besonderen Fällen kamen aber auch noch höhere Verzinsungen vor 2. Besonders dann etwa, wenn es galt, Familien¬ mitglieder, die nicht mehr selbst in der Firma mitarbeiteten, dafür zu entschädigen, daß man sie nicht mehr am vollen Gewinn repartieren ließ 3, oder auch dann, wenn man sich eine hohe Person besonders verpflichten wollte 4. Die Einlagen wurden durchaus als Schuldtitel der betreffenden Firma betrachtet . Auch bezüglich der Haftung den Gesellschaftsgläubigern gegenüber, wie wir gleich sehen werden. Sie wurden in das sogenannte Wechselbuch der Firma eingetragen, weil dafür von seiten der Firma ein Solowechsel ausgestellt wurde. Als sich zu Anfang des 16. Jahrhunderts - die antikapitalistische Tendenz der Zeit gegen die übermächtigen Kapitalkonzentrationen richtete, wie sie in den großen Handels¬ gesellschaften vor sich gingen, da wandte man sich auch gegen die Aufnahme von festverzinslichen Einlagen, von Zinsgeldern. In einem an den deutschen Reichstag gerichteten Gutachten 5von 1522 wird vorgeschlagen, es solle verboten werden, daß hinfüro die 1 Strieder , Inventur der Firma Fugger aus dem Jahre 1527, S. 8; Ehrenberg, Zeitalter der Fugger. I. Bd., S. 124, 391; W. Möllenberg, Die Eroberung des Weltmarktes durch das Mansfelder Kupfer, S. 28. 2 Cfr . z. B. M. Jansen, Jakob Fugger der Reiche, S. 149. Die Verzinsung von 10 und 12 %, die die Loitzen Vornahmen, war ungewöhnlich und wohl schon ein Zeichen des nahenden Falliments der Firma. Hering a. a. O., S. 90. 3 Beispiel: Eh r en b erg a. a. O., I. Bd., S. 232. 4 Beispiel : M ö 11e n b e r g a. a. O., S. 28, 107. 6 Abgedruckt in den deutschen Reichstagsakten jüngere Reihe, Bd. III, S. 556 Anm. 3. Vgl. auch das unten im Abschnitt,,Ent¬ stehung der Aktiengesellschaft“ über diesen Punkt Gesagte. 2 102 Drittes Buch: Monopole, Kartelle und Aktiengesellschaften im Mittelalter. Handelsgesellschaften „frembd gelt, gestalt ains -Wechsels und da man gelt von gelt gibt, in irer geselschaft nemen und anlegen oder damit handeln, sondern allein mit i r e m zugelegten gelt hantieren solten.“ Es scheint, als ob auch zu Anfang des 16. Jahrhunderts noch der größte Teil der festverzinslichenEinlagen von den Verwandten und Freunden der Gesellschafter gestellt wurde, kleinere Depositen rührten — aus Gefälligkeit angenommen — von Bediensteten der Gesellschafter her. Im allgemeinen muß es in den ersten Jahr¬ zehnten des 16. Jahrhunderts auch in Augsburg noch nicht üblich gewesen sein, von jedermann Depositen, selbst auch in kleinen Quanten anzunehmen. Wenigstens regt sich der gut unterrichtete Clemens Sender weidlich darüber auf, daß Ambrosi Höchstetter Depositengelder nahm, wo er sie bekam. Der gleichzeitige Augs¬ burger Chronist schreibt: „ Zu Ambrosius Höchstetter haben fürsten, graffen, edel, burger, bauren, dienstknecht und dienstmägt ir gelt, was sie haben gehapt, gelegt und von dem 100 genomen5 fl. Mengen baurknecht und die nit me haben gehapt dann 10 fl., die haben es im in sein geselschaft geben; haben gemeint, es sei inen gantz wol behalten und haben darzu ain järliche nutzung. Diser Hoech¬ stetter hat ain zeit lang in seiner geselschafft zechenhundert tausent fl. verzinst (die gemein sag ist gewesen, er lieg geren) kain mensch hat gewist, daß er sovil geltz verzinst hat “ 1. Es wird sich fragen, ob die Wechsel, die die Depositengläubiger der großen Gesellschaften erhielten, jederzeit an Dritte weitergegeben werden konnten, ob sie also die Eigenschaft von Obligationen, wie sie unsere heutigen großen Aktiengesellschaften auszugeben pflegen, schon angenommen hatten . Das wäre ein bedeutsamer Schritt auf die „Kommerzialisierung“ des Wirtschaftslebens gewesen, auf eine Entpersönlichung, eine Versachlichung von Wertpapieren schon im 16. Jahrhundert hin a. Die Frage kann hier nur angeschnitten werden. Jedenfalls sind Fuggersche Teilschuldverschreibungen schon um 1540 „ ein kuranter Handelsartikel“ an der Antwerpener Börse gewesen. „Die Fugger hatten , um ihre großen Antwerpener Geschäfte ausführen zu können und sich relativ billig Geld zu 1 Die Chroniken der deutschen Städte. 23. Bd. Leipzig 1894, S. 219. 2 Vgl . Sombart, Die Juden und das Wirtschaftsleben. Kap. VI besonders S. 74 ff. Erstes Kapitel: Deutsche Aktiengesellschaften vornehmlich des 16. Jahrh. 103 verschaffen , angefangen , von Messe zu Messe oder auch auf zwei Messen bei den Kaufleuten (besonders den süddeutschen ) in Ant¬ werpen Anleihen von jener Art aufzunehmen , welche man euphe¬ mistisch „Depositen “ nannte , obwohl es wirkliche Darlehen waren. Die Fugger zahlten dafür jährlich ca. 9 % , während sie ihrerseits mit dem Gelde in Antwerpen ca. 12—13 % erzielten “ 1. Die Obli¬ gationen wurden kurz „ Fuggerbriefe “ genannt und erfreuten sich großer Beliebtheit . In ihnen jedenfalls , vielleicht aber auch in den Zinsgeldern anderer Firmen war die moderne Obligation vorbereitet. Übrigens hatten sich im Verlaufe des 16. Jahrhunderts z. B. in Danzig schon die Schuldverschreibungen der Stadt zu richtigen Inhaberpapieren weiterentwickelt . Bürgermeister und Ratmannen als Verweser des gemeinen Gutes „sampt den anderen verordneten Ordnungen der kgl. stadt Dantzigk “ versprachen regelmäßig dem Gläubiger und dessen Erben , „oder wer diesen brief mit ihrem guten willen innehat" den Hauptstuhl , d. h. das Kapital jährlich an bestimmter Stelle zu bestimmter Zeit mit einem bestimmten Satz zu verzinsen , mit dem Zusatz „wollen auch [die Genannten ] in allen zufelligen nöten aller steur , Schatzungen , dienst, auch alle andere gefahr , nachteil und schaden , von welcher herschaft oder burgermeistern und rat sampt den andern verordenten Ord¬ nungen der kgl. stadt Dantzigk auf solche geld geleget werden mochte oder wie daz geschehen kan oder mag , in allem ohne alle entgeltnusse schadlos und frei halten “ . Eine bestimmte Kündigungs¬ frist wurde ausbedungen , zuweilen auch die Kündigung erst nach Ablauf einer gewissen Frist für zulässig erklärt und die Währung festgelegt , in welcher der Hauptstuhl abzutragen war 2. Die kommanditistische Beteiligung spielt in der süddeutschen Handelswelt des 15. und 16. Jahrhunderts eine hervorragende Rolle nicht . Doch ist sie nicht gerade selten gewesen 3, wie schon aus dem Privileg hervorgeht , das Friedrich III . im Jahre 1464 der Stadt 1 Ehrenberg, Zeitalter der Fugger . I. Bd . S. 148. 2 Zum Jahre 1587; alles nach Max Foltz, Geschichte des Danziger Stadthaushaltes . Danzig 1912. S. 293. 3 Vgl. auch Ott Rulands Handlungsbuch ed. K. D. Hassler, Stuttgart 1843, S. 15 1. ..Item daz ich Ott Ruland enpfangen hab von dem Walthasar Ramsteiner zu Nürnberg 200 reynisch gülden, die soi ich im anlegcn zu gwin und Verlust auf sein wagnus . . 104 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. Nürnberg verlieh 1. Darin heißt es : „Mehr so ordnen wir von des handeis und kaufmannschaft wegen : Welche person , burger oder burgerin der vorgemelten . stadt Nürnberg ein nehmlich summa gelds mit geding in ein gesellschaft legen, daß sie solch geding halten und dem nach kommen sollen. Welch obgemelt person aber ihr gut und gelt in gesellschaft thun und legen ohn geding, sondern zu gewinn und Verlust und doch für sich selbst die handtierung der gesellschaft nit pflegen zu handeln , ob und wann dieselbe gesellschaften durch ungefall oder sonsten Verlust leiden und in schulden fallen würden und dieselbe schulde von dem hauptgut, das sie alle in der gesellschaft hetten , nicht mochte bezahlet werden, so sollen dieselben personen , die, als vorstehet , ihr gut und gelt unverdingt in gesellschaft hetten , nicht mehr zu bezahlen pflichtig und schuldig sein, denn allein so viel als sich nach anzahl ihres zugelegten hauptguethes gebieren und damit der übrigen schulden ganz entledigt und auch alle andere ihr liaab und guth , wo sie die hetten , deshalb von allermänniglichen , unangelangt unaufgehalten und unbekümmert sein und bleiben . . . .“ Keutgen 2 hat die in dem ersten Satze berücksichtigten Personen als stille Gesellschafter bezeichnen wollen. Davon kann nicht die Rede sein. Die Bürger oder Bürgerinnen von Nürnberg, die „ ein nehmlich summa gelds mit geding in ein gesellschaft legen “, sind Depositengläubiger der betreffenden Firma . Geding ist hier die Verabredung eines festen Zinses auf das hergeliehene Kapital 3. Die Depositengläubiger sind im zweiten Satze denen gegenübergestellt , die „ ohne geding , sondern zu gewinn und vertust “ Geld inseriert haben und doch nicht in der Gesellschaft mitarbeiten . Bei der Haftungsfrage , die den Gegenstand der Ürkunde bildet , scheiden dann auch die im ersten Satze genannten Depositengläubiger völlig aus . Mit Recht , denn sie sind ja nicht Mitgesellschafter , sondern Gläubiger der Gesellschaft. Wenn wir oben sagten , daß im süddeutschen Frühkapitalismus des 15. und 16. Jahrhunderts diejenigen Firmen die Hauptrolle 1 Abgedruckt in Woelckers Historia diplom . Norembergensis. Nürnberg 1738 , S. 682 und (ziemlich fehlerhaft ) in L ü n i g , Reichs¬ archiv 14, S. 127. 2 a . a . O., S. 606. 3 Vgl. J . und W . Grimm, Deutsches Wörterbuch , IV . I, 1. S. 2027. Erstes Kapitel: Deutsche Aktiengesellschaften vornehmlich des 16. Jahrh. 105 spielten , in denen eine Reihe von verwandten und verschwägerten Kaufleuten ihr gesamtes verfügbares Kapital zusammenlegten , so mußten wir hinzufügen , daß es auch nicht an solchen Gesellschaften fehlte , die, „von vornherein auf freier Vereinbarung beruhend “, nur einen Bruchteil der Kapitalien der Vertragschließenden um¬ faßten . Vielfach sahen sich die großen Familienfirmen aus der Entwicklung ihres Geschäftes und der Entwicklung der wirtschaft¬ lichen Verhältnisse heraus gezwungen , solche gelegentliche , be¬ sondere Gesellschaften miteinander oder mit anderen Kapitalisten einzugehen . Ich denke hierbei zunächst besonders an jene Gesell¬ schaften , die sich bildeten , weil die einzelne Firma das Risiko eines be¬ stimmten Geschäftes nicht allein tragen wollte oder konnte . Erwähnt seien etwa die Finanzgeschäfte mit Fürsten 1. Bei manchen solchen Finanzgeschäften , z. B. den „ Silberkäufen “ und den „Kupfer¬ käufen “ mit den Habsburgern als den Landesherren von Tirol, mußten auch deshalb oft mehrere große Gesellschaften zu einer Gelegenheitsgesellschaft zusammen treten , weil die Habsburger mehreren Firmen gegenüber Verpflichtungen hatten , die sie durch Gewährung von „ Silberkäufen “ auf einmal befriedigen wollten 2. Ich denke ferner an Gesellschaften von großen Firmen , die sich bildeten , um ein Unternehmen gemeinsam zu führen , das sich für die einzelne Firma zu führen nicht lohnte . So taten sich ver¬ schiedene große süddeutsche Firmen , die als Gewerken in den alpinen Bergbau eingedrungen waren , zu sogenannten Pfennwerthandelsgesellschaften zusammen , um die Bergarbeiter mit Proviant (Pfennwert ) und Arbeitsmaterial (z. B . Unschlitt zum Geleuchte und Eisen zu den Gezähen , Werkzeugen ) zu versehen 3. 1 Bei Ehrenberg , Zeitalter der Fugger, finden sich so viele solcher Vergesellschaftungen genannt, daß auf eine Aufreihung weiterer Beispiele, wie sie mir aus Literatur und gedruckten und ungedruckten Quellen (besonders den Handlungsbüchern der großen Firmen) zur Verfügung stehen, hier füglich verzichtet werden kann. 2 Über die gelegentlichen Vergesellschaftungen, die aus solchem Anlaß die Fugger mit verschiedenen anderen süddeutschen Geschäfts¬ häusern Vornahmen, vgl. jetzt am besten Jansen, Jakob Fugger der Reiche, in dem Kapitel: Jakob Fugger in Tirol, S. 79 ff. 3 Vgl . Z y c h a in Vierteljahrsschriftfür Sozial- und Wirtschafts¬ geschichte 5. Bd. (1907), S. 256 ff. ; Strieder, Fuggerinventur, S. 70 : „Unslit- und eysenhandl zu Swatz. Capital darinn fl. 1300, dartzue man auf Jorg negstkünfftig 2 jar nutzung soll zallen, die wir anslagen auf fl. 130 summa 1430.“ 106 Drittes Buch: Monopole . Kartelle und Aktiengesellschaften im Mittelalter. Eine hervorragende Bedeutung für den Bergbau erlangten die Tochtergesellschaften der großen, in der Montanindustrie arbeitenden Firmen des 16. Jahrhunderts . Diese Tochtergesellschaftenwurden gewöhnlich „ Gesellschaften des Berg- und Schmelzwerkhandels“ genannt. Wir lernten bereits früher die 1565 in Tirol gegründete Gesellschaft des „ Jenbacher Berg- und Schmelzwerkhandels" kennen, an der neben den Fuggern zunächst auch die Langenauer, die Haug und die Katzbeck erheblichen Anteil hatten 1. Schon vorher (1526) war unter Beteiligung der Fugger, des Benedikt Burkhart , des Christoph Herwart, unter Beteiligung auch von Anton und Hans Bimmel die Gesellschaft des „ Schwazer Berg-, Schmelz- und Pfennwerthandels“ gegründet worden 2. Wohl die berühmteste Tochtergesellschaft zweier großer Farniliengesellschaften des Frühkapitalismus bildet das Handels¬ unternehmen, das von 1495—1525 die Fugger in Gemeinschaft mit der ungarischen Familie Thurzo betrieben. Bekanntlich drangen die Fugger mit Hilfe dieser Verbindung in den reichen ungarischen Bergbau ein, um ihn fast ein Menschenalter zu beherrschen.' Über die Organisation der Firma Fugger-Thurzo, über ihre Umsätze und Gewinne sind wir jetzt durch Max Jansen 3 genau unterrichtet. Die beiden Familien bildeten eine Handelsgesellschaft„ des gemeinen ungarischen Handels“ (die Fugger natürlich mit einem viel größeren Kapitaleinschuß), die völlig losgelöst von der übrigen Handels¬ tätigkeit der beiden Familien basiert war. Der „ gemeine ungarische Handel“ verkaufte seine Bergwerksprodukte an die Fuggersche Handelsgesellschaft und ebenso an die Thurzo, die sie beide dann auf eigene Faust weiterverkauften. Umgekehrt verkauften die Fugger ihrer Tochterfirma, dem „ gemeinen ungarischen Handel“, Waren zum Weiterverkauf (Seiden- und Wollstoffe, Edelsteine usw.) oder zu Geschenken für die ungarischen Herren. Die Beispiele für die Durchsetzung der süddeutschen frühkapitalistischen Wirtschaft mit Handelsgesellschaften, die nicht familiengesellschaftlichen Charakter trugen, sondern von vornherein auf freier Vereinbarung nicht verwandter Wirtschaftssubjekte be¬ ruhten, sind hier aus dem Gebiete des Erzhandels bzw. Bergbaus 1 Die näheren Literaturnachweise bei Zycha a . a. O., 5. Bd. (1907), S. 281. 2 Daselbst. 3 Jakob Fugger der Reiche, S. 150 ff. 1 Erstes Kapitel : Deutsche Aktiengesellschaften vornehmlich des 16. Jahrh . 107 gewählt worden. Es geschah das deshalb, weil dort, wie wir bereits früher sahen, besonders fortgeschrilteneWirtschaftsformenherrschten und deshalb Exempel solcher gelegentlicher Verbindungen großer Firmen leicht zu finden waren. Aber man darf nicht glauben, daß sie in anderen Handelszweigen fehlten. Eine bedeutende Rolle spielten sie beispielsweise auch im Gewürzhandel des 16. Jahr¬ hunderts. Bekanntlich hatte sich die portugiesische Krone den kolonialen Gewürzhandel als Kronrecht reserviert \ Auf diese Weise erlangte der König von Portugal besonders auf dem WeltPfeffermarkt ein unbestreitbares Monopol. Wer Pfeffergroßhandel treiben wollte, mußte mit dem König von Portugal einen „Pfeffer¬ kauf“, d. h. einen Kontrakt auf Pfefferlieferung bzw. -Abnahme eingehen2. Natürlich gegen Vorauszahlung des größten Teiles des Wertes der betreffenden Lieferung. Wir kennen ja bereits vom Erzhandel her die Bedeutung der „Käufe“ als fundiertes Anleihe¬ system der öffentlichen Gewalten. Zu Gewürzabnahmekontrakten mit dem König von Portugal haben sich im Verlaufe des 16. Jahr¬ hunderts pngemein häufig süddeutsche Handelsgesellschaften zu gelegentlicher Vergesellschaftung zusammengetan. Namentlich seit die portugiesische Regierung die indischen Gewürze gleich nach Antwerpen bringen ließ und hier ihre großen Lieferungskontrakte, ihre „Käufe“ abschloß, bildete das Pfefferspekulationsgeschäfteine häufige Veranlassung von Konsortienbildungen deutscher Kauf¬ leute in der Scheldestadt. In den siebziger Jahren des 16. Jahrhunderts hat dann Konrad Rott , ein Augsburger Großkaufmann, durch die Übernahme eines Gesamtkontraktes von der portugiesischen Krone den ganzen Pfeffergroßhandel in seine Hände zu bekommen versucht. Rott verpflichtete sich im ersten Jahre seines großen Pfefferkaufs 12 000 Zentner, in den folgenden je 20 000 Zentner der kostbaren Ware zum Preise von 34 Dukaten pro Zentner abzunehmen. Dafür hatte er dem König von Portugal eine Anleihe von mehreren hundert¬ tausend Dukaten zu mäßigem Zinssatz zu gewähren. Die Anleihe sollte durch Pfefferlieferungen, allerdings erst im letzten Jahre des laufenden Kontrakts allmählich abgezahlt werden. Als besondere 1 Für das Folgende vgl. K. H ä b 1e r , Konrad Rott und die Thüringische Gesellschaft in : Neues Archiv für sächsische Geschichte 16 (1895), S. 177 ff. 2 Häbler, Konrad Rott , a. a. O., S. 180. 108 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften ira Mittelalter. Vergünstigung wurde es angesehen, daß Rott „ein Fünftel des Kaufpreises dem König in alten portugiesischen Schuldbriefen er¬ legen durfte, die zur Zeit von ihren Besitzern zu weniger als dem halben Werte zu haben waren; auch sollte er einen Teil der Zahlung in Teer, Tauwerk und anderen zum Schiffsbau nötigen Artikeln liefern, die man in Lissabon aus den Ostseeländern zu beziehen pflegte.“ Trotz dieser Erleichterungen in der Zahlungsweise war der Kontrakt K. Rotts eines der waghalsigsten Spekulationsgeschäfte des 16. Jahrhunderts . Bald zeigte sich auch die Unfähigkeit des Augsburgers, das Unternehmen allein durchzuführen. Nachdem er sich vergeblich bemüht hatte , ein deutsches Konsortium zusammen¬ zubringen, trat er im April 1576 dem Giacomo dei Bardi & Co. dre: Achtel seines Kontraktes ab. Noch mehr war Konrad Rott auf die Beteiligung fremden Kapitals bei dem noch erweiterten Gesamtpfefferkontrakte an¬ gewiesen, den er bald darnach beim Regierungsantritt König Heinrichs von Portugal übernahm 1, und mit dessen Hilfe der spekulationslüsterne Augsburger nun erst recht ein Weltmonopol für den Pfefferhandel an sich zu bringen versuchte. Rott teilte jetzt von vornherein den gesamten Geldwert, den seine Verträge repräsentierten, in 30 Anteile. Von diesen behielt er 12L, für sich. Von dem Reste trat er 10 Anteile an portugiesische, 7}<>an italienische Firmen ab. Außerdem aber ging Rott noch mit dem Kurfürsten von Sachsen die sogenannte Thüringische Pfefferhandelsgesellschaft ein, eine spezielle Vergesellschaftung, die den Zweck verfolgte, in Leipzig den Pfefferverkauf für „Deutschland, die Niederlande, Ostland und Polen“ zu konzentrieren, mit deren Hilfe sich aber zugleich der Augsburger Kaufmann Kredit und bares Geld zu ver¬ schaffen wußte. Es kann bei Häbler nachgelesen werden 2, wie die Gesellschaft des Thüringer Pfefferhandels organisiert war. Wir kommen auch an anderer Stelle darauf zurück, wie mit Hilfe eines Kartells zwischen der oben genannten internationalen Gesellschaft und der Thüringer Gesellschaft die Absatzgebiete unter hoher Konventionalstrafe abgegrenzt waren, hier lag mir nur daran, die gelegentlichen Tochtergesellschaftenzu erwähnen, die sich aus der Rottschen Muttergesellschaft abzweigten. 1 Häbler a. a. O., S. 185 f. 2 Hä b 1e r a. a. O., S. 191. 3 Erstes Kapitel: Deutsche Aktiengesellschaften vornehmlich des 16. Jahrh. 109 Neuerdings hat Möllenberg auf die Mansfeldischen Saiger¬ handelsgesellschaften als eine eigenartige Form der Kapitalvergesellschaftung des endenden 15. und des 16. Jahrhunderts hin¬ gewiesen. Also auf die Gesellschaften der Hütten Schwarza und Mansfeld (gegründet 1472 mit 6000 fl. Einlage ), der Hütte Arn¬ stadt (gegründet 1502 mit 31 500 fl. Kapital ), der Hütte Leutenberg (gegründet 1524 mit 70 000 fl. Kapital , das in der nächsten Zeit bis auf 120 710 fl. erhöht wurde ), der Hütte . Gräfenthal , der Hütte Luderstadt , der Hütte Steinach und der Hütte Eisfeld (ge¬ gründet 1546). Die Tätigkeit aller dieser Handelsgesellschaften war im wesentlichen dieselbe. Sie schossen einen Teil des in ihnen in¬ vestierten Kapitals den kleinen Unternehmern im mansfeldischen Kupferbergbau , den sogenannten Hüttenmeistern 1 vor . Diese verpflichteten sich dafür , der Gesellschaft das innerhalb einer festgesetzten Zeit produzierte Rohkupfer zu einem genau vereinbarten Preise zu liefern . Das auf diese Weise erworbene Rohkupfer 2 wurde sodann von den Saigerhandelsgesellschaften in eigenen großen Saigerhütten — naeh diesen sehr kostspieligen industriellen Anlagen 2 führten die Gesellschaften zumeist ihre Namen — gesaigert , d. h . mit Hilfe eines Zusatzes von Blei von dem darin reich enthaltenen Silber getrennt . Das Silber ging größtenteils in die Münze, das Kupfer zumeist nach Nürnberg an die größeren oder kleineren industriellen Verbraucher , besonders aber auch nach Frankfurt a. M. und nach Antwerpen 4. Hier wurde es von der 1 „Hüttenmeister “ sind nach Möilenberg kleinere Unternehmer, die Bergteile muteten , die ihnen zugemessenen Felder ausbeuteten und die gewonnenen Erze einschmolzen . Sie errichteten zu diesem Zwecke entweder eigene Schmelzöfen (Erbfeuer ), oder sie pachteten Öfen von den Grafen von Mansfeld (Herrenfeuer ), die den Hütten¬ meistern gegenüber zu Ende des 15. Jahrhunderts als Unternehmer allmählich zurücktraten. 2 Vielfach erwarben die Gesellschaften auch noch auf andere Weise Rohkupfer . Sie kauften den Regalherren , also den Grafen von Mansfeld , das Kupfer ab , das von den Gewerken an sie abgegeben werden mußte (sogenanntes Zehntkupfer ). 3 Zu einer auf etwa 7000 Zentner Jahresproduktion berechneten Saigerhütte gehörten 8 Schmelzöfen , 12 Saigeröfen , 3 Garherden, 3 Treibherden und 2 Dörröfen. 4 Unter den Antwerpener Kunden der Leutenberger Gesellschaft nennt Möilenberg neben den Fuggern u . a . „ Hans und Jorg Herbart “ . 2* 110 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. großen niederrheinischen Kupfer- und Messingindustrie in Empfang genommen, die in Aachen ihren Mittelpunkt hatte , sich aber bis Köln und Antwerpen hin erstreckte, Insgesamt wurden um 1531 auf allen „Mansfeldischen“ Saigerhütten ca. 24 000 Zentner Kupfer jährlich produziert. Davon gingen laut einer Angabe Christoff Fürers 10 000 Zentner nach Nürnberg und 14 000 Zentner nach Frankfurt a. M. und in die Niederlande. Der Osten Europas kam als Absatzgebiet für Mansfeldisches Kupfer nicht in Betracht. Hier dominierten die Fugger mit ihrem ungarischen Kupfer. Die innere Organisation der Saigerhandelsgesell¬ schaften bietet in mancher Hinsicht ein anderes Bild, als es die meisten uns bekannten Handelsgesellschaften Mittel- und Süd¬ deutschlands zu Anfang des 16. Jahrhunderts zeigen. Die starke Teilhaberschaft nicht kaufmännischer Elemente (Adliger und Ge¬ lehrter), die jährliche Generalversammlungder Teilhaber, die jähr¬ liche Bilanz und Gewinnausschüttung1, der Mangel einer be¬ stimmten Zeitdauer und der mehr öffentliche Charakter der Ge¬ sellschaften®, das und manches andere ist hier zu nennen. Es bleibt zu untersuchen, inwieweit wir es hier mit Wandlungen auf die bedeutungsvollste wirtschaftliche Organisationsform der späteren Zeit, mit Wandlungen auf die Aktiengesellschaft hin zu tun haben. Zweiter Abschnitt. Die Entstehung der Aktiengesellschaft. Wir erinnern uns nach den vorangehenden Exemplifikationen, die leider den durchgehenden Gang der Untersuchung etwas unter¬ brechen müssen, wieder an den zuerst betonten Gegensatz im WirtDie Firma hat nichts , wie Möllenberg vermutet , mit den Herbrot zu tun . Gemeint sind vielmehr die Augsburger Hans und Georg Herwart . Vgl . Strieder, Zur Genesis des modernen Kapitalis¬ mus , S. 122. 1 Generalrechnung von 1532 : Item so haben wir uns gesellschafter der saigerhutten unter Leittenberg und Aysfelt samptlich vergleicht und vcrainichet , das man soi einem jeden pro raitto austaylen und raichen auf jedes hundert aylf guide . . . 2 Dieser offenbart sich z. B . darin , daß in den Gesellschafts¬ verträgen für den Fall von Streitigkeiten unter den Kontrahenten ein öffentliches Organ , etwa der Rat von Nürnberg , als Schiedsrichter Erstes Kapitel : Deutsche Aktiengesellschaften vornehmlich des 16. Jahrh . 111 l> schaftsleben des süddeutschen Frühkapitalismus im 16. Jahrhundert und des deutschen Hochkapitalismus im 19. Jahrhundert . Hier sahen wir die Führerschaft bei der Aktiengesellschaft, dort bei großen „Familiengesellschaften“. Nun ist es über allen Zweifel erhaben, daß die Aktiengesellschaftals Massenerscheinung erst für die Wirtschaft des 19. Jahrhunderts ihre überragende Bedeutung erlangt hat , aber für den Historiker fragt es sich doch auch, wann und wo tritt die neue Form der Kapitalbeschaffung zuerst oder doch zuerst häufiger auf. Nun ist es wiederum fraglos, daß zuerst das 17. Jahrhundert in den kolonialen Handelsgesellschaften(der holländisch-ostindischen Kompagnie usw.) Aktiengesellschaften in größerer Anzahl hervorbrachte, Bildungen, von denen aus sich die Entwicklung in ununterbrochener Überlieferung bis zu den Aktien¬ gesellschaften unserer Tage verfolgen läßt, aber ist damit gesagt, daß im 16. Jahrhundert und früher nicht schon vereinzelte Aktien¬ gesellschaftsbildungen vorkamen? Im besonderen fragt es sich für die deutsche Wirtschaftsgeschichte: Hat der mächtige Aufschwung, den das süddeutsche frühkapitalistische Wirtschaftsleben im 16. Jahr¬ hundert nahm — ein Aufschwung, der nur im 19. Jahrhundert eine Parallele findet -—, nicht schon aktiengesellschaftliche Organisations¬ formen hervorgebracht, längst ehe in den Niederlanden und in England die Aktiengesellschaft eine erste Blütezeit im 17. Jahrhundert erlebte? Bis zu dem Erscheinen von Karl Lehmanns Buch: „Die ge¬ schichtliche Entwicklung des Aktienrechtes bis zum Code de com¬ merce“ (Berlin 1895) galt es als opinio communis, daß die Heimat der modernen Aktiengesellschaft Italien sei L Die St. Georgsbank in Genua und seit Goldschmidt2die genuesischen Maonen(Kolonial¬ gesellschaften) wurden bis dahin allgemein als die ältesten Kapitalussoziationsformenangesehen, die Grundprinzipien der viel später erst vollständig ausgebildeten Aktiengesellschaftenthielten. eingesetzt wird . Man beachte demgegenüber die Ängstlichkeit , mit der die alten Gesellschaften die Öffentlichkeit von sich abzuschließen sch mühten . Vgl . z. B . den Fnggerscheu Gesellschaftsvertrag von 1494, jetzt abgedruckt bei Jansen, Jakob Fugger der Reiche S. 262 ff ., besonders S. 266 . Dazu S. 32. 1 Nähere Angaben über die Begründung dieser Lehre und den geringen Widerspruch , den sie fand , bei K . L e h m a n n , a . a. O., S. 4. 2 L . Goldschmidt, Universalgeschichte des Handelsrechts, 295. ?12 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. Karl Lehmann will demgegenüber die Entstehung der modernen Aktiengesellschaft um einige Jahrhunderte später ansetzen. Für ihn sind die Kolonialgesellschaften des beginnenden 17. Jahrhunderts die ersten Aktiengesellschaften von Bedeutung für die moderne Entwicklung dieses Instituts . Also die niederländisch-ostindische Kompagnie, die englisch-ostindische Kompagnie, die niederländisch¬ westindische und so fort. Zwar leugnet Lehmann nicht, daß die St. Georgsbank in Genua seit dem Jahre 1409 wenigstens eine Aktiengesellschaft war 1, aber er meint, eine Geschichte der Aktien¬ gesellschaft könne sich nicht bei der Tatsache beruhigen, daß im Anfang des 15. Jahrhunderts in einer Stadt Italiens aus einer Ver¬ schmelzung von Staatsgläubigergruppen eine Bank hervorging, die durch eine von der Not der Lage erzwungene Ersetzung des Zinses durch Dividenden zur Aktienbank sich umwandelte. Es gälte zu untersuchen, ob darin der Ausgangspunkt für unsere heutige Aktien¬ gesellschaft vorliege, oder ob es sich um eine singuläre Begebenheit handelt, deren Einfluß nicht nachweisbar ist. Lehmann vertritt schroff die Ansicht, daß ein Zusammenhang zwischen den Aktiengesellschaften des 17. Jahrhunderts und den genannten italienischen Bildungen nicht bestehe. Im „Namen, Gegenstand des Unternehmens, Struktur “ usw. liege in den Bildungen des 17. Jahrhunderts „etwas ganz Neues vor“. Nun muß aller¬ dings zugegeben werden, daß der Ausdruck Aktie, der in Holland zur Bezeichnung der Anteile an der neuen Art von Erwerbsgesell¬ schaften üblich wurde, auch in den meisten übrigen europäischen Ländern sich durchsetzte 2; aber es soll doch nicht unterlassen werden, darauf hinzuweisen, daß dies teilweise ziemlich spät und unter Verdrängung älterer autochthoner Ausd r ü cke für dieselbe Sache geschah. In Frankreich taucht erst in den sechziger Jahren des 17. Jahrhunderts an Stelle des älteren „part “, „portion“, die Bezeichnung „action“ auf 3. In Deutsch¬ land ist in den ältesten Octrois, d. h. den obrigkeitlichen Privi¬ legierungen und Bestätigungen der Aktiengesellschaften, von 1 Die von Goldschmidt (a . a . O., S. 295 f.) untersuchten und als Aktiengesellschaften erklärten Maonen von Chios und Cypern hält Lehmann für keine Aktiengesellschaften , S. 17 ff. 2 Am zeitigsten in den Ländern einer jungen selbständigen, wirtschaftlichen Kultur , in Schweden und Dänemark. ! Lehmann , a . a . O. S. 9. Erstes Kapitel : Deutsche Aktiengesellschaften vornehmlich des 16. Jahrh . 113 „Portionen“, „Anteilen“ im Sinne von Aktien die Rede. Auch findet hier „die selbst weit später nicht völlig überwundene Auf¬ fassung der Aktie als einer Schuld in der Bezeichnung dieser Ur¬ kunde als „Obligation“, „Obligationsbrief“ Andeutung“ 1. Eng¬ land gar hat sich seinen Ausdruck „share“ bis heute bewahrt. Auch das folgende verdient gegen Lehmanns Beweisführung eine gewisse Beachtung: Das Wort Aktien wird in Deutschland schon in der Mitte des 16. Jahrhunderts für Inhaberpapiere gebraucht. Im Abschied des Reichstages von Augsburg (1551) heißt es in § 78 ff.: „Neben dem so erfindt sich, daß auch die Juden solche ihre unbilliche Schulden und Anforderungen, die sie auf den armen Christen mit höchsten Beschwerden und unziemlichen Vorteil erlangt, anderen Christen verkaufen und die Ver¬ schreibungen auf die Kauffer stellen lassen, welche in die armen, übervorteilten Schuldner zu dem hefftigsten dringen und sie etwan gar von Haus und Hoff vertreiben. Diesem zu begegenen sind wir ..... dahin entschlossen, daß die Juden hinfürter kein Verschreibung oder Obligation vor jemands anders dann der ordentlichen Oberlceit, darunter der contrahirend Christ gesessen, auffrichten.“ Dann folgt der für uns hier wichtigste Passus: . ..... Es soll auch kein Christ hinfürter einem Juden sein Action und Forde1VilctorRing , Asiatische Handelskompagnien Friedrichd. G. Ein Beitrag zur Geschichte des preußischen Seehandels und Aktien¬ wesens, Berlin 1890, S. 238. Interessant ist folgende Definition des Begriffes Aktie, wie sie sich bei P. J. Marperger Neueröffnetes ( Kaufmannsmagazin, 2 Bde., 4. Aufl., Hamburg 1765, Bd. I, S. 14 ff.), also einem kaufmännischen Fachmann findet: „Actie, Actien ist in Holland wie auch in Engelland und Dänemark der Verkauf der Obli¬ gation auf diejenigen Capitalien, die jemand in der Ost- und West¬ indischen Compagnie hat. Worauf denn die Obligation ihren Nahmen Verliert und Actie genennet wird.“ Der folgende Passus in dem¬ selben Artikel bei Marperger zeigt, wie umständlich damals noch der Verkauf der Aktien war: „Zum Einkauf der Actionen oder Actien bedient man sich eines Mäklers“ und wenn das Geschäft ab¬ geschlossen, „so lasset der Verkäufer solche gleich in dem Buche der Compagnie dem Käufer zuschreiben, unterzeichnet auch zugleich unter seiner Hand vor denen Herrn Directoribus eine Quittance, kraft welcher er sein Recht dem Käufer überträgt, hingegen muß dieser die Parthey gleich in Banco abschreiben lassen oder der Trans¬ port ist null und nichtig.“ Strieder , Studien ?.. Gesell , kapitiiliat , Orgamsalionsformen . 8 .8 S 114 Drittes Buch : Monopole, Kartelle und Aktiengesellschaften im Mittelalter. rung gegen einen anderen Christen ab kaufen oder ein Jud als Schuldgläubiger einem anderen Christen solche Actionen und Forderungen in einigem Weg cediren oder einigs Contracts-weiß zustellen bey Verlust derselben Forderung“ 1. Auf die Bedeutung, die der Wunsch der Juden, ihre Forderungen (d. h. diejenigen, die sie an Christen hatten ) zu übertragen, ohne die geringste Spur ihres früheren Besitzers an ihnen zu lassen, für die Verbreitung des Inhaberpapiers haben mußte, hat S o m b a r t neuerdings wieder aufmerksam gemacht 2. Aus den oben an¬ geführten Verordnungen sieht man, wie häufig die Juden schon um die Mitte des 16. Jahrhunderts das reine Inhaberpapier an¬ wendeten. Hier handelt es sich uns speziell um die Bezeichnung actio. Es wäre verkehrt, uns darauf hinzuweisen, daß in den ge¬ nannten Quellenstellen das Wort actio wohl für ein Inhaberpapier (was die Aktie wohl ist), aber für eine Schuldverschreibung (was 1 Die Bestimmungen sind in der Reichspolizeiordnung von Frankfurt (1577), Titel XX , § 4 wiederholt. In Tirol wurden die Verordnungen kräftig gehandhabt , vergl. Mim, a. a. O., I. Bd., S. 425. In Sachsen hat sich das kaiserliche Verbot nicht durchführen lassen und wurde 1715 (Deeisiv-Befehl Friedrich Augusts vom 5. No¬ vember ) aufgehoben. „ Allemaßen wir aber bei anderweiter der Sachen Ucberlegung der Billigkeit allerdings gemäß und zur Beförderung des Commercii wie auch des Wcchselrechts am verträglichsten befunden, die ..... Frage dahin zu erörtern , daß da euern Anführen nach der Reichs-Abschied de anno 1551 zusamt der Policei-Ordnung wegen des Verboths derer Jüdischen Cessionen an Christen in unsern Landen zu einer durchgehenden Observanz nicht gediehen, vielmehr auf die Validitaet dergleichen Handlungen bei dem Oberhof¬ gerichte zu Leipzig gesprochen worden, es in Zukunft noch ferner also gehalten und alle Cessiones derer Schuldverschreibungen nicht weniger derer Wechsel-Briefe und Steuerscheine, sie geschehen gleich von Christen an Juden oder von diesen an jene, sie seien gerichtlich oder extrajudicialitcr geschehen, bei Kräften verbleiben und vor gültig erachtet werden sollen . . . Codex Augusteus I, S. 1190. (Die erwähnten „ Steuerscheine “ sind sächsische Landschaftsschuld¬ verschreibungen .) Erwähnt mag hier wenigstens werden , daß auch Papst Paul IV. in seiner berühmten , gegen die Juden gerichteten Bulle ihnen die Ausstellung fingierter Kontrakte verbietet . § 6: „Seu Christianos quoquo modo gravare aut contractus fictos vel simulatos celebrare.“ Bullarium diplomatum et privilegiorum sum¬ morum romanorum pontificum . 6. Bd. Augustae Taurinorum , 1860, 8. 499. 2 Die Juden und das Wirtschaftsleben , S. 86. Erstes Kapitel : Deutsche Aktiengesellschaften vornehmlich des 16. Jahrh . 115 die Aktie doch nicht ist) gebraucht wird. Dem muß entgegengehalten werden, daß selbst im 18. Jahrhundert noch in Deutschland die Auffassung der Aktie als einer Schuld nicht völlig überwunden war, was in ihrer Bezeichnung als „Obligation“ und „Obligationsbrief“ zum Ausdruck kommt V So könnte also das im 16. Jahrhundert in Deutschland für einen Inhaberschuldbrief gebrauchte „actio“ auch für die Urkunden, die wir heute Aktien nennen, anstandslos ge¬ braucht worden sein. Ob es tatsächlich dafür gebraucht worden ist, das müssen noch nähere Studien lehren. Nun ist ferner schon betont worden und soll auch in diesem Zusammenhänge nicht geleugnet werden, daß erst seit den hol¬ ländischen und englischen Gründungen des 17. Jahrhunderts eine ununterbrochene Kette von uns bekannten Aktiengesellschaften bis auf unsere Zeit zu laufen beginnt. Aber es wäre doch methodisch durchaus verfehlt, aus einer Unkenntnis von Aktiengesellschaften zwischen der — auch von Lehmann als Aktiengesellschaft an¬ gesprochenen— St. Giorgio-Bank von Genua und den kolonialen Aktienkompagnien des 17. Jahrhunderts den Schluß zu ziehen, es hätten in . der Zwischenzeit keine Aktiengesellschaften existiert. Karl Lehmann zieht diesen Schluß nicht. Für ihn bleibt die Frage, ob im 15. und 16. Jahrhundert aktiengesellschaftliche Kapilalassoziationen existiert haben, offen, weil sie ihm irrelevant erscheint. Der genannte Forscher sieht erst mit den Aktiengesellschaften des 17.Jahrhunderts das Charakteristikum der neuen Vergesellschaftungsform gegeben, die Spekulation. Wohl, meint er, „gab es in Italien loca com perarum, sie wurden im Verkehr gehandelt und ihr Kurs war ein schwankender, aber dieses Schwanken war doch nur durch die Bewegungen des Weltmarktes und die finanzielle Lage des Schuldners bedingt. Von Mißbräuchen wie beim Aktienhandel erfahren wir nichts, und sie waren durch die Natur der loca, wenn nicht ganz ausgeschlossen, so doch erheblich eingeschränkt. Es waren ihrer Grundlage nach nicht Dividenden-, sondern Renten¬ papiere. Aber acht Jahre nach der Gründung der niederländischostindischen Kompagnie begegnet bereits ein Edikt gegen die Miß¬ bräuche des Aktienhandels, und überall, wo die neue Form auftritt, stoßen wir auf dieselben Erscheinungen, die ungeheuren Kurs1 Ringi a. a. O., S. 238. Auch Schm oll er in seinem Jahrbuch , 17. Bd ., S. 988. 8 116 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. Schwankungen, das Wechseln des Angebots und der Nachfrage, die Spekulation auf Steigen und Fallen, die Spielwut, das Jobber¬ tum !“ 1 Einer solch hohen Einschätzung des Spekulationselementes bei der Entstehungsgeschichte der modernen Aktiengesellschaft kann ich mich nicht anschließen. Die Tatsache der Erweckung der Spekulation war doch nur ein akzessorisches Moment hei dem Entstehen der neuen Kapitalassoziationsform. Daß die Speku¬ lation nicht natur not wendigerweise an die Aktien¬ gesellschaft geknüpft ist, kann man schon daraus erkennen, daß es eine ganze Reihe von Aktiengesellschaften gab und gibt, die niemals Gegenstand einer Spekulation wurden und werden. Man denke an die vielen Aktiengesellschaften, die dem Gemeinwohl, der Belehrung oder dem gewerblichen Gesamtinteresse eines Ortes dienen. Von den Aktiengesellschaften des 17. Jahrhunderts machte nur eine ganz kleine Anzahl ausgezeichnete Geschäfte und eröffnete damit der Spekulation glänzende Aussichten2. Aber von der Mehrzahl der neuen Gründungen konnten kaum und nur mit großer Mühe der interessierten Regierungen die Aktien abgesetzt werden. Die Zeichner brachten -— oft sehr unfreiwilligerweise— das Stamm¬ kapital als ein Opfer auf, das sie dem Staate darreichten. An eine Spekulation war kein Gedanke. So wird man denn die Behauptung aufstellen können: Es gab und gibt Aktiengesellschaften mit und ohne Spekulationsgeschichte. Die Tatsache, daß infolge glänzender Geschäfte und daraus resul¬ tierender starker Nachfrage nach ihren Aktien schon an einzelne Aktiengesellschaftendes 17. Jahrhunderts die Spekulation heran¬ gebracht wurde, ist gewiß interessant. Diese Erscheinung zog auch die Blicke der Mitwelt und der nachlebenden Wirtschaftshistoriker auf die betreffenden Institute, aber die Tatsache der hier und da in ihrem Gefolge sich einstellenden Spekulation berührt nicht eigentlich das Wesen der neuen Gesellschaftsform. Die Spekulation ist viel älter als die Aktiengesellschaften des 17. Jahrhunderts. Wir wissen aus verschiedenen Beispielen, wie die Spekulation schon im 16. Jahrhundert in den Niederlanden blühte. Für Waren1 Lehmann , a. a. O., S. 24 f. 2 Es waren namentlich die Institute , die ihre Tätigkeit auf dem die ganze niederländische bzw . englische Nation interessierenden Felde des kolonialen Handels entfalteten. Erstes Kapitel : Deutsche Aktiengesellschalten vornehmlich des 16. Jahrh . 117 Spekulation sei auf den portugiesisch-indischen Pfefferhandel ver¬ wiesen, außerdem auf die Objekte, die A. Sayous nennt 1. Auf die Spekulation in Wertpapieren, z. B. in den berüchtigten Rent¬ meisterbriefen haben schon Pirenne und andere aufmerksam gemacht 2. Eine Spekulation mit Wertpapieren hatte es in Deutsch¬ land dann spätestens seit dem 15. Jahrhundert schon (in Berg¬ werkskuxen) gegeben3. Das wesentlich Neue bei der Aktien¬ gesellschaft war die neue Art der Kapitalassoziation4. Mit dem Aufkommen der Aktiengesellschaftbeginnt ein neuer Abschnitt in der Geschichte der gesellschaftsweisen Unternehmung, in der Ge¬ schichte der wirtschaftlichen Assoziationsformen. In der Aktien¬ gesellschaft wird, wenn ich mich so ausdrt'rcken darf, die Form der Kapitalbeschaffung demokratisiert5, verallgemeinert, popularisiert. 1 „La spéculation dans les Pays -bas au XVIe siècle ." In Journal des économistes 1901. 2 H. Pirenne, Geschichte Belgiens , III . Bd ., übersetzt von Fritz Arnheim , Gotha 1907 , S. 348 f. Für Italien vgl . R . P ö h 1m a n n , Die Wirtschaftspolitik der Florentiner Renaissance und das Prinzip der Verkehrsfreiheit , S. 86. 3 Cfr. O p e t , Zeitschrift für Bergrecht 34, S. 308 Anm . 3. Vgl. auch H o p p e , a. a. O., S. 74 ff . Vgl . aber besonders die Bestimmungen über den Kuxhandel , wie sie in Spezialordnungen der Landesfürsten des 16. Jahrhunderts über Bergsachen niedergclegt sind . Zum Bei¬ spiel für Sachsen, gesammelt im Codex Augusteus, II . Teil , S. 140 , 212 usw . Ich habe im Anhang einige dieser Be¬ stimmungen , wie sie von den vereidigten Kuxhändlern gehandhabt wurden , abgedruckt . Für Böhmen vgl . die zweite Joachimstaler Bergordnung de anno 1541 , II . Teil , Art . 91 ; Schmidt, Samm¬ lung Österreich . Berggesetze , I, 1, S. 268 ; A. Salz, Geschichte der böhmischen Industrie der Neuzeit , S. 26 . Nach O. Hué, Die Berg¬ arbeiter . Historische Darstellung der Bergarbeiterverhältnisse von der ältesten bis auf die neueste Zeit , I. Bel., Stuttgart 1910 , S. 126 wurde auch in Pfalz -Zweibrücken (1565 ), in Saalfeld (1575), in HessenKassel (1616 ) usw . gegen die betrügerischen Kuxkränzler vorgegangen. 4 „Das Wesen der Aktiengesellschaft liegt in der Kapital¬ zusammenfassung und damit der Erhöhung der Leistungsfähigkeit des zersplitterten Kapitals einzelner kleinerer oder sogar größerer Besitzer .“ W . Wygodzinski, Einführung in die Volkswirt¬ schaftslehre , Leipzig 1912 , S. 71. 5 Eine „ Demokratisierung “ tritt etwa seit Ende des 18. Jahr¬ hunderts auch in den großen Finanzunternehmungen zutage . Bis dahin waren die Geldgeschäfte mit den Staatsoberhäuptern von einer verhältnismäßig kleinen Anzahl reicher Geldmänner mit eigenen 118 Drittes Buch : Monopole . Kartelle und Aktiengesellschaften im Mittelalter. Was schon bei der Ausbildung der kapitalistischen Gewerkschaft im Bergbau in einer für die Verbreitung kapitalistischen Geistes bedeutungsvollen Art geschehen war: die Hereinziehung weiterer vermögender Kreise (Adliger, geistlicher Korporationen, Gelehrter usw.) in die kapitalistische Produktion und in den kapitalistischen Handel, die fortschreitende Erfüllung der Gesellschaft mit kapita¬ listischem Geiste, dasselbe vollzog sich auch bei der Verbreitung der neuen Form der Erwerbsgesellschaft. Und zwar wurden die neuen Kapitalisten nicht zur „risikofreien“ Anteilnahme in der Form von festverzinslichen Einlagen herangezogen, sondern zur Beteiligung zu Gewinn und Verlust (des eingeschossenen Kapitals). Die Voraussetzung dieser „Demokratisierung“ ist natürlich die, daß einmal genügend kapitalkräftige und genugsam vom kapita¬ listischen Geiste erfaßte 1Personen vorhanden sind, die nicht die Möglichkeit hatten , im eigenen Geschäft ihre Anlage suchenden Kapitalien unterzubringen. Entweder weil sie kein eigenes Geschäft hatten (also Nichtkaufleute waren), oder weil das eigene Geschäft nicht mehr Kapital gebrauchen konnte usw. Vielfach hat in der ersten Entstehungszeit der Aktiengesell¬ schaften. die öffentliche Gewalt den betreffenden Gesellschaften— für Privilegierungen, besonders Monopole, die sie ihnen zuteil werden ließ — die Verpflichtung auf erlegt, den Beitritt allen Landes¬ kindern usw. offen zu halten. Also die genannte „Demokrati¬ sierung“ unterstützt . So war der holländische Typus der Aktien¬ gesellschaft ein „ halb im öffentlichen, halb im Privatrecht wurzelndes Verbandsgebilde“2. Beispiele städtischer obrigkeitlicher Offenhaltung Mitteln (oder doch mit Mitteln, die ihr persönlicher Kredit zu¬ sammengebracht hatte) geführt worden. Aristokratisches Prinzip! Höchste Repräsentanten: die großen italienischen Bankiers des Mittelalters und des 16. Jahrhunderts, die. großen süddeutschen Firmen im Zeitalter der Fugger, dann im 17. und 18. Jahrhundert besonders jüdische Geldmänner! Im 19. Jahrhundert wird der Bankier immer mehr nur der Vermittler der großen öffentlichen Anleihen, an denen sich jetzt hunderte, tausende kleiner Geldbesitzer beteiligen. „Demokratisches“ Prinzip! 1 Die anderen nicht vom spiritus capitalisticus durchseuchten Kreise begnügten sich mit der Verwendung ihrer Kapitalien als Rentenanlage. 2 Lehmann, Die geschichtliche Entwicklung des Aktien¬ rechts, S. 8, 30, 33. Erstes Kapitel : Deutsche Aktiengesellschaften vornehmlich des 16. Jahrh . 119 einer Aktiengesellschaft für alle •— kapitalkräftigen ! — Mitglieder der Stadtgemeinde werden wir gleich noch kennen lernen . Es soll im Zusammenhang mit der Aufreihung einiger deutscher Aktien¬ gesellschaften des 16. Jahrhunderts geschehen . Vorher aber noch ein Wort über die Frage , wie stellte sich die Wirtschaftsethik des 16. Jahrhunderts (die, wie wir sahen , noch allgemein die des Mittel¬ alters war) zu den neuen Bildungen ? Da ist es nun interessant zu beobachten , wie die Tatsache , daß die kanonische Lehre sich gegen die Einlagen zu festem Zins in die Handelsgesellschaften wandte, — ohne es zu wollen — der Entstehung und Verbreitung der Aktiengesellschaften , dieser eminent kapitalistischen Gebilde, Vor¬ schub leistete . In dem folgenden Falle können wir den angedeuteten ursächlichen Zusammenhang zwischen Wirtschaftsethik des Mittel¬ alters und Aktiengesellschaftsbildung deutlich erkennen . Als es in den siebziger Jahren des 16. Jahrhunderts galt , die Steyrer Eisen¬ handelskompagnie , die wir als Aktiengesellschaft nachweisen werden, zu gründen , da wurde von deren Befürwortern auch mit betont, daß durch die neue Gründung einer Forderung der strengen Wirtschaftsethiker entgegengekommen würde . Die Steyrer Privatleute könnten das Geld , das sie bisher bei den Eisenverlegern auf Inter¬ esse, d. h. zu festem Zins stehen hatten , zu Gewinn und Verlust in die neue Gesellschaft einschießen . Sie entgingen damit der Ver¬ urteilung durch die „Prädikanten , die stark gegen die Zinsen predigten “ 1. Tatsächlich erhielt die schon ältere kanonistische Verurteilung der Einlage von Geld zu festem Zins in die Handels¬ gesellschaften 2 im Verlaufe des 16. Jahrhunderts einen starken Bundesgenossen in der antikapitalistischen Bewegung jener Zeit. Wiederholt wurde auf Reichstagen usw. der Antrag eingebracht, „man soit kain gelt in die gesellschaft fünf von hundert nemen noch geben “ 3. Des längeren heißt es in einem Gutachten einer Reichstagskommission (1522/23) : Es sei üblich , „an kaufleut zu 1 Aus der Denkschrift eines der Hauptfreunde der neu zu gründenden Gesellschaft, des Hofkammerrats Adam Wucherer, vgl. F. M. Mayer, Das Eisenwesen zu Eisenerz in den Jahren 1570 bis 1625 in Mitteilungen des historischen Vereins für Steiermark, 33. Heft (1885), S. 172. 2 Siehe unten S, 121. 3 Deutsche Reichstagsakten jüngere Reihe , III . Bd., S. 566. Vgl. auch daselbst S. 557 Anm. 3. 120 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. iren kaufhendeln gelt auf zins zu entlelien .“ Das dürfe nicht ge¬ schehen . Es müsse reichsgesetzlich geboten werden , „ daß kein gelt oder gcltswert in irgend einem handel oder kau fin annsgewerb, er sei groß oder klein , gelegt , entlehnt oder eingenommen werde davon man , on ivagniß gewinns oder verlusts gelt oder zins nehme oder gebe . Ob auch ainiche geselschaft oder sondere personell solch entlehnt gelt itzt dergestalt bei inen im handel hetten , die¬ selben sollten dasselb in ainer zeit , wie hernach begriffen steht, von ihnen thun und bezalcn . Dann eben , wie solichs ungötlieh und wucherlich , also ist es auch gemeinem nutz nachteilig und schedlich , und soll derhalb die straff den lehner und entlehnet’ zugleich verbinden “ . 1 a . a. O., S. 584 t . Interessant für die Auffassung der groß¬ kaufmännischen Kreise über diese Dinge ist die Erwiderung , die Peutinger in seinem Gutachten de anno 1530 (Manuskript der Augs¬ burger Stadtbibliothek Cod . 2° Aug . 386 , Fol . J 96 vff .) gibt . Dort heißt es : „ Item aliud assertae consultationis medium , quod mercatores et societates in negotiationes suas nullam pecuniam pro annuo censu acceptare deberent etc . non solum est contra ius commune , quod hisdem non prohibet in eorum vei earum negotiationes pecunias recipere vel ad competentem censum vel mutuo vel alio debito modo etiam et si non ad partem , damni vel lucri , cum tempore illo nunc graviori non omnibus convenit partem vel lucri vel damni expcctare vel bona immobilia emere , sed multo commodius suam pecuniam ad honestum censum collocare , et cum voluerit ad sc retrahere , ut commodius possint filiis ct filiabus suis in dotibus et donationibus propter nuptias providere. Item inveniuntur etiam plurcs honesti viri ex statibus etiam nobilium , civium , orplianorum et aliorum artificia non exercentium nec scientium , qui nec serviunt nec se aliter educare vel enutrire valent , nisi cum iactura capitalis vel ex censibus vel redditibus et si habent aliquando ultro pecuniam paratam quam ad census ordi¬ nare vellent , hos tamen commode emere non possunt , cum bona immobilia per quosdam alios cum venditioni exponuntur semper emuntur et ita in magno pretio , quod nunc rarissime quis bona im¬ mobilia saltem debito pretio coemere poterit . Et si vellent alias pecunias suas ad perpetuos census coram aliis magni vei inferioris status collocare , id propter varios casus et sic cum magno gravamine cogerentur facere adeo quod se in periculum non solum ratione census sed etiam capitalis constituerent ct debita reemptione carere cogerentur. Quis autem debite huius modi honestis hominibus cum tali impio medio praejudicare vellet vel deberet et adeo ut non possent cum honestis mercatoribus super pecunia propria ad annuum censum con- Erstes Kapitel: Deutsche Aktiengesellschaften vornehmlich des 16. Jaluh. Aus 121 Auch die Monopolienkommission des Reichstags von Augsburg (1530) wandte sich gegen die Aufnahme von Zinsgeld durch die großen Handelsgesellschaften1. Und so ging es noch lange fort. Vergeblich hat der vielverkannte Dr. Eck in einer Reihe von öffentlichen Disputationen versucht , die Einlagen in Handels¬ gesellschaften zum festen Zinssatz von 5 % unter dem schon älteren kanonischen Zinstitel des sogenannten contractus trinus (contractus quinque de centum) 2 zu verteidigen und zu all¬ gemeiner Anerkennung in dem wirtschaftsethischen Urteil seiner Zeit zu bringen. Vergebens wies der genannte Gelehrte auf die starke Verbreitung dieses Geldgeschäftes hin. Es werde, führte er aus, in Augsburg seit vielen Jahren angewendet von Männern und Frauen , deren Gewissenhaftigkeit nicht bestritten werden könne, von einer Menge ehrenwerter Bürger , die überall des besten Rufes und hoher Achtung sich erfreuten , von Frauen¬ klöstern , von gelehrten und rechtskundigen Männern. Und das geschähe seit mehr als 40 Jahren , so daß sich selten ein wohl¬ habender Mann daselbst finde, der nicht persönlich und dessen Eltern nicht, auf solche Weise Geld hingegeben oder empfangen hätten 3. Es war selbst keine Übertreibung, wenn ein anderer Ver¬ teidiger des contractus quinque de centum , der Augsburger trahere , et sic cogere eosdem , se de proprio capitali educare ac in iacturam agere , quod etiam esset contra publicam utilitatem, destructio et perditio plurium statuum imperii Germanicae nationis. Etiam posito , quod societatibus inhiberetur , ne pecunias in negotiationes pro annuo censu acceptarent , quod tamen esset ex¬ presse contra ius commune ut ostensum est , tamen non possent inhiberi concambia . Sicut etiam antequam census in usum venerunt , ob¬ servatum fuit , et si non per census , liceret tamen alia via contrahere .“ 1 K. E . Förstemann, Urkundenbuch zur Geschichte des Reichstags zu Augsburg vom Jahre 1530 , 2 Bde ., Halle 1835 , II . Bd ., S. 199. 2 In welcher Art man kasuistisch das 5prozentige Zinsgeld als moralisch erlaubt durch eine Auflösung dieses Geldgeschäftes in drei Kontrakte zu rechtfertigen suchte , siehe J . Schneid , „ Dr . Eck und das kirchliche Zinsverbot “ , Historisch -politische Blätter 108 . Bd. (München 1891 ), S. 255 f. 3 Dr . Ecks Tractatus de contractu trino , fol . 124 b , 149 b. Cod . Manuscr . Nr . 125 fol . Universitäts -Bibliothek München . Vgl. Historisch -politische Blätter 108, S. 571 . Auch König, a . a. O., S. 105. 122 Drittes Buch : Monopole, Kartelle und Aktiengesellschaften im Mittelalter. Dr . utriusque jur . Sebastian Ilsung , Richter des Schwäbischen Bundes und Mitglied des Augustinerordens , behauptete , das Geld¬ geschäft des Contractus trinus sei in der ganzen Christenheit üblich . Trotz alledem vermochte Eck und seine Freunde den Widerstand der allzu strengen Wirtschaftsethiker gegen das fünf¬ prozentige Zinsgcld nicht zu brechen . Sein mannhaftes wissen¬ schaftliches Eintreten für eine freiere Auffassung des Kredit¬ verkehrs hat ihm nur boshafte Verleumdungen und den Ruf eines von den Fuggern bestochenen Skribenten eingebracht und teilweise bis heute bewahrt . Im letzten Drittel des 16. Jahr¬ hunderts scheint eine besonders scharfe Beurteilung des Zinsgeldes die Oberhand bekommen zu haben . Wir hörten oben ja schon von seiner Bekämpfung durch die Prädikanten . „ Im Jahre 1565 kam der Contractus trinus vor das Forum der Provinzialsynode von Mailand und wurde verworfen . Diesem Beschlüsse trat die Synode von Bordeaux im Jahre 1583 bei . Drei Jahre später verurteilte auch Sixtus V. in der Bulle .Detestabilis ' den Vertrag, nachdem noch Pius V. für die Herausgabe der Werke des Kanonisten Navarrus , welcher den Kontrakt verteidigt hatte , ein Privi¬ leg erteilt und Gregor XIII . die Widmung seines Hauptwerkes angenommen hatte “ 1. Zweifellos hat dabei nicht nur in dem einen eben von uns er¬ wähnten Falle die werdende Aktiengesellschaftsform von der Strömung gegen festverzinsliche Depositen Nutzen und Förderung gezogen. * * * Ehe wir uns nun auf die Suche nach faktischen Kapital¬ assoziationen im 16. Jahrhundert begeben , die auf dem Aktien¬ gesellschaftsprinzip aufgebaut sind , gilt es in erster Linie, noch das folgende zu beachten . Es wäre durchaus verkehrt , die Kenn¬ zeichen, die Merkmale unserer Aktiengesellschaften des 19. und 20. Jahrhunderts in ihrer Gesamtheit von den Aktiengesellschaften des 16. Jahrhunderts fordern zu -wollen. Die Arbeiten zur Ge¬ schichte der niederländischen , der englischen , der preußischen und anderer Aktiengesellschaften des 17. und 18. Jahrhunderts beweisen mit aller nur wünschenswerten Deutlichkeit , daß wichtige Elemente, 1 Historisch -politische Blätter 108 , S. 809 ; auch F . X . Funk, Geschichte des kirchlichen Zinsverbots . Tübingen 1876 . S. 58 ff. 4 Erstes Kapitel : Deutsche Aktiengesellschaften vornehmlich des 16. Jahrh . 123 mit deren Hilfe wir heute die Aktiengesellschaften von anderen Kapitalassoziationen unterscheiden können , in der Frühzeit der Geschichte des Aktienvereins fehlten 1. Zum Beispiel fehlte die leichte Übertragbarkeit der Aktie . Wenn auch die Aktie „regel¬ mäßig übertragbar war , so tritt die Inhaberaktie doch erst seit dem Ende des 17. Jahrhunderts auf und bildet auch im 18. Jahr¬ hundert noch die Ausnahme . Vorherrscht die Namensaktie . Es bedarf der Umschreibung auf den Erwerber , und hin und wieder reserviert sich die Gesellschaft ein Zustimmungsrecht “ 2. Noch bei den asiatischen Handelskompagnien Friedrichs des Großen ist die Veräußerung der Aktien (auch Anteile , Portionen genannt ) ein relativ umständlicher Prozeß . Die Aktien waren in einem Aktien¬ buche verzeichnet . „Die Ubertragungsform ist verschiedenlichst geregelt : Wiederholt ist Umschreibung im Aktienbuche schlechthin verlangt ; selbst die Erschwerung , daß die Übertragung persönlich oder durch notariell Bevollmächtigten im Aktienbuche eingezeichnet werden muß , andernfalls aber der Vertrag auch bei Auslieferung der Aktie ungültig ist , wird verordnet . Dabei wird , sofern die Umschreibung stattfindet , bald gefordert , daß ein Kompagnieleiter den Transport auf den Rücken der Aktie notiere , bald , von seiten eines Aktionärs , für ausreichend erachtet , daß die Aktie in blanco indossiert sei“ 3. Auch das Merkmal eines festen und ewigen Grundkapitals ist den Aktiengesellschaften in der ersten Hälfte des 17. Jahrhunderts durchaus noch nicht allgemein eigen, ebensowenig wie in jener Zeit schon die Zerlegung desselben in gleiche Anteile vorkommt . In der holländisch -ostindischen Kompagnie stand es laut Gründungsoctroi jedem Aktienzeichner frei, nach zehn Jahren sein Kapital aus der Gesellschaft zu nehmen . In ähnlicher Weise haben notorische Aktiengesellschaften des 17. oder 18. Jahrhunderts oft nicht die 1 Vgl . für das folgende die Ausführungen Schmollers in seinem Aufsatz „Die Handelsgesellschaften des 17. und 18. Jahrhunderts, hauptsächlich die großen Kompagnien “ , Schmollers Jahrbuch 17. Jahr¬ gang (1893), S. 959 ff., besonders S. 987 ff. •Lehmann , a. a. O., S. 26. 3 Ring, Asiatische Handelskompagnien Friedrichs d. Gr., S. 238 f. Schmoller, a. a. O., S. 989. 4 Le h m a n n , a. a. O., S. 44, 34, 35; R i n g , a. a. O., S. 235 f. ; Schmoller, a. a. O., S. 989. 124 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. -—heute obligatorischen— Merkmale der jährlichen General¬ versammlung, der jährlichen Gewinnausschüttung , der be¬ liebigen Übertragbarkeit der Aktie auf jedermann 1usw. Gingen so den Aktienvereinen des 17. und 18. Jahrhunderts Kennzeichen ab, die wir heute als diesen Institutionen obligatorisch ansehen, so hingen andererseits diesen frühzeitlichen Aktiengesell¬ schaften oft noch Elemente an, die die Fortentwicklung als rudi¬ mentär abstieß. Zum Beispiel findet sich die Nachschußpflicht, die heute die Aktiengesellschaft von der Gewerkschaft unterscheiden hilft. „ Sei es die limitierte Nachschußpflicht, sei es die illimitierte mit oder ohne Abandonierungsrecht“ 2. Mit anderen Worten soll alles das heißen: Wir müssen uns vergegenwärtigen, daß Aktien¬ vereine (wie alle Institutionen der Geschichte) eine lange Ent¬ wicklung durchzumachen hatten , ehe sie auf die Stufe gelangten, auf der sie uns liéute begegnen. Das gilt für die Aktiengesellschaften des 17. und 18. Jahrhunderts , es gilt natürlich erst recht für die Aktiengesellschaften des ausgehenden Mittelalters und des 16. Jahr¬ hunderts, die wir im folgenden untersuchen wollen. Gustav Schmoller hat in seinem oben genannten Aufsatz „Die Handelsgesellschaften des 17. bis 18. Jahrhunderts , hauptsächlich die großen Kompagnien“, der einen Teil seiner bedeutenden Artikelserie „Die geschichtliche Entwicklung der Unternehmung“ 3 bildet , die wichtigsten Züge zu¬ sammengestellt, die den großen Handelskompagnien des 17. und 18. Jahrhunderts gemeinsam waren 4. Man wird bei einem Ver¬ gleich des dort Ausgeführten mit dem im folgenden hier Gebotenen finden, daß die charakteristischen Merkmale der Aktiengesellschaften des 17. und 18. Jahrhunderts durchaus auch schon bei den Aktien¬ gesellschaften des 16. Jahrhunderts vorhanden waren. Namentlich zeigte sich auch bei letzteren schon — auf welche Erkenntnis, wie ersichtlich, in diesem ganzen Werk besonderer Wert gelegt ist —, daß die Mitwirkung der öffentlichen Gewalt bei der Entstehung der neuen kapitalistischen Organisationsformvon der größten Be1 Im 17. und -18. Jahrhundert durften die Aktien oft nur an Landeskinder übertragen werden. “Lehmann , a. a. O., S. 24, 47 ; Ring, a. a. O., S. 236. 3 Schmolle rs Jahrbuch für Gesetzgebung; Verwaltung und Volkswirtschaft im Deutschen Reiche, Jahrg. 14 —Al1890 ( —1893). 4 A. a. O., Jahrg. 17 (1893), S. 985 ff. Dorther auch die folgenden Zitate. Erstes Kapitel : Deutsche Aktiengesellschaften vornehmlich des 16. Jahrh . 125 deutung war. Die Handelszweige, welche die Aktiengesellschaften auch schon des 16. Jahrhunderts zeitigten, betrafen „ Geschäfte of greater and more general utility, wie Adam Smith sagt, die in den Kreisen ihrer Vaterstadt , ja häufig im ganzen Staate mit Interesse verfolgt wurden, aber anderseits doch nicht als eigentliche Ge¬ meinde- oder Staatsangelegenheit erschienen.“ Es handelt sich auch im 16. Jahrhundert schon „ um ein neues Mittelglied, ein neues eigenartiges Organ, das zwischen die älteren Formen der privaten Unternehmung und Gemeinde und Staat sich einschiebt.“ Schon die Aktiengesellschaft des 16. Jahrhunderts hat bei ihrer Entstehung „den Charakter einer halb öffentlichen, halb privaten Organisation; sie ruht rechtlich auf einer Anerkennung durch die Staatsgewalt, auf einem Privileg oder Octroi, wie man es damals hieß.“ Dies vorausgeschickt, wird das folgende besser beurteilt werden können. Dritter Abschnitt. Aktiengesellschaften im steiermärkischen österreichischen Eisenerzhandel und ober¬ 1. Die Ausbeute der reichen Erzlager des steirischen Erzberg bei Leoben geschah schon im Mittelalter von zwei Punkten aus, von Innerberg, dem heutigen Eisenerz, und von Vordernberg. Die Berechtigung zur Ausübung des Berg- und Hüttenbetriebes stand in beiden Gebieten erblich gewissen Bürgern der Märkte Vordern¬ berg und Innerberg, den sogenannten Radmeistern, zu. Die Vordernberger Radmeister verkauften ihr Eisen nach Leoben(Stcicrmark), die Innerberger zumeist nach der Stadt Steyr in Oberösterreich. Der Verkauf mag zuerst gegen Barzahlung erfolgt sein. Mit der Zeit aber gewährten die Kaufleute der genannten Städte den meisten Radmeistern Vorschüsse (Verlag) auf das bestellte Produkt 2. Der Kaufmann sicherte sich auf diese Weise die Lieferung der ihm 1 L. Bittner, Das Eisenwesen in Innerberg -Eisenerz bis zur Gründung der Innerberger Hauptgewerkschaft iin Jahre 1625. Im Archiv für Österreich. Geschichte, Bd. 89, S. 451 ff. A. v. P a n t z , Die Innerberger Hauptgewerkschaft 1625—-1783. In Forschungen zur Verfassungs- und Verwaltungsgeschichte der Steiermark , VI . Bd., 2. Heft , Graz 1906. 2 Bittner , a. a. O., S. 514 ff. 1 126 Drilles Buch: Monopole . Kartelle und Aktiengesellschaften im Mittelalter. nötigen Warenmenge, die Radmeister sicherten sich den Absatz, und, was die Hauptsache für die ärmeren unter ihnen war, sie erhielten die Betriebsunkosten vorgelegt. Dieser Einfluß der Kaufmannschaft der Städte Leoben und Steyr auf die Eisenproduktion wurde nicht geringer als seit dem 14. Jahrhundert mit dem Fortschritte der Technik eine Arbeits¬ teilung in dem Verhüttungswesen eintrat . Ursprünglich erfolgte nämlich die Verhüttung des Eisens einschließlich der Herstellung von Weicheisen und Stahl am Berge durch die Radmeister. Mit der Zeit aber wurden die feineren, zuletzt genannten Prozesse in besonderen Hammerwerken besorgt, die in der näheren und ferneren Umgebung von Innerberg bzw. Vordernberg von sogenannten Hammermeistern angelegt worden waren. Es ist hier nicht unsere Aufgabe zu untersuchen, aus welchen Bevölkerungselementcnsich die Hammermeister zusammensetztenh Es genüge zu wissen, daß sie in den meisten Fällen auf die Dauer nicht kapitalkräftig genug waren, um ohne Vorschüsse ihrer kaufmännischen Abnehmer den Verlag an die Radrneister zu zahlen. Wohl verlegte jetzt also viel¬ fach der Hammermeister den Radmeister, aber den ersteren verlegte doch wiederum der Kaufmann zu Steyr, dem er für die Verlagssumme fertiges Eisen zu liefern hatte 2. Und nicht selten kam es vor, daß dieser Steyrer Kaufmann, der so als Verleger auftritt , gleichzeitig nun seinerseits wiederum ein Verlegter war. Häufig schloß er mit den Kaufleuten in den österreichischen Eisenniederlagsplätzenund Legorten, wohin er sein Eisen brachte, oder auch mit reichsdeutsch en 1 Bittner meint , die ersten Hammerwerke seien wohl von Radmeistern angelegt worden , mit der Zeit aber wären die Rad¬ meister außerstande gewesen , beide Betriebe zugleich zu führen und hätten deshalb die Hämmer selbständigen Besitzern überlassen . Es ist nicht gesagt, daß die von Bittner S. 506 Anm. 1 genannten Eisen¬ erzer Bürger, die von den Grundherren das Hecht erhalten hatten, Eisenhämmer anzulcgen, Rndmeislcr waren. Die Tatsache, daß noch im 16. Jahrhundert dort Rad - und Hammerwerke zugleich im Besitze ein und derselben Familie waren , läßt sich auch so erklären, daß der Hammerwerksbesitzer dem Radmeis 1er — was die Regel war - - Vorschuß gegeben hatLe , dieser dann nicht Erz liefern konnte und so das Radwerk in die Hände seines Verlegers geriet (S . 516 ). 2 Bittner, a . a . O., S. 531 , 542 ; hier Näheres über die Ver¬ lagsformen. 1 Erstes Kapitel: Deutsche Aktiengesellschaftenvornehmlich des 16. Jahrh. 127 Handelsleuten Lieferungskontrakte ab, bei denen er sich bedeutende Vorschüsse gewähren ließ x. Zu manchen Zeiten traten die nicht in Steyr wohnhaften Kauf¬ leute aber auch wieder direkt mit den innerbergischen Radmeistern in Verbindung. So wissen wir, daß zu Anfang des 16. Jahrhunderts die großen süddeutschen Handelshäuser und Gesellschaften mit den Radmeistern gegen Vorschuß Verlagskontrakte abschlosseu, in denen diese sich zur Eisenlieferung verpflichteten. Das Roh¬ eisen, das die Kaufleute auf solche Weise an sich brachten, ließen sie dann meistens in den inländischen Hammerstätten — nun natürlich auf dem Wege des reinen Lohnwerkes — weiter ver¬ arbeiten. Manchmal besaßen sie auch eigene Hämmer 2. In den vordernbergischen Gebieten vollzog sich der Geschäfts¬ verkehr zwischen Radwerk und Hammerwerk von Anfang an durch Vermittlung der Leobener Kaufleute. Die Hämmer waren hier zu weit von dem Berge entfernt, als daß die Hammermeister selbst regelmäßig den Einkauf aus den Radwerken besorgen konnten. Die Leobener Kaufleute sprangen in die Lücke, sie nahmen den Radmeistern (zumeist natürlich auf dem Wege des Verlagssystems) ihr „Halbprodukt“ ab, verkauften es weiter an die Hammermeister, um es schließlich nach Fertigstellung wieder zu erwerben3. Die Leobener allgemeine (Aktiengesellschaft Eisenhandelsgesellschaft. gegründet um 1415 .) Zunächst war der Eisenverlag im vordernbergischen Gebiete von jedem der Leobener Eisenhändler auf eigene Faust getrieben worden. Höchstens daß sich Handelsgesellschafteneinzelner dabei bildeten. Aber schon um 1415 entstand eine neue Form der Erwerbs1 B i 11 n e r , S. 609 . Es wäre eine dankbare Aufgabe , die verschiedenen Verlagssystemsformen , die in der Wirtschaftsweise des Mittelalters und des 16. Jahrhunderts eine große Rolle spielen, einmal monographisch zu behandeln. 2 Bi 11 n e r , S. 542. 3 „Nur einen geringen Teil und vorzugsweise nicht stahlhaltiges Eisen durften sie schon Anfang des 15. Jahrhunderts in ihren eigenen Hämmern verarbeiten .“ Bittner, S. 514 . Einen Teil des Eisens durften die Eisenhändler von den Hammerwerken auf dem Wege des Lohnwerks für sich verschmieden lassen . Den größten Teil dagegen mußten sie für alle Hamniermeister frei zum Verkauf stellen. Bittner, a . a. O., S. 514 Anm . 2. 3 128 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. gesellschaft im Leobener Eisenhandel . Am 25. Mai 1415 bestätigte Ernst , Erzherzog zu Österreich , Steiermark , Kärnten und Krain, Graf zu Tirol usw., daß Richter , Rat und Bürger der Stadt Leoben ,,ainer solchen ainung überain worden seind, daß sy das eisen aus beeden pergen auf ainen gemainen pfening und nuz arbaiten , kaufen und verkaufen sollen und wellen“ 1. Deutlich ist in einer Urkunde desselben Fürsten vom 25. Dezember 1421 (datiert Bruck an der Mur) gesagt , daß es sich um eine „ Commune “ handelt , in die,jeder¬ mann sein gelt legen inüge nach seinen statten [d. h. Vermögens¬ verhältnissen ] und auch davon aufhebe den gewün , der davon gefölt “ 2. Herzog Friedrich der Jüngere bestätigte am 29. Januar 1439 die Leobener Eisenhandelskompagnie auf fünfzehn weitere Jahre . Die Bestätigung enthält einen interessanten Passus , der offenbar gegen eine Tendenz der Beherrschung der Gesellschaft durch wenige ganz reiche Bürger gerichtet war . Es wurde darin bestimmt , daß niemand mehr als 100 Pfund Pfennige in die Commune einschießen dürfe 8. Uber die Schicksale der Leobener Eisenhandelskompagnie und 1 Aus dem Privilegienbuch der Stadt Leoben , Fol . 42. 2 Privilegienbuch der Stadt Leoben , Fol . 47b . A . v . Muchar, Geschichte des Herzogtums Steiermark , 7. Bd . (Graz 1864 ) hat bei dieser Urkunde die Datierung irrtümlich aufgelöst , da damals der 25 . Dezember in der fürstlichen Kanzlei als Jahresanfang galt . Es muß also richtig heißen : 25. Dezember 1421 . Auch die andere Urkunde , die sich auf die Leobener Eisenkommune bezieht , hat Muchar falsch datiert ; muß heißen 12. Dezember 1421 . Der darin genannte Landschreiber heißt L . Stubiar. 3 Vgl. Jos . Chmel, Geschichte Kaiser Friedrich IV ., I. Bd ., Hamburg 1840 , S. 392 Anra . 1. „ Daz wir durch aufnemens willen unsrer stat ze Leuben und sunderlich umb ainen gemainen nucz aller unser burger daselbs denselben unsern bürgern vergunnet und erlaubt haben wissentlich mit dem brief , daz si ain commaun mit dem kauf des geslagen eisens und der maeß daselbs halten sullen ..... und dann darumb der kauf von denselben unsern bürgern umb ainen gemainen phenning beschehen soi , doch daz die radmaistcr und arbaiter , so solchs geslagen eisen und meß verkaufen mit der bezalung des gelts nicht gesaumbt werden . Und in dasselb commaun soi und mag ain jeder gesessner burger daselbs zu Leuben sein gelt legen als vil er dann wil nach seinen statten [Vermögen ], doch über hundert phunt phenning nicht und auch davon aufheben den gewin , der davon gevellet, nach geleicher anzal ungeverlich .“ 81 Erstes Kapitel: Deutsche Aktiengesellschaften vornehmlich des 16. Jahrh. 129 über ihr.e Organisation im einzelnen dürfte namentlich aus dem schönen Archiv der k . k. steiermärkischen Statthalterei zu Graz reichliches Material zu schöpfen sein. Eine lohnende Monographie zur Geschichte der Unternehmung scheint mir hier gegeben . Uns muß an dieser Stelle der Hinweis auf die neue Unternehmungsform genügen , die sich in der Leobener Eisenhandelskompagnie zeigte. Die Einzelheiten der Organisation solcher allgemeiner Eisenhandels¬ kompagnien ergeben sich aus der folgenden Geschichte der all¬ gemeinen Steyrer Eisenhandelskompagnie , über die wir besser unter¬ richtet sind. Die Gründung der handelskompagnie Steyrer allgemeinen - Aktiengesellschaft Eisen¬ 1582 ( ). Im Gegensatz zu Leoben , wo sich, wie wir sahen , früh eine allen kapitalbesitzenden Bürgern zugängliche „allgemeine “ Eisen¬ handelsgesellschaft bildete , hat Steyr , die Beherrscherin des innerbergischen Eisenwesens , erst im 16. Jahrhundert eine entsprechende Kapitalassoziation gesehen K Bis dahin hatten kapitalkräftige Steyrer Bürger den Verlag der Hammer - bzw. Radmeister , sowie den Weiterverkauf des auf dem Wege dieses Verlagssystems ge¬ wonnenen Eisens einzeln oder in den üblichen Gesellschaftsformen des Mittelalters betrieben 2. 1 Wenn wir dem Bericht des kenntnisreichen Hans Steinberger aus Schladming vertrauen dürfen 1 Hans Steinberger war zu einem Gutachten über die Frage der wirtschaftlichen Zweckmäßigkeit einer Steyrer allgemeinen Eisenhandelsgesellschaft aufgefordert worden. Das interessante Schriftstück , in dem er seine Aufgabe zu lösen ver¬ suchte , und auf das wir später noch näher zu sprechen kommen , ist abgedruckt bei V . P r e u e n h u b e r , Annales Styrenses , Nürnberg 1740, S. 297 ff. Übrigens hatten die Steyrer schon 1531 einmal vor¬ gehabt , „ die Eiscnhandlung in ain Handt und gemaine Gesöllschaft zu bringen “. Mitteilungen des historischen Vereins für Steiermark, 33. Heft , S. 173. aAls „ zum Exempel so etwan ein Hammerwerck in 6, 7 und 8 tausend Gulden Verlag bedarff , und es nun nicht jeden gemeinen Bürgers Vermögen ist einen solchen Verlag zu tun , da möchten 2— 4 zusammen legen und eine besondere Gesellschaft anrichten , damit sie ein Hammer -Werck verlegen und einen Handel führen und er¬ schwingen könnten .“ Aus dem Gutachten Steinbergers . Preucnhuber, a. a. 0 ., S. 301. Strieder , Studien z. Geßch. kapitalißt . Organisationeformen , 9 1 130 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. Zunächst war dabei die Unternehmungsorganisation so be¬ schaffen gewesen, daß ' jeder Bürger an dem Verlag sich beteiligen konnte und daß selbst Mitglieder des Adels in den Verband der Stadtgemeinde einlraten , um daran teilnehmen zu können. Mit dem Verlaufe des endenden 15. und 16. Jahrhunderts jedoch bildete sich ein abgeschlossener Stand von Verlagsherren heraus. Das waren größtenteils eingewanderte Familien, die den zu Ende des 15. Jahrhunderts gesunkenen Wohlstand der Stadt wieder hoben und selbst dabei reich wurden. In ihren Händen ruhte besonders seit den vierziger Jahren des 16. Jahrhunderts völlig der Eisen¬ verlag. Die übrigen Bürger waren von dem Eisenhandel und Eisen¬ verlag ausgeschlossen. Eine gesamte erwerbsgescllschaftliche Ver¬ einigung der Verlagsfirmen fand zunächst nicht statt . Jede arbeitete im Eisenverlag und Eisenhandcl auf eigene Faust 2. Erst 1582 trat hierin eine wichtige Änderung ein, die nun auch der Stadt Steyr eine „allgemeine Eisenhandelskompagnie oder Sozietät“ brachte, wie sie Leoben vor mehr als 114 Jahrhunderten schon besessen hatte. Wenn man sich fragt, warum die bisherige Organisation des Steyrer Eisenhandels und Eisenverlags aufgegeben wurde, so muß man zwei Ursachenkomplexe unterscheiden. Einmal drängten die Bürger von Steyr, die nicht zu der führenden, den Eisenhandel und Verlag innehabenden Ratsklique gehörten, zu einer Öffnung 1 Bittner , a. a. O., S. 540 f. Es gab etwa 20—30 solcher Vcrlagsfirmen in Steyr . Manche von ihnen besaßen „auch Radund Hammerwerke , in deren Besitz sie wohl zumeist durch die Zahlungsunfähigkeit zahlreicher Rad - und Hammermeister am An¬ fänge des 16. Jahrhunderts gekommen waren.“ Die Verlagshäuser waren durch Interessengemeinschaft miteinander verbunden , zumeist auch verwandt und verschwägert und beherrschten den Rat voll¬ kommen . Viele brachten es zu bedeutendem Reichtum , den sie dann in Grundbesitz und Rittergütern anlegtcn und zur Gewinnung von Adelspatenten benutzten. 2 Über das Wesen und die Organisation der sogenannten Ge¬ sellschaft des gestreckten Stahls ist aus der Literatur kein Aufschluß zu gewinnen. Möglicherweise läßt sich aus Archivmaterial der Nach¬ weis erbringen , daß wir cs auch in dieser Gesellschaft (die Sachfirma deutet , wenn auch nicht unbedingt sicher, darauf hin) mit einer ähnlichen Organisation zu tun haben , wie die Steyrer Eiscnhandclskompagnie eine wurde . Etwas Näheres über die Tätigkeit der interessanten industriellen Groß Unternehmung der Gesellschaft des gestreckten Stahles weiter hinten. Erstes Kapitel: Deutsche Aktiengesellschaften vornehmlich des 16. Jahrh. 131 der bisherigen Schranken im Eisenverlag. Schon 1511 hatte einer der Artikel der mit dem Stadtregiment unzufriedenen Handwerker¬ schaft das befürwortet 1. Nun hätten aber solche Wünsche der niederen Stadtbevölkerung niemals durchdringen können, wenn nicht im Verlaufe des 16. Jahrhunderts im Eisenverlage Mißstände eingerissen wären, die ein Eingreifen der österreichischen Regierung notwendig erscheinen ließen. Der Verlag der Hammermeister bzw. der Radmeister war über den kapitalistischen Interessen der Ver¬ leger in Unordnung geraten 2. Eine Anzahl der ärmeren Rad- und Hammermeister konnte keinen Verleger finden, andere arbeiteten mit Defizit. Die Folge war eine starke Verbitterung unter den Rad- und Hammermeistern und die Erkenntnis auf Seiten der Regierung, da'ß man von dem bisher geübten System, einzelnen Kapitalisten den Verlag zu überlassen, abgehen müsse. In jenem Übereifer, der den Merkantilismus namentlich in seiner Frühzeit oft kennzeichnet, trat Erzherzog Karl — wir werden diesem Herrscher noch bei anderen Verstaatlichungen begegnen — mit dem Projekt hervor, den „gesamten Rauheisenhandel (Eisen¬ verlag' und Verkauf) auf landesfürstliche Kosten zu betreiben.“ Erst als man ihn auf rechtliche Bedenken und besonders auf das wirtschaftlich unrationelle eines solchen Verfahrens aufmerksam machte 3, stand er davon ab. Dafür glückte es der Regierung nach langem Drängen und nach ausgedehnten Unterhandlungen mit dem Steyrer Rat , den Eisenhandel (Verlag und Großverkauf) an eine neu zu gründende, allgemeine Eisenhandelskompagniezu bringen, also an eine Kompagnie, zu der die Teilhaberschaft in beliebiger Höhe jedem kapitalbesitzenden Bürger offenstehen sollte. In beliebiger Höhe wenigstens zunächst und solange es nicht sicher war, ob das nötige Kapital gezeichnet werden würde. Für später ist in dem Statutenentwurf eine Limitierung der Einlagen Vor¬ behalten. Sie sollte gegebenenfalls so vor sich gehen, daß die Bürgerschaft in drei Reichtumsklassen eingeteilt und für jede eine bestimmte Obergrenze der Einlage festgesetzt wurde. Eine solche Limitierung sollte geschehen, „damit der Reich den Unvermugigern 1Gemeine „ Stadt könnte durch solchen Handel in groß Auf¬ nehmen kommen , wo solcher auf dieselbe geleitet würde .“ P r e u e n huber, a. a. 0 ., S. 195. 8 Bittner , S. 599 ff. 3 B i 11 n e r , a. a. O., S. 600 Anm . 2. 9* 132 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. hierinnen wo diese Gesellschaft nütz- und gewinnlich sein würde, nicht engen oder mit zu villem Leggeld an seiner Nahrung hindern möge“ L Es war der Regierung nicht leicht gemacht worden, die neue allgemeine Kompagnie durchzusetzen. Zum Teil aus egoistischen, zum Teil aus altruistischen Gründen hatte man sich in Steyr und sonst der Neuerung widersetzt. Auf die Mängel alles Kompagnie¬ wesens hatte in feinen Bemerkungen voll tiefer ökonomischer Sach¬ kenntnis besonders Steinberger aufmerksam gemacht. Er hatte betont, „daß in Gesellschaften nicht so genau und treulich hausgehalten wird, als wohl jeder sonsten für sich selbsten thut , sondern die tägliche Erfahrung giebt es, daß man weit schlechtere Ordnung führet, schwere Unkosten aufgehen läßt, als einer allein, daß also immer einer auf den andern wartet, einander nicht folgen, die wichtigsten Rathschläge oft fürziehen und viel Hirten selten wohl hüten . Und ob man schon lang flicket und mit Verschreibungen den Sachen helfen will, so geschieht es doch gemeiniglich, daß Nachlässigkeit, Unfleiß und Liederlichkeit die Oberhand behalten.“ Käme es aber vor, daß „ die Regierung der Gemeinschaft in ordent¬ licher und fleißiger Leut Hände kommt“, so dächten diese oft nur an ihren eigenen Vorteil und versuchten „mit Hilfe etlicher, die sie an sich hängen und die im Handel verwandt seyn, ihre Mit¬ verwandten hart zu drucken, wie dann schon große Gesellschaften hiedurch zerfallen seyn.“ Schließlich wies Steinberger auf die demoralisierende Wirkung des Verdienstes ohne Arbeit in den vollkommenen Kompagnien hin. „Und was den andern Punkt anbetrifft, daß es nemlich um eine vollkommene Compagnie was schönes sey, gestalten ihrer viel Bürger ihr Geld darein legen könnten, hätten ihren Gewann jährlich zu gev'arten und könnten sich also gar fein nähren und ruhig leben. So ist hierauf diss meine Ant¬ wort : Daß solches ein rechtes Verderben ist der edlen Jugend und groß Verderben vieler Leute. Als zum Exempel, weil die Bürger der Stadt Augspurg unverdrossen gereist, Gewerb und Handthierung mit vieler Mühe geführet, hat Gott ihre Arbeit gesegnet, daß sie in groß Aufnehmen erwachsen. Da aber ihrer viel von Mühe und Arbeit abgelassen, ihr Geld auf Interesse angelegt und davon gelebet haben, da ist bey ihnen nichts als Müßiggang, Faulheit, Pracht, Stoltz, Wollust und Geldverthun erfolget und da hernach die Inter1Näheres vgl. § 3 des Statuts. Erstes Kapitel : Deutsche Aktiengesellschaften vornehmlich des 16. Jahrh . 133 essen bey den Potentaten nachgelassen, so ist nicht allein das Ver¬ derben der Stadt erfolget, sondern es haben auch die Bürger zur Handthierung und Arbeit den Lust nebst der Erfahrung und Ge¬ schicklichkeit verlohren und gehet bis dato noch mit schwehrer Mühe zu, daß sie sich in Mühe und Arbeit begeben und sich dardurch wieder erholen. Eben also kan auch die Stadt Steyer zu viel höherer Wohlfahrth und Aufnehmen an Mannschaft und Vermögen steigen, so ihrer viel arbeiten, durch Reisen und Handthierung ihre Nahrung suchen, als wann sie sich auf das bloße Interesse oder Gewinn, den ihnen andere erwerben sollen, begeben und verlassen“ 1. Alle gut- und auch schlechtgemeinten Abratungen von der Durchführung einer allgemeinen Steyrer Eisenhandelskompagnie fruchteten nichts. Ihre Eröffnung wurde nur um so eifriger be¬ trieben. Aus Beratungen eines weiteren und eines engeren Aus¬ schusses der Bürgerschaft von Steyr und zweier kaiserlicher Ab¬ gesandter ergab sich ein Statut (Hauptordnung genannt) 2 für die neue Gesellschaft, das wohl im wesentlichen angenommen wurde. Das Statut legte etwa folgende Organisation der Gesellschaft fest. Der neuzugründenden Kompagnie wurde der Eisenverlag und Eisenvertrieb als Monopol staatlicherseits 3 übertragen (§ 1). Das heißt : der Eisenverlag und der Eisenhandel wurde den privaten Eisen¬ händlern entzogen und der Kompagnie Vorbehalten. Die bis¬ herigen privaten Träger dieser Geschäftszweige erhielten die Auf¬ forderung, bis zum Martinstag 1582 mit den von ihnen verlegten Hammermeistern abzurechnen. Das Eisen, das sie bis dahin noch in Händen hatten , durften sie, falls es die Kompagnie nicht über¬ nahm, noch privatim verkaufen (§ 16). Später konnten sie ihre Kapitalien, sofern sie sie nicht anderweit anlegten, nur noch in der Kompagnie arbeiten lassen. Der Verlag der Hammermeister durch die neuzugründende Kompagnie hatte gemäß den Bestimmungen der staatlichen Eisenordnungen zu erfolgen, wie sie bisher für die Privathändler gegolten hatten 4. 1 Preuenhuber, Annales Styrenses, S. 300/1. 2 Ich habe die wichtigsten Teile des Statuts im Anhänge dieses Buches abgedruckt. 3 Eine besondere landesfürstliche Verordnung sollte bei Beginn der Gesellschaft das Nötige publizieren (§ 16). 4 Ich gehe hier nicht näher auf die Verlagsordnung ein. Näheres bei Bittner. 134 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. AlsName für die Gesellschaft ist der Ausdruck „Compagnia oder Gemeinschaftseisenhandlung “ in dem Statutenentwurf ge¬ braucht. Auch die Bezeichnungen „ Gemeine Eisenhandlung“, „Völlige Gesellschaft“ , „ Gemeinwesen“ und ähnliche kommen vor. Das oben genannte Gutachten Steinbergers spricht von der „voll¬ kommenen Kompagnie“. Die hervorragende Rolle, die die Stadt Steyr als solche in der Gesellschaft spielte, — wir kommen hierauf noch zu sprechen — drückt sich in der Bezeichnung aus : „Eisen¬ handel bei der Stadt so unter dem Namen und Titel der Stadt Steyr solle ausgehen.“ Die Kompagnie sollte, wenn irgend möglich, für ihre Buchhalterei und Kasse im Rathaus eine „wohlbewahrte“ Schreibstube erhalten. Ihre Schriftstücke (mit Ausnahme der Obligationen) hatte sie mit einem eigenen „Hand-Petschaft “ zu siegeln. Auf dem Siegel war das Wappen der Stadt, ein Panther, zu sehen und die Unterschrift zu lesen: „ Stadt Steyr und die ge¬ meine Gesellschaft der Eisenhandlung allda“ (§ 7). Der Beitritt in die Gesellschaft sollte jedem geschworenen Bürger von Steyr, gleichgültig, ob er Kaufmann oder Handwerker war, offenstehen1, der imstande war, ein Kapital — zumeist wird statt dieses Ausdrucks, der auch schon üblich ist, die Bezeichnung Leggeld gebraucht — von mindestens 100 fl. einzuschießen. Es war gleichgültig und wohl auch schwer zu kontrollieren, ob der Bürger Einlage „aus eigenem Gut geschehe“ oder von „sonsten anderwärts aufgebracht“ war. Die Höhe der Einlage war nach oben unbegrenzt. Kleinere Einlagen als 100 fl. wurden nicht angenommen. Jeder Gesellschafter erhielt einen „sonderen gefertigten Schein“, der, mit dem Siegel der Kompagnie gesiegelt und vom Buchhalter unterzeichnet, seine Mitgliedschaft in der Gesellschaft und die Höhe des eingelegten Kapitals bestätigte 2. War die Konstituierung der Gesellschaft erfolgt, so sollten neue Mitglieder nur jeweils am Jahresschlüsse nach Abschluß der Jahresrechnung aufgenommen werden. Wenigstens in den ersten vier Jahren. Später sollte die Aufnahme neuer Mitglieder nur alle zwei Jahre nach Schluß des Geschäftsjahres erfolgen können. 1 Vgl . § 5 und 6 des Statuts. Auch die holländischen Kom¬ pagnien des 17. Jahrhunderts waren zuerst nur Holländern offen. Hansische Geschichtsblätter 1911, S. 239. 2 Nach§ 6 ist dem Texte des Statutentwurfs das Formular eines solchen Mitgliedschaftsscheinesbeigefügt. Cfr. Anhang. Erstes Kapitel: Deutsche Aktiengesellschaftenvornehmlich des 16. Jahrh. 135 Meldeten sich nach Beginn der Gesellschaft zu. viele Kapitalisten zur Mitgliedschaft in der Kompagnie, so daß es denjenigen, die zuerst eingetreten waren und das Risiko des Anfangs auf sich ge¬ nommen hatten , nachteilig und der Gesellschaft schädlich erschien, dann sollte es in der Macht des Vorstandes der Gesellschaft liegen, die Beitrittswilligen abzuweisen (§ 6). Die Einlagen waren vier Jahre unkündbar (§ 4). Dann mußten sie, wenn der Gesellschafter nicht in der Kompagnie verbleiben wollte, halbjährlich gekündigt werden. Nur im Fallè erweislicher und unumgänglicher Not und unter der Voraussetzung, daß es der Gesellschaft möglich und unschädlich war, konnte ein Gesellschafter seine Einlage ganz oder teilweise früher zurückziehen. Natürlich wurden dann dem ausscheidenden Gesellschafter die Verluste, die die Gesellschaft während seiner Teilhaberschaft gehabt hatte , ab¬ gezogen. Im Falle des Todes eines der Gesellschafter sollte ebenfalls der Witwe oder den Kindern oder auch den Gläubigern auf Wunsch die Einlage schon vor dem Ablauf der vier Jahre ausgehändigt werden 1. Die Aushändigung geschah nach halbjähriger Kündigung an den „treuen Brieffs - Inhaber ’\ Und zwar im Falle die Kapitalseinlage 500 fl. nicht überstieg, in Jahresfrist, sonst im allgemeinen in vierjährigen Raten 2. Auf Wunsch konnte auch in zwei Jahren die gesamte Rückzahlung erfolgen. In beiden Fällen partizipierte selbstverständlich der ausscheidende Gesellschafter nur für die in der Kompagnie verbleibende Summe pro rata am Gewinn. Vorausgesetzt, daß die Erben Bürger von Steyr waren. Im anderen Falle oder wenn die Erben die Stadt verließen, nahmen sie für den Rest der Einlage nicht mit am Gewinn teil, sondern die Einlage wurde ihnen mit 5 % pro anno verzinst. Wollten dagegen die Erben eines verstorbenen Teilhabers oder die Gläubiger eines Teilhabers das ihnen zustehende Kapital in der Gesellschaft stehen lassen, so war ihnen das unverwehrt. Waren sie Bürger, so nahmen sie am Gewinn und Verlust teil wie die anderen Gesellschafter auch; wenn nicht, wurde ihr Kapital mit 5 % ver¬ zinst (§ 14). 1 Unter Abzug natürlich auch hier der Verlustquote an den „gar ungewissen und für verlohrn befundenen Schulden". 2 Vgl . hierüber außer § 4 auch § 14, wo eine teilweise Wieder¬ holung des in § 4 Gesagten sich befindet. 6 136 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter, Außer den Einlagen der Gesellschafter sollte das Kapital der Kompagnie aus festverzinslichen Anleihen (Obligationen, Schuld¬ verschreibungen) 1aufgebracht werden 2. Für diese Anleihen und für die regelmäßige Zinszahlung hafteten die Güter der Stadt Steyr und die Güter der Gesellschaft unbedingt 3. Der Darleiher oder der Inhaber der Obligation war, im Falle er sein Geld zurückziehen wollte, zu halbjähriger Kündigung verpflichtet, wie anderseits die Kompagnie ihm sechs Monate vorher die Abstoßung ansagen mußte. Als Darlehen wurden auch die Mündelgelder, die in städtischem Gewahrsam wären, in die Gesellschaft gelegt und mit 5 % verzinst (§ 11). Im übrigen dürften diese Darleiher wohl dieselben Kauf¬ leute sein, die früher mit den privaten Steyrer Eisenhändlern ihre Vorschußkontrakte abgeschlossen hatten 4. Zu Leitern der Geschäfte der Kompagnie wurden vom Hat vier Teilhaber çler Kompagnie gewählt, zwei davon mußten Mit¬ glieder des Rats sein, zwei nicht. Nach zweijähriger Dienstzeit schieden die zwei älteren dieser Häupter der Gesellschaft aus ihrem Posten und wurden durch zwei neue Kompagniemitgliederersetzt 5. Die vier Leiter sollten „der Gesellschaft Häupter“ sein. Sie sollten „die Obhandt“ über das ganze Gesellschaftswesenhaben. Die gleich noch zu erwähnenden Beamten der Gesellschaft waren ihnen untergeben und zu Gehorsam verpflichtet. An gewissen Tagen und Stunden hatten sich die Leiter allesamt oder doch nach Ver¬ abredung je zwei von ihnen in der Schreibstube der Kompagnie aufzuhalten. Sie hatten zuzusehen, „was allerorts vorgefallen und was zu handeln von nöten“, um dann das Notwendige zu veranlassen. In wichtigen Fragen konnten sie sich noch einige andere Mitglieder der Kompagnie kooptieren. Eine Besoldung des „Aufsichtsrates“ 1 Wie das ja auch heute bei unseren Aktiengesellschaften ge¬ schieht. 2 Vgl . § 9 und vorher gelegentlich Gesagtes. In § 9 ist das Formular einer solchen Schuldverschreibung gegeben. 3 Sollte ein Darlehensgeber lieber nur die Garantie der Stadt haben wollen, so solle die Kompagnie der Stadt einen Revers aus¬ stellen, auf Grund dessen sie der Stadt gegenüber die Mithaftung mit ihren Gütern übernahm (Ende von § 9). 4 Siehe oben S. 126f. und Bittner, a . a. O., S. 609 ; Pantz, a. a. O., S. 6 ff. 6 Während der ersten vier Jahre sollte diese Neubesetzung tun¬ lichst unterbleiben. Erstes Kapitel : Deutsche Aktiengesellschaften vornehmlich des 16. Jahrh . 137 erfolgte nicht, aber alljährlich erhielten die Leiter eine „gebürliche Verehrung“ für ihre gewiß nicht geringe Mühewaltung. Außer den vier Leitern sollten aus der Reihe der vornehmsten und tüchtigsten Gesellschafter sodann zwei Kassierer erwählt werden. Nach Ablauf eines Jahres wurde einer von ihnen durch einen neuen Kassierer ersetzt, so daß der Kontinuität der Geschäfts¬ leitung wegen immer ein neuer und ein alter Kassenführer amtierte. Die Kassierer hatten bei ihrem Amtsantritt dem Rat als Vertreter der Gesellschaft Handgelöbnis abzulegen, daß sie ihre Stellung treu¬ lich ausfüllen und nicht mit den Gesellschaftsgeldern eigennützige Zwecke verfolgen wollten. Jeder der zwei Kassierer hatte einen anderen Schlüssel zur Kasse, die mit zwei verschiedenen Schlössern verwahrt war, so daß sie nur stets gemeinsam öffnen konnten. Auch die Kassierer erhielten keine feste Besoldung. Nur eine „Verehrung“ wurde ihnen zuteil, deren Höhe sich nach dem Ge¬ schäftsgang und ihren Dienstjahren richtete. Das Rechnungswesender Kompagnie hatte ein Buchhalter zu versehen. Er sollte womöglich ein Steyrer Bürger sein. War unter der Bürgerschaft kein für einen solchen Posten tauglicher Mann zu finden, so durfte es ein Auswärtiger sein. Doch sollte er bald unter die Bürgerschaft eintreten. Ein besonderer Treueid wurde ihm abgenommen, sein Geschäftsbereich ihm durch eine eigene In¬ struktion vorgezeichnetL Jede Geschäftstätigkeit außerhalb der Kompagnie, die der Gesellschaft zum Schaden gereichen konnte, war dem Buchhalter verboten. Als Lohn erhielt er „eine billige und ziemliche Jaresbesoldung“. Außer dem Buchhalter waren vier festbesoldete Beamte (Diener, Faktoren, Händler) für die Kompagnie tätig. Ihnen fiel die Haupt¬ arbeit in der Gesellschaft zu. Zwei von ihnen sollten den Verlag an die Hammermeister abführen, das Eisen in den Hämmern auf seine Güte prüfen (Beschau) und dann in die Magazine der Gesell¬ schaft bringen, die am Ennsufer zu Steyr errichtet waren. Die zwei anderen Faktoren übernahmen sodann den Vertrieb des Eisens im In- und Auslande. Es war ihre Pflicht, die großen Eisenmärkte zu Linz, Freistadt und Krems zu besuchen, um hier und anderwärts Abschlüsse mit den Großabnehmern zu vereinbaren; sie mußten 1Die Instruktion entstand aus einem Auszug der entsprechenden Paragraphen der Hauptordnung. 138 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. Gelder für geliefertes Eisen kassieren und zur Kasse der Kom¬ pagnie bringen usw. usw. Die vier Faktoren hatten wieder eigene Diener unter sich, auch mußten sie sich Pferde halten und andere Ausgaben bestreiten. Die Bezahlung dieser Spesen hatte von ihrem Jahresgehalte zu erfolgen. Der Buchhalter, die Faktoren, aber auch die Kassierer waren während ihres Dienstes von allen städtischen Ehrenämtern befreit, damit sie ihre ganze Arbeitskraft und Zeit in das Wohl der Gesellschaft stellen konnten. Auch die festbesoldeten Angestellten durften und sollten ihre Kapitalien in der Kompagnie anlegen, damit sie um so mehr an deren Wohl¬ ergehen interessiert waren. Buchhalter, Kassierer und Faktoren hatten sich den Anord¬ nungen der vier Leiter der Kompagnie unbedingt zu fügen, sie sollten wichtige Handlungen erst nach Beratschlagung mit ihnen vornehmen. Der Buchhalter insbesondere mußte den Leitern so oft sie es verlangten eine Bilanz vorlegen. Bedurfte er Hilfspersonen, so hatte er sie, auf eigene Kosten anzustellen und für ihr Wohl¬ verhalten zu haften. Ebenso hatten die vier Faktoren den Leitern genaue Rechnung über ihre Geschäftstätigkeit zu geben. Auf Grund der Berichte der Buchhalter, Kassierer und Faktoren mußten die Leiter vierteljährlich einen Bericht über den Geschäftsgang dem Rat der Stadt Steyr zustellen (Ende von § 10). Am Jahresende traten die vier Leiter, unterstützt von noch vier anderen Gesellschaftern, die aus der Zahl der kapitalstärksten Teilhaber zu wählen waren, mit den Faktoren und dem Buch¬ halter zur Aufstellung und Kontrahierung der Jahresbilanz zu¬ sammen (§ 12). Dem Rat war eine Abschrift der Bilanz zu über¬ geben. Auf Grund der Bilanz erfolgte spätestens 14 Tage nach ihrem Abschluß die Gewinnverteilung (§ 13). Die Empfänger hatten zu quittieren. Nur im Falle der Not und mit Zustimmung und Wissen aller Gesellschafter durfte die Gewinnausschüttung verzögert werden. Die Gesellschaft behielt sich vor, später Änderungen des Statuts vorzunehmen, wenn es in ihrem und der Stadt Steyr Interesse notwendig erschiene. Doch sollte hierzu das Einverständnis des Landesherren erforderlich sein und die staatliche Eisenordnung nicht dabei verletzt werden (§ 18). Namentlich behielt sich die Gesellschaft eine gelegentliche Erweiterung ihres Geschäftskreises vor. Wenn es ihr angebracht erschien, sollte später auch die ge- 1 Erstes Kapitel : Deutsche Aktiengesellschaften vornehmlich des 16. Jahrh . 139 samte Eisen- und Stahlverarbeitungsindustrie von Steyr unter ihr Monopol fallen (§ 17). D. h. die Kaufleute, die bisher die Messer, Sägen, Sicheln und Nägel herstellenden Handwerker verlegt und deren Produkte verkauft hatten , sollten dieses „ Gewerbebetriebs“ verlustig gehen und ihre Kapitalien — wenn sie wollten — in die Kompagnie geben. Vorläufig scheiterte die Forderung an der Unmöglichkeit, viele Tausende von Gulden auch noch für diesen Verlag aufzubringen und an dem Widerspruch der Interessenten. So erklärten die Messer- usw. Verlagskaufleute sich nur unter der Bedingung zum Beitritt in die allgemeine Kompagnie bereit, daß ihnen der Messer- usw. Verlag und Verkauf offen bliebe x. Dagegen wurde die „ Gesellschaft des gestreckten Stahls“ der allgemeinen Kompagnie einverleibt (§ 1 des Statuts). Die „ Ge¬ sellschaft des gestreckten Stahls“ war um 1516 gegründet worden 2. Sie schloß, unter erzherzoglicher Genehmigung, zum Zwecke des Verlags von Vorderkernstahl mit den Hammermeistern von Weyer und Umgebung Verlagsverträge ab, „wonach diese allen von ihnen in den welschen Hämmern aufgebrachten Vorderkernstahl nicht in Zainhämmern verarbeiteten, sondern der Gesellschaft verkauften, die für die alleinige Bezugsberechtigung einen höheren Preis zahlte.“ Den auf dem genannten Verlagswege erkauften Vorderkernstahl ließ die „ Gesellschaft des gestreckten Stahls“ in kleinen von ihr erbauten Streckhämmern in der Umgebung Steyrs zu Scharsachstahl, Sensenknüttel, Schwertschrott und anderen feineren Stahl¬ sorten verarbeiten, um sie dann weiter zu verkaufen. Alle Rechte der „ Gesellschaft des gestreckten Stahles“ gingen nunmehr auf die allgemeine Steyrer Eisenhandelskompagnieüber. Ihre Funktionen und Geschäfte wurden von letzterer übernommen. Die „ Gesellschafter des gestreckten Stahles“ erhielten für die Kapitalien, die sie bisher in ihrer Gesellschaft hatten arbeiten lassen, entsprechende Anteile an der allgemeinen Kompagnie. 1 Die Messerverlagskaufleute waren zu einer Vereinigung"der sog. „Einigen Messerhandlung" verbunden. Bittner, a. a. O., S. 608. Eine Untersuchung über die Art ihrer Assoziation wäre als Beitrag zu einer Geschichte der Unternehmung im 16. Jahr¬ hundert hoch willkommen. Ebenso muß noch die Organisation der „V erwandten der Rohr - und Büchsenhandlung zu Steyr “ (Bittner, a . a. O., S. 558 Anm. 3) untersucht werden. 2 Das folgende nach Bittrier, a. a. O., S. 541 f., 519. 140 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. Die „ Gesellschaft des gestreckten Stahls“ muß ihrer Auflösung in die allgemeine Kompagnie des Eisenhandels zugestimmt haben. Sie erscheint neben der Stadt Steyr — die das bisher von ihr schon im Eisenverlag angelegte Kapital einzuschießen versprach — und neben elf bisherigen Eisenverlegern sowie 62 anderen Bürgern als erste Anteilszeichnerin an der neuen Kompagnie1. Auch an der sogenannten kleinen Kompagnie, die der größeren Nachfolgerin die Wege ebnen sollte, erscheint die Gesellschaft des gestreckten Stahls sofort beteiligt. Weil nämlich die genannten gezeichneten Summen nicht genügten, um die eigentliche Kompagnie sofort ins Leben zu rufen, eine Reihe von unverlegten Hammermeistern aber nach Kapital verlangte, um nicht feiern zu müssen, so sollte eine kleine Kompagnie sofort in Tätigkeit treten 2. Als Termin des Beginnes der großen Kompagnie wurde der Martinstag (11. No¬ vember) des Jahres 1582 bestimmt. Ob dieser Termin eingehalten werden konnte, ist aus dem geringen Quellenmaterial, das mir zur Verfügung stand, nicht ersichtlich; jedenfalls arbeitete die große Kompagnie bald darauf in dem Rahmen, den ihr das hier länger besprochene Statut gab 3. Wir brauchen darauf nicht näher ein¬ zugehen. Für uns erhebt sich die Frage : Sind die Leobener Kom¬ pagnie und die Steyrer die einzigen derartigen Gesellschaften jener Zeit gewesen oder lassen sich noch Spuren anderer Unternehmungen namhaft machen, in denen uns schon wichtige Prinzipien der später erst voll ausgebildeten Aktiengesellschaften entgegen treten ? Aus dem schon oben erwähnten Gutachten Steinbergers ist zu erkennen, daß in einer Reihe anderer Städte Österreichs bereits ähnliche „vollkommene Kompagnien“ existierten bzw. bestanden hatten , wie sie für Steyr zur Zeit der Abfassung dieses Gutachtens 1 Die 11 Eisenhändler hatten 35 000 fl. gezeichnet, die 62 anderen Bürger 33 000 fl. Die Mehrzahl gerade der reichsten Eisenhändler hatte sich noch nicht zu Einlagen bereit erklärt, andere hatten üb§r die vorläufig gezeichnete Summe von 3000—6000 fl. hinaus für später größere Kapitalien in Aussicht gestellt. 2 Der ebenfalls erhaltene Statutenentwurf der kleinen Kom¬ pagnie (Stadtarchiv Steyr) gibt uns über deren Organisation Aus¬ kunft. —• Als die Gelder für die zu gründende englisch-ostindische Kompagnie nicht sofort einkamen, wurde zunächst eine Teilgesell¬ schaft, eine sogenannte subordinate association geschaffen. Leh¬ mann, a. a. 0 ., S. 38/9. 3 Bittner , a. a. O., S. 609 f. 1 Erstes Kapitel : Deutsche Aktiengesellschaften vornehmlich des 16. Jahrh . 141 im Werden war. Steinberger führt als Beispiele Hall, Ausser (wohl Aussee), Istria oder Idria ( ?), Schweitz ( ?) und Gasstein an L Die Untersuchung, inwieweit wir es hier mit Kompagnien zu tun haben, die ähnlich wie die Steyrer organisiert waren, muß, da die Literatur nichts bietet, der weiteren archivalisehen Forschung überlassen bleiben. Es soll aber schon hier nicht unterlassen werden, darauf hinzuweisen, daß es sich in den Steinbergerschen Angaben offenbar teilweise um Großgewerkschaften handelt. Das geht auch aus einer Denkschrift des Hofkammerrat Wucherer hervor, der die Fusion der Eisenhändler von Steyr in „aine Compania und Gesöllschaft“ seit 1575 aufs eifrigste betrieb 2. Wucherer führte darin aus : Auch die Salzwerke zu Aussee , Hallstadt und Hall im ( Inntal) seien in vielen Händen gewesen, bis Kaiser Friedrich diese Werke „in a i n Handt gebracht und ist solche Verenderung ohne Schaden zu merklicher hohen Vermehrung des Kammergutes ersprossen und bishero standhafft erhalten.“ Auch habe der Erz¬ bischof von Salzburg etliche Bezirke, zumal die von G a s t a i n , „in aine Gemainschaft und Hand gezogen“. Ebenso sei in der Stadt Passau der Salzhandel in „ aine gemaine bürgerliche Gsöllschafft gezogen“. Wahrscheinlich handelte es sich in Passau um eine ähnliche Gesellschaft, wie sie in München Anfang des 16. Jahrhunderts existierte. In einem Aktenstücke des Lindauer Stadtarchivs heißt es über letztere: „Dann vor jarn in weiland vermellz rneins gn. hern hertzog Aulbrechten regiment haben die von Mönchen die gemainen saltzfertker gar vast gedruckt und den handel gar gros und weitläuff gemacht. Nämlich ain gemaine buchs gehalten ; darein hat ain jeder burger zu Mönchen, der kain handtwerclc gedriben und nit aigen saltz herauf in dis land gefurt, legen mugen 100 oder 50 gülden nach gestalt seins vermugens, doch nit minder noch mer. Darüber sind siben man ver¬ ordnet gewesen, die haben gar ain große anzal saltz durch ire be¬ stellten knecht gefurt, ain mergklichen nutz daran gehept und das saltz in ain solchen aufschlag, darinn es noch ist, gebracht“ 3. 1 Freue nh über , Annales Styrenscs , S. 300, 301. s Franz Anton Mayer, Das Eisenwesen zu Eisenerz in den Jahren 1570—1625. Mitteilungen des historischen Vereins für Steiermark , 33. Heft (Graz 1885), S. 172. 3 Manuskript im Stadtarchiv Lindau i. B., Loc. 100, 6. 41 142 Drittes Buch: Monopole, Kartelle und Aktiengesellschaften im Mittelalter. Vierter Abschnitt, Die Iglauer Tuchhcmdelskompagnie. Das deutsche Städtchen Iglau an der böhmisch-mährischen Grenze, im Mittelalter durch seinen Bergsegen berühmt, erfreute sich seit alters auch eines bedeutenden Tuchgewerbes. Als im 16. Jahrhundert der Absatz zurückging, suchte die Zunft durch die übliche Beschränkung der Gesellenzahl, der Stücke, die der einzelne weben durfte usw., den wirtschaftlich schwachen Meistern ein Minimaleinkommen zu garantieren. Weil aber alle diese Maß¬ nahmen versagten, mußte der Mangel an Absatz und zugleich der Mangel an Betriebskapital bei einzelnen Mitmeistern der Zunft durch ein anderes Mittel beseitigt werden 4. Um den Fernabsatz, zu dem Geld und Unternehmungslust gehörte, zu organisieren, um auch den Druck zu beseitigen, den die Tuchkaufleute auf die ärmeren Webermeister durch Vorschüsse von Materialien, Wolle, Alaun, Röte usw. ausübten, beschloß man die Errichtung einer Gesellschaft, die Verlag und Absatz der Iglauer Tucherzeugnisse übernehmen sollte und die zugleich den Einkauf der Wolle besorgte. Das Statut der Kompagnie wurde von Kaiser Rudolf II. am 17. Juni 1592 bestätigt . Diese Bestätigung (in tschechischer Sprache) ist im Iglauer Stadtarchiv erhalten. Leider konnte aber weder dort noch in Prag 2, Brünn oder Wien 3 das Statut („der Plan“) selbst von mir aufgefunden werden, so daß wir bei der Darstellung der Organisation der Iglauer Tuchhandelskompagnieauf gelegentliches anderes Aktenmaterial angewiesen waren. Wie aus einem Schreiben der Vorsteher der neuen Gründung vom 15. August 1598 deutlich zu erkennen ist 4, war die Iglauer Tuchhandelskompagnie eine direkte Nachahmung der Steyrer all1 Vgl . für das folgende Karl Werner, Urkundliche Ge¬ schichte der Iglauer Tuchmacherzunft, Leipzig 1861, S. 60 ff. 2 Wo ich im Landesarchiv des Königreichs Böhmen und im k. k. Statthaltereiarchiv nachfragte. 3 Wo ich im k. k. gemeinsamen Finanzarchiv nachforschte und im k. k. allgemeinen Archiv des Ministeriums des Innern (dem Archiv der ehemaligen böhmischen Hofkanzlei), sowie im k. u. k. Haus-, Hof- und Staatsarchiv brieflich um Auskunft bat. Auch eine An¬ frage im mährischen Landesarchiv zu Brünn war vergeblich. 4 Manuskript im Stadtarchiv zu Iglau, Copeybuch VII in der Mitte ungefähr des starken, nicht paginierten Bandes. Erstes Kapitel: Deutsche Aktiengesellschaftenvornehmlich des 16. Jahrh. 143 gemeinen Eisenhandelskompagnie1. So dürfte wohl ihre Organi¬ sation. der steyrischen, die oben ausführlich geschildert worden ist, ziemlich ähnlich gewesen sein. Wir hätten es also auch hier mit einer Aktiengesellschaftzu tun. Der Zutritt zu der Gesellschaft war allen kapitalhesitzenden Bürgern der Stadt Iglau erlaubt 2, gleichgültig, ob sie reich oder arm waren oder dem Mittelstand angehörten. Den ärmeren Be¬ völkerungsklassen war dadurch der Beitritt erleichtert, daß man ihnen nahelegte, zu zweien, dreien oder zu mehreren zusammen¬ zutreten, um das Legegeld, das also offenbar eine feste, runde Summe ausmachte, aufzubringen3. Trotz dieser und anderer Be¬ mühungen der Gesellschaft, genügend Kapital für ihr Unternehmen aufzubringen, mußte man auch in Iglau wie in Steyr festverzinsliche Obligationen ausgeben4. Die Geschäftstätigkeit der Iglauer Aktiengesellschaft bestand im wesentlichen in folgendem: Sie kaufte zunächst in Böhmen, Mähren usw. Wolle auf; zum Teil gegen Vorschußzahlung an die Wollproduzenten. Sodann verkaufte die Kompagnie die Wolle an die Iglauer Tuch- und Hutmacher, denen sie dafür ihre fertigen Waren abnahm (bei den ärmeren wiederum natürlich Verlag!). Die so erworbenen Waren verkaufte die neue Gesellschaft auf Messen und Märkten bis tief nach Ungarn und Siebenbürgen hinein. 1 Die Kompagnie, heißt es dort, habe genau wie ,,auch z.u Steuer [Steyr] die Eysen-Compania, aus mangl des starcken Verlags billich aufgericht müssen werden“. Einige arme Weber wären gezwungen gewesen, Verlag zu nehmen. Mit der Zeit aber hätten die Privat¬ leute des schlechten Geschäftsganges wegen den Verlag nicht mehr übernehmen wollen, so sei die Kompagnie nötig geworden. 2 Laut obengenannter Bestätigung Kaiser Rudolfs II. 3 „Da einer allein des Vermögens nicht gewesen sich ihrer 2, 3 und mehr gesessene burger haben mögen miteinander behelfen und ihr capital ihnen einzubringen vergünstiget worden.“ Aus einem Bericht „der Verwalter einer ersamen Gesellschaftshandlung zu Iglau“ an Herrn Hans Löbl, Landeshauptmann in Österreich ob der Enns und Adam Gerninger, Vitztum daselbst, 15. August 1598. Manuskript im Ratsarchiv Iglau. Deutsches Copeybuch VII , un¬ gefähr in der Mitte des starken, nicht paginierten Bandes. 4 In dem eben genannten Bericht heißt es, man habe sich „in und außer landts umb lehenschaft bewerben“ müssen. Vornehme Kaufleute aus Steyr, Prag und aus anderen Städten hätten der Ge¬ sellschaft namhafte Summen gegen jährlich zu zahlende Zinsen geliehen (viele Tausend Gulden). 144 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. Ein eigentliches Monopol hatte die Iglauer Kompagnie nicht. Nach wie vor gab es Tuchhändler in Iglau, die auf eigene Faust Tuche von den Webern einkauften. Freilich „jene Tuchmacher, welche ihre Tücher durch die Gesellschaft abgekauft wünschten, mußten sich verpflichten, nur mit dieser, sowohl was den Woll¬ einkauf als auch den ferneren Absatz ihrer Tucherzeugnisse betraf, in Verbindung zu bleiben und von niemand anderem Rohprodukte einzukaufen oder an keinen anderen Waren zu verkaufen.“ Wie zu erwarten war 1, hat die Geschäftsgebarung der Kom¬ pagnie und ihr anfänglich guter Profit die Gegner mit dem Geschrei „Monopol“ und mit der Heranziehung der betreffenden Reichstags¬ abschiede auf den Plan gerufen. Man warf der Gesellschaft be¬ sonders vor, daß sie daran schuld sei, daß die Wolle eine exorbitante Preissteigerung (von 15—16 fl. pro Zentner auf 28—30 fl. pro Zentner) erfahren habe. Die „Verwalter einer ersamen GesellschaftHandlung zu Iglau“ wußten aber die Angriffe gut zu parieren. Zunächst machten sie darauf aufmerksam, daß Kaiser Rudolf II. im Jahre 1592 ihre Gesellschaft und deren Statuten bestätigt habe. Und „quod imperatori Romano sive (ut textus habet ) principi placet , legis vigorem habeat ac ab omnibus observari debe t “. Außerdem aber betonten die Direktoren der jungen Aktiengesellschaft, daß die Paragraphen der Reichslagsabschiede, die von Monopolen handelten, absolut keinen Bezug auf ihr Unternehmen hätten . Dort würde „von monopoliis und gesellschafften tradiert , die mit dem unserm tuchhandel gar in kheinem similtudine nit stehen et exemplum valde claudicat. Denn die monopolia aida inhibirt werden, mit welichen ein fürkauf geübet wirdt. Da doch von uns aus die waren vielen leuten zu nutz verkauft werden, die in der stadt Iglau von derselben selbst inwonnern selbsten gemacht werden. Dazu wird das geldt vor specerey und englische tuch aus dem heiligen römischen reich in andere weit abgelegene lender weggefürt. Davor Igler Luch das geldt allein in majestät lendern Beheimb und Marhern [Mähren] und derselben inwohner als herrn, grafen ..... vor wolle von tuchmacher wieder ausgeben und da allein verbleiben 1 Es war im 16. Jahrhundert direkt Mode geworden, gefährliche Handclskonkurrcnten auf Grund der Reichsgesetze als Monopolisten anzuklagen . Cfr. R . Ehrenberg, Hamburg und England im Zeitalter der Königin Elisabeth , S. 159. Erstes Kapitel : Deutsche Aktiengesellschaften vornehmlich des 16. Jahrh . 145 thuet .“ Ihre Gesellschaft sei also keine Monopolgesellschaft im Sinne der Reichstagsabschiede , sondern eine „ ehrliche Gesellschaft “, die auch deshalb die kaiserliche Bestätigung erfahren habe. Der Protest der Gegner, die hauptsächlich in den Kreisen der österreichischen Gewandschneider (ob der Enns ) zu suchen sind, vermochte nicht die Auflösung der Kompagnie zu bewirken. Noch 1620 muß sie bestanden haben 1. Und so fest blieb die Er¬ innerung an ihre Wirksamkeit bei den folgenden Generationen haften , daß man 1725 die Neugründung einer Iglauer Tuchhandels¬ kompagnie auf dieselben Prinzipien basieren konnte , die' man im Jahre 1592 schon der alten gegeben hatte . Nur insofern fand eine Änderung statt , daß jetzt nicht nur Iglauer Bürger , sondern jeder¬ mann , der dazu kapitalkräftig genug war, an dem Unternehmen teilnehmen konnte 2. Fünfter Abschnitt. Die Gesellschaft des Amberger Zinnblechhandels. Das bedeutende Amberg -Sulzbachsche Eisengebiet im bayrischen Nordgau , das durch die großen Einigungen seiner Hammermeister schon seit der Mitte des 14. Jahrhunderts für die Geschichte wirtschaft¬ lich interessanter Organisationsformen von Bedeutung wurde 3, erlebte im 16. Jahrhundert die Gründung einer Zinnblechhandelskompagnie auf aktiengesellschaftlicher Grundlage . Offenbar ging die Anregung zu der Gründung der Kompagnie von der staatlichen Gewalt aus 4. ‘Werner , a. a . O., S. 69. UVerner , a. a. O., S. 113 f. 3 Vgl. Ludwig Beck, Die Geschichte des Eisens , I. Bd ., S. 766 ff ., II . Bd ., S. 665 ff. Jetzt auch E . H . Knauer, Der Bergbau zu Amberg , in Mitteilungen aus dem Stadtarchiv Amberg, 2. Heft , Amberg 1913 , S. 9 ff. 4 Der Chronist Michael Schwaiger , der 1538— 1561 Bürger¬ meister der Stadt Amberg war , berichtet : „ Anno 1534 hat seine churfürstliche Gnaden in Amberg eine Gesellschaft der Zinnblech¬ händler aufrichten lassen und geboten , daß alle Blechhammermeister ihr Bodenblech und Dünneisen nirgends anders , dann gegen Amberg, zu den Zinnpfannen geben sollen , welcher Zinnpfannen jetzt 4 allda seind , hat jede ihren Zinnblechmeister und 4 oder 5 Gesellen , seind mehrenteils Burger und beweibet , welche Weiber auch fast ihre stete Arbeit mit dem Reiben und Abwischen der Bleche haben . Diese Bleche werden , wann sie verzinnt sind , auch eines Theils schwarz Strieder , Studien z. Gesch . kapitalist . Organisationeformen . 10 2* 146 Drittes Buch : Monopole, Kartelle und Aktiengesellschaften im Mittelalter. In einem interessanten Schreiben 1vom 14. November 1533 erklärte Pfalzgraf Friedrich der Stadt Amberg , er gedenke in seinem Fürstentum der Pfalz „hieoben in Bayern " einen Blech¬ handel aufzurichten und der Stadt Amberg „ vor anderen gnädiglich zu vergönnen “. Die Amberger Bürger möchten „ denselben Blech¬ handel eine zeitlang selbst verlegen “. Er , der Pfalzgraf , aber ge¬ dächte „etlich Geld zu ihnen in berührten Handel zu legen.“ Wenn die Amberger sich nicht mit der Gründung einverstanden erklärten , dann war Friedrich entschlossen , „ sich des Handels selbst zu unterfangen , oder aber einen andern auf eine Anzahl Jahre [wie er sich mit dem betreffenden Kapitalisten vereinigen würde] den Blechhandel zu führen vergünstigen .“ Die von Friedrich gewünschte Amberger Blechhandelskompagnie ist noch im Jahre 1533 zustande gekommen . Der Pfalzgraf und sein „Hofgesind “, d. h. seine Räte , waren in hervorragender Weise daran beteiligt . Es hatten eingeschossen a: Herzog Friedrich zu Bayern ........ 1000 fl. rh . in Münz Hans von Slamerstorff , Hofmeister . . . . 200 ,, ,, ,, ,, Kanzler Dr . Melchior Soyter (und „ sofern von nöten ein merers “) ........ 200 ,, ,, „ ,, Doktor Hartmann ............ 200 „ ,, ,, „ Chammermeister zu Neumarkt Philip Schelm von Bergen erstens .......... 200 „ „ „ „ und auf Pfingsten ............ 500 „ „ „ „ Melchior von Harstal ........... 100 ,, „ ,, ,, Berthold Mulbegk ............ 100 ,, „ „ ,, Rentmeister ............... 150 „ „ „ ,, Erasmus Nadler (Canzleiverwal ter zu Amberg) 100 „ „ „ „ Kotzing ................. 100 „ „ „ Die von den Räten des Pfalzgrafen gezeichneten Summen scheinen freilich nicht alle sofort eingezahlt worden zu sein. Am in Fäßlein eingeschlagcn und jede Sort mit der Stadt Amberg Wappen, auch des Blechzinnmeisters Zeichen , gebrannt , alsdann in Frank¬ reich , Niederland , Italien , auch in der Frankfurter , Leipziger , Linzer und anderen Messen , und sonderlich auf Nürnberg geführt , ferner in Türkei und Insul , da sie ohne Zweifel hoch werden gehalten . . .“ 1 Kgl. bayrisches Kreisarchiv Amberg . Archiv -Abteilung Amberg Stadt Fase . 326 , Nr . 11. 2 Kgl.bayr . Kreisarchiv Amberg . Amberg Stadt , Fase . 326 , Nr . 32. Erstes Kapitel : Deutsche Aktiengesellschaften vornehmlich des 16. Jahrh . 147 6. August 1534 mußten die Verordneten der Gesellschaft des Am¬ berger Zinnblechhandels an Pfalzgraf Friedrich die Bitte richten, dafür zu sorgen, daß diejenigen seiner Räte , die mit der Einzahlung im Rückstände wären , das bei dem Faktor bis zum Bartholomäitag (24. August ) nachholten . Wer bis dahin einzahlte , sollte an der Dividende teilnehmen , als hätte er zu Pfingsten eingezahlt . Wer nicht , könne nicht mehr in die Gesellschaft kommen 1. Wie hoch sich demgegenüber die Anteile der Amberger Bürger¬ schaft beliefen, habe ich leider aus den Akten nicht feststellen können . In einer Denkschrift des Amberger Rates vom Jahre 1533 ist die Hoffnung ausgesprochen , ca. 4000 fl. zusammenzubringen. Zunächst war offenbar die Begeisterung in der Bürgerschaft für die neue Gründung nicht sehr groß . Der Aufforderung , bis Mit¬ fasten die Einlagen vorzunehmen , mußte die Drohung hinzugefügt werden : „wo ainer oder mer in dem seumig und in der zeit nit legen würde , der oder die sollen hinfürter nit mer zugelassen werden “ . Man muß bedenken , daß bereits in Amberg die „ Große Gesellschaft des Eisenbergwerks “ bestand , eine privilegierte Großgewerkschaft, in die jeder Bürger Geld zu Gewinn und Verlust einschießen konnte 2. ] 1 . c. Fase . 326, Nr . 25. 2 Damit die Gesellschaft des Eisenbergwerks nicht von einigen wenigen Kapitalisten beherrscht wurde, war festgesetzt , daß nur eine bestimmte Summe von jedermann eingeschossen werden dürfte. Scharfe Maßregeln hinderten schon im 15. Jahrhundert die Um¬ gehung dieser der Allgemeinheit zugute kommenden Bestimmung. Da heißt es z. B. : „Es sollen 2 eeleuth für ainen tail oder ain person geacht werden. Wellich manns - oder Weibspersonen aber verwittibt sein, der yede wirt auch für ainen tail angenommen. Desgleichen werden die unmündigen kinde , die in Verwaltung der vermundtschaft und von ieren eitern vertailt sein und aigne guter haben , ir sein vil oder wenig, fur ainen tail zugelassen. Dartzu mögen sich derselben kinde eitern , vater und inueter wo si mit den kinden , wie obstet, vertailt , si sein verheurat oder nit , solhen einlegens auch geprauchen . . . . Man und weib, die mit gefaßter hanndt wollen sitzen und sich yedes seins guets selbst geprauchen , . . . yeder mit dem einlegen in sonders zutzelassen , ist aus bewegenden Ursachen abgestellt . Und vermittels gütlicher gnaden sein wenig unter uns, si hetten sollich zwifachtig leggelt ..... Es wirt aber von gemaines nutzes wegen und im peßten damit der perg nit allein under den vermöglichen stegk erlassen Kgl . . .“ . bayr . Kreisarchiv Arnberg, Amberg Stadt Fase . 31, Nr. 50. 10 * 148 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. Freilich konnte schon 1464 die Großgewerkschaft nicht mehr alle Einschüsse verwerten , es mußte eine Kapitalreduktion statt¬ finden , die aus folgender Tabelle ersichtlich ist : • 1464 waren an der sogenannten großen Gesellschaft des Eisen¬ bergwerks zu Amberg beteiligt 1: Stadt Arnberg . . 1000 (800) Hans Hufnagel . . 98 (90) 500 97 Hans Becherer . . Hans Clopfer . . . (50) (300) Lienhart Rurer . . 500 (90) (400) Markart Fuchsin . 95 Hans Hubmair . . 500 (400) Contz Cantzier . . 68 14 (54) Michael Hecker . . 59014 (470) Hans Breytenloer . 500 (400) — Fritz Urspringer . 500 (400) LIeinz Mock . . . 200 — Hans Witrer . . . 500 (300) Contz Altmeister . 200 — Lorenz Modler . . 500 (400) Albrecht Heuptel . 200 — Heinrich Heydens Jorg von Riechen . 100 — Kinder..... 445 70 (340) Thoma Tyeffenbach Heintz Witrer . . 34814 (300) Hayden Gropp und — Hans Freißlich . . 433 70 (330) Dyetz..... — Felix Freißlich . . 43814 (330) Hans Volkmayr 70 — Nicolaus Bachmann 410 (347) Herr Jacob Wechsler 70 Hermann OchselEngelhart Alt— meister . . . . 341 (156) inayr...... 70 — Hans Weiß . . . 315 (240) Jorg Schondel . . 70 — Seitz Moler . . . 30914 (250) Alt Badei Beck . . 70 — Hans Streubl . . . 26314 (208) Gregory Alhart, . . 55 — Ott Bühler . . . . 256 (208) Marckart Beumel . 55 — Jorg Meinhart . . 321 (252) Ulrich Eckkell . . 50 — 219 Merten Mendlin . . 30 Dietz Sayler . . . (200) — 180 25 Heintz Forsterin . Jung Hans Witrer. (163) Hans Totzier . . . 140 (127) St . Merteins Zehend— Conrat Meiner . . 132 (110) lut....... 100 Sobald schon in den ersten Jahren der Geschäftstätigkeit der 1533/34 gegründeten Amberger Blechhandelskompagnie die Ge¬ schäfte der Firma sich recht gut anließen , drängte auch das bürger¬ liche Kapital kräftig heran , so zwar, daß es schon bald abgelehnt bxw. bis auf eine Vergrößerung der Aufgaben der Gesellschaft 1 Kgl . bayr . Kreisarchiv Amberg , Amberg Stadt Fase . 31, Nr . 28. Die eingeklammerten Zahlen bedeuten die Einlagen nach der Kapital¬ reduktion. Erstes Kapitel : Deutsche Aktiengesellschaften vornehmlich des 16. Jahrh . 149 vertröstet werden mußte 1. Um 1556 sollen mehr als 150 Personen in der Gesellschaft gewesen sein 2; ein anderes Mal wird allerdings nur von 80 Teilhabern gesprochen. Die Außenstände der Gesellschaft waren schon bald nach der Gründung nicht unbedeutend . 1540 schuldeten ihr 18 Blech¬ hammermeister insgesamt 7700 fl. Der größte dieser Schuldner stand mit 744 fl. zu Buche . Der kleinste mit 110 fl. Außer¬ dem hatten die Amberger Blechzinnmeister 2000 fl. Verlags¬ schulden 3. Die erste sichere Nachricht über die Anzahl der Teilhaber und die Höhe des gesamten in der Gesellschaft des Zinnblechhandels zu Amberg investierten Kapitals konnte ich aus dem Jahre 1614 feststellen 4. Am 15. Februar dieses Jahres waren ca. 25 000 fl. zu Gewinn und Verlust von 117 Gesellschaftern eingezahlt . Die kleinsten Beiträge lauteten auf 25 fl., die höchsten auf 450 fl. Letztere Summe war freilich nur von zwei Teilhabern , von der Stadt Amberg und der Stadt Neumarkt , investiert worden . Außerdem waren „uff Interesse “, d. h. als Depositen zu festem Zins, 11 850 fl. in der Gesellschaft . Diese Summe setzte sich aus 13 Posten von 250—5000 fl. zusammen . Den höchsten , nur einmal vorkommenden Posten von 5000 fl. hatte das kurfürstliche Pfennigmeisteramt (Finanzministerium ) gegeben , den nächstgrößten (1000 fl.) Hans König , der Pfennigmeister , aus seinen privaten Mitteln. Es versteht sich bei der heftigen Abneigung des 16. Jahr¬ hunderts gegen jede starke Betonung des kapitalistischen Geistes von selbst , daß auch die Gesellschaft des Amberger Zinnblech¬ handels sich den Vorwurf des unerlaubten Monopols gefallen lassen mußte . Interessanter als die Anklage ist deshalb auch die Ver¬ teidigung , die die Verordneten der Gesellschaft an den Ankläger 1 Dem Hammermeister Paul Hegner wurde die Investierung von 800 fl. versagt. Man gab ihm den Trost, „wo sich initier Zeit zutruege, daß mer anlagen gelts zu ainem z i n k a u f oder anderem nottürftig würden“, so sollte Hegner und andere Hammermeister Kapital einschießen dürfen. Tatsächlich hat sich die Gesellschaft zeitweise mit dem Gedanken getragen, den Schlackenwalder Zinn¬ kauf —- wir kommen später hierauf zurück — zu übernehmen. 2 Kgl . Kreisarchiv Amberg, Amberg Stadt Nr. 106/327. 3 .a a. 0 „ Nr. 79/327. * a. a. 0 ., Nr. 158/329. 150 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. (Wiwold von Wirsberg ) richteten h Man hätte sich, führten sie aus , des Anwurfs „als ob der Zinnblechhandel allhie monapollisch“ sei, nicht versehen . Bekanntlich wäre der genannte Handel durch den Pfalzgrafen Friedrich mit großer Mühe und Kosten zu Wohl¬ fahrt , Nutz und Frommen von Land und Leuten wieder in die Pfalz gebracht worden , nachdem er zuvor eine gute Anzahl Jahre mit sonderlichem Nachteil der Pfalz und ihrer Einwohner in fremden Landen gestanden . Und doch würden die Roherze , woraus das Blech hergestellt werde , in der Pfalz gewonnen . Und charakteristisch schließen die Aufsichtsräte der Gesellschaft wie folgt : „ Ob nun das, wo ein landsfürst mit denselben , den seinen, darunder auch spittal und andere almussen -heusser , dergleichen nit wenig wittib , wayssen und andere notturftige personen begriffen , mit gueter , erbarer Ord¬ nung und handtierung hielft , auch hiertzu die seinen, solcher Ord¬ nung zu geleben , mandirt , für monapollischs angezogen werden wille, das würdt einem , wo es an gepuerende ort gelangen soit , noch vil weniger gegen andern hohen potentaten , welche die handthirungen in ire aig'ne chamergefelle ziehen, beschwerdtlich zu verantworten steen ..... “ Solange sich die Gesellschaft der Gunst des Kurfürsten erfreute, konnten Klagen über ihren Monopolismus ihre Entwicklung nicht hindern . Offenbar machte die Gesellschaft in der zweiten Hälfte des 16. Jahrhunderts recht gute Geschäfte und konnte nicht un¬ erhebliche Dividenden zahlen . Man sieht das aus der Tatsache, daß sich das Kapital in die Gesellschaft drängte ; größtenteils aller¬ dings vergeblich . Die Amberger Kirchturm -Wirtschaftspolitiker , die das Wort in dem Unternehmen führten , waren nicht willig und wohl auch nicht fähig , die Gesellschaft und ihren Handel voll zur Entfaltung zu bringen . Ihre mißgünstige Eifersucht besorgte , viele neue Teilhaber könnten den Nutzen der alten Teilhaber schmälern. Aus solchen Erwägungen heraus petitionierte die Gesellschaft schon 1540 an den Pfalzgrafen , er möge den Ausschluß der „Aus¬ länder “ gestatten . Sie würden gern diejenigen Amtleute und Räte seiner fürstlichen Gnaden , mit deren „ Darlegung und Wagnis “ die Gesellschaft erstlich in „ den Gang und das Werk gebracht “ und bisher erhalten worden sei, soweit sic noch im Fürsten1 7. September 1553. Kgl. Kreisarchiv Amberg. Amberg Stadt, NT. 127/328. Erstes Kapitel: Deutsche Aktiengesellschaften vornehmlich des 16. Jahrh. 151 tum wohnhaft waren , in der Gesellschaft des Amberger Zinn¬ blechhandels sehen. Aber anders verhielt sich die Sache mit den¬ jenigen , die sich außerhalb der Pfalz , in fremden Herrschaften häuslich niedergelassen hatten . Die strichen nur die Dividende ein, nützen könnten sie der Gesellschaft und ihrem Handel absolut nichts . Darum sei es nicht anders als billig, wenn man sie aus der Sozietät entfernte 1. Ob das durch Rückgabe ihrer Anteilscheine an die Gesellschaft geschehen sollte oder durch freien Verkauf der¬ selben geschehen durfte , muß dahingestellt bleiben , jedenfalls läßt sich aus den Akten der Nachweis führen , daß in einem Falle ein freier Verkauf stattfand 2. Auch später noch haben die Einwohner der Pfalz und ins¬ besondere die Amberger Bürger die Gesellschaft des Zinnblech¬ handels für ihr Reservat betrachtet und dadurch natürlich deren Entwicklung zu einer größeren Bedeutung hintangehalten . Noch in der „ Reformation “ der Statuten der Eisenblechhandelskompagnie (1595) heißt es : „Und weilen der bandel seiner churfürstlichen Gnaden angehörigen und underthanen zue nutz und vortheil an¬ gesehen, sollen sie [die neuernannten Verordneten der Gesellschaft] ehisten erkhundigen was vor frembde , die im churfürstenthum Pfaltz gar nicht gesessen, gelt im handel liegen haben , denselben das ihre abgelegt und hingegen seiner churfürstlichen Gnaden angehörige und underthanen an der frembden statt , doch mit der maß zuegelassen , daß solcher, wie er vilen zu vortheil gemeint, auch vilen und nicht wenigen personen gegönnet werden und darumb künftig wo einer oder ander aus seiner churfürstlichen Gnaden räthen , dern wittibin oder waisen gelt in den handel zu legen begerte , von deren keinem mer als 400 fl., einem canzleyund rathsverwanten bei den statten , deren wittibin oder waisen 300 fl., einem burger oder andern underthanen , deren wittibin oder waisen 200 fl. anzulegen gestattet und wer jetzo höher summen in handel bette , dieselben uff jetzt gemelte summen gerichtet , das übrig hinausgegeben und den andern aus obigen zugelassen werden. 1 Kgl. Kreisarchiv Amberg. Arnberg Stadt , Nr. Sl/327. 2 „So wissen wir doch, daß unser geschwcig, die Nadlerin selig als sie sich von Ambergk ghen Ingolstadt zuthun fürhabens ire hauptsumma im blechhandel einem andern verkauft und solches ist zugelassen worden.“ (zum Jahre 1564). Kgl. Kreisarchiv Amberg. Amberg Stadt , Nr. 137/328. 1 152 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. Dabei doch achtung zu geben und zu verhüten ist , im fall ein vater, der gelt im handel gehabt , abstürbe dessen wittib hernach von khindern abgetheilt und die im handel liegende summa der khinder einem zugetheilt würdt , daß nicht über das der wittib ein sonderbar einlag zu thun nachgeben werde “ L Interessant ist das Verhältnis der Gesellschaft zum Pfalzgraf und zur öffentlichen Gewalt überhaupt . Offenbar war die Gründung in erster Linie mit der Initiative der Regierung zu danken gewesen. Der Pfalzgraf und die kapitalkräftigen seiner Räte waren auch, wie wir sahen , mit bedeutenden Summen beteiligt . Wo immer es möglich war , unterstützte die Regierung die Gesellschaft mit Rat und Tat . Das Wichtigste war dabei die Monopolstellung im Eisen¬ blechgroßhandel , die der Gesellschaft eingeräumt wurde . Alle Blechhämmer „ der Reviere Amberg und Sulzbach “ mußten ihr Blech an die Gesellschaft liefern , die es dann in ihren Zinnpfannen verzinnte und nach Fertigstellung vertrieb . Aber auch in der Ge¬ schäftsführung im einzelnen erfreute sich die Gesellschaft der tätigsten Beihilfe der Regierung . Als es z. B . galt , den Absatz¬ kreis der Gesellschaft des Zinnblechhandels zu erweitern , da schrieb Pfalzgraf Friedrich an den Juden Simon nach Frankfurt a. M. und erbat sich genaue Auskunft , wie die Nürnberger Zinnblech¬ händler bisher den Absatz in Frankfurt und Antwerpen organisiert hätten 2. Durch ein besonderes Schreiben bemühte sich auch der Pfalzgraf , den tüchtigen Kaufmann Hans Steinhäuser als Faktor für die Gesellschaft zu gewinnen 3 usw. 1 Kgl. Kreisarchiv Amberg. Amberg-Stadt, Nr. 157t/328. 2 Aus dem Jahre 1537. Kgl. Kreisarchiv Amberg, AmbergStadt, Nr. 53/326. Die interessante Antwort des Juden, der wahr¬ scheinlich auch sonst mit dem Pfalzgrafen in Geschäftsverbindung stand, empfiehlt einen Frankfurter Kaufmann Heinrich Wixstetter. Derselbe habe bisher von den Nürnbergern Blech gekauft und nach Antwerpen, eventuell weiter nach England, Frankreich usw. ver¬ kauft. Wixstetter sei bereit, nach Amberg zu kommen, um zu sehen, ob er mit der Gesellschaft in Geschäftsverbindungkommen könne. 3 Steinhäuser stellte folgende Bedingungen: 1. Entlastung von allen städtischen Ehrenämtern; 2. für Kredite, die er mit Wissen des „Ausschusses“ gewähren würde, ist er nicht haftbar; 3. keine Ver¬ pflichtung, größere Reisen zu machen; 4. die Vergütung für Reisen, die Beauftragte Steinhäusers ausführen, zahlt die Gesellschaft; 5. zum Auf- bzw. Abladen des Bleches wird ihm ein Diener gestellt; 6. als Gehalt erhält Steinhäuser jährlich 100 fl. 1 Erstes Kapitel : Deutsche Aktiengesellschaften vornehmlich des 16. Jahrh . 153 Später sind diese Bemühungen dem Landesherrn dann wieder zugute gekommen . Schon im Jahre 1550 konnte sich Pfalzgraf Friedrich 4000 fl. aus der Zinnbleclihandelsgesellschaft ein Jahr lang unverzinst „ fürleihen “ lassen Und seit 1595, seit der „Reformation “ der Statuten der Gesellschaft , nahm der Kurfürst sogar „ allen jars den 15. Pfennig an dem gewinn “ für sich in Anspruch 2. Über die innere Organisation der Gesellschaft des Amberger Zinnblechhandels ist etwa das folgende zu sagen. Wir sahen bereits, daß jeder Bürger von Amberg und Sulzbach das Recht hatte , in die Gesellschaft einzutreten . Dasselbe R.echt stand den Hammer¬ meistern und den Hofleuten des Herzogs zu. Der Austritt konnte nach einjähriger Kündigung jederzeit erfolgen 3. Außer mit den Anteilen der Aktionäre arbeitete die Gesell¬ schaft mit festverzinslichen Einlagen . Diese Einlagen waren fünf Jahre unkündbar , wenigstens von seiten der Gläubiger . Die Ge¬ sellschaft dagegen durfte alljährlich nach vorangegangener viertel¬ jährlicher Kündigung die Einlagen aufsagen 4. 1 Nach Amberger Akten. 2 Kgl. Kreisarchiv Amberg'. Amberg-Stadt , Nr . 157t/328. 3 „Zum andern soll ein jeder gewergkh und mitleger sein erlegt gelt, welcher Zeiten er will, aufzusagen und zu erfordern haben und keiner bedranngt werden, dasselbig lennger, dann sein gefalcn stet, ligen zu lassen. Doch daß solich aufsagen ein jar lang vor der bezalung beschehe und erst nach erscheinung des jars sol der so auf¬ gesagt hat , haubtgelts und gewinnung ob es dieselben ertragen hett, auch der Verlust gewarten .“ Aus der „ Ordnung im plechhandel auf des durchl . hochgeb. fürsten u. lierrn , herrn Friedrichen , pfaltzgraven bei Rhein u. hertzogen in Baiern meines gn. herrn verpesserung furgenomen (1534)“ . Kreisarchiv von Amberg. AmbergStadt , Nr. 326/25. Auch die folgenden Zitate sind der „ Ordnung“ von 1534 entnommen. 4 „Zum dritten ob etlichen ir einlag auf gewin und abgang wolte beschwerdlich sein, die sollen mit irer einlag auf gewondlich gepurendt Verzinsung angenomen werden. Doch daß keiner sein einlag in 5 jarn, den negsten von dem lag seiner einlag an zu rechnen, abzufordern hab . Aber die gewergkhen sollen jeden jars solche einlag mit bezalung ausstendiger verfaluer gülten zu erledigen macht haben . Und daß ainem jeden ein virtl jar sollich ablosung vor der abgekliundten zeit wissentlich gemacht werde und dann die ablosung auf selbige abgekhundte Zeiten beschehe.“ 154 Drittes Buch : Monopole, Kartelle und Aktiengesellschaften im Mittelalter. Die Geschäfte der Gesellschaft besorgten zwei (eventuell auch mehr ) Faktoren gegen festes Gehalt . Dieselben sollten durch Einlagen besonderes Interesse an der Entwicklung der Firma haben 1. Die oberste Leitung batte ein Aufsichtsrat (ein „rath “ heißt es in den Urkunden der Gesellschaft ), der die Rech¬ nung abnahm und andere wichtige Obliegenheiten der Gesellschaft besorgte 2. Natürlich traf die Errichtung der Ambergei ' Zinnblechhandels¬ gesellschaft schwer die in Nürnberg gesessenen Großhändler 3, die das Produkt der Amberger und Sulzbacher Blechhämmer bis dahin empfangen und zu Zinnblech verarbeitet weiter vertrieben hatten. Die Nürnberger mußten daran denken , sich mit der Amberger Gesellschaft auf irgend einer Grundlage zu vereinigen . Sie ver¬ suchten 4 eine Vereinigung der beiderseitigen Interessen auf die Weise zustande zu bringen , daß sie sich bereit erklärten , die gesamte Produktion von der Amberger Zinnblechhandelsgesellschaft zu übernehmen . Die Gesellschaft sollte alles Blech, gezinntes und ungezinntes , das in ihre Hände gelangte , den Nürnbergern und. sonst niemandem verkaufen und frei nach Nürnberg transportieren. Ausgenommen war das Blech , das der Landesfürst und seine Freunde und Verwandten für eigenen Gebrauch bedurften . Das 1 „Zum vierdten sollen mit allerseits der gewergkhen vorwissen zwen oder mer ditz handls zu ausrichtern und factoren umb zimblicli belonung , die auch mit einlegung irs gelts dem handl verwanth sein.“ 2 „Daneben ein rath , in antzal etlicher personell, rechnung aufzunemen und die gemainen täglichen furfallend hendl abzurichten, .....die auch mit kaufen und verkaufen der plech, zines und ander notturft nach irem guetbedungkhen handln mögen und dartzu sich befleißen zu Frangkhfort , Leibtzigkh und ander enden erfarung zu machen , damit sollich plechwerch statlich und mit nutz mögen vertriben , und wie sie fur guet ansehen, in Verwechslung ander warhen oder pfenbarten gebracht werden. Wurden aber aine oder mer Sachen inen beschwerdlich zu verrichten furfallen , dan mögen die factoren und erwellt räthe ander mer ir gewergkhen, so vil sie der¬ selben im fall der notturft gehaben mögen, zu inen erfordern und nach rath derselben handln .“ 3 Die Großhändler waren die Verleger der Nürnberger Ziriner. 4 Das folgende alles nach einem Manuskript des Amberger Kreis¬ archivs , Abt . Amberg Stadt , Fase . 326/25 . Datiert Neumarkt , 2. Sept. 1534. Erstes Kapitel : Deutsche Aktiengesellschaften vornehmlich des 16. Jahrh . 155 sollte die Gesellschaft frei liefern dürfen . Die Nürnberger Zinner und ihre Verleger verpflichteten sich demgegenüber ganz von dem Zinnen abzusehen und der Amberger Gesellschaft ihre Zinnarbe'iter zuzuweisen , damit, in Amberg noch „4 oder 5 zinpfannen uffgericht“ werden könnten . Auch ihre Werkzeuge und noch vorhandenen Roh¬ materialien stellten die Nürnberger der Gesellschaft zur Verfügung, wie sie sich auch anheischig machten , bis zu 1000 fl. als festver¬ zinsliches Depositum zu Verlagszwecken der Gesellschaft zur Ver¬ fügung zu stellen. Für den Kontrakt war vorläufig eine Dauer von fünf Jahren vorgesehen . Die Preise wurden für die ganze Vertragsdauer fest¬ gesetzt . Jedoch hatte , im Falle das Blech auf - oder abschlüge, alljährlich eine entsprechende Preisänderung einzutreten. Es war nicht zu verkennen , daß eine derartige Abmachung auf alleinige Lieferung auch dem Interesse der Gesellschaft des Am¬ berger Zinnblechhandels entsprach . Die Gesellschaft hatte ja zunächst keine Erfahrung im Absatz des Blechs in Frankfurt , Ant¬ werpen usw. Wenn sich aucli dieser Mangel korrigieren ließ, blieb noch immer der Nachteil , daß die Gesellschaft keine Rückfracht aus Frankfurt , Antwerpen usw. zu verladen gehabt und so die Spesen wesentlich verteuert hätte . Außerdem galt es, das folgende zu beachten . Wenn man sich nicht mit den Nürnbergern zu¬ sammentat , so würden diese natürlich das unverzinnte Zinnblech von den Sulzbacher und Amberger Hammermeistern zu erlangen suchen . Sie würden diesen mehr bieten , als die Gesellschaft an sich zu geben nötig hatte . Das mußte die Preise des Roheisen¬ blechs in die Höhe treiben und außerdem die Hammermeister allen obrigkeitlichen Verboten zum Trotz zum heimlichen Verkauf ihrer Produkte nach Nürnberg veranlassen . Dagegen hörte jeder heim¬ liche Verkauf der Hammermeister und jede Weiterung mit ihnen auf, sobald in Nürnberg keine Zinner mehr existierten. Auch der weitere Vorteil , der sich für die Amberger Zinnblech¬ handelsgesellschaft aus einem Kontrakte alleiniger Lieferung nach Nürnberg ergab , leuchtete leicht ein. Die Nürnberger hatten sich erboten , das Blech sofort bei Lieferung bar zu bezahlen . Dadurch konnte „ ermelter handl mit 5000 oder 6000 gülden so statlieh und wol, als wenn er frei und unverbunden sein soit , mit 20 000 gülden betriben und verlegt werden und 100 gülden so vil als sonsten 300 ungeverlich nutzs ertragen .“ 156 Drittes Bach: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. Es ist aus den Akten nicht ersichtlich , ob der Pfalzgraf er¬ laubte , daß die Amberger mit den Nürnbergern „ einen contract und verainigung uffrichteten “ oder ob er wünschte , daß der Handel frei getrieben werde und die Gesellschaft gehalten sein sollte , „die plech wo ihr gevellig oder gelegen zu verkaufen “ . Eine Geschichte der Amberger Gesellschaft des Zinnblechhandels muß Aufschluß darüber geben 1. Hier kam es mir lediglich darauf an , zu zeigen, wie man auch in dem vorliegenden Falle darauf bedacht war , eine unnötige Konkurrenz auszuschalten und wie man durchaus imstande war , die ökonomischen Vorteile eines solchen Vorgehens bis in die letzten Konsequenzen hinein zu ermessen. Zweites Kapitel. Kartelle des 14. bis 18. Jahrhunderts. Fast allgemein ist heute unter den Nationalökonomen die Ansicht verbreitet , daß Unternehmerverbände , die man als Kartelle, Syndikate usw. bezeichnet , eine Erscheinung des 19. Jahrhunderts, besonders der zweiten Hälfte dieses Zeitraumes seien und nur in einigen wenigen Fällen in England noch in das letzte Viertel des 18. Jahrhunderts hineinragten 2. Ganz vereinzelt und schüchtern nur fragte man sich, ob diese späte historische Ansetzung der Entwicklung des Kartellwesens mit 1 Die Amberger Zinnblechhandelsgesellschaft hat übrigens Schule gemacht . Fiir den sächsischen Eisenhandel sollte um das Jahr 1557 eine Gesellschaft aufgerichtet werden, wie „ dann in der Pfalz “ eine war . „ Diese geselsehaft solte alle hammerschmide verlegen und alles eisen in ire hende bringen .“ Weimar, Staatsarchiv Reg. T. fol. 506 a. 2 R . Lief mann, Die Unternehmerverbände (Konventionen, Kartelle ), ihr Wesen und ihre Bedeutung . Freiburg i. Br . 1897, S. 135 ff. Von der sonstigen Literatur seien nur noch — weil in gewissem Sinne die opinio communis darstellend — die Artikel „Kartelle " des Handwörterbuchs der Staatswissenschaften und des Wörterbuchs der Volkswirtschaft genannt . Für die meinen Aus¬ führungen zugrunde gelegte Definition der Kartelle vgl. J . Landes¬ berg e r , Gutachten über die Frage : Welche Maßregeln emp¬ fehlen sich für die rechtliche Behandlung der Industriekartelle ? Ver¬ handlungen des 26. deutschen Juristentages , II . Bd . (Gutachten ). Berlin 1902, S. 296 f. Zweites Kapitel : Kartelle des 14. bis 18. Jahrhunderts . 157 den Tatsachen der Wirtschaftsgeschichteim Einklang stände. So sprach Ad. Menzel— auf dem römischen Rechte und den Reichs¬ tagsabschieden des 16. Jahrhunderts fußend — die Ansicht aus, daß für Handel, Handwerk und Transportwesen Kartelle schon im Altertum und im Mittelalter existiert haben müßten 1. Deutlich genug heißt es ja in dem Gesetze Kaiser Zenos, De monopoliis et de conventu negotiatorum illicito vel artificum ergolaborumquenec non balneatorum prohibitis illicitisque pactionibus: „ Jubemus ne quis cuiuscumque vestis aut piscis vel pectinum forte aut echini vel cuiuslibet alterius ad victum vel ad quemcumque usum perti¬ nentis speciei vel cuiuslibet materiae pro sua auctoritate, vel sacro iam elicito aut in posterum eliciendo rescripto aut pragmatica sanctione vel sacra nostrae pietatis adnotatione, monopolium audeat exercere, neve quis illicitis habitis conventionibus conjuraret aut pacisceretur, ut species diversorum corporum negotiationis non minoris, quam inter se statuerint, venumdentur. Aedificiorum quoque artifices vel ergolabi aliorumque diversorum operum pro¬ fessores et balneatores penitus arceantur pacta inter se componere, ut ne quis quod alteri commissum sit opus impleat aut iniunctam alteri sollicitudinem alter intercapiat : data licentia unicuique ab altero inchoatum et derelictum opus per alterum sine aliquo timore dispendii implere omnique huiusmodi facinora denuntiandi sine ulla formidine et sine iudiciariis sumptibus. Si quis autem monopolium ausus fuerit exercere bonis propiis spoliatus perpetuitate damnetur exilii. Ceterarum praeterea professionum primates si in posterum aut super taxandis rerum pretiis aut super quibuslibet illicitis placitis ausi fuerint convenientes huiusmodi sese pactis constringere, quinquaginta librarum auri solutione percelli decernimus: officio tuae sedis quadraginta librarum auri condemnatione multando, si in prohibitis monopoliis et interdictis corporum pactionibus com¬ missas forte, si hoc evenerit, saluberrimae nostrae dispositionis con¬ demnationes venalitate interdum aut dissimulatione vel quolibet vitio minus fuerit exsecutum2. Emil Steinbach hat auf eine 1 Schriften des Vereins für Sozialpolitik 61, S. 32. 2 Corp. iur . civ . Cod . IV , Tit . 59. Peutinger übersetzt und interpretiert in seinem früher genannten Gutachten über das Kupfer¬ syndikat (Msc . Augsburger Stadt -Bibi . Cod . 2° Aug . 398, Bl . 193 r) die auf Kartelle bezügliche Stelle des Zenonianischen Gesetzes wie folgt : „ . . . daß niemand fur sich selbs oder aus zugebung kuniglich * 158 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. humoristisch gehaltene Hinweisung aut schon ältere Gesetze Roms gegen kartellistische Verabredungen hingewiesen1. Sie findet sich zu Anfang des dritten Aktes der „Captivi“ des Plautus, dort wo der hungrige Parasit Ergasilus seinen Plan wie folgt darlegt: Nunc barbarica lege eertum ’st jus meum omne persequi. Qui consilium iniere, quo nos victa et vita prohibeant, His diem dicam , inrogabo multam , ut mihi coenas decem Meo arbitratu dent , quum cara annona sit ; sic egero 2. Ebenso deutlich wie das römische Recht sprechen verschiedene Reichstagsabschiededes 16. Jahrhunderts das Kartellierungsverbot aus 3. Hier war also von Menzel bei einiger Beachtung schon der naheliegendsten Quellen die Situation nicht zu verkennen. Wenn Menzel dann aber, der allgemeinen Ansicht entgegenkommend, meint, neuartig sei die Erscheinung im 19. Jahrhundert auf dem Gebiete der Großindustrie und neuartig seien die Organisationsformen, unter denen uns heute die Kartelle entgegentreten, so ist der eine Teil dieser Ansicht so irrig wie der andere. Ad 2 habe ich in einem Aufsatz des „Historischen Jahrbuchs“ 4 gezeigt, wie stark sich die Organisationsform eines Kartells aus dem Jahre 1743 den .heutigen nähert, ja in ihrer Kompliziertheit viele von ihnen über¬ ragt. Ad 1 hat H. Levy für England nachgewiesen, daß es dort bereits im frühen 18. Jahrhundert Kartelle in der Großindustrie macht ainicher war und kaufmannsgut in Vermischung mit anderen begeben oder handeln soi und sonderlich mit andern sich nit zu vertragen ainich war und kaufmanns¬ gut dermaßen und nit anderst zu verkaufen, dann laut irs Vertrags Satzung ze thun. Dann die oder dergleichen vertrage als unzimlich geacht werden sollen , die weil doch sollichs dem gemainen nutz widerwertig .“ 1 E m i 1 Steinbach, Der Staat und die modernen Privat¬ monopole . Vortrag , gehalten in der Wiener Juristischen Gesell¬ schaft am 17. Dez . 1902 . Wien 1903 , S. 18. 2 „Jetzt werde ich sicherlich mein volles Recht nach dem aus¬ ländischen Gesetze verfolgen . Diesichver abredet haben, uns das Leben zu verteuern , werde ich verklagen , und ich werde als Buße begehren , daß sie mir zehn Mahlzeiten nach meiner Aus¬ wahl geben , obwohl jetzt alles teuer ist . Das werde ich tun .“ 3 Wir kommen noch darauf zu sprechen. 4 Ein Kartell deutscher Kaufleute aus dem Jahre 1743 . Histo¬ risches Jahrbuch 1911 , S. 49— 02. 4 Zweites Kapitel : Kartelle des 14. bis 18. Jahrhunderts . 159 gab 1. Für die Thüringer Glasindustrie konnte W. S t i e d a schon auf ein interessantes Preiskartell aus dem Jahre 1735 auf¬ merksam machen 2. Neuerdings aber zeigt uns das schon öfter genannte Buch von W. Möllenberg, ebenso wie M ücks Publikation, daß bereits die deutsche Großindustrie des 16. Jahr¬ hunderts vollständige Kartellbildungen kannte und durchzuführen wußte. Der erstgenannte Autor hat uns im siebenten Kapitel seines Buches mit den interessanten, schließlich von Erfolg ge¬ krönten Kartellierungsversuchen Christoff Fiirers im Mansfelder Saigerhandel bekannt gemacht 3. Seit Christoff Fürer gegen den hartnäckigen Widerstand des konservativen Jakob Weiser d. Ä. von Nürnberg ein Syndikat der Hüttengesellsehaften Gräfenthal, Schwarza, Steinach, Arnstadt, Luderstadt, Eisfeld und Leutenberg zustandegebracht hatte , stiegen die Dividenden der einzelnen Ge¬ sellschaften bedeutend. Leider sind wir nur über die Gewinne einer von ihnen, der Leutenberger, im Zusammenhang unterrichtet . Die Dividenden dieser Gesellschaft hatten 1527 und 1531, also vor Gründung des Syndikats, noch 11 % betragen, 1535 kamen 14 %, 1536 sogar 19 % zur Verteilung und 1537 konnten auf 100 fl. Anteil nicht weniger als 22 fl. 9 gr. Gewinn ausgeschüttet werden. Auf ein Kupfersyndikat um die Wende des 15. Jahrhunderts hatte vor längerer Zeit bereits Ehrenberg aufmerksam ge¬ macht . Zu einer bisher unbeachteten Kartellverabredung kam es auch bei Gelegenheit des früher schon in einem anderen Zusammenhänge 1 H . L e v y , Monopole , Kartelle und Trusts in ihren Be¬ ziehungen zur Organisation der kapitalistischen Industrie , S . 08. 2 W . S t i e d a , Ältere deutsche Kartelle . In Schmollers Jahrbuch XXXVII (1913 ), S. 725 ff . Dort hat Stieda zusammen¬ gestellt , was an älteren deutschen Kartellbildungen (zumeist in der allerletzten Zeit ) bekannt geworden ist . Auch auf die Kartell¬ bildungen im Holland des 17. Jahrhunderts ist hier im Anschluß an A' ndré - E. S a y o u s (Les ententes de producteurs et de com¬ merçants en Hollande au XVIIe siècle . Mémoire lu à l’Académie des sciences morales et politiques le 7. sept . 1901 . Neue verbesserte Ausgabe Paris 1908 ) hingewiesen. 3 Über die späteren Schicksale des Kartells vgl . auch W . Möllenberg a . a . O., S. 125 . Der Syndikatsvertrag der Hüttenwerke ist abgedruckt bei M ück a . a . O., I . Bd . Urkunde Nr . 288 ; vgl . auch I, S. 110. 4 Das Zeitalter der Fugger , I, S. 396 ff ., 417 ff. 1 160 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. erwähnten großen Pfeffermonopolprojektes des Konrad Rott h Wir sahen, daß der spekulationsfroheAugsburger zum Zwecke der Durchführung eines Weltmonopols in Pfeffer mit portugiesischen und italienischen Kaufleuten und außerdem mit der sog. Thüringer Gesellschaft sich verbündete. Dabei sollte ein Gebietskartell ver¬ hindern, daß sich die Kontrahenten selbst eine lästige Konkurrenz machten. Konrad Rott übernahm es, „die Absatzgebiete der Parteien so zu begrenzen, daß den Portugiesen Spanien, Portugal, Frankreich und England, den Italienern Jtalien mit seinen Inseln, ihm selbst und der Thüringischen Gesellschaft aber Deutschland, die Niederlande, Ostland und Polen zur Ausbeute überlassen wurden.“ Jede Partei, die Pfeffer außerhalb ihrer Handelssphäre verkaufte, sollte als Konventionalstrafe der durch die Übertretung der Kartell¬ bestimmungen geschädigten Partei 10 Dukaten für jeden verkauften Zentner zahlen 2. }{t if. Ich habe in dem vorhin genannten Artikel „Ein Kartell deutscher Kaufleute aus dem Jahre 1743“ die These aufgestellt, daß Kartelle im 16. Jahrhundert eine häufige Erscheinung des westeuropäischen Wirtschaftslebens wären, nachdem schon das Mittelalter eine Anzahl derartiger Unternehmerverbände in den Kulturländern Europas gesehen hatte . Diesen Satz gilt es zunächst mit weiterem Material zu belegen. Wenn wir dabei zuerst von einigen Kartellen des Mittelalters sprechen wollen, so muß zu¬ vörderst an die beiden Syndikatsbildungen erinnert werden, die wir oben bereits kurz in einem anderen Zusammenhänge erwähnten. An das Salzvertriebssyndikat des Jahres 1301 und an das Alaun¬ syndikat vom Jahre 1470. Dazwischen mögen einige Kartellbildungen im hansischen Wirtschaftsgebiet kurz mit Erwähnung finden. Das Salzvertriebssyndikat vom Jahre 1301. Die Untersuchung der Wechselwirkung von öffentlicher und privater Wirtschaft bildet ein interessantes, wenn auch bisher kaum beachtetes Problem der europäischen Wirtschaftsgeschichte des 1 Vgl . oben S. 107—108. 2 Hä b 1e r in Neues Archiv für sächsische Geschichte 16 (1895), S. 191. Dazu Falke, Die Geschichte des Kurfürsten August von Sachsen in volkswirtschaftlicher Beziehung, S. 308. VIII 161 Zweites Kapitel : Kartelle des 14. bis 18. Jahrhunderts. Mittelalters. Es fragt sich dabei insonderheit: Ist die private Wirt¬ schaft, seit sie rationell, mit Buchführung usw., getrieben wurde, den Spuren der Wirtschaftsführung öffentlicher Organe gefolgt oder ist die Entwicklung umgekehrt vor sich gegangen? So zwar, daß die fiskalische Wirtschaftsführung sich die Errungenschaften der privaten Wirtschaftskunde anzueignen verstand ? Die Lösung dieses Problems ist dadurch nicht erleichtert, daß man etwa die ältesten bekannten Geschäftsbücher von Verwaltungskörpern mit den ältesten auf uns gekommenen Handlungsbüchern von Kaufleuten vergleicht 1 und aus der größeren oder geringeren Fähigkeit 1 Also etwa Rechnungsbücher der Päpste oder der italienischen Kommunen des Mittelalters mit Handlungsbüchern italienischer Kaufleute. Einige alte, bisher edierte Keclinungsbücher von VerwalLungskörpern nennt G. Brambilla in seinen „Storia délia ragioneria italiana" (1901). Vgl. Sombart, Moderner Kapitalis¬ mus I, S. 393. Seitdem sind noch mehrere bekannt geworden. Die ältesten bisher edierten oder in der Literatur erwähnten Handlungs¬ bücher großer italienischer Firmen sind etwa die folgenden (vgl. auch Huvelin, L’Histoire du droit commercial, Revue de Syn¬ thèse historique Vil (1903), S. 347 f.) : Für das 13. Jahrhundert: Ein Handelsbuch einer Florentiner Wcchslerfirma, von dem San¬ ti n i Bruchstücke , auf das Jahr 1211 bezüglich, veröffentlicht hat (P . Santini, Frammenti d’un libro di banchieri fiorentini . . . . In Giornale storico della lettcr . italiana X (Turin 1887), S. 161 ff.). Für die Wende des 13. und des 14. Jahrhunderts: Die Bücher der Peruzzi und Alberti 1292—1343 (vgl. S. L. P c r u z z i , Storia del commercio et dei banchieri di Firenze dal 1200—1345 (Firenze 1868). Ferner die des Guido delF Antella vom Jahre 1298 ff. (herausgeg. von Polidori, Arch . stör, liai., 1. Serie, IV [1843], S. Off.). Vgl. dazu: Ricordi di un mercante fiorentino del XIII ° secolo scritti in volgare su tavolette cerate esistenti nel r. archivio di stato in Firenze. Die der Bardi in Florenz von 1310 an. Die Bücher be¬ finden sich im Besitz der Familie Ginori Lisci in Florenz (cfr. L i v i a. a. O., S. 2 Anm. 1). Die der Buonsignori vor 1344 (G. Arias, Studi e documenti di storia del diritto, S. 37—67). Die des Miliadusso Baldiccione im 14. Jahrhundert (Ricordi di Meliadus Baldiccionc de’ Casalberti Pisano ed. Bonaini in Arch. storico ital., 1. Serie, VIII1845 ( ), Append. 7, 9, 17, 68). Für die Handlungsbüeher der Medici vgl. H. Sieveking, Die Handlungsbücher der Medici (Sitzungsbericht d. K. Akademie d. Wissenschaften in Wien, Bd. GLI, Wien 1905). Auch schon daselbst Anzeiger der historisch-philos. Klasse vom 3. Dezember, Jahrg. 1902, Nr. XXV . Derselbe Forscher hat auch auf die Geschäftsbücher einiger venezianischer Firmen des 15. Jahrhunderts, namentlich der Gebr. Soranzo, ausführlicher hinStriedei -, Studien 7. Gesoh . kapitaUsfc , Organisatiousformen . 11 162 Drittes Buch: Monopole , Kartelle lind Aktiengesellschaften im Mittelalter. rechnerischer Erfassung geschäftlicher Vorgänge, die sich bei der einen oder der anderen Gruppe offenbart, also aus der fort¬ geschrittenen Buchführungstechnik, auf eine Priorität im obigen Sinne schließt. Das wäre methodisch durchaus verkehrt. Denn wir können ja niemals wissen, ob das Vergleichsmaterial, das uns zur Verfügung steht, wirklich typisch ist, d. h. demjenigen Grad entspricht, den die BuchfQhrungskunde in der öffentlichen Ver¬ waltung bzw. der privaten Unternehmung im allgemeinen in der betreffenden Zeit erlangt hatte. Zu ähnlichen Fragestellungen über die Wechselwirkungenvon privater und öffentlicher Wirtschaftstätigkeit gelangt man auch in der Geschichte des Kartellwesens. Die ältesten Kartelle, die ich naehweisen kann, sind fiskalischer Natur . Sie wurden von den Pächtern öffentlicher Wirtschaftsobjekte unter hervorragender Beteiligung der öffentlichen Gewalt abgeschlossen. Auch hier wäre es aber ein methodischer Fehler, die Möglichkeit leugnen zu wollen, daß diesen „fiskalischen“ Kartellen, die uns zufällig bekannt geworden sind, solche privater Unternehmer vorausgegangen sein können. Die Kaufleute hatten allen Grund, Abmachungen wie Kartelle, die der Wirtschaftsethik der Zeit mit ihrer scharfen Betonung des „ge¬ rechten Preises" entgegenstanden, geheim zu halten. Oft wird es gar nicht zu schriftlichen Fixierungen der Konvention — die auf uns gelangen konnten — gekommen sein. Nichts wäre verkehrter, als hier quod non est in actis, non est in mundo zu schließen. Freilich gewiesen, nachdem schon in den achtziger Jahren der italienische Gelehrte Best» auf diese Schätze des venezianischen Staats¬ archivs aufmerksam gemacht hatte. H. Sieveking, Aus venezianischen Uandlungsbüchern. Ein Beitrag zur Geschichte des Großhandels im 15. Jahrhundert in Schmollers Jahrb. f. Gesetz¬ gebung, Verwaltung und Volkswirtschaft, Bd. XXV (1901), S. 1489ff. ; Bd. XXVI (1902), S. 189 ff. ; Bd. XXV , S. 1499 f. erwähnt Sieve¬ king einige genuesische Handelsbüchcr, besonders ein Hauptbuch der Bank S. Giorgio. Vgl. dazu desselben Verfassers „Genueser Finanzwesen“, Freiburg i. Br. 1898—99. Auf die GeschäftsbUcherScliätze des Ragusancr Staatsarchivs (seit Anfang des 14. Jahr¬ hunderts) hat neuerdings wieder C. Leyerer aufmerksam ge¬ macht (Österreich. Handclsschulzeitung 1913, August- u. November¬ heft). Für das außerordentlich reichhaltige kaufmännische Archiv des Franc Dntini (f 1410) zu Prato bei Florenz vgl. meine Aus¬ führungen in Vierteljahrsschrift für Soz. u. Wirt.- Gesell. 10 (1912), S. 442 ff. 163 Zweites Buch : Kartelle des 14. bis 18. Jahrhunderts. für wahrscheinlich halte ich die Nachbildung der fiskalischen Kartelle nach privaten Vorbildern nicht. Die öffentlichen Gewalten be¬ durften dessen kaum. Sie hatten in den Handelsverträgen Vor¬ bilder genug, wie man eine lästige Konkurrenz ausschalten und sich gütlich über wirtschaftliche Beziehungen einigen konnte. Es mutet einen fast wie eine Abmachung moderner Kaufleute an, die in schwerer wirtschaftlicher Depression den Konkurrenzkampf auf¬ geben, wenn man etwa die Handelsvergleichsverhandlungenliest, die im Jahre 1181 Pisa und Lucca miteinander schlossen. „Unter dem Druck langdauerader Hungersnot und schwerer Seuchen, die. Italien damals heimsuchten, reichten sich die bisher so unversöhnlichen Gegner die Hand zu ernstlichem Frieden nicht nur, sondern zu so engem Bunde, daß beide Städte in mancher Beziehung fast als ein Staats wesen erscheinen konnten“ 1. So mußte z. B. der aus der Münze jeder der beiden Städte sich 'ergebende Reingewinn zu gleichen Teilen an Lucca und Pisa verteilt werden. „Ebenso sollte fortan auch der Reingewinn aus dem in beiden Städten erhobenen Ufer¬ zoll und dem Salzmonopol, sowie aus dem nur in Pisa bestehenden Monopol auf Eisen und Eisenerze, solange ein solches vorhanden sein würde, endlich auch der Reinertrag aus dem Pisanischen See¬ zollamt (decatia) zu gleichen Teilen unter die vertragschließenden Städte geteilt werden“ usw. 2 Es braucht kaum gesagt zu werden, daß öffentliche Gewalten, die solcher Art Handelsverträge eingingen, keine kaufmännischen Vorbilder brauchten, wenn sie für ihre fiskalischen Industriegegenstände Kartelle abgeschlossen haben. Auch die Tatsache, daß staatliche Monopole für gewisse Handelsgegenstände, z. B. für Salz, Eisen usw., schon früh im Mittelalter existierten 3, zeigt 1 Adolf Schaube, Ilandelsgeschichtc der romanischen Völker des Mittelmeergebietes bis zum Ende der Kreuzzüge . München und Berlin 1906 , S. 650 f. 2 Schaube a . a , O., S. 651, 3 Vgl. für Monopole in den italienischen Staaten des früheren Mittelalters Schaube, Register sub Monopol , dazu George Yver, Le commerce et !es marchands dans l’Italie méridionale au XIII e et au XIV e siècle . Paris 1903 . Yver hat (a . a. O., S. 24 f.) mit Re.cht darauf hingewiesen , daß die Anjous in Sizilien im 13. und 14 . Jahrhundert — wie auch ihre staufischen und normanischen Vorfahren ■ — tüchtige Geschäftsleute waren , die ihren Wirtschaft11 * 164 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. übrigens, daß man staatlicherseits die Vorteile des Monopoliums — auf die ja jedes Kartell hinausstrebt — wohl zu würdigen verstand. Vielleicht ist es kein Zufall, daß das älteste Kartell fiskalischer Werke, das mir bekannt geworden ist, gerade auf dem Gebiete des Salzhandels gegründet wurde, auf jenem Gebiete also, auf dem vielfach eine staatliche Monopolbewegungseit langem existierte. Zum Zwecke der Ausschaltung der gegenseitigen Konkurrenz, die sich die dem König von Neapel gehörigen Salinen in der Provence und die königlichen französischen Salzwerke von Aigues-Mortes und der area maritima von Aigues-Mortes seit längerer Zeit in Südfrankreich gemacht hatten , kam es um das Jahr 1301 zu Ver¬ handlungen zwischen den beiderseitigen Pächtern dieser staatlichen Salinen. Im Aufträge des Königs von Neapel resp. im Aufträge seines Pächters (der bekannten Florentiner Bankfirma Bardi) leitete Bonacursus de Tecco 1die Syndikatsverhandlungen, im Aufträge des französischen Königs besonders sein Salzpächter Albizi Franzesi, ebenfalls einer bekannten Kaufmannsfamilie von Florenz angehörig 2. Leider scheinen sich, wie eine Anfrage im Staatsarchiv von Neapel ergab, keine anderen urkundlichen Niederschläge dieser Verhand¬ lungen erhalten zu haben, als die Aufforderung König Karls II. von Neapel an den Seneschall der Provence, die Verhandlungen schleunigst durch einen Syndikatsvertrag zum Abschluß zu bringen. (Neapel, 6. Dezember 13013.) In dem Schreiben ist ausdrücklich ausgesprochen, daß das Kartell zwischen den beiderseitigen Höfen, also nicht nur zwischen den Pächtern der königlichen Salinen, abgeschlossen werden sollte. Als einziger Zweck der Vereinigung ist ohne Umschweife der Nutzen liehen Vorteil im staatlichen Eigenhandel usw. so gut wie der kapita¬ listische Kaufmann zu wahren verstanden. 1Tecco „ Bonaccorsi“ im Jahre 1299 als Mitglied der Handels¬ gesellschaft der Bardi erwähnt bei Y v e r a. a. 0 ., S. 297. * Vgl. u. a. für die Franzesi: Otto Mcltzing, Das Bank¬ haus der Medici u. seine Vorläufer. Jena 1906. S. 42 ff. 3 Das Schreiben des Königs ist erwähnt und iin Exzerpt wieder¬ gegeben bei R. Davidsohn, Forschungen zur Geschichte von Florenz. III. Teil. Berlin 1901. Nr. 382. Im Anhang habe icli das interessante Schriftstück nach einer freundlich übersandten Ab¬ schrift des Staatsarchivs von Neapel in extenso wiedergegeben. Zweites Kapitel : Kartelle des 14. bis 18. Jahrhunderts . 165 des Fiskus genannt 1. Wenn dabei von dem Kartellvertrag als einem „Tractatus societatis“ gesprochen ist, so bedeutet hier Ge¬ sellschaft nicht Handelsgesellschaftim technischen Sinne, sondern eine Vereinigung im weiteren Sinne, eine „societas communis vendi¬ tionis“ wie es in dem Briefe des Königs von Neapel an seinen Seneschall in der Provence heißt. Auch Konrad Peutinger bezeichnet in seinem Gutachten über das Kupfersyndikat von 1498 ff. dieses notorische Kartell als „societas cupri“ 2, als „ Gesell¬ schaft des Kupfers“ oder auch als „ Gesellschaft und Vereinigung“. Peutinger führt aus, die beteiligten Firmen hätten „ain sondere gemaine gesellschaft und vertrage etlicher anzal kupfer und allain derselben war beschlossen und gemacht“. Genau so hat auch der Ausdruck „ Gesellschaft“ in der Neapolitaner Urkunde eine weitere Bedeutung. Vielleicht wird aus französischen oder neapolitanischen Archiven Licht über die Wirksamkeit des provençalischen Salz¬ vertriebssyndikats vom Jahre 1301 verbreitet werden können. Wir mußten, um den Abschluß dieses Buches nicht allzuweit hinaus¬ zuschieben, auf so weite und dem Risiko des Versagens so stark ausgesetzte Archivstudien verzichten 3. * * * Wie Florentiner Kaufleute die Pächter der fiskalischen Salinen waren, zwischen denen der erste uns' bekannte Kartellvertrag ab¬ geschlossen wurde, so waren auch Florentiner Kaufleute die ersten privaten Kartellisten, von denen uns die Literatur berichtet. Bekanntlich wurden im Verlaufe des 13. Jahrhunderts die italienischen Großkaufleute, namentlich die Florentiner, den staat1 „Magna utilitas utriusque curiae “ und ähnliche dreimal in dem kurzen Schreiben. Wendungen 2 Vgl . das interessante „consilium in causa societatis cupri" Peutingers, das auch die Grundlage bildete für Ehren¬ bergs Schilderung des Kartells. Msc. in der Augsburger Stadt¬ bibliothek 2° Aug. 398 fol. 189r—198v. Dazu jetzt König a. a. O., S. 109. 3 Y v e r , der in seinem Buche „ Le commerce et les marchands dans l' Italie méridionale au XlIIe und XlVe siècle “ die ausgebildete Wirtschaftspolitik der Anjous und die Handelsgeschäfte der sizilischen Krone sehr ausführlich behandelt , ist leider das Kartell von 1301 entgangen , sonst wäre er vielleicht bei seinen Archiv¬ forschungen der interessanten Erscheinung nachgegangen. 166 Drilles lïuch : Monopole, Kartelle und Aktiengesellschaften im Mittelalter! liehen Gewalten immer unentbehrlicher. In Rom und Neapel, in Frankreich und England waren die italienischen Kaufleute die Hauptgläubiger der Krone. Ohne ihre Kreditgewährungen war schon damals die Staatsmaschine kaum mehr in Gang zu halten. In ihren Händen befanden sich die besten und einträglichsten Ein¬ nahmen der reichsten europäischen Staaten der damaligen Zeit 1. Im Königreich Neapel bedeutet die Periode zwischen dem Tode Heinrich VIII . und der Ankunft des Herzogs von Kalabrien in Toskana die Zeit der höchsten Blüte der Florentiner Hochfinanz2. Die Bardi und die Peruzzi, denen sich bald noch die Acciajuoli zugesellten, machten damals Ihre besten Geschäfte. Namentlich auch deshalb, weil die genannten Firmen bald einen unnützen Konkurrenzkampf glücklich vermieden. Nach Yver existierte etwa vom Jahre 1316 an ein Syndikat, das alle wichtigen Finanz¬ geschäfte des Königreichs monopolisierte. Dem Syndikat der drei genannten Geldmächte schloß sich nach 1330 noch das Haus der Bonaccorsi an. Näheres über das Syndikat hat Yver leider nicht beigebracht. Ihm muß auch die literarische Verantwortung über¬ lassen bleiben, ob es sich in den Abmachungen der genannten Firmen um ein wirkliches Syndikat oder nur um gelegent¬ liche Konsortialbeteiligungen handelt. Erwähnt soll aber doch werden, daß S c a c c i a in seinem „Tractatus de commercio et cambio“ (Ausgabe Frankfurt a. M., 1648, S. 300) an der Stelle, wo er von Monopolen handelt, Verabredungen von Kauf¬ leuten erwähnt, in denen diese sich gegenseitig verpflichten, den Fürsten nur zu einem festbestimmten Prozentesatz Anleihen zu ge¬ währen 3. 1 Vgl. besonders O. M e 11 z i n g , Das Bankhaus der Medici und seiner Vorläufer und die dort genannte Literatur . Dazu be¬ sonders Schaube, Handelsgeschichte der romanischen Völker, und G. Yver, Le commerce et les marchands dans l’ Italie méridio¬ nale au XlIIe et au XlVe siècle . Eine recht brauchbare Zu¬ sammenfassung der Wirksamkeit der italienischen Hochfinanz des Mittelalters bietet auch der bereits genannte Aufsatz von Ku¬ lischer, Warenhändler u . Geldausleilier im Mittelalter. 2 Y v e r a . a . O., S. 308. 3 „ Quando mercatores divites , scientes extare principes qui pro aliqua urgenti necessitate quaerunt pecunias ad cambium con¬ veniunt de non dando illas , nisi pro tanto pretio , quod esset iniustum et excessivum .“ 1 5 Zweites Kapitel : Kartelle des 14. bis 18. Jahrhunderts. Kartellbildungen gebiet im hansischen des Mittelalters. 167 Wirtschafts¬ Für Deutschland sind wir zuerst von Unternehmerverbänden aus dem hansischen Wirtschaftsgebiet unterrichtet . Schon im Jahre 1309 hören wir von einem Wachsring , den deutsche nach England handelnde Kaufleute abgeschlossen haben sollen a. Es handelte sich um eine Verabredung 2, der zufolge die Zufuhr russischen Wachses nach England hintangehalten werden sollte, damit der Wachspreis in die Höhe ging. Aber nicht nur die Hansen suchten sich im Mittelalter gelegent¬ lich den Markt durch kartellistische Verabredungen zu verbessern, ebenso handelten ihre flandrischen und holländischen Geschäfts¬ freunde . So wird z. B. in einer Beschwerdeschrift des deutschen Kaufmanns zu Brügge aus dem Jahre 1417 Klage darüber geführt, daß „die Fläminger die Preise der Waren untereinander fest ver¬ einbarten , so daß keiner sie billiger als der andere gebe“ 3. In den sechziger oder siebziger Jahren muß sich dann in Leiden ein Kartei ! der dortigen großen Tuchhändler gebildet und zeitweise den dortigen Markt beherrscht haben 4. Es handelt sich bei dieser kapitalistischen Organisation um keine Handelsgesellschaft wie v. B e 1o w 6 meint . Wenn es in den auf die genannte Bildung 1 KarlKunze , Hanseakten aus England 1275—1412 . Halle a . S. 1891 . Urkunde Nr . 40 . Dazu Walter Stein, Die deutsche Genossenschaft in Brügge und die Entstehung der deutschen Hanse. Hansische Geschichtsblätter 1908 , S. 430 ff. 2 Die Ausdrücke compositio , collusio , ordinatio werden als Be¬ zeichnung für den Ring gebraucht. 3 „Vorlmer so maken de borgers van Brügge endracht uppe ere gud , dat . se dat alle to enem pryse vorkopen moten , so dal . erer eyn des nicht beteren kop geven mach , den de. andere , dat dem copmanne lo grotem vorvange unde hindere is an der copenschop , de he van en kopet .“ In : Die Hanserezesse und andere Akten der Hanse¬ tage von 1256— 1430 . Bd . VI (1889 ), Nr . 400 , § 10. Vgl. auch E . D a e n e 11 , Die Blütezeit der deutschen Hanse . Hansische Geschichte von der 2. Hälfte des 14 . bis zum letzten Viertel des 15. Jahrhunderts . 2 Bde . Berlin 1905/06 . II . Bd ., S. 428. 4 Die Belege aus den Hanserezessen zitiert bei D a e n e 11 a . a . O., II , S. 429 , und in dem in Anm . 5 zitierten Aufsatz von B e 1 o w s , S. 10. 5 „Großhändler und Kleinhändler im deutschen Mittelalter .“ 1 168 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. bezüglichen Urkunden der Hanserezesse heißt : „ dat siek etlike van en [d. h. einige Leidener Kaufleute ] tosamen gheworpen unde selschop ghemaket hebben “, so ist hier Gesellschaft nicht gleich Handelsgesellschaft , sondern gleich Vereinigung im weiteren Sinne, eben gleich Kartell. Den Hansen war das Kartell besonders noch deswegen lästig, weil es nicht nur die Preisbildung des Leidener Tuches auf den niederländischen Märkten beherrschte , sondern auch das gesuchte Leidener Fabrikat in Hamburg , Lübeck und in den östlichen Städten zu niedrigeren Sätzen verkaufte , „ so daß der hansische Zwischen¬ händler in seiner Heimat in keiner Weise zu konkurrieren ver¬ mochte “ \ sondern einfach ausgeschaltet war. Das Alaunkartell des Jahres 1470. Um das Alaunkartell vom Jahre 1470 ganz zu verstehen , müssen wir zunächst einen Blick auf die Entwicklung des Welthandels in Alaun werfen 2. Bis nach der Mitte des 15. Jahrhunderts kam für den Welthandel so gut wie ausschließlich der Orient als Alaun¬ produzent in Frage . In Alexandria stapelte sich nubischer und arabischer Alaun auf , in Aleppo mesopotamischer und armenischer, besonders aber in Konstantinopel strömten „ die Produkte von Thrazien , einzelnen griechischen Inseln (Lesbos) und vor allem des ungemein alaunreichen Kleinasien zusammen “ 3. Von den italieni¬ schen Levantehandelsmächten erlangte Genua die größte Bedeutung für den Alaunhandel . Der klugen Unterstützung , die die ligurische Hauptstadt im vierten Kreuzzug den griechischen Paläologen zuteil werden ließ, verdankte sie für fast zwei Jahrhunderte ein nahezu In : Jahrbücher für Nationalökonomie und Statistik . III . Folge. 20 . Bd . (1900), S. 10. 1 v. Below a. a . O., S. 10. 2 Alaun wurde namentlich für die Tuchfärberei und zur Leder¬ bereitung im Mittelalter in Mengen gebraucht und gehörte zu den wertvollsten Artikeln des mittelalterlichen Welthandels. 3 G. von der R o p p , Zur Geschichte des Alaunhandels im 15. Jahrhundert . Hansische Geschichtsblätter 1900 , S. 122 . Im wesentlichen nach W . H e y d , Histoire du commerce du Levant au moyen -âge . 2 Bde . Leipzig 1885/86 . Franzos , vermehrte und ver¬ besserte Ausgabe des zunächst in deutscher Sprache erschienenen Werkes . II . Bd -, S. 565 ff. 6 Zweites Kapitel : Kartelle des 14. bis 18. Jahrhunderts . 169 vollständiges Monopol für den Welthandel in Alaun. Dies um so mehr, als genuesische Familien von 1275—1455 im Besitz der besten und ausgiebigsten Alaungruben, der von Phokaea (am nördlichen Eingang des Busens von Smyrna) waren 1. Das Vordringen der Türken im östlichen Becken des Mittel¬ meeres, das 1453 mit der Eroberung von Konstantinopel einen vorläufigen Abschluß fand, änderte die Lage des Alaunmarktes von Grund auf. Die Türken wurden jetzt die Beherrscher der besten Alaungruben der Welt, und ganz Europa mußte ihnen in der enorm hohen Pacht, die die italienischen Pächter dem Sultan zu zahlen hatten , tributpflichtig werden. Erst wenn man dieser Tatsache eingedenk ist, versteht man den Jubel, der die ganze Christenheit durchdrang, als 1461 im Kirchenstaate bei Tolfa, unweit Civitavecchia, mächtige Lager eines vorzüglichen Alaunsteines gefunden wurden 2. Seit dem Frühjahr 1463 wurde in Tolfa gearbeitet, bald angeblich von 8000 Menschen3. Die Ausbeute hatte Pius II. als Monopol einer Gesellschaft, der sog. Societas aluminum, über¬ tragen, die Joli, de Castro, der Entdecker von Tolfa, mit dem Genuesen Barth , de Framura, Skriptor der apostolischen Briefe, und Ca?l de Gaetanis von Pisa bildete. Der erste Pachtkontrakt ist bisher leider nicht aufgefunden4, aber Gottlob hat dessen Ver¬ längerung (auf neun Jahre , beginnend mit dem 1. November 1465) aus den Akten des vatikanischen Archivs auszugsweise mitgeteilt B. Der Vertrag hat in seinem Inhalt eine große Ähnlichkeit mit den Kontrakten , in denen im 16. Jahrhundert die Fugger und andere Finnen die Pacht der spanischen Quecksilberbergwerke von Almaden übernahmen. Die Tolfaer societas aluminum hatte den Betrieb der Gruben vollständig auf eigene Kosten zu führen, alle Bauten vorzunehmen usw. Dafür stand ihr im Kirchenstaat das alleinige 1 Noch das Libell of englishe policye (um 1436 ) schildert die Genuesen als Bringer des für die englische Tuelifabrikation unent¬ behrlichen Alauns. 2 Durch den päpstlichen Finanzbeamten Johannes de Gastro Vgl . die anschauliche Schilderung in den Denkwürdigkeiten Papst Pius II . (Enea Silvio Piccolomini ), in Übersetzung mitgeteilt bei Gottlob, Camera apostolica und von der Ropp. 3 Gottlob, Camera apostolica , S. 282. 4 Er trat im November 1462 in Kraft ; vgl . G. Zippel, L ' allume di Tolfa e il suo commercio , a . a . O., S. 21 und 437. 6 Jetzt im Wortlaut abgedruckt bei Zippel , a . a. O., S. 438 ff. 170 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. Alaungewinnungsrecht zu. Die Gesellschaft lieferte als Entgelt 30 000 Cantare (Zentner = 150 röm. Pfund) Alaun zu dem geringen Preise von % Dukaten für jeden Zentner an die päpstliche Kammer ab. Die Bezahlung durch die Camera apostolica hatte bei der Übergabe der Ware und an Ort und Stelle zu erfolgen, sie konnte in bar oder durch Rücküberlassung der Ware geschehen; in letzterem Falle wurde der Engros-Marktpreis angerechnet und die Gesell¬ schaft übernahm die den Händlern von der Kammer gewährten Lieferungsbedingungen. Nach Ablauf des Kontrakts war die päpst¬ liche Kammer gehalten, „alle Gebäude, Anlagen und Betriebs¬ gerätschaften der Gesellschaft zu einem durch zwei von beiden Teilen ernannte Sachverständige festgesetzten Preise käuflich zu erwerben“ 1. Als im Frühjahre 1466 einer der Gesellschafter der Tolfaer societas aluminum ausschied und dafür das Bankhaus der Medici eintrat, wurde unter dem 1. April 1466 ein neuer Pachtvertrag (wiederum auf neun Jahre) 2verabredet . Die Gesellschaft über¬ nahm zu den Betriebskosten in den Tolfaer Gruben jetzt auch noch die sämtlichen Vertriebsspesen (Spedition des Alauns in die päpst¬ lichen Magazine zu Civitavecchia, Schiffsverfrachtung des Alauns und seine Überführung in die Handelsplätze). Der Gewinnanteil der päpstlichen Kammer wurde fixiert, er „bestand jetzt nicht mehr in der freien Lieferung einer bestimmten Quantität des gewonnenen Produkts, die dann erst durch die Kammer selbst in den Handel kam, sondern die Gesellschaft übernahm auch den ganzen kauf¬ männischen Teil des Geschäfts und bezahlte der Kammer für jede Cantare des verkauften Alauns 2 Dukaten in Gold“. Betrug der Verkaufspreis mehr als 3 Dukaten, so fielen von dem Mehr zwei Drittel an die Kammer, ein Drittel an die Gesellschaft. Jeder Verkauf an die Händler konnte nur aus den Magazinen in Civita¬ vecchia geschehen3. Dabei mußte stets ein päpstlicher Kammerbeamter zugegen sein. Die Kurie versprach in dem Vertrag, in der 1 Gottlob a. a . O., S. 283 f. 2 Jedoch stand dem Papst das Recht zu , den Vertrag in den ersten 30 Monaten seiner Gültigkeit zu kündigen . Die wichtigsten Teile des Kontrakts abgedruckt bei Z i p p e 1 a . a . O., S. 405 Anm . 1. Der Wortlaut in Rom , Archivio di Stato ; Depositeria generale della crociata 1464— 1475 cc. 29— 32. 3 Vgl. die Casa de contratacion in Portugal und Spanien. Zweites Kapitel : Kartelle des 14. bis 18. Jahrhunderts. 171 ganzen Christenheit den Verkauf des türkischen Alauns zu unter¬ sagen, und der Gesellschaft„ alle dieserhalb nötigen Patente , Bullen, Breven usw. taxfrei auszustellen“ 1. Schiffe, die dem päpstlichen Verbot entgegen mit türkischem Alaun befrachtet betroffen wurden, sollten angehalten und ihre Ware konfisziert werden. Von dem Erlös hatte die päpstliche Kammer zwei Dritteile, die Gesellschaft ein Drittel zu beanspruchen. In demselben Verhältnis sollten auch die Kosten für die Ausrüstung jener Schiffe geteilt werden, die die Alaunflotte beschützten und „ der Durchführung des alleinigen Ver¬ kaufsrechtes der Gesellschaft dienten.“ Die Ausbeuten der Tolfaer Alaunwerke erreichten sofort eine bedeutende Höhe 2. Der jährliche Reingewinn für die apostolische Kammer wird von Zeitgenossen Pauls II . auf etwa 100 000 Dukaten angegeben. Er betrug im Jahre 1471 für 70 000 Cantare Alaun, deren Verschiffung das Haus Medici übernommen, 140 000 Dukaten. Im folgenden Jahre, 1472, verschifften die Medici wiederum 70 000 Cantare. Es wurden ihnen darauf 30—32 000 Dukaten, die sie schon vorher für Zwecke des Türkenkrieges hergeliehen hatten, angerechnet und 80 000 Dukaten sollten sie wieder innerhalb vier Jahren bezahlen3. Bei den hohen Profitraten, die die Tolfaer Alaungruben der apostolischen Kammer und natürlich auch den Pächtern abwarfen, ist das folgende zu beachten. Schon vor der Entdeckung der Tolfaer Gruben hatte Italien und seine Inseln etwas Alaun ge¬ liefert. Die bekanntesten Fundplätze waren auf der Insel Ischia bei Neapel, in Pozzuoli und Volterra 4. Von größerer Bedeutung scheint zur Zeit der Entdeckung der Tolfaer Gruben nur der Betrieb auf Ischia gewesen zu sein; von hier drohte also dem päpst¬ lichen Alaunmonopol eine empfindliche Konkurrenz. Um sie aus¬ zuschalten, begann Paul II. mit König Ferdinand (Ferrante) von Neapel, dem Regalherrn der Ischianer Alaungruben, Verhandlungen, die zu dem Kartell vom 11. Juni 1470 führten 5. Das Kartell wurde 1 Gottlob a . a . O., S. 286 . Seine Rechtfertigung erhielt das päpstliche Monopol dadurch , daß seine Erträgnisse für die Zwecke der Cruciata , d . h . für die Türken - und Hussitenkriege ausgesetzt wurden . Ibidem S. 289 ff ., 294. 2 Zahlen bei Gottlob a . a . O., S. 287. 3 Gottlob a. a . O., S. 288. 4 Zi p p e 1 a . a . O., S. 11 ff ., 34. 5 Vgl. Gottlob a . a . O., S. 296 , und Z ippel a . a . O., S. 34 ff. 1 172 Drittes Buch : Monopole , Kartelle und Aktiengesellschaften im Mittelalter. zwischen den Generalkommissarender Cruciata und dem Bevoll¬ mächtigten König Ferrantes, einem Neapolitaner Kaufmann namens Aniello Perotto abgeschlossen und von Papst und König be¬ stätigt . Es sollte zunächst für die Dauer von 25 Jahren gelten 2. Das Syndikat wurde in der ausdrücklich ausgesprochenen Absicht abgeschlossen, den Preis des italienischen Alauns auf der Höhe zu erhalten. Deutlich heißt es in dem Protokolle, das die Bevoll¬ mächtigten Pauls II. und P’errantes von Neapel aufnahmen: „ . . . ac novissime idem Sanctissimus dominus noster attente con¬ siderans eiusdem aluminis precium eidem sancto operi dicatum ex eo diminui plurimum, quia alumen aliud ex minera Ischana, ad serenissimum principem dominum Ferdinandum regem Sicilie perti¬ nentem ad easdem mundi partes deferebatur communiter, ad quas cruciate alumen delatum fuerat aut deferri sepius contingebat, ita ut per concursum et h abundantiam alumi¬ nis precium utri usque impediretur et viles¬ ceret, ordinatumque ipsius sancte cruciate subsidium demum 3 minime proveniret et prefatus serenissimus dominus rex cognos¬ cens hec eadem requisiverit suam Beatitudinem, libenter Sua Sanctitas annuit et consensit, ac pro 4communi utilitate aut utriusque commodo et utilitate cruciate ac reipublice praedictarum, ad conventiones, pacta et capitula modo et forma infrascriptis per supra et infrascriptos reverd. dominos cardinales deveniendum censuit, laudavit et mandavit . . .“ Als Bezeichnung für das Kartell werden die Ausdrücke societas, compagnia, intelligentia, aber auch conventio, unione usw. ge¬ braucht. An einer Stelle des Kartellstatuts heißt es, die beiden Alaunbetriebe Tolfa und Ischia sollten während der Vertragsdauer 1 Zippel vermutet wohl mit Recht in diesem Vertrauten des Königs von Neapel den Pächter der Ischianer Alaun¬ gruben. 2 Das interessante Kartellinstrument ist uns in mehreren Ab¬ schriften erhalten . Die vatikanische hat A . T h e i n e r im 3. Bande seines „ Codex diplomaticus dominii temporalis sanctae sedis “ (Rom 1862 ), S. 463 ff., abgedruckt . Korrekter ist die Handschrift , die im Archivio di Stato zu Rom ruht („ Depositeria délia cruciata 1464 bis 1475 “ , Fol . 1— 16). Die folgenden Zitate sind dieser Handschrift entnommen. 3 Th e i n e r hat deinde. 4 Meine Abschrift aus dem Archivio di Stato hat pio. Zweites Kapitel : Kartelle des 14. bis 18. Jahrhunderts . 173 sein „ uno corpo overo anima “ 1. Tatsächlich „ kontrollierten “ sich die beiden Ala imwerke so scharf wie nur irgendein modernes Syndikat. Jeder der beiden Kontrahenten hatte einen ständigen Kommissar in den Alaungruben bzw. in den Alaunniederlag ^n des anderen . Die Kommissare waren im Besitz von Schlüsseln zu allen Magazinen. Ihnen mußte Rechnung gelegt werden , wieviel Alaun gewonnen und verschifft wurde . Der Zweck dieser Kontrolle war die Durch¬ führung des strikten Verbotes , Alaun anders als auf dem durch das Kartell vorgeschriebenen Wege zu verkaufen 2. Denn erst mit dem Verkaufe begannen die die freie wirtschaftliche Entschließung der Kontrahenten einschränkenden Bestimmungen des Syndikats. Die Produktion , der Betrieb , blieb jedem der beiden Kontrahenten überlassen , ebenso der Transport des verkaufsfertigen und des verkauften Minerals (inklusive Versicherung , Verzollung usw.) 3. Die gemeinsame Aktion der Kartellkontrahenten setzte erst beim Verkaufe ein. Während der 25 Jahre der Kontraktsdauer durfte kein Alaun von einer der Kartellparteien selbständig und ohne Vorwissen der anderen verkauft werden . Alle Verkaufs¬ abschlüsse leiteten zwei Abgeordnete der Kontrahenten , ein päpst1 Anima hat Theiner, meine Abschrift aus dem Archivio di Stato hat maona . Über die Gesellschaftsform der Maonen vgl . be¬ sonders H . Sieveking, Genueser Finanzwesen vom 12. bis 14. Jahrhundert . Freiburg i. Br . 1898 , S. 43 und sonst . L. Gold¬ schmidt, Universalgeschichte des Handelsrechts , S. 295 . Am Ende des 15. Jahrhunderts bedeutet maona wohl schon jede compagnia di traffico o quasi voglia altra società di guadagno (Rezasco ). 2 „Che non si possa da questo di ayante per alchuna de le parte vendere ne fare vendere a particulare persona alcuna quantita de alumi de quelli fossino fatti o se facesseno ne lc alumcre de la camera apostolica et la Maiesta de Signore Re . Ma tutti allumi fatti et ehe se faranno nel ditto tempo de soppra , se abbi a reservare et navicare et vendere per uso de ia compagnia et per declaratione de cadauna de le parte , nel reame al ’ alumere de la Maiesta dei Signore Re abbia arrestare per nome de la camera apostolica uno commissario et a Oivitavecchia a lc alumere de nostro Signore , uno altro per la Maiesta dei Signore Re , li quali cadauno habbia a teniro una de lc ehiave de cadauno de li magazini et bon conto de li allumi ehe se feranno et ehe se navicharauo .“ 3 „Che cadauna de le parte per la sua mitta habbia a pagare seperatamente lc spese in fare fare li sui alumi , navicare et ogni altra spesa occorresse , ct similiter tenire sui conti seperatamente de in¬ trata et spesa .“ Und ferner : „Facendcsse assecurare una de lc 3* 174 Drittes Buch: Monopole , Kartelle und Aktiengesellschatten im Mittelalter. licher und ein königlicher Zu jedem Auftrag steuerten die päpst - ' liehen Gruben (Tolfa) und die königlichen (Ischia ) je die Hälfte bei 2. Auch dann hatte das zu geschehen , wenn einmal der Ver¬ kaufsabschluß nur von einem der Abgeordneten geschehen war :i. Ausgenommen waren von dieser Halbierung des Verkaufsquantums vorläufig Brügge und Venedig . Die päpstliche Kammer hatte nämlich mit „gewissen Kaufleuten “ eine Konvention geschlossen, lrut der sie den Verkauf des Alauns , der augenblicklich in Brügge (ca. 60 000 Cantare ) und Venedig (ca. 20 000 Cantare ) lagerte , auf einige Zeit unterlassen (soprassedere ) sollte . Ging der Verkauf weiter , so sollte der König von Neapel , der selbst kein (Ischaer) Alaun in Brügge und Venedig lagern hatte , zunächst für die 60 000 bzw , 30 000 Cantare mit einem Sechstel am Reingewinne beteiligt werden . Waren diese Quanten aber verkauft , so trat auch hier die Bestimmung in Kraft , daß Ischia und Tolfa je zur Hälfte zu liefern hatten KKonnte einer der Kontrahenten in seinen Aiaunparle per lu sua mitta lo possi lare et utile et spesa sia de la parte se l'ara assecurare .“ 3 „Che dicti alumi dove se ordenarà siano naviehati in cadauno loco si habbino a vendere per clui deputati , uno per la Sanctitä de nostro Signore et camera apostolica , l’ al Iro perlaMaiesta dei Signore Re et non si possi vendere per altri ." 2 „Che Luito lo allume ehe nel sopradiclo tempo se consumera overo se navicliara per diverse parte dei mundo per consumptione et uso de quello , la mitta se nitenda et debia essere de quelli alluini de le aluinere de la SancLilà dei nostro Signore et de la camera apostolica , et 1’altra micta de quello de le aluinere de la maiesta dei Signore Hc . Et de celero quando se habbi a navicare alume per qualuneha parte dei mundo , la micta sc habbia a levare da Civitavecchia per la parte de la camera apostolica et 1’altra micta de le aluinere dei Signore Hc per la parte sua .“ 3 cl . quello vendesse uno s’intenda ia micta de ia vendita esser per conto dd nostro Signore et de la camera apostolica etiam sci fussi facto per ei deputato de la maiesta dcl Signore Re , et ita e converso , e 1’ altra micta de ia maiesta de Signore Re .“ 4 So möchte ich die Bestimmungen des Kartellstatuls auffassen, die im Wortlaute folgendermaßen lauten : „ Et per havere de la Sanctità de nostro Signore et ia camera apostolica certa compositione per certo tempo cum io illustre Signore duca de Borgogna de retinere bene fomite le sue provineie et dominio de alumi et quelli non si possino vendere ad piu precio de libre quaLro et meza la charicha. I.a dicta compositione se debbia observare in omnibus suis partibus * Zweites Kapitel : Kartelle des 14. bis 18. Jahrhunderts. 175 werken nicht so viel produzieren , um seine Hälfte zu den Verkäufen .beisteuern zu können , so trat der andere Kontrahent mit einer größeren Lieferung (natürlich dann auch mit entsprechend größerem Gewinne) ein 1. Für irgendwelche Schadenersatzansprüche der Konsumenten bei Lieferung von schlechtem oder weniger gutem Alaun haftete nicht das Kartell , sondern der Kontrahent , der das betreffende reklamierte Produkt geliefert hatte 2. Die Preise , zu denen die Kartellkontrahenten verkaufen sollten, waren genau festgesetzt . Schloß einer der Beauftragten billiger ab, so hatte seine Grubenherrschaft der anderen den Gewinnausfall zu ersetzen 3. Kamen solche Unregelmäßigkeiten nicht vor , so wurde der Reinertrag aus dem Verkaufsgeschäfte zu gleichen Teilen aii die beiden Kontrahenten verteilt 4, Jeder der Vertragschließen¬ et similiter la compositione facta cum certi merehadanti de soprasedere ad vendere de alumi navicato fin questo di, maxime a Bruza circa cantara sexanta millia a Venetia circa cantara trenta millia, ne li quali loci la maiesta de Signore Re non ha sui alumi, se contenta la sanctità prefata de nostro Signore et camera apostolica che ne li dicti loehi, fino sera fornito vendersi dicti alumi, la maiesta de Signore Re debbia participare per la sexta parte dei utile , detractone prima le vere spese costano dicti alumi de cavedale ad epsa camera aposto¬ lica, et, fornito sera de vendere essi alumi , se habbia a navicarc in dicti loehi per mitta et similiter vendersi ut supra per mitta ne li altri loehi navigasse et ex nunc vendassi per mitta et questa com¬ positione principia baver locho a die stipulati contractus ." 1 „Et perche de sopra è ditto che cadauna de le parte debbia ponere la mitta de li alumi etiam si caso fusse che ad una de le parte li manchasse li allumi , o non podesse supplire del suo allume fabricato ne sue allumere fîno a la mitta per qualuncha accidente podesse advenire , î’altra parte possa supplire et per quella rata se ponesse piu , habia quelia parte supplisse a tirai ' tanto piu ." 2 „Se a caso fosse ehe o nel navicare o fabricare allume o per qualunche altro respecto esso allume retenesse frustro o non fussi bono et mercantile in modo clic nel vendere se havesse ad esser dif¬ ferentia de precio et casi simile , el damno ne seguisse sia damno de la parte sonno ditti allumi ." 3 „Et dicti allumi non si possino vendere ne piu ne meno di quello li sera deputato li precii de la camera apostolica et de la maiesta del Signore Re ; et si pur accadessi che per minor precio fosse vendito, per quello tale fussi facto tale vendita , si habbi a refare di sui beni quello meno fussi vendito , et per cadauno di quelli serano deputati a recevere et vendere dicti alumi ." 4 „El retracto veramente de li alumi se habhia a dividere per * 176 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. den war verpflichtet, den anderen von etwaigen fraudulösen Machen¬ schaften seiner Beauftragten unverzüglich zu unterrichten 1. Die Beauftragten waren gehalten, bei ihren Abschlüssen auf Kauf gegen Geld zu dringen und nicht auf Tausch gegen Waren sich einzulassen. Offenbar geschah das der schlechten Verrechnung wegen, die sich bei Annahme von Tauschwaren ergeben hätte . Auch lange Kredit¬ gewährungen sollten die Beauftragten tunlichst vermeiden. Ein Jahr ' (l) durfte die Obergrenze sein, natürlich mit der nötigen Bürgschaftssetzung2. Der Papst verpflichtete sich, alljährlich — wie früher schon geschehen war —- das Verbot des Kaufs und Verkaufs von türkischem Alaun der ganzen Christenheit einzuschärfen. Schiffe, die türkischen Alaun mit sich führten, sollten mit samt der Ladung denen gehören, die sie kaperten. Wollten die Kaperer den Alaun an das Kartell verkaufen, so sollte das geschehen können, falls die Ware gut war und wenn sich die Betreffenden mit der Hälfte des Marktpreises begnügten. Wenn nicht, mußte der Alaun unter Obhut der Be¬ auftragten des Kartells liegen bleiben, bis das Kartell zu Ende war 3. Es versteht sich von selbst, daß die Kartellkontrahenten ihre Konvention durch allerlei Maßnahmen vor dem Bruch zu schützen sich bemühten. Das Karteilinstrument wurde z. B. von den Be niitta , zoè , la mitta sia de la Sanctità di nostro Signore et l’altra initia de la maesta de Signore Re .“ 1 „La camera apostolica per el suo e la maiesta del Signore Re per il suo promettino relevare l’altra parte senza damno et similiter de l’altra fraude o matichamenti fossino commessi per cadauno delli deputati , videlicet cadauna de le parte per il suo .“ 2 „Che tutte vendite di alumi se faranno , si liabbino a fare , per quelli scrano deputati a vendere , a dinari contanti et non abbarato, ne se posse vendere a piu longo tempo de anno uno vel circa , tamen cum idonea fidciussione et non aliter .“ 3 „Ogni anno se liabbi a fare generale prohibitione de li allumi de li infideli et dicti allumi con li navili siano dati in preda a quelli li prendesscro ; ne li prendit .ori possino essere astrecti da niuna de le parle fare gratia ne alchuna misericordia a quelli fossino presi. Et volendo dare li allumi essendo boni et mcrcantili a la compagnia per la mitta mono de quello se vendera ne li lochi dove se condurano, la compagnia li debbia acceptare ; non volendo darii , si possino lenire ma non vendere in quello locho dove capilareno sotto custodia de li deputaLi de la compagnia fin al fine de dicta compagnia , et finito el tempo , li possino vendere ." Zweites Kapitel : Kartelle des 14. bis 18. Jahrhunderts . 177 vollmächtigten des Papstes und des Königs feierlichst beschworen. Ein Notariatsakt , der von dem ganzen Vorgang aufgenommen wurde, gibt uns genau darüber Auskunft. Die Bevollmächtigten gelobten in ihrem Schwur für ihre Auftraggeber alle und jede ein¬ zelne Bestimmung des Kartellkontraktes während der verabredeten 25 Jahre einzuhalten. Als Unterpfand setzte die eine Partei (Tolfa) alle Güter der Cruciata und der Camera apostolica, die andere (Ischia) alle Besitzungen des Königs von Neapel. Jede Über¬ tretung der Bestimmungen des Syndikats sollte mit 50 000 Dukaten di camera bestraft werden. Die Zahlung dieser Buße solle aber keineswegs von dem Vertrag entbinden. Schon vor Abschluß des Kartells mit dem König von Neapel hatte die Kurie Maßnahmen ergriffen, um sich für ihr Tolfaer Alaun ein Monopol— wenigstens in der Christenheit — zu sichern. Des allgemeinen Verbots, türkischen Alaun einzuführen, gedachten wir bereits, aber hiermit begnügte sich die apostolische Kammer noch nicht. Es galt, um dieses Verbot erst richtig wirksam zu machen, die einzelnen besonders stark alaunverbrauchenden Staaten bzw. die einzelnen großen Alaunhändler in das päpstliche Interesse hineinzuziehen. Das geschah für Venedig dadurch, daß die aposto¬ lische Kammer und die Generalkommissare der Cruciata mit Bartolomeo Giorgio (Bartolo Zorzi), dem bedeutendsten venetianischen Alaunhändler jener Zeit, einen alleinigen Verkaufskontrakt in Tolfaer Alaun für bestimmte Gebiete abschlossen (1. Februar 1469) 1. Die Firma übernahm von der Camera apostolica 18 000 Cantare Alaun innerhalb dreier Jahre zu fest bestimmtem Preise und verpflichtete sich, jährlich 6000 Cantare davon aus der päpstlichen Niederlage, in Venedig abzunehmen. Das alleinige Ver1 Gottlob a. a. O., S. 297. Z i p p e 1 a. a. O., S. 47. Schon die italienischen Firmen, die vor der Entdeckung von Tolfa den türkischen Alaun nach Europa einführten, hatten mit ihren Haupt¬ abnehmern monopolistische Lieferungskontrakte abgeschlossen. So beklagten sich in den vierziger Jahren des 15. Jahrhunderts die Hansen darüber, daß die „Lombarden“ mit gewissen Brügger Kauf¬ leuten verabredet hätten, nur ihnen Alaun, und zwar in bestimmter jährlicher Menge zu liefern. Die Folge war eine enorme Preis¬ steigerung dieser Ware. Cfr. E. D a e n e 11 , Die Blütezeit der deutschen Hanse. Hansische Geschichte von der 2. Hälfte des 14. bis zum letzten Viertel des 15. Jahrhunderts. 2 Bde. I. Bd., S. 398 f. und die dort zitierte Literatur. Strieder , Studien z. Gesch . kapituliet . Organisationeformen . 12 178 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. kaufsrecht der Firma erstreckte sich außer auf alles venetianische Gebiet auf Friaul, die Mark Treviso, die Lombardei und die Romagna bis nach Fano im Süden. Über die Alpen hinaus reichte es in das Gebiet des deutschen Kaisers und des Herzogs von Österreich hinein. Nirgends in den genannten Gegenden durfte die Camera apostolica Tolfaer Alaun verkaufen oder verkaufen lassen. Wir können es hier unterlassen, die Frage zu beantworten, wie sich mit diesem Kontrakt zwischen der Camera apostolica und dem Venetianer Kaufmann Bartolomeo Giorgio die Tatsache vereinigt, daß Paul II. im Jahre 1470 der Republik Venedig be¬ deutende Quantitäten von Alaun überließ 1. Uns kommt es hier nur darauf an, die Bemühungen zu kennzeichnen, die sich die Camera apostolica machte, um ein Monopol des Tolfaer Alauns überall auch faktisch durchzuführen. Diese Bemühungen waren in dem Kontrakt mit Bartolomeo Giorgio bei weitem nicht erschöpft. Besonders in England und in Burgund, den beiden damaligen Hauptkonsumenten des Alauns, setzten um dieselbe Zeit Versuche der Kurie ein, genügende Sicher¬ heit für die ausnahmslose Verwertung von Tolfaer Alaun zu er¬ halten. Als päpstlicher Nuntius eilte der Bischof Stephan von Lucca im Frühjahr 1466 nach England, um Eduard IV. und die englischen Städte in das päpstliche Interesse zu ziehen und Verträge ab¬ zuschließen2. In seiner vom 18. März 1466 datierten Instruktion wurde der Nuntius angewiesen, „er solle vom Könige ein strenges Verbot alles fremden Alauns fordern und mit ihm einen Alaun¬ lieferungskontrakt auf sechs bis zehn, eventuell auch mehr Jahre abschließen. Er dürfe den Papst und die apostolische Kammer auf die Lieferung der für alle Gebiete und Städte des Königreichs alljährlich benötigten Alaunvorräte nach London verpflichten und den Marktpreis auf 4 Pfund Flandrischer Groschen verabreden. Gleichzeitig sollte die Bulle über die geistlichen Strafen, die allen Übertretern des päpstlichen Alaunmonopols angedroht seien, in England publiziert werden.“ Leider sind wir über den Erfolg der Sendung des Bischofs Stephan von Lucca nicht unterrichtet. Genaueres wissen wir dagegen von einer gleichen Mission, die um dieselbe Zeit der Nuntius Lucas de Tolentis bei Karl dem Kühnen 1 Vgl . Zippel a. a. O,, S. 48 ff. 2 Gottlob a. a. O., S. 297f. Zippel , S. 396 ff. 1 * 8 Zweites Kapitel : Kartelle des 14. bis 18. Jahrhunderts. 179 von Burgund auszuführen hatte . Hier kam es zu dem Abschluß eines Kontraktes , der besonders folgende wichtige Bestimmungen enthielt. Karl der Kühne untersagte mit Zustimmung der General¬ räte und der Staatskommissare auf zwölf Jahre in seinen burgundischflandrischen Ländern den Import und den Konsum alles nichtrömischen Alauns 2. Nur das päpstliche Produkt, das durch den Faktor der Medici in Brügge, Tommaso Portinari 3, oder andere indirekte Beauftragte der Kurie eingeführt wurde, sollte in Flandern, Hennegau, Brabant , Friesland usw. verkauft, gekauft und ver¬ wendet werden 4. Auch alle fremden Surrogate für Alaun waren streng verboten 6. Karl der Kühne verpflichtete sich, der Kurie bei der Durchführung geistlicher Strafen gegen die Übertreter des päpstlichen Monopols seinen weltlichen Arm zu leihen. Demgegenüber übernahm der päpstliche Nuntius für seinen Auftraggeber folgende Verpflichtungen: Die Camera apostoiica bzw. die Pachtgesellschaft der Tolfaer Alaungruben durfte in den den Herrschaftsgebieten des Herzogs benachbarten Ländern Alaun unter keinen Umständen billiger verkaufen als in Karl des Kühnen Reich. Der Verkaufspreis des Tolfaer Alauns wurde für Burgund, Flandern usw. auf 4 % Pfund flandrischer Groschen pro Brügger Caricum herabgesetzt 6. Davon gingen6 solidi (1 Pfund = 20 solidi) in die Kasse der herzoglichen Finanzverwaltung. Die päpstliche Kammer und ihr Tolfaer Pächter hatten dafür zu sorgen, daß in 1 4. Mai 1466 . Im Wortlaut veröffentlicht bei Th ein er a . a . O., III , S. 452 ff. 2 Das Edikt ist erhalten (24. Juni 1468 ). Gottlob, S . 298 Anm . 2. 3 Uber diesen Mann sind wir durch die zwei folgenden Auf¬ sätze , die auch auf den Alaunhandel Bezug nehmen , ausführlich unterrichtet . G. von der Ropp, Zur Geschichte des Alaun¬ handels im 15. Jahrhundert . Hansische Geschichtsblätter . Jahrg. 1900 . S. 119 ff . O. Meltzing, Tommaso Portinari und sein Konflikt mit der Hanse . Hansische Geschichtsblätter . Jahrg . 1906, S. 101 ff. 4 „in nostris dominiis iacentibus in partibus septentrionis .“ 6Interdicere „ omne commercium terre specierum et aliarum mixturarum , quas loco aluminis opifices introduxerunt , ac ipsi sub gravissimis penis prohibere , ne deinceps huius modi mixturis utantur ." T h e i n e r a . a . O. Vgl . auch Z i p p e 1 a . a . O., S. 390 Anm . 3. 8 Also ein ziemlich günstiger Abschluß . Cfr . Zippel, S . 391 Anm . 1. 12 * 6 180 DrittesBadi : Monopole , Kartelle und Aktiengesellschaften im Mittelalter. ihrer» Brügger Magazinen stets genügende Vorräte verkaufsfertigen Alauns sich befanden. Der von der Kurie gewünschte Zweck eines alleinigen Ver¬ brauches von Tolfaer Alaun in Flandern wurde auch durch den Kontrakt vom 4. Mai 1466 nicht erreicht. Um so weniger, als diese Abmachungen von seiten der flandrischen Regierung bald nicht mehr eingehalten wurden 1, Fremder Alaun machte dem Tolfaer in Brügge bald erhebliche Konkurrenz. Dasselbe Bild gewahren wir in England 2 und besonders auch in Venedig3 seit dem Verlaufe der siebziger und achtziger Jahre. Die Einfuhr des türkischen Alauns erlangte trotz aller kirchlichen Zensuren wieder eine große Bedeutung für die europäischen Großmärkte in Alaun. Außerdem wurden vielfach einheimische Alaungruben in- den europäischen Staaten — z. B. in Frankreich 4, in Böhmen usw. —•entdeckt und ausgebeutet. Man darf behaupten, daß die erste Blüte des Tolfaer Alaungeschäftes mit Pauls II. Tod endete. Seit Sixtus IV. ging der Absatz und damit natürlich auch die Einnahmen für die Camera apostolica resp. die Cruciata zurück. Von Nachteil war es auch, daß Sixtus IV. sich mit den Pächtern der päpstlichen Alaungruben, den Medici, überwarf. Seitdem führten verschiedene andere große italienische Bankfirmen den Tolfaer Alaunbetrieb und -vertrieb. Unter Sixtus die Genuesen Domenico Centurioni und Giovanni de Auria & Co., unter Alexander VI. Paolo Rucelai & Co,, unter 1 Näheres Gottlob a. a. O., S. 299, 303. Z i p p e 1 a. a. O., S. 392 ff. 2 Gottlob a. a. O., S. 301 ff. Zippel a. a. O., S. 397. 8 Gottlob a. a. O., S. 300, 303. 4 Gottlob a. a. O., S. 304. Zippel a. a. O., S. 400. ,,En Languedoc, Louis XII . a donné, en 1504, à un Italien Domenico Baldini, le droit d’ouvrir des mines d’alun, vitriol et soufre quel¬ que part qu’il s’en puisse trouver.“ Imbart de la Tour, Les origines de la Réforme. Paris 1905/09, I. Bd., S. 232 f. Im Jahre 1507 wurden neue Alaungruben entdeckt, so daß schon 1512 der französische König die Einfuhr fremden, d. h. römischen Alauns verbieten konnte. Diese Störung der finanziellen Kreise der Kurie war einer der Gründe des Bruches Julius II. mit Frankreich und der großen Allianz gegen Frankreich. Imbart a. a. O., S. 233. 6 Schon 1470 gab König Wenzel dem UnterhofmarschallValentin das Privileg am Dorfe Preilep (Pf’ilep) auf Gold, Silber und besonders auf Alaun zu bauen. Salz, Geschichte der böhmischen Industrie, S. 171. 1 Zweites Kapitel : Kartelle des 14. bis 18. Jahrhunderts. 181 Julius II . der berühmte Agostino Chigi, unter Leo X . Andrea Bellanti L Entsprechend dem viel geringeren Preis , der auf den Welt¬ märkten des beginnenden 16. Jahrhunderts für Alaun gezahlt wurde 2, waren natürlich auch die Pachtsummen gesunken , die die genannten Nachfolger der Medici der Camera apostolica zahlten. Die obenerwähnte Firma Andrea Bellanti , die 1513 auf zwölf Jahre die Pacht übernahm , erlegte dafür die Summe von jährlich 15 000 Dukaten . Wir wissen, daß dieselbe Gerechtsame Paul II . mehr als 100 000 Dukaten jährlich eingebraeht hatte. Nun hat Gottlob mit Recht seine Verwunderung darüber ausgesprochen , daß trotz der Reformation , welche die letzLe Möglich¬ keit beseitigte , ein päpstliches Alaunmonopol mit religiösen Mitteln wenigstens noch liier und da durchzuführen , dennoch in den vierziger oder fünfziger Jahren des 16. Jahrhunderts der aposto¬ lischen Kammer wieder erhöhte Einnahmen aus der Pacht der Tolfaer Alaungruben erwuchsen . Tatsächlich „übernahm Messer Bindinello , Erbe des Agostino Sauli, im Jahre 1553 die Tolfaer Gruben auf zwölf Jahre für den jährlichen Zins von 21 250 Scudi und von 1557 an bezahlte er sogar jedes Jahr 34 250 Scudi an die päpstliche Kammer “ 3. Es wird sich fragen , wie war eine solche Wertsteigerung der päpstlichen Alaungruben in Tolfa denkbar ? Vielleicht bietet das folgende die Möglichkeit einer späteren Beantwortung dieser inter¬ essanten Frage und die Anregung , aus den Akten des vatikanischen Archivs und des römischen Staatsarchivs die Geschichte der kapi¬ talistischen Organisationsformen im Alaunhandel des 16. Jahr¬ hunderts und darüber hinaus zu verfolgen. In einer Bittschrift , die Cristoff von Gerndorf , der Monopol¬ inhaber des böhmischen Alaun - und Kupfervitriolhandels im Jahre 1 Gottlob a . a . O., S. 299 f. und mit einigen Abweichungen Zippel a . a . O., S. 413 ff. 2 „Im Jahre 1506 kostete der Zentner Alaun , für den man früher in Civitavecchia 3 Goldgulden gelöst hatte , ebendort nur mehr 28— 30 solidi , nicht einmal Dukaten , und Julius II . sah sich genötigt , um die englischen Händler noch in Civitavecchia festzuhalten , den Preis sogar auf 20 lind 22 solid ! herabzusetzen .“ Gottlob a . a . O., S. 305. 3 Gottlob a . a . O., S. 305. 4 182 Drittes Duell: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. 1540 an den deutschen König Ferdinand richtete 1, erwähnte er die Tatsache, daß „die Schotzische Geselschaft das romanische und hispanische Alaun in einer Hand habe und den Preis dieser Ware hochhalte“. Zweifellos ist mit der „ Schotzischen Gesellschaft“ die Firma Schetz in Antwerpen, eines der bedeutendsten Großhandels¬ häuser des 16. Jahrhunderts 2, gemeint. Danach hätte also diese Firma durch die vereinigte Herrschaft über die beiden damals wichtigsten Alaunwerke Europas ein Monopol für diese Ware be¬ sessen. Wenigstens für die Niederlande, für Italien und England, also die unzweifelhaft auch damals noch wichtigsten Gebiete für den Alaunverbrauch. Daß gerade ein Antwerpener Handels¬ haus dieses Monopol schuf, ist verständlich, wenn wir hören, daß schon im Jahre 1491 Philipp der Schöne und Maximilian I. der Stadt Antwerpen den Stapel für den Import und Verkauf alles Alauns in den Niederlanden verliehen3. Begreiflich ist es auch, daß die Schetz ein Alaunmonopol in die Hand zu bekommen suchten. Die bei den großen Firmen des 16. Jahrhunderts allgemein übliche Neigung zu Monopolisierungsversuchen war bei der Handels¬ gesellschaft Schetz, wie es scheint, in besonders starkem Maße vorhanden. So wissen wir, daß sie auch z. B. in Galmey ein Monopol durchzuführen verstand . 1 Manuskript des F . A . Wien . Cr. v . Gerndorf von Hohenelb hatte ein Verfahren erfunden , um aus dem Grubenwasser von Kutten¬ berg in Böhmen Alaun und Kupfervitriol zu gewinnen . König Ferdinand gab ihm ein Privileg vier Jahre hindurch allein das neue Verfahren anwenden zu dürfen . Außerdem erhielt Gerndorf ein Monopol für die Alaungewinnung in Böhmen und in Ferdinands Erblanden . F . A. Wien . Böhmen , Kuttenberg , 1. April 1540 . Vgl. die Mandate Ferdinands , durch die verboten wurde , ausländisches Alaun und Kupfervitriol in Böhmen und den inkorporierten Ländern zu verkaufen . F . A. Schmidt a . a . O., I, 2, Nr . 45 , 52 , 66 . Zu Gerndorfs Tätigkeit im böhmischen Alaunbergbau vgl . auch Graf Kaspar Stern borg, Umrisse einer Geschichte des böhmischen Bergbaus . 2 Bde . in 3 Abteilungen . Prag 1836/38 . I, 2, S. 83 ff. Danach S a 1 z a . a . 0 ., S. 172. 8 Vgl . Ehrenberg, Zeitalter der Fugger . Register. 3 E . Rach fahl, Wilhelm von Oranien und der nieder¬ ländische Aufstand . 2 Bde . in 3 Abteilungen . Halle 1906/08 . I . Bd ., S. 322. 4 R a c h f a h 1 a . a . O., I. Bd ., S. 607 . „Pareillement ceulx de Namur ont reinonstré que la Majesté avoit mis entre les mains des Zweites Kapitel : Kartelle des 14. bis 18. Jahrhunderts . 183 Von der Tatsache der Vereinigung der Tolfaer und der spanischen Alaunproduktion in der Hand der Firma Schetz aus, ist nun meines Erachtens die erneute Wertsteigerung der päpst¬ lichen Alaungruben von Tolfa leicht verständlich. Je mehr das Monopol der Schetz den Alaunpreis hob, um so mehr mußte natur¬ gemäß auch die Tolfaer Pachtsumme — nach Ablauf des laufenden Kontraktes — steigen. Kartellbestrebungen im süddeutschen Früh¬ kapitalismus (besonders im 16. Jahrhundert). Wenn man fortgeschrittene wirtschaftliche Organisationsformen der Vergangenheit, also z. B. Kartelle, in Deutschland sucht, so wird man naturgemäß in erster Linie seinen Blick auf die Zeit des 16. Jahrhunderts zu richten haben, auf das Zeitalter der Fugger und Welser, auf jene Zeit deutscher weltwirtschaftlicher Macht¬ entfaltung, die erst im 19. Jahrhundert von uns wieder überholt wurde. Schon die gesetzgeberischen Maßnahmen, die sich von Reichs¬ und Landtagen, von Kreis- und Stadtratsversammlungen aus gegen Kartellversuche erhoben, und die vorangehenden Klagen der Be¬ troffenen dort, lassen den Schluß auf eine relativ starke Verbreitung hoirs de Erasmus Schetz , par réserve, la marchandise de cahnine, pierre fort nécessaire aux chauldronniers de leur pays, de sorte qu’ilz achetoyent maintenant XVIII patars ce qu’ilz souloyent avoir pour six patars: requérantz aussy que à tèle réserve fust pourveu et remédié par les estatz.“ Margarete v. Parma, die Regentin der Niederlande, verlängerte am 29. Mai 1562 den Pachtvertrag der Gebrüder Schetz aus Antwerpen über die Galmeibergwerke im Herzog¬ tum Limburg um weitere 6 /> Jahre . In dem Vertrag heißt es aus¬ drücklich, daß die Schetz die Pacht schon länger innehatten: „Van weghen Coenrad Schets ende consorten is ons verthoent gewest, hoe dat Gaspar Schets, heere van Grobbendonck, ende zynen andere broeders over lang h en tyt in pachte , gehouden hebben die mynen van de calmynbergen van onsen lande ende hertoochdomme van Lymborch . . .“ Der interessante Kontrakt ist ab¬ gedruckt bei R. A. Peltzer, Geschichte der Messingindustrieund der künstlerischen Arbeiten in Messing (Dinanderies) in Aachen und den Ländern zwischen Maas und Rhein von der Römerzeit bis zur Gegenwart. Zeitschr. d. Aachener Geschichtsvercins, 30. Bd. (1908), S. 437 ff. 184 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. dieser Erscheinung zu. Das Gleiche gilt von den häufigen Ver¬ urteilungen, die die Institute der Kartelle, Syndikate usw. durch die Sittenprediger des 16. Jahrhunderts erfuhren. Auf beide Quellenerwähnungen müssen wir zunächst unsere Blicke lenken, ehe wir zu einer Besprechung der tatsächlich uns bekannt ge¬ wordenen Kartelle übergehen. Gesetzgebung und Kartelle im 16. Jahrhundert. Wenn sich die offizielle Gesetzgebung des 16. Jahrhunderts — übrigens, wie wir sahen, ohne Erfolg und an den maßgebenden, führenden Stellen auch ohne wirklichen Ernst — gegen die Monopole wandte, so verstand sie darunter auch die Kartelle mit. Das geht schon deutlich aus den deutschen Reichstagsverhandlungenund aus den sich daran anknüpfenden Debatten hervor. Es genügt hier, auf die folgenden Stellen hinzuweisen. In einem Kommissionsbericht des sog. kleinen Monopolaussclmsses des Reichstages zu Nürnberg (1522/23) ist davon die Rede, daß die Gesellschaften „heimlich verstant mit einander machen, wie sie iderlei irer war geben wolten, damit ir keiner den andern zu wolfeilem kauf ver¬ ursacht oder dringe“ 1, Ich weiß nicht, was man unter den so charakterisierten heimlichen Verabredungen der Kaufleute anders verstehen will als Kartelle. Leider ist die Edition der deutschen Reichstagsakten erst bis zum Jahre 1522 fortgeschritten, so daß wir auch die Reichstags¬ verhandlungen über „Monopole“ nur bis dahin genauer verfolgen können. Die kurzen, zusammenfassendenAbschlüsse der Verhand¬ lungen aber, wie sie uns in den Sammlungen der Reichstagsabschiede vorliegen, sagen nicht mit der wünschenswertenKlarheit, ob unter den Monopolisten auch Kartellisten mit zu verstehen sind. Immerhin zeigen schon Interpretationen des 17. Jahrhunderts , die sich auf (kanonische) Rechtslehrer des 16. Jahrhunderts berufen, daß unter denjenigen Kaufleuten, die sich der „Monopolitas“ schuldig machen, auch Kartellkontrahenten zu verstehen sind. So definiert Cieorg Friedrich Schuster in seiner Dissertatio juridica in pragmaticam imp. rom. german. sanctionem de monopoliis ad tit . XVIII . reforma1 Vgl. Deutsche Reiehstagsakten. Jüngere Reihe. III . Bd., S. 589 . Dazu vergi . S. 573 , Zeile 18— 26 ; S. 581 , Z. 5 ff . ; S. 585, Z. 30 ff. 3* Zweites Kapitel : Kartelle des 14. l)is 18. Jahrhunderts . 185 tionis politicae de anno, 15481als Monopolisten auch die „plures alii, qui licet non directe, per obliquum tamen privati compendii ergo commerciorum libertatem restringunt, variis conven¬ tionibus , pactis ac collusionibus , videlicet de non emendo vendendove ultra vel infra certum p r e t i u m“. Bestätigt wird unsere Interpretation und die Ansicht, daß die Gesetzgebung des 16. Jahrhunderts unter Monopolien die Kartelle mit einschloß, durch folgende Verordnungen der Landesgesetzgebung. ïn einer sächsischen Gesetzesverordnung vom Jahre 1534 heißt es: „Es sollen auch die Ritterschaft und die Städte fleißig Achtung haben, damit die Wollen-Käuffer und Vorkäuffer sich nicht versammlen und einigs Kauffs sich vereinigen, wie sie die Wolle und nicht anders kauffen oder verkauften wollen; und wo sie solche Vereinigung der Wollen-Käuffer und Vorkäuffer befinden, die sollen von jeder Obrigkeit darum gebührlich gestrafft werden und die Städte solches alle Schaar- und Wollen-Märckte ausruffen lassen 2.“ Es handelt sich hier um Einkäuferkartelle, wie sie uns auch sonst aus dem 16. Jahrhundert und früher bekannt sind. Ich erinnere nur an die Verhältnisse im böhmischen Bergbau. Dort kauften Erzkäufer von den Gewerken und Häuern das Erz auf. Schon die Constitutiones juris metallici Wenzels IL (1283—1305) tadelten die Kartellierungen der Erzkäufer zur Drückung der Preise, die oft dabei vorkamen 8. Später bedrohte die Kuttenberger Ord¬ nung diese Kartelle mit strengen Strafen. Etwas näher sind wir auch unterrichtet über die kartellistischen Verabredungen der süddeutschen Salzhandelsleute. Im ersten Drittel des 16. Jahrhunderts hatten die Salzendter, d. h. die Salzhandelsleute, in Wasserburg und Traunstein, aber auch wohl in anderen Legstädten des Reichenhaller Salzes, unter 1 Gießen 1686 , S. 18. Die Dissertation ist unter Nicol . Thilelilus angefertigt. 2 Aus: „ Letzterer Pragischer Vertrag zwischen Land und Städten des Markgrafentums Ober -Lausitz , das Justiz - und Policeiwesen u . a . betreffend nebst König Ferdinand I. Confirmation dar¬ über , 15. September 1534 .“ In Codex Augusteus , III . Teil , S. 44. 3 Adolf Zycha, Das böhmische Bergrecht des Mittelalters auf Grundlage des Bergrechts von Iglau. I. Bd ., S . VIII ; S. 171 Anm . 115. 2 Bde. Berlin 1900. 186 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. sich ein Kartell abgeschlossen, welches das Angebot in der Weise regelte, „daß der eine der Kaufleute nach dem Verkauf seines Quantums Salz mit der Abholung einer weiteren Fracht solange wartete, bis auch der nächste und dieser bis der dritte usf., bis jeder sein Salz verkauft hatte “ 1. Auch die Münchener Salzendter hatten ein Statut , wonach jeder nur ein bestimmtes Quantum Salz einführen durfte ohne Rücksicht auf den jeweiligen Bedarf2. Nun waren zwar die Salzendter oder Salzfertiger, wie man anderwärts sagte, in einer Zunft organi¬ siert, aber es wäre sehr verkehrt, diese Kaufleute etwa als Hand¬ werker anzusprechen und ihre obengenannten Verabredungen als Zunftbestimmungen aus der Reihe der Kartelle zu streichen 3. Welch stark kapitalistischer Geist in den Salzfertigern herrschte, erkennt man aus dem folgenden. Die Salzendter verlegten die SalzFuhrleute auf eine sehr „fortgeschrittene“ Art und Weise. Sie gaben Vorschuß in Gestalt von Geld, Tuch oder Eisen usw. und brachten dadurch die Bauersleute (das waren die Fuhrleute zumeist) so stark in Abhängigkeit von sich, daß sie sie verpflichten konnten, Salz erst dann zu holen bzw. weiter zu führen, wenn es den Salz¬ fertigern geeignet erschien. Dabei zeigten sich übrigens auch die schlimmsten Seiten des sog. Trucksystems. Eine Salzausgangs¬ ordnung der Regierung mußte bestimmen, daß die Salzendter, „wenn sie überhaupt schon vorher den Bauern die Spesen der Salzexpedition in Naturalien reichen wollten, doch Getreide, Pferde, Eisen, Tuch u. a. Pfennwerte nicht teurer abgeben durften als sie derzeit im Preise stehen“ 4. Um mehr als lediglich eine Zunftabmachung lokaler Natur handelt es sich auch bei der Konvention sächsischer Gerber, die den Gegenstand erregter Debatten des Dresdener Landtags von 1527 bildete 5. Schon deshalb müssen wir hier ein Kartell an1 F . X . E b e r 1 e , Die Organisation des Reichenhaller Salzwesens unter dem herzoglichen und kurfürstlichen , Produktions - und Handelsmonopol . Münchener Dissertation 1910 , S . 72. 2 A. a . O., S. 73. 3 Vgl. Liefmann, Die Unternehmerverbände , S . 136. ‘Eberlea. a . O., S . 172. 6Zum „ siebenden , solte guth achtung zu geben sein auf die gerber , die durch ire voraynigung am leder den schustern theurung einführen . Daraus fleust , daß die schuster die schuen auch vortheuren müssen .“ Leipziger Ratsarchiv . Tit . II . A . Nr . 1. * Zweites Kapitel ; Kartelle des 14. bis 18. Jahrhunderts . 187 nehmen, weil es sich offenbar um eine intei lokale Abmachung der sächsischen Gerber handelte. Prinzipiell ist bei der Fragestellung „Zünfte und Kartelle“ zu beachten, daß die Innungsmitglieder sehr wohl über ihre Zunft¬ bestimmungen hinaus Kartelle schließen konnten 1. Besonders unter den sog. Handelszünften wird das oft vorgekommen sein. Gegen die Verabredungen der Handwerker, soweit sie den Rahmen der — von der städtischen Obrigkeit gutgeheißenen — Zunft¬ bestimmungen überschritten und kartellistischer Natur wurden, richteten sich bereits die Bestimmungen des Breve dell’ ordine del mare délia città di Pisa vom Jahre 13432, worin es heißt: „Non si possa nè debbia fare . . . alcuno monupolio . . . di lavorare u di non lavorare u vero per certo pregio tanto. “ Noch deutlicher sprechen die Antimonopolgesetze des 16. Jahrhunderts das aus, worauf es uns hier ankommt. Und ebenso die Lehren der Wirtschaftsmoralisten jenes Zeitalters. So heißt es bei Salicetus3: „Delendum et illud statutum [est] quod vetat opus ab uno coeptum ab alio perfici posse absque consensu eius qui inchoaverit.“ Und in ähnlicher Weise sind in der Aurea practica des Johannes Berberius 4 diejenigen „conventiones, congregationes und conspira¬ tiones“ als moralisch unerlaubt erklärt, laut denen der einzelne Handwerker desselben Gewerbes nicht billiger verkaufen dürfe als Bl . 76. „ Der 7. Artikel , die gerber belangende acht der ausschuß von nöthen einsehung zu thuene , daß sie nicht aus irem voraynigten und beschlossenen kaufe den gemeinen schustern einen verderb¬ lichen aufsatz machten . . . .“ Desgleichen Bl . 91 f. 1 Noch heute kommt das vor ! „ Im lokalen Handwerk und Kleinhandel findet man nicht selten solche im privaten Kreise (Innung , Klub usw .) vereinbarte Kartellierungen , die . . . sich von den festen , durch Konventionalstrafen gesicherten , großen Syndi¬ katen kaum unterscheiden .“ Wörterbuch der Volkswirtschaft. Artikel : „ Unternehmerverbände .“ Auf einige kartellistische Hand¬ werkerabmachungen , die allerdings die Bestätigung der städtischen Obrigkeit erhielten und deshalb nach der allgemeinen Ansicht nicht zu den Kartellen gerechnet werden können , hat F . Eulenburg hingewiesen . In Viertèljahrssehrift für Sozial - und Wirtschafts¬ geschichte , 2. Bd . (1904 ), S. 270. 2 Ediert Firenze 1857 , S. 478. 3 Vgl. Benv . Straecha, Tractatus de mercatura seu merca¬ tore , S. 283 der Ausgabe Venedig 1575. 4 Coloniae Agrip . 1576 , S. 107. 188 Drittes Buch: Monopole, Kartelle und Aktiengesellschaften im Mittelalter. der andere, oder laut denen er nicht die Arbeit vollenden darf, die ein anderer angefangen hat . Es ist genau derselbe Standpunkt, auf dem auch die Polizeiordnung des heiligen römischen Reiches von 1548 steht, wenn sie in Tit. XXXVI bestimmt: „Und nach¬ dem die Handwerker in ihren Zünften und sonst zu Zeiten sich miteinander vereinigen und vergleichen, daß einer seine gemachte Arbeit oder Werk in feilem Kauf nicht mehr oder weniger ver¬ kaufen soll, dann der ander und also einen Aufschlag oder Steige¬ rung machen, daß diejenigen, so derselben Arbeit nothdürftig sein und kaufen wollen, ihnen die ihres Gefallens bezahlen müssen etc. meinen wir hiemit sonstlich und wollen, daß solches von den Oberkeiten hinfüro keineswegs geduldet oder gestattet , sondern gebiihrlichs Einsehens gethan werde. Wo aber darüber von Handwerckern geschehe, daß alsdann die Qberkeit dieselben nach Gestalt der Sachen unnachläßlich strafen sollen1.“ Sittenlehre und Kartelle im 16. Jahrhundert. Wie ernst es die christliche Kirche des Mittelalters mit ihrer strengen Wirtschaftsethik nahm, das erkennt man aus dem breiten Raum, den wirtschaftsmoralische Auseinandersetzungenin den In¬ struktionen für Beichtväter, in den sog. Summae confessorum de casibus conscientiae oder den Summae poenitentiae einnehmen. Namentlich seit dem 15. Jahrhundert wuchsen dabei die Fragen, was „justum pretium“ sei und was nicht, was von kaufmännischer Geschäftstätigkeit unter die pravitas usuraria falle und was nicht, dermaßen an, daß besondere Monographien von den Kanonisten darüber geschrieben werden mußten („Tractatus de usuris et con¬ tractibus mercatorum“ und ähnlich betitelt). Th . Muther 2 und nach ihm Rod . Stintzing 3 haben bereits in den sechziger 1 Neue Sammlung der Reichsabschiede , II , S. 605 . Es bleibt zu untersuchen , ob wir es in den „ Bündnissen und Vereinigungen der Handwerker gegen die Bürger “ , die Maximilian I. im Jahre 1518 annullierte , mit Kartellen zu tun haben . Vgl . F . B . v . B u c h holtz, Geschichte der Regierung Ferdinands I., VIII . Bd. S. 254. 2 Th . Muther, Aus dem Universitäts - und Gelehrtenleben im Zeitalter der Reformation . Erlangen 1866 . S . 154 ff. 3 Roderich Stintzing, Geschichte der populären Litera¬ tur des römisch -kanonischen Rechts in Deutschland am Ende des 15 . und im Anfang des 16. Jahrhunderts . Leipzig 1867 . S . 540. Zweites Kapitel : Kartelle des 14. bis 18. Jahrhunderts . 189 Jahren des 19. Jahrhunderts auf die Bedeutung dieser Literatur¬ gattung für die Erkenntnis der verschiedenen kaufmännischen Ge¬ schäfte jener Zeit aufmerksam gemacht. Die in den Summen und Wuchertraktaten niedergelegten wirt¬ schaftsethischen Anschauungen dienten nun nicht nur den Geist¬ lichen zur Anleitung für die Beratung in Gewissensfragen im Beicht¬ stühle, auch den Predigern auf den Kanzeln der großen Städte boten sie die Richtschnur für ihre Forderung einer strengen alt¬ ruistischen Wirtschaftsmoral. Vielfach haben ja die großen sozialen Prediger des 15. und 16. Jahrhunderts selbst auch vielverbreitete Wuchertraktate verfaßt; z. B. der vielleicht bedeutendste, jeden¬ falls der wirksamste von ihnen, Johann Capistrano 1. Wie steht es nun — so fragen wir für unsere vorliegende Unter¬ suchung —- mit der Erwähnung von Kartellen und Syndikaten in den genannten Literaturgattungen des Mittelalters und der be¬ ginnenden Neuzeit? Sind den Moralisten jener Zeit diese Er¬ scheinungen einer kapitalistischen Wirtschaftsordnung bekannt? Ich begnüge mich im folgenden damit, einen Auszug aus einer Predigt Geilers von Kaisersberg zu geben. Schon aus seinen Aus¬ führungen dürfte zur Genüge ersichtlich sein, daß Kartelle eine häufige Erscheinung des 16. Jahrhunderts waren. Erscheinungen, die trotz der Heimlichkeit, mit der die Kaufleute dabei zu verfahren pflegten, doch auch der breiteren Öffentlichkeit nicht entgingen. In der fraglichen Predigt, „Wie ein frommer Kaufmann sein soll“ betitelt, handelt Geiler zunächst von den eigentlichen Mono¬ polisten. Da führt er aus : „. . . heissen Monopoli, die da ein War allein feil hond und haben wellen. Und über semlichs, so erwerben sie ein Freiheit, Brief und Sigel von eim Fürsten im Land oder von eim Künig. Das seind die rechten Monopoli, die ein Ding allein verkaufen wellen.“ Den eigentlichen Monopolisten stellt Geiler sodann die Karteilisten gegenüber: „Die andren Monopoli seind, die nit ein Ding wellend allein verkaufen, aber sie stupfen 2 mit einander umb das gelt (de precio ) , wie sie es geben wellend , also und anders nit. Und d i e Monopoli heisse ich Stupfer, als da sie etwan miteinander stupfen. . . . Also stupfen dise die war also ze geben 1 Muther a. a. O., S. 155 f. 2 =heimlich etwas verabreden. * 190 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. und nit anders bei seinem Eid 1. Dy seind minder denn dy ersten . [Diese] wellend den Gewin allein hon und nieman darf es feil hon , denn sie. Sy stont allein im Trog als ein Mor 2, die kein andre Suw hinein wil lassen . Also wellen sie die War allein hon und yedermann der muss sein Liecht von irem Liecht anzünden . Das thunt [jene ] nit , sie stupfen numer zesamen , dass keiner ein Ellen desThuchs , oder was es ist , wölfler 3 gebe denn also. Er mag es wol türer geben , aber nit wölfler. Und wenn sie es schon uff ein zimlich Gelt setzen und die Leut nit übermessen , noch so seind es Monopolistüpfer. Warumb ist das Stupfen unzimlich ? Darum , es hat ein Schein und scheint wie es ein erber Ding sei, und ist doch dem gemeinen Nutz schedlich . Wie ist das ? Es nimt dem Merck 4 sein Freiheit. Es ist hie und anderswo ein freier Mergt , darumb so soi iederman sein Kaufmanschatz mögen geben wie er welle. Dy Freiheit nimt das Stupfen hinweg . Wann er hat gestupft und geschworen das also zegeben und nit wölfler aber wol thürer . Zu dem andern, so ist es schedlich dem gemeinem Man wenn ein Ding ze geben hat sein zall wie er es geben wil oder mag und er dennocht hat erbern Gewinn daran . An dem gelt mage er auf und abe gon, mee oder minder neminen umb ein phennig oder zwen und bestott er dennocht wol darbei . Nim dz Exempel : Ich setz dass ein Thuchman , der nit gestupft hat , der setzt für sich und schlecht an , dass er ein Ellen wol mag geben umb fier Schilling Pfennig . Und ob er es eins Pfennigs neher gebe, so hat er dennecht ein erbern Gewinn; wann der Gewinn ist nit gesetzt auf ein Oertle 6, oder auf ein Fierteil eines Oertlis . Es gat uff und ab . . . . Es kumpt ein guter Fründ, dem will ers eins Pfenniges neher geben , dann umb die vier Schil¬ ling . Das mag er thun wenn er nit ist Monopolus, ein Stupfer und nit gestupft hat . Wenn er aber gestupft hat , so gethar er seinez Frünt den Pfennig nit nachlon ; wann er wer meineidigk . Wann 1 Der Eidschwur kommt bei älteren Kartellen oft vor. „Iurata fide constituunt ut nullus eorum nisi tanti vendat. S c a c c i a , Tractatus de commerciis et cambio. Frankfurter Aus¬ gabe 1648. S. 300. 2 = Sau. 3 D. h. wohlfeiler. 4 Markt. 6 Ein Ort ist der vierte Teil eines Guldens. Zweites Kapitel : Kartelle des 14. bis 18. Jahrhunderts . 191 er hat gestupft, ein Ellen nit neher ze geben denn eben umb die vier Schilling. Darum ist das Stupfen schedlich dem gemeinen Nutz.“ Auch unter den Handwerkern jener Zeit kommen nach Geiler solche kartellistische Verabredungen vor. Verabredungen, die, wie die oben schon von uns erwähnten, über die obrigkeitlicherseits zugeiassenen Zunftbestimmungen hinaus gingen, auf privater Ab¬ machung der Unternehmer beruhten und die deshalb unter den Begriff der Kartelle fallen. Hören wir Geiler von Kaisersberg: „. . . Die 4. Monopoli sein die Bader und die Scherer, die ein Statut machen und stupfen zesamen niemans ze baden noch ze scheren, denn eben umb ein semlich Gelt. Das soll nit sein. Es ist unrecht. Es stot im Text keiserlichs Rechten de balneatoribus 1. Die 5. Stupfer, das sein die Murer und Zimmerleut; die stupfen ze¬ samen: Wenn einer ein Werck angefahet, so gethar das Wergk keiner ausmachen. Es soi keiner dem andern in sein Werck gönn. Darum wenn einer eim ein Werck verdingt hat, so macht er ein Gerüst dar ; so hat er an eim andern Ort auch ein Gerüst, und also werdent Biderleut umb getriben, wan keiner gethar dem andern in sein Werck ston, das er angefangen hat . Die 6. Stupfer seind die Schneider (sectores). Wenn einer ein Rock schneidet, so legt er in dorthin und treibet in umb wan es gethar niemands in aus¬ machen. Die 7. Stupfer seind die auch im Text stont, die die Hüsser ferleihen und die Gedenck2in der Mess. . . . “ Bei der moralischen Bewertung macht nun Geiler keinen Unterschied zwischen dem „reinen" Monopolisten und dem Kartell¬ listen. Beider Tun erscheint ihm als gleich unmoralisch im höchsten Grade, als Todsünde. Uns interessiert hier nur, wie der einfluß¬ reiche Prediger über die Karteilisten urteilt : „Auch die andern, die do stupfen ein Ding also zu geben und nit umb minder . . . thunt wider das natürlich fernünftig Gesatz, auch wider das keiserlich und bäpstlich Gesatz, und ist bei grossen Penen, dem Keiser fallen, ferboten 3. Aus denen Stücken allen nemmen die cristenlichen Lerer , dass es Totsünd sei .“ 1 Gesetz Kaiser Zenos siehe oben. 2 vielleicht= Stände. * Geiler zeigt sich genau unterrichtet über die Strafbestim¬ mungen, wie sie auf den Reichstagen gegen die Monopolisten erlassen worden waren. Er führt sie genau in der genannten Predigt an. 192 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. Die Begründung , die Geiler diesem Verdikt gibt , strahlt in deutlich klarer Weise die ganz soziale, absolut unindividualistische Wirtschaftsethik des christlichen Mittelalters wider . Die einzelnen Glieder der menschlichen Gesellschaft sind dem großen sozialen Prediger — und der Wirtschaftsethik des Mittelalters überhaupt — wie die einzelnen Glieder am menschlichen Körper . Ein Glied hat für und mit dem anderen zu wirken und zu arbeiten zum Wolile des Ganzen . Ist es dazu nicht gewillt oder fähig , so muß un¬ verzüglich Remedur geschaffen werden . ,,Du sihest in eim Leib des Menschen , dz ein Glid dem gantzen Leib dienet ; mein Aug, das sicht den Füssen , die Füss gond und tragen den gantzen Leib. Der Mund isset dem Magen, der Mag nimpt die Speiss und teilt es dem gantzen Leib aus und allen Glidern . Und hettest du ein Klotzen uff der Achsslen ston , der dem gantzen Leib schedlich wer und ziig an sich davon andere Glider leben solten , du schnittest in hinweg und sprechest , was soi er da zu ston . Also sag ich : Wir hier zu Strassburg seind alle ein Leib und wir seint Glider . Ist nun ein Glid ein Kaufman , ein Stiipfer , der den andern Glideren schedlich ist . . . ein semlichen Glotzen soi man abhauwen und dannen thun,“ Die Predigt Geilers klingt in eine kräftige Mahnung namentlich an die Beichtväter aus , unnaclisichtlicb gegen Monopolisten und Karteilisten zu wirken : „Das hab ich euch sagen wollen , dass ir sehet wie die Juristen also genow hinzu reden . Die Theologi , die thunt es aber nicht . Darumb so red ich ungern von der Matery . . . . Nun muss man es dennocht auch sagen, es ist not . Es ist nit genug einem Kaufman , dass er spricht , ich hab von den Dingen nüt gewisst . Er ist nicht entschuldiget , er soit es gewisst, haben und die Juristen gefragt haben , die es bass wissen weder die 1 Theologi. Sehen auch die Beichtveter zu der Kaufleuten ; die solten die Ding auch wissen. Und die Ding , die ich gesagt hab , die schreiben weder Münch noch Pfaffen , aber gross treffenlich Lüt , als König und Keiser .“ Ich denke , die Ausführungen Geilers von Kaisersberg dürften zum Beweise der These genügen , daß Kartelle im 16. Jahrhundert eine nicht seltene wirtschaftliche Erscheinung waren . In dem dritten Kapitel dieses Buches werden noch einige uns tatsächlich i als die. 193 Zweites Kapitel : Kartelle des 14. bis 18. Jahrhunderts. bekannt gewordene Kartellbestrebungen der Montanindustrie auf¬ geführt werden . Zunächst folgen hier einige Blätter aus der Kartell¬ geschichte des 17. und 18. Jahrhunderts . Ich hoffe, die Liste durch spätere Archivstudien noch sehr verlängern zu können. Analekten zur Kartellgeschichte des 17. und 18.Jahrhunderts. Salzkartelle im 17. Jahrhundert. Eine tiefer eindringende Geschichte des europäischen Salz¬ handels und der Salzpröduktion zu Beginn der Neuzeit wird eine Menge Kartelle und kartellartiger Verabredungen zwischen Re¬ gierungen , die das Salzwesen monopolisiert hatten , aber auch zwischen Unternehmern , die im Salzhandel tätig waren , ans Licht fördern . Hier muß ich mich damit begnügen , auf einige Beispiele aus dem 17. Jahrhundert hinzuweisen 1, Zu interessanten Kartellabmachungen , bei denen wir etwas länger verweilen wollen, kam es in der Mitte des 17. Jahrhunderts zwischen der bayrischen Regierung und der Tiroler (österreichischen) Regierung , als Monopolinhabern der Reichenhaller bzw. Haller (Inntal ) Salzproduktion und des dortigen Salzhandels 2. Die Kon¬ kurrenz des bayrischen und Tiroler Salzes war sehr alt . Sie wurde den Einnahmen der Regierungen aber besonders seit der Zeit schädlich und gefährlich , seit reiche Salzgroßhändlcr von Mem¬ mingen , Lindau , Basel usw. die Sachlage auf geschickte Weise aus¬ zunutzen verstanden 3. 1 F . X . Eb erle, Die Organisation des Reichenhaller Salz¬ wesens unter dem herzoglichen und kurfürstlichen Produktions - und Handelsmonopol , Münchener Dissertation 1910 , erwähnt schon für das 16. Jahrhundert Abmachungen der Herzoge von Bayern (Reichen¬ hall ) mit benachbarten Salzproduzenten , die den Zweck verfolgten, die gegenseitige Konkurrenz auszuschalten . Cfr . S. 74, 86 . Ferner S. 78, 80. Dazu : J . G. Lori, Sammlung des bayerischen Berg¬ rechts . München 1764 . Nr . CLXI , S. 359. 2 Als Name für das Kartell kommt in den gleichzeitigen Akten der Ausdruck „ Convention , Intelligenz “ etc . vor. 3 Vgl. Hans Ockel, Die Entstehung des landesherrlichen Salzmonopols in Bayern und seine Verwaltung im 17. Jahrhundert. In Forschungen zur Geschichte Bayerns . 7. Bd . (1899 ), S. 26; Eb erle a . a . O., besonders S. 111 ff . Schon im 16. Jahrhundert hatte man die preisdrückende Wirkung der bayerisch -tirolischen Salzkonkurrenz erkannt und zu einem Kartell geraten . Die bayerische Strieder , Studien z. Geach . kftpitaliat . Organiaationsformen . 13 194 Drittes Buch: Monopole ) Kartelle und Aktiengesellschaften im Mittelalter. Die Kaufleute schlossen mit den genannten Regierungen Kon¬ trakte ab, in denen sie sich verpflichteten, jährlich so und soviel Hunderte oder auch Tausende Faß Salz abzunehmen. Sie setzten dann das Salz teils auf eigene Rechnung ab, teils lieferten sie es auf Konto einer Stadt oder Gemeinde an diese. Es handelt sich in den folgenden Ausführungen zumeist um Salzvertrieb nach dem Bodensee, dem Breisgau, den Schwarzwaldgegenden und besonders in die schweizerischen Kantone. Natürlich kontrahierten die Salzgroßhändler mit derjenigen Re¬ gierung, die ihnen die billigsten Preise und die günstigsten Zahlungsund Lieferungsbedingungen machte. Die Folge war ein harter Konkurrenzkampf zwischen Reichenhall und Hall im Inntal und naturgemäß ein niedriges Preisniveau. Nach verschiedenen früheren Versuchen, dem abzuhelfen, nach heftigen gegenseitigen Repres¬ salien auch der beiderseitigen Regierungen, kam es im Jahre 1649 zu einem Kartell der Beherrscher der Reichenhaller und Plaller Salzproduktion. Das Kartellstatut wurde am 5. August 1649 auf einem Vertretertag in Rosenheim beschlossen1. Es sollte vor¬ läufig vom 1. Dezember 1649 bis zum gleichen Datum des Jahres 165! gültig sein und enthielt namentlich folgende Bestimmungen. Tirol verpflichtete sich, sein Haller Salz nicht mehr bis Lindau, sondern nur noch bis Reutte zu führen, und „allda die Haubtniderlag anzestellen“. Bayern versprach, das Reichenhaller Pro¬ didit nicht über seine Landsberger Salzniederlage hinaus dem Bodensee zuzuführen. Die Preise, zu denen die beiden Kontrahenten Hofkammer äußerte sich 1591 folgendermaßen darüber : „ Diss [d. h. das bayerische ] salz hat auch fürnemblichen an dem Intalischen . . . einen sondern feind , Ursachen und wo dieselben Zusammenstößen, so mues ains dem andern mit dem kauf weichen , da doch festlich der abschlag des kau Fs niemant dan dem auslendischen kaufmann zu nuz kumbt . Demnach ratsamlich geachtet , daß disfals mit irer fürstlichen durchlauclvt ertzherzog Ferdinand ain sonde rer verstaut gemacht werde , dan , wo m ' a n s 1 c li destwegen bederseits vergliche , könte beden salzen von jar zu jar noch ein furnemer hoherung ervolgen und um sovil leichter das jezige kaufgelt vor abschlag oder ringerung bestendig erhalten werden .“ Stieve, Zur Geschichte des Finanzwesens . Sitzungsberichte d. Akademie d. Wissenschaften zu München . 1881 . I, S. 50 . Vgl . auch Hirn a . a . 0 ., 1, S. 571 ff. 1 Es ist im Anhang dieses Werkes abgedruckt. Zweites Kapitel : Kartelle des 14. bis 18. Jahrhunderts . 195 das Salz zu liefern haften, wurden genau festgelegt. Ein chur¬ bayrisches oder Reichenhaller Fäßchen Salz von drei Scheiben oder 7 '/z Fiederl durfte nur zu 9 fl. 30 Kr. Reichswährung verkauft, ein Tiroler halbes Fäßchen von 11/2 Hallischeu Fudern nur zu 10 fl. Reichswährung abgegeben werden. Unter den stipulierten Preisen durfte auf keinen Fall Salz von den kartellierten Unternehmern in den Handel gebracht werden. Wohl aber über diesen Preis hinaus, wenn die Gelegenheit sich bot. ln diesem Falle war der betreffende Kartellkontrahent verpflichtet, davon dem anderen Mitteilung zu machen. Dazu sollten folgende Zahlungsbedingungen von beiden Kontrahenten unverbrüchlich gefordert werden. Bei Abschlüssen (Kontrakten s. o.) hatte der Salzgroßhändler sofort ein Viertel der Kaufsumme in bar in Reichswährung zu entrichten. Die übrigen drei Viertel zu je einem Viertel in den nächsten darauffolgenden drei Bozener Märkten. Wenn ein Bozener Markt etwa gleich zwei bis drei Wochen nach Abschluß eines Salzkontraktes fallen sollte, dann „kbann die selbige Marcktfrist erst auf den negst darauf volgenten andern Markht anfangen“. Jede Umgehung der festgesetzten Preise durch Zugaben, lange Kreditgewährung usw. war den Kartellkontrahenten streng ver¬ boten. Über die genannten einzelnen Bestimmungen hinaus sicherten sich die kartellierten Regierungen zu, „guet Correspondenz ze halten was für Hindernussen und Ungelegenhaiten zu beeder chur- ur.d ertzfürstlichen Herrschaft Schaden und Steckhung des Saltzverschleis firgehen“ würden. Insonderheit wollte man sich treulich darüber unterrichten, wenn die auswärtige Konkurrenz, d. h. das burgundische, lothringische und französische Meersalz1 in die Absatzgebiete der Kartellkontrahenten eindringe. Sehr rigoros waren die Bestimmungen des Kartells gegen die1 Das sog . Baie - oder Boysalz . Baiesaiz war ursprünglich Salz, das an der seichten Bai von Bourgneuf hinter der Insel Noirmoutiers, unmittelbar südlich von der Loiremündung , gewonnen wurde . Das dortige Salz hieß bei den hansischen Kaufleuten des Mittelalters einfach das Baiesaiz . Vgl . D . Schäfer, Die Hanse . Bielefeld und Leipzig 1903 . S. 46 . Später wurde Baiesaiz alles an der franzö¬ sischen , spanischen und portugiesischen Küste aus Meerwasser ge¬ wonnene grobkörnige , schwärzliche Salz genannt . E b e r 1 e a . a . O., S. 39. 13 * 196 Drittes Buch: Monopole, Kartelle und Aktiengesellschaften im Mittelalter. jenigen Salzgroßhändler, die wegen rückständiger Zahlung oder aus anderen Gründen sich mit dem einen der Kartellgenossen zer¬ worfen hatten und nun an den anderen zum Zwecke des Salzkaufs herantraten . Dem „böswilligen“ Käufer durfte solange keine Ware verabfolgt werden, „ bis er beglaubte Schein fürweisen konnte, dass er hierumb gebihrundte Satisfaction erstattet und alles richtig gemacht habe“ 1. Zur Überwachung der genauen Ausführung der Kartellbestim¬ mungen war es jedem der Kontrahenten erlaubt, in Lindau oder sonstwo auf seine Kosten Kontrolleure anzustellen. Der Kartellkontrakt von 1649 ist in den nächsten Jahrzehnten immer wieder erneuert worden, obwohl öfters Klagen laut wurden, in denen sich die Kontrahenten einer zeitweisen Umgehung seiner Bestimmungen beschuldigten. Um solche Umgehungen zu er¬ schweren oder womöglich ganz zu verhindern, erhielt das Statut von 1649 im Jahre 1677 einige wichtige Zusätze 2. Es sei hier 1 Vgl. auch hierzu die Ergänzungen zu dem Kartellkontrakt vom 5. August 1649 , festgestellt zu Kufstein am 20. September 1677. Original im Münch . Allg . Reichsarchiv Loc . Tirol (fürstl . Grafschaft ). 19. i'asc . Bl . 101 f. „ Inmaßen auch mehrernante höchste Heiser wo ain oder des anderen Thails Contrahenten mit Erlag der Fristen oder in andere Weeg wider Verhoffen ermanglen wurden , auf alle mögliche und gedeyliche Mitl den Ermangelnden zur Schuldigkeit und Observanz des Contracts zu halten bedacht sein und aneinander die verhilffliche Handt denen vorigen Verstentnussen gemeß pieten sollen und wollen .“ 2 Ergänzungen zu dem Kartellkontrakt vom 5. August 1649, festgestellt zu Kufstein am 20 . September 1677 . Original im Münchener Allgemeinen Reichsarchiv Loc . Tirol (fürstl . Grafschaft ). 19. fase . Bl . 99 ff. — „ . . . Damit dis desto gewisser bederseits beschehe , man sich dahin nachbarlich und freundlich verstanden hat, daß khonftig , ehevor ainiger Salzcontract , wann es auch nur 1000 oder 500 Saltz -Veßl antreffete , von ain oder anderer Hofcamer abgehandlct und geschlossen wurde , man einander in wehrenden Tractaten zeitlich und vor dem Schluß von allem communication und parte geben , auch wann darwider erhöbliche Erynnerung weren. solche ain Thail von dem anderen für guet annemben . . . . Inmaßen baide hochlob ]. Heiser einander hiemit crefftiglich versprechen, vicissim aufrichtig zu communicieren , wann es fir ratlisam befunden wurde , daß ainichen Saltz -Contrahenten , welche namhaffte Conträct oder gleichsam den ganzen Salzverschleis in gewisse Landt und Orth annemben etwas wegen irer Wagnus , Verlag und Gefahr einzuwilligen, * Zweites Kapitel : Kartelle des 14. bis 18. Jahrhunderts. 197 wenigstens der eine hervorgehoben, der die kartellierten Unter¬ nehmer zwang, von allen schwebenden Kontrakten, auch wenn es sich nur um Lieferung von 500 Fäßchen Salz handelte, einander Mitteilung zu machen. Zu den größten und wohl auch berechtigtsten Vorwürfen, die man heute den Kartellen macht, gehört die Anklage, daß die Kartelle oft, nachdem sie sich durch Ausschaltung der inländischen Kon¬ kurrenz einen Monopolpreis gesichert haben, an das Ausland 1 billiger verkaufen und so die eigenen Volksgenossen den Ausländern gegenüber benachteiligen. Die genannte Tendenz der Kartelle von heute läßt sich schon in der älteren Kartellgeschichte nachweisen. Beispielsweise auch in der Geschichte des Salzkartells von Reichen¬ hall und Hall im Inntal. Seit alters hatte das burgundische Salz dem bayrischen und Tiroler Salz in der Schweiz usw. eine kräftige Konkurrenz geboten. Um die Mitte des 17. Jahrhunderts ging Burgund soweit, daß es zur Vergrößerung seines Salzhandels ins Ausland zum Selbstkosten¬ preis lieferte 2. Wenn also die bayrischen und öster¬ reichischen Kartellisten in Lothringen, im Elsaß, im Sundgau, ebenso über den Gotthardpaß hinaus ,,im Welsch- und WalliserLand“ die burgundische Konkurrenz schlagen und selbst auch in Burgund Absatz finden wollten, so mußten sie mit dem Preise für diese „fernen“ Lieferungen heruntergehen. Tatsächlich ergänzten die bayrischen und österreichischen Kartellkontrahenten am 5. Ok¬ tober 1651 und später ihren erneuerten Vertrag von 1649 dahin, daß sie sich erlaubten, den Verkaufspreis für das Salz „in die Ferne“ niedriger anzusetzen. Alles Salz, das über Bern, Basel, Solothurn und Freiburg im Uchtland hinaus gen Burgund und Lothringen, über den Gotthard ins Welsch- und Walliserland, über die Furca daß ein lobl. Hofcamer der anderen hivor ir Sentiment durch Schreiben oder Abgeordnete mit verthreulicher Apertur aller Umbstent zuverstendigen und hernach mit denen Salz-Contrahenten auf jenige Weiß zuschließen wie bayde Hofcamer es unanimi consensu für guet erachten werden. Außer dessen aber und ohne beeder lobl. Hofcamer Einwilligung kheinen Salz-Contrahenten einicher Forti, Nachlaß, Eingab, Schänckhung, Ergrößerung der Vaß oder anders sub ullo exeogitabili nomine nit beschehe.“ 1 Gegen dessen Konkurrenz im Inlande die Karteilisten zumeist durch die heimischen Schutzzölle gesichert sind. 2 Eberle, a. a. O., S. 112. 1 198 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. ins Elsaß und den Sundgau ging, durfte zu Landsberg bzw. Reutte um 30 Kreuzer billiger abgegeben werden als für den näheren Vertrieb verabredet worden war . Allerdings mußte durch glaubwürdige Urkunden der genannten schweizerischen Städte zweifellos bezeugt werden , daß dieses Salz nirgends anders wohin als in „dieselbige ferne Länder vertriben worden sei“ K Natürlich waren solche Maßnahmen nur geeignet , den Kon¬ kurrenzkampf zwischen bayrischem und Tiroler Salz einerseits und burgundischem andererseits um so heftiger entbrennen zu lassen, bis schließlich auch zwischen ihnen eine gütliche Vereinigung und eine gewisse Ausschaltung der Konkurrenz in Form eines Kartells erfolgte . Auf Ersuchen der Fermiers généraux , der Pächter des burgundischen Salzwesens zu Salins, beschickten die bayrische und die Tiroler (österreichische ) Regierungen als Monopolinhaber der Reichenhaller bzw. Haller (Inntal ) Salinen eine Salzverschleiß¬ konferenz zu Kempten im Allgäu , auf welcher am 3. November 1659 folgender Kartellvertrag 2 zustande kam : Zwar hatten die Fermiers „mit dem Orth und Stand “ Bern auf 4 y2 Jahr und dann mit Solothurn , Freiburg in Üchtland und der Grafschaft Neu¬ burg auf sechs Jahre Salzlieferungskontrakte abgeschlossen , die sich nicht aufheben ließen , aber die Fermiers verpflichteten sich doch, dafür Sorge zu tragen , daß durch diese genannten vier Orte „ alles Salz, so sy von denen burgundischen Fermiers jerlich nemen weithers nit als in jedes Orths oder Standts aignem District und Gebieth verbraucht und in khein ander Gebieth weder in Vessln noch dem Ausmass nach verkhauft werde “ 3. Für das Salz, das die Fermiers in andere Kantone oder Gebiete als die vorhin genannten führten und selbst oder durch andere ver¬ kauften , verpflichteten sie sich, einen Mindestpreis von 19 guten 1 Ich habe diese wichtige Ergänzung des Kartellstatuts von 1649 , wie sie am 5. Oktober 1651 zu Kufstein beschlossen wurde , im Wortlaut im Anhänge abgedruckt. 2 Im Anhänge abgedruckt. 3 Die Fermiers généraux hatten demgegenüber verlangt , die bayrischen und Tiroler Komparenten sollten sich verpflichten, „weder im Vaß noch dem Ausmeß nach in obbemelten Orth und Cantonen khain Reichenhall - noch Hall -Intalisches Salz “ zu ver¬ kaufen . Die Forderung wurde aber von den beiden Regierungen abgeschlagen und war dann fallen gelassen worden. 1 Zweites Kapitel : Kartelle des 14. bis 18. Jahrhunderts . 199 Schweizer Gulden pro Fäßchen loco Solothurn zu nehmen \ Dabei durfte nur bares Geld und kein anderer Wert oder Zahlungsmittel für das Salz von den Fermiers angenommen werden. Die Zahlungs¬ fristen wurden folgendermaßen fest stipuliert : Der vierte Teil der Kaufsumme war sofort bei Abschluß des Kaufs zu erlegen; die übrigen drei Viertel mußten in den drei folgenden Jahresquartalen bezahlt werden. Streng war es den Kartellkontrahenten verboten, den Salzhandelsleuten beim Abschluß der Salzkontrakte besondere Vergünstigungen irgendwelcher Art, direkt oder indirekt, zu ge¬ währen. Insbesondere waren Zugaben, Verlängerungen der Zah¬ lungsfristen usw. strengstens untersagt. Es wäre interessant, aus Münchener usw. Archivmaterial das Schicksal unseres internationalen Kartells genauer zu verfolgen. Hier kann diese Forscherarbeit nicht gemacht werden. Hier muß ich mich begnügen, an einem Beispiel darauf hinzuweisen, wie auch in dem Kartellwesen des 17. Jahrhunderts die Frage nach der ethischen Erlaubtheit derartiger Verabredungen eine Rolle spielt. In einer Erneuerung des Kartells von 1649 aus dem Jahre 16862 ist unverhüllt anerkannt, daß der Zweck des Kartells der war, die Kammergefälle, d. h. die Finanzeinnahmen, „beider höchsten Häuser“ zu vermehren und zu verbessern2. Das Mittel zu diesem Zweck- war darin gegeben, daß man den Salzpreis in die Höhe schraubte. Ethisch begründet wird diese monopolistische Preis¬ politik nun mit folgenden Argumenten. Noch niemals sei es mehr vonnöten gewesen, die fürstlichen Einnahmen zu erhöhen, als gerade jetzt , „alldieweilen mehrist dise hochpreislichiste beide Heuser als mechtigste Säulen der wertisten Christenheit ihre eisseriste chrefften lobwirdigist angegriffen, damit dem grausamben Erbfeindt in seinem weltkhindigermassen gehabten bluetbegirigen Vorhaben hat khönnen Hinterung gemacht werden und ohne disem so gewaltigen Widerstandt diser 2 Potentaten das ganze Hail des 1 „weihlen das Reichenhall - und Hall - Intalisch Salz bis nacher dem Baslischen und Solothurnischen auch selbiger Enden wenigst bis auf 22 Gulden gedachter Wehrung steigen thut .“ 2 „Seitemahlen das Absechen haubtsächlich dahin gerichtet ist, daß baide hegste Heuser einander (als ohne das vielfeltig bluetsverwahndt ) die Handt pietten und beiderseits Camergeföll helfen vermehren und verbessern .“ Kgl . Bayr . Allgem . Reichsarchiv München a . a . O., Bl . 147 ff. 1 200 Drittes Buch : Monopole , Kartelle und Aktiengesellschaften im Mittelalter. auserwöhlten Volckhs Christi hegstens periclitiert hete . Welche Ge¬ fahren verrer abzuwenden es annoch unbeschreiblichen costen er¬ fordern wird et . Solchen grossen Costen aber beide Hofcamer billichist aus eben jenigen Mitlen suechen muessen , die ihnen von dem Allmechtigen und der Natur vor andern Landschaften vermuetlich zu dem Ende seint aus getlicher Vorsichtigkheit zuegeaignet worden , damit sye hieraus den nervum belli gerendi pro ecclesia Christi erziechen und bestreiten khönnen . Und nun also dar sich herfür thuet , dass solche köstliche Kriegsarmatura wider so mechtigen Feindt allen jenigen christlichen Völckhern zu guetem und ihrer Conservation geraichet , welchen dise stattliche Gottesgab des payrischen und Tyrolischen Salzes verkhauft und zuegefiehret wirdet , denen volglich auch nicht schwer fallen solle, einen mehrern Preiss hierfir zu betzallen , indeme das gelt zu allgemeinen Heil und ad conserva¬ tionem totius wissentlich . . . appliciert wirdet V‘ Kartelle in der Calwer Zeugindustrie hunderts. des 17. Jahr¬ Ein Kartell aus dem frühen 17. Jahrhundert erwähnt Walter Troeltsch in seiner „ Geschichte der Calwer Zeughandels¬ kompagnie “ 2. Im Archiv des Inneren zu Ludwigsburg 3 ist uns der „interessante Entwurf eines Preiskartells zwischen .einer ge¬ samten Ferbergesellschaft zu Pforzheim und Cahv‘ von Laurenti (10. August ) 1620 erhalten , der ursprünglich geheim gehalten , 1657 von der markgräflich badischen der Stuttgarter Regierung mit¬ geteilt wurde , um die frühere Handelsfreiheit zwischen beiden Städten zu beweisen. Danach sollten die Kontrahenten auf drei Jahre bei den gefärbten Waren bestimmte Verkaufspreise einhalten . Übertretungen waren erstmals mit Konfiskation , im Wiederholungsfälle mit Ausschluß aus der Handelsgemeinschaft be¬ droht . Auch ein Minimallohn für das Färben der rohen Zeuge 1 A. a . 0 ., Bl . 147 ff. 2 Die Calwer Zeughandelskompagnie und ihre Arbeiter . Studien zur Gewerbe - und Sozialgeschichte Altwürttembergs . Jena 1897. S. 30 . „ Zeuge “ sind glatte , schmale , wenig oder gar nicht gewalkte Gewebe aus langhaariger Wolle. 3 Fase. 65 der Akten der Calwer Färberkompagnie . Laut gütiger Mitteilung von Prof . Troeltsch. 1 Zweites Kapitel : Kartelle des 14. bis 18. Jahrhunderts . 201 war in Aussicht genommen. Um eine Umgehung der Preisverab¬ redungen zu verhindern, sollte jeder Färber die Geldsorten nicht höher in Zahlung nehmen \ als sie angeschlagen und verrufen waren.“ Hauptsächlich der gleichzeitigen Münzwirren wegen, die eine Gebundenheit der Preise unmöglich machten, kam die Konvention nicht zustande. Von neuen Kartellierungsversuchen der Pforzheimer und Calwer Zeughändler hören wir erst wieder im Jahre 1668. Aus diesem Jahre „ist eine Vereinbarung zwischen den Zeughändlern beider Orte vorhanden, die sich auf die Bedingungen des Verkaufs gewisser Waren in Leipzig und beim polnischen Handel, in Nürnberg und Augsburg, bezieht“ 2. Kartelle in Brandenburg im 17. Jahrhundert. Die folgenden Bestimmungen Friedrich Wilhelms aus einem Rezeß vom 26. Juli 1653 (§ 44) beweisen, daß auch im Brandenburgischen schon unter dem Großen Kurfürsten Kartellierungs¬ versuche vorkamen: „ Demnach wir auch berichtet worden, dass die Hopfen-Führer sich untereinander wie hoch sie den Hopfen ein¬ kaufen wollen, verbinden, und wer dawider handelt unter sich strafen : So wollen wir solch schädliche Monopolia nicht dulden, sondern durch öffentliche Edicta verbiethen, auch dem Magistrat jedes Orths anbefehlen, hierunter mit Fleiss zu inquirieren und die Delinquenten gebührlich darüber strafen. Und weil auch Klagen eingekommen, dass die Tuchmacher einen Preis der Wolle setzten, was sie nemlich den Edelleuten, Priestern, Bauern und Hirten vor einen Stein Wolle geben wolten, als wurde ferner in gedachten Recesses Anhang § 5 gesetzet: An denen Monopoliis und dass etzliche Handelsleute und Handwerker wegen des Korns-, Viehs- und Woll-Kauf etc. zu Schaden ihres Nechsten unziemliche Verknüpfungen machen, 1 Vgl. hierzu die analoge Bestimmung in den §§ 15 und 16 des Kartellstatuts der Naumburger und Weißenfelser Bettfedernhändler aus dem Jahre 1743 . Historisches Jahrbuch 1911 . S. 60. 2 Troeltsch a . a . O., S. 96 Anm . 3. Für die Aktenstücke vgl . Archiv des Inneren zu Ludwigsburg , Fase . 23 (1668 ) der Akten der Färberkompagnie , Stück 1 und 2 . Nach gütiger Angabe von Prof . Troeltsch. 202 Drittes Buch : Monopole, Kartelle und Aktiengesellschaften im Mittelalter. tragen wir keinen Gefallen, es soll auch solches hiemit verbothen sein. . . .“ 1 Begründet wird das Kartellierungsverbot durch den Großen Kurfürsten mit einem charakteristischen Hinweis auf die „Freiheit der Commerden“, die dadurch verhindert würde. Wir sahen früher, daß ein Sittenprediger des beginnenden 16. Jahrhunderts , Geiler von Kaisersberg, genau denselben Grund für seine Bekämpfung der Syndikate mit geltend machte. Kartellbildungen industrie in der böhmischen des 18. Jahrhunderts. Glas¬ Zum besseren Verständnis einiger Kartellerscheinungen in der böhmischen Glasindustrie des 18. Jahrhunderts sei das folgende vorausgeschickt: Das böhmische Glas hatte sich im Verlaufe der zweiten Hälfte des 17. Jahrhunderts zu einem Welthandelsartikel emporgeschwungen2. Diese Ausweitung des Absatzes war in erster Linie das Verdienst eines (zumeist aus bäuerlichen Kreisen hervorgehenden) unternehmungs- und wanderlustigen Glashändler¬ standes, eines Hausierertums 3, das zunächst mit dem Glaspack, mit der Glaskiepe, auf dem Rücken, dann mit der Schubkarre und schließlich mit Fuhrwerk und zu Schiff das böhmische Glas in die Nähe und in eine immer mehr wachsende Ferne absetzte. Nach Norddeutschland, „nach Polen und den Ostseeländern, nach Ruß1 Vgl. P. J. Marperger, Neueröffnetes Handelsgericht Hamburg , o. J, , S . 330. 2 Vgl. für das folgende besonders Edmund Schebek, Böhmens Glasindustrie und Glashandel . Quellen zu ihrer Geschichte. Prag 1878. 3 Wir können es hier dahingestellt sein lassen , aus welchen Be¬ völkerungselementen die böhmischen Glashändler hervorgingen . Zum Teil wohl aus dem Veredelungsgewerbe , aus den Kunsthandwerken, die , um die Glashütten sich lagernd , berufsmäßig das „ Malen , Ver¬ golden , Schneiden , Reißen “ des Rohglases besorgten . ( In dieser Veredelung bestand lange Zeit der Vorzug , das Monopol der böhmi¬ schen Glaswaren .) Zum Teil aber gelangten auch ursprünglich betriebsfremde Frachtführer in die Hän dl erschaff , indem sie sich mit den akkumulierten Verfrachtungsgewinnen als Grund - und BetriebsKapital selbständig machten . Schließlich aber dürfte wohl Salz recht haben , wenn er die Glashändler meist als Familienangehörige, als jüngere Söhne usw . der Glasproduzenten , der Glashüttenmeister anspricht . Salz, Geschichte der böhmischen Industrie , S. 241 ff. 1 Zweites Kapitel : Kartelle des 14. bis 18. Jahrhunderts. 203 land bis Moskau und vielleicht weiter, nach Holland, Italien, Ungarn und Siebenbürgen, nach der Moldau und Wallachei bis nach Adria¬ nopel hinab ziehen die kühnen Männer aus Nordböhmen. Das Meer steckt ihren Fahrten keine Grenzen. Von Stralsund segeln sie nach Riga, von Hamburg nach London und von Varna nach Konstantinopel; Kopenhagen und Stockholm werden aufgesucht und über Archangel in wenig Jahren viel ,hundert Tausend Glas“ in das Innere von Rußland vertrieben. Frühzeitig müssen sie auch mit ihren Waren an den Küsten von Portugal und Spanien— von Cadix wird es aus dem Jahre 1691 ausdrücklich berichtet — ge¬ landet, sein, welche Länder später nebst Holland die Hauptemporien ihres überseeischen Handels werden sollten V‘ Mit der allmählich wachsenden Ausdehnung des böhmischen Glashandels über einen großen Teil Europas ergab sich zweierlei für die Fortentwicklung der böhmischen Glasindustrie besonders Wichtiges. Einmal erfolgte eine ungeahnte Vermehrung der Glas¬ hütten in ganz Böhmen 2und ein Eindringen auch fremder Kapita¬ listen in die Produktion. Gleichzeitig aber ging der böhmische Glaswarenhandel — wenigstens in seiner bedeutungsvollsten Spitze 3 in — einen Meß- und Markthandel und bald zu der Er¬ richtung dauernder fester Faktoreien, besonders in den wichtigsten Seehandelsplätzen, über. Im Verlaufe des endenden 17. und des 18. Jahrhunderts wurde schnell hintereinander „der Küstensaum des europäischen Festlandes von St. Petersburg bis Konstanti¬ nopel mit solchen Niederlassungenbesetzt. Man findet deren ver¬ zeichnet zu St. Petersburg, Reval, Libau, Riga, Kopenhagen, Lübeck, Hamburg, Bremen, Amsterdam, Leyden, Haag, Rotter¬ dam, Dordrecht, Middelburg, Bordeaux, San Sebastian, Bilbao, Santander, Ferrol, Coruna, St. Jago, Vigo, Oporto, Lissabon, Sevilla, Cadix, Malaga, Valencia, Alicante, Barcelona, Marseille, Livorno, Neapel, Palermo, Ancona, Triest, Konstantinopel. Von den Nieder1 Schebek a . a . O., S. III. 2 Schon zu erkennen aus Balbinus , Miscellanea historica regni Bohemiae . Pragae 1679 . I, Cap . 21. Vgl . Schebek a . a . O., S. XI ff. 3 Natürlich trieben noch immer die untersten , erst im Empor¬ steigen begriffenen Schichten des böhmischen Glashändlertums mit der Glaskiepe auf dem Rücken oder dem Schubkarren ihr Gewerbe im Umherziehen. 1 204 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. lassungen aus, zu denen noch einige in Binnenstädten, wie zu Madrid, Valladolid, Mailand, Lyon, Paris, Nancy, Straßburg, Brüssel, Amersfort, Utrecht, Leeuwarden und Frankfurt a. M. kamen, ver¬ sorgten die Glashändler die inneren Märkte und streckten ihre Fühler immer weiter über die See hinaus. Es wurden sogar einige Etablissements in fremden Weltteilen, als zu Smyrna, Beyrut, Kairo, Mexiko, Baltimore und Neuyork, errichtet L“ Eine derartige Ausbreitung des Flandels mit böhmischen Glas¬ waren war nur einzig und allein dadurch möglich geworden, daß die Händler sich zu jenen bedeutungsvollenGlashandelskompagnien zusammengeschlossenhatten , die den böhmischen Glashandel in seiner Blütezeit beherrschten. Die Arbeitsteilung in den Handels¬ gesellschaften war die folgende: Das Gesellschaftshaupt, oft mit dem Familienhaupt identisch, blieb zu Hause, besorgte den Ein¬ kauf der Waren von den Glashüttenmeistern, die Verpackung, Buch¬ führung, Berechnung usw. Die jüngeren Teilhaber zogen hinaus in die fremden Faktoreien und leiteten von dort aus den Vertrieb weiter. Bald erweiterte sich dabei der Umkreis der Geschäfte mancher böhmischen Glashandelskompagnien noch bedeutend. Um allen Ansprüchen der Glaskäufer entgegenkommen zu können und um keine fremde Konkurrenz in die Höhe kommen zu lassen, wurden zunächst die Glaslager der Kompagnien nach jeder Richtung hin komplettiert. So „mit bairischem Tafelglas, mit Thüringer und englischen Glaswaren, später auch mit Paderborner, Münzthaler, holländischem, belgischem und französischem Glas“. Dann aber wurde überhaupt der Aktionskreis über diesen Glashandel hinaus erweitert. Außer Glas wurden mitunter noch andere Artikel böhmischen Ursprunges, namentlich Leinwand, in den Nieder¬ lagen geführt. In Portugal ging man nach dem dortigen Glas¬ einfuhrverbote ganz zu Leinwand über. In „ Spanien warf man sich auch auf holländische Thonwaren, auf Remscheider Eisen waren , auf Nürnberger, englische und andere Waren, was man insgesamt unter der Benennung „Kramerei“ be¬ griff. Ähnlich wurde es in Amsterdam, und vielleicht auch ander¬ wärts gehalten. Dazu kamen noch die Artikel, die man in Tausch für Glas annahm, wie Tabak in Spanien, Pelzwerk und Juchten in Rußland“ 2. So war aus dem Handel des böhmischen Glas1 Sc h e b e k a. a. O., S. V. 2 Schebek a. a. O., S. Vf. Zweites Kapitel : Kartelle des 14. bis 18. Jahrhunderts . 205 hausierertums ein nennenswerter Zweig des Welthandels er¬ wachsen 1 Auf Grund des Vorausgeschickten ist es uns möglich, zu einer kurzen Darstellung der aus dem Arbeitsgebiet der böhmischen Glas¬ industrie und des böhmischen Glashandels uns bekannt gewordenen Kartellbestrebungen überzugehen . Eine eigenartige Konven¬ tion schlossen im Jahre 1715 (10. Oktober ) alle böhmischen Glasproduzenten (Verleger) und Glashändler , die nach Portugal exportierten . Wir müssen bei dem Vertrage , der diese Verabredung festlegte , etwas länger verweilen: In der Arrenga wird ausgeführt , daß noch vor zehn Jahren ein sehr gutes Geschäft nach Portugal zu machen war . Jeder Handelsmann sei auf seine Rechnung gekommen , von dem reich¬ lichen Verdienst wäre „sowohl der Verleger als der Glasarbeiter jederzeit richtig bezahlt worden “. Dagegen sei jetzt das portu¬ giesische Geschäft in denkbar schlechter Verfassung . Wenn man nach Abschluß des Friedens auf Besserung hoffen wolle, so sei es nötig , die unredlichen Elemente auszuschalten , die sich im böhmi¬ schen Glasgeschäft in Portugal breit gemacht hätten . Das seien unehrliche Händler , die von den Verlegern auf Kredit Waren nähmen , in Portugal dann verschleuderten . Der Verleger werde zumeist nicht oder nicht völlig bezahlt , zugleich werde aber auch den ordentlichen Händlern eine empfindliche Konkurrenz geboten. Um hier Wandel zu schaffen , habe man bedacht , „dass alle andere Professionen , Commercien und Handelschaften in allen Ländern und Städten durch nichts anderes , als durch gewisse Statuten , Articul und Gesetze ihren Anfang genommen , durch diesen in ihrem Stand und Flore , auch in geliebter Ordnung erhalten worden “. So müßten also auch die böhmischen Glashändler , die nach Portugal Handel trieben , sich „ Statuten “ setzen ', eine „ Confoederation und Gemeinschaft “ untereinander und mit den für den portugiesischen Export in Frage kommenden Glasproduzenten schließen . Die wesentlichen Punkte des aus solchen Erwägungen hervorgehenden Kartellstatuts lassen sich wie folgt zusammen¬ fassen : 1. Jeder , der nach Portugal oder Algarbien Glashandel aus den allein in Betracht kommenden vier böhmischen Flerrschaften (Oberliebich , Bürgstein , Böhmisch -Kamnitz , Neuschloß ) treiben wollte , mußte sich „bei der Ausfuhr bei seinem Verleger , wo er das 206 Drittes Buch : Monopole, Kartelle und Aktiengesellschaften im Mittelalter. • Glas nimmt, “ schriftlich verpflichten , den Kartellvorschriften ge¬ treulich nachzukommen . Er hatte dann „ eine Attestation und Zeugenschafl , daß solches geschehen “ sei, mit nach Portugal zu bringen. 2. Bcx seiner Ankunft in Portugal hatte der Händler den Kartellgenossen das Attest des heimischen Verlegers vorzuweisen und noch einmal zu geloben, den Kartellbestimmungen gemäß sein Geschäft zu treiben. 3. Keiner der Kartellkontrahenten durfte — bei 100 Reichs¬ tag Konventionalstrafe — „einiges Glas an einen solchen Menschen, der di<;:es wiederum zu unserm Schaden verhausieren “ wollte, verkaufen. 4. Sofern einem der Kartellgenossen „ eine Noth zufallen sollte , dass er sein Waar verkaufen und die Logi verlassen müsste “, solle er einem der Kontrahenten der „ Konföderation und Ge¬ meinschaft “ die Restbestände seines Warenlagers anbieten . Er¬ klärt sich keiner davon zur Übernahme bereit , so' übernimmt das Kartell als solches käuflich die Waren und verteilt sie unter seine Mitglieder. 5. Den Outsidern , die sich nicht den Kartellkontrahenten an¬ schließen , vielmehr ihr Glas „verschwenderisch an ein wiederum damit hausierendes Gesindel verkaufen “, wird Erzfeindschaft zu¬ gesichert . Namentlich soll „ einem solchen Menschen von keinem Verleger einiges Glas mehr ausgefolgt werden “. Im § 6 wird Vorsorge getroffen , daß die Bestimmungen des Kartells nicht von böswilligen Komparenten umgangen werden können . So soll z. B. jedem Kartellmitglied zwar natürlich erlaubt sein, „ Glas kistenweis aus dem Königreich Portugal in andere Länder zu verführen “, jedoch soll kein Scheinexport Vorkommen, d . h. keiner darf sein Glas gerade nur vor die Stadt führen und „solches an bemeltes Gesindel verkaufen “, welches es hernach wiederum in die Stadt bringe und zum Schaden der kartellierten Firmen verhausiere ' . 1 Die Hinzufügung „ auch mit dem expressen Reservat , daß die bisherige holländische Compagnie, mit dieser portugie¬ sischen Gemeinschaft nicht eonfundiret und von derselben das Glas nach Belieben nacher Portugal eingeführt werden möchte , sondern präcise was von Haus attestirter vermög 1. und 2. Punctes dahin abgeschicket wird , soll es darbei festiglich sein Bewenden haben “ , 1 Zweites Kapitel : Kartelle des 14. bis 18. Jahrhunderts . 207 In § 7 wird den böhmischen Glasproduzenten, den Glas¬ verlegern, der Dank dafür abgestattet , daß sie durch das Ver¬ sprechen, nur an die kartellierten Firmen liefern zu wollen, das Zustandekommen des Kartells überhaupt erst ermöglichten. Der Dank bestand darin, daß die Karteilkontrahenten sich verpflichteten, die Verleger tunlichst vor Verlusten zu bewahren. § 8 bestimmte über die Verteilung der einlaufenden Strafgelder das folgende. Ein Drittel davon soll zu religiösen Zwecken, ein weiteres Drittel für die Bedürfnisse des Kartells verwendet werden. Das letzte Drittel versprechen die Kartellierten an diejenige der obengenannten vier böhmischen Herrschaften abzuliefern, deren Untertan jeweils die Strafe zu zahlen hatte. In § 9 behielten sich die Kartellkontrahenten im Notfälle eine Abänderung der Statuten vor. In § 10 erfahren wir, daß sie bei den „vier hohen herrschaft¬ lichen Ämtern (dann zu Dato sonsten von keinem Ort einiges Glas nach Portugal abgehet)“ um Genehmigung ihrer Kartellstatuten mit Erfolg nachgesucht hatten . Leider sind wir nicht darüber unterrichtet , ob das Kartell die von seinen Kontrahenten gewünschte Wirkung zeitigte. Einige Jahrzehnte später kam es im Gebiet der böhmischen Glasindustrie wiederum zu einer Syndikatsbildung. Allerdings vereinigten sich diesmal nicht Glasproduzenten und Glashändler zu gemeinsamem Vorgehen gegen eine Schleuderkonkurrenz der Giashändler; vielmehr schlossen sich die böhmischen Glasproduzenten (Glashüttenmeister, d. li. Besitzer der Glashütten) gegen die Gesamt¬ heit ihrer Abnehmer zusammen. In einer Beschwerdeschrift, welche die Glashändler auf den Herrschaften Kamnitz, Bürgstein, Reich¬ stadt , Neuschloß, Ober-Liebich, Tetschen und Meistersdorf gegen die dortigen Glashüttenmeister an die Regierung abgehen ließen 2, ist mir nicht ganz verständlich . Namentlich müßten nähere spezielle Untersuchungen Aufklärung darüber verschaffen , ob mit . der „ holländi¬ schen Kompagnie “ ein ähnliches Kartell , wie das hier besprochene, der nach Holland handelnden böhmischen Glashändler zu ver¬ stehen ist. 1 Die Bestätigung der Genehmigung ist dem Kartellinstrument am Schlüsse beigefügt . Schebek a . a . O., S. 363. 2 Die Beschwerdeschrift ist uns mit einem dazugehörigen Gut¬ achten des Direktoriums in publicis et cameralibus erhalten . Original 208 Drittes Bach: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. heißt es: „Die Glashüttenmeister [d. h. die Besitzer der Glas¬ hütten ] haben anno 1739 in dem Stadtl Czistitz [Cechtitz], Czaslauer Kreises eine, ihnen, Glashändlern, nachtheilige Bündnuss er¬ richtet , vermög welcher sie einstimmig das Glas in höhern Preis zu geben und kleinere Gattung und Maass zu machen und einige Gattungen gar nicht mehr zu verfertigen unter einer Straf von 100 Kremnitzer Dukaten beschlossen und erst neulich abermalen das Glas um 30 Kr. künftig theurer zu geben erinnert hätten .“ Auf Vorstellungen der Regierung hin antworteten die Hütten¬ herren : „Die Einverständniss wegen Erhöhung des Glaspreises wäre von darum gepflogen und die Straf dabei ausgemessen worden, weilen viele Glashüttenmeistere schlechte Glaswaaren verfertigt, hierdurch das commercium geschwächet und einer dem andern geschadet hätte .“ Wenn sie durch ihreVereinbarung höhere Preise zu erzielen beabsichtigt hätten , so wäre das in der Geschäftslage begründet gewesen. Die Rohmaterialien seien teurer geworden, z. B. sei Pottasche sehr im Preise gestiegen usw. Die Entscheidung der Regierung steht noch ganz und gar auf dem kartellfeindlichen Standpunkte der mittelalterlichen Wirtschaftsethik, ein Standpunkt, den sich die österreichische Gesetzgebung bekanntlich bis heute bewahrt hat . Da heißt es in dem Votum der Regierung vom 10. August 1750: „Es streite wider die rationem commercii wann die Fabrikanten über die-Erhöhung des Preises ihrer fabricatorum Parti cular-Verträge errichten und dardurch nicht allein die Handels¬ leute, sondern auch das Publicum bedrucken, mithin ist kein An¬ stand, derlei conventiones nicht allein pro praeterito zu cassiren, sondern auch pro futuro zu verbieten. Sollte aber ein oder der andere Professionist schlechte Ware machen und damit zu schleudern anfangen, mithin die Ware in discreditu bringen, so stehet denen Mitmeistern frei, darüber bei denen Vorgesetzten Instanzien die Remedur zu suchen.“ Kartelle in Schweden , Frankreich im 18. Jahrhundert. und Sachsen Auf ein recht bemerkenswertes Syndikat aus der ersten Hälfte des 18. Jahrhunderts in Schweden macht Beck aufmerkim k. k. Archiv des Minist, des Innern zu Wien. Bohemica IV. F . 1595—1791. Abgedruckt bei Schebek a. a. O., S. 365ff. Zweites Kapitel : Kartelle des 14. bis 18. Jahrhunderts. 209 sam 1. Um den darniederliegendenEisenhandel zu kräftigen, wurde 1745 vom schwedischen Reichstag „ der Beschluß gefaßt, die Hütten¬ besitzer zu veranlassen, unter sich Summen zusammenzuschießen und, auf diese gestützt, sich gegenseitig einen gewissen niedrigsten Verkaufspreis zu garantieren (Syndikat, Ring). Zur Verwaltung der Fonds und Ausführung der Maßnahmen, welche zur Erhaltung und Durchführung dieses Abkommens nötig waren, wurde ein Aus¬ schuß mit dem bleibenden Sitze in Stockholm erwählt. Das ganze Institut erhielt den Namen „Eisenkontor“ (Jernkontor). Die Ab¬ gabe zur Bildung des Fonds wurde auf 1 Kupfertaler (10 Pf.) von jedem Schiffspfund, das zur Wage gebracht wurde, festgesetzt; nach einem späteren Abkommen mußte der Käufer diese Abgaben erlegen. Mit diesen Summen sollten auf den großen Eisenmärkten Einkäufe gemacht werden, wenn der Preis unter die bestimmte Grenze zu fallen drohte. Auch sollten die Hüttenbesitzer, wenn sie nicht an ihren Preis kamen, ihr Eisen deponieren und Summen zu 4 % Zinsen darauf aufnehmen. Die vier Bevollmächtigten wurden jährlich in einer Versammlung vier Brak-Sozietät 2 gewählt. Gleich beim ersten Markte zu Gothenburg trat das neue Institut mit großem Erfolge in Aktion. Es gingen im ersten Jahre über 300 000 Kupfertaler ein. Der ursprüngliche Plan wurde später, 1766, dahin abgeändert, daß man den Einkauf auf den Märkten fallen ließ, dagegen Hütten¬ besitzer, die aus Mangel an Betriebsfonds zu niedrigen Preisen verkaufen mußten, durch Vorschüsse unterstützte , was sich um so besser durchführen ließ, als das Eisenkontor nach und nach ein großes Vermögen gesammelt hatte und ihm von den Reichsständen( !) überdies ein Kredit von 900 000 Mk. zu geringem Zins bei der Hauptbank eröffnet worden war.“ Das „Eisenkontor“ hat lange Zeit bestanden und sehr viel für Schwedens Bergbau und Hütten¬ wesen geleistet. Es wäre zu wünschen, daß der hochinteressanten Organisation eine besondere Monographie von sachkundiger Seite gewidmet würde. 1 Beck, Geschichte des Eisens . III . Bd ., S. 1103— 1 109. Nach M. Meyer, Eisenhüttenwesen in Schweden 1829 . S. 21. 2 Eine Vereinigung der schwedischen Hütten - und Hamincrherren mit der Absicht , neben Wahrung ihrer eigenen Interessen das Eisenliüttengewerbe in Schweden zu fördern . Beck a . a . O., III . Bd ., S. 1102. 14 Strieder , Studien z. Gesch . kapitalist . Organisalionsfonnen. 210 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. Eine Anzahl von Kartellen französischer Kaufleute und Fabri¬ kanten erwähnt Germain Martin in seiner „ Geschichte der französischen Industrie im Zeitalter Ludwigs XV.“ 1Wiederum, wie so oft, ist es die Montanindustrie, die auch hier die fortgeschrit¬ tensten kapitalistischen Organisationsformen gezeitigt hat . ln der Mitte des 18. Jahrhunderts gewahrt mau, daß die verschiedenen Bergwerks-Unternehmer und Besitzer der Languedoc Konventionen miteinander abgeschlossen haben, laut denen sie die Kohle reihum zu festbestimmtein Preise verkaufen 2. Martin erwähnt noch einige andere Industriekarteile. Hier sei darauf hingewiesen, daß es nach Sa vary im Frankreich des 18. Jahrhunderts auch Handelskartelle in größerer Anzahl gab. Dabei muß es freilich dahin¬ gestellt bleiben, oh der gelehrte kaufmännische Theoretiker Ver¬ einbarungen von nur kürzerer oder auch von längerer Dauer im Auge hat 3. Aus der sächsischen Wirtschaftsgeschichtedes 18. Jahrhunderts mögen wenigstens zwei Kartellbildungen erwähnt werden. Im Jahre 1772 einigten sich die Plauenschen (innungsmäßig organi¬ sierten) Textilwarenhändler mit ihren Innungsverwandten von Ölsnitz, MübJtroff, Elsterberg und Pausa zu folgender Konvention. 1 G. Martin, La grande industrie en France sous le règne de l.ouis XV . Paris 1900 . S. 228 fl. 2 Ma v l i il a . a . O., S. 229. 51 . a c c) u c s S a v a r y , Le parfait négociant etc . 2 Bde. 2. Auf ], Genf 1752 . I. S. 395 . . . les plus puissans marchands font ensemble des sociétés anonymes ( !), ou inconnues . . . qui ayant, accappeté ex acheté dans le pays des autres petits marchands , toutes leurs marchandises pour les porter aux foires et marchés , y mettent tel prix qu 'ils veulent et par ce moyen , il faut que ceux qui veulent acheter passent par leurs mains , à moins de s’en retourner sans rien acheter . Ces sortes de sociétés sont à proprement parler des mono¬ poles , qui sc font contre le bien public et qui renversent l’économie du commerce . J ’ai vû autrefois dans des foires pareilles choses arriver ; les marchands qui étoient pour vendre se tenir fermes et ne la donner de concert qu ’à un même prix . . .“ Oft kamen dann Gegenkartelle der Käufer zustande , Savary, 1 . c. S. 395 . Inter¬ essant ist es, daß auch nach der strengen Wirtschaftsethik des 16. Jahr¬ hunderts solche Gcgenkartelle als Notwehr ethisch erlaubt waren. „Quando vero venditores monopolium facerent , tunc juste emptores quasi vim vi repellentes possent convenire inter se , ut nullus nisi certo pretio emeret ." S c a c c i a , a . a . O., S. 300 , nach Sot . de iusticia et iure lib . 6 qu . 2 articul . 3 col . 3 in fine. 1 Zweites Kapitel : Kartelle des 14. bis 18. Jahrhunderts . 211 Vor Beginn jeder Leipziger Messe wurde für jede Gattung Waren ein Minimalpreis festgesetzt , unter welchem keiner der Kon¬ trahenten verkaufen durfte . Dagegen war es den betreffenden Firmen erlaubt , über den festgesetzten Preis hinaus zu Verkaufen, soviel sie konnten und wolltenL Guten Quellen 2 zufolge hatte diese Konvention sofort die günstige Folge, daß der eingerissenen Warenverschleuderei ein Ziel gesetzt und ein gesunder Absatz ein¬ geleitet wurde 3. Die Tendenzen der Ausschaltung eines unnötigen Konkurrenz¬ kampfes (mit seiner preisdrückenden Wirkung ), wie sie in der zweiten Hälfte des 18. Jahrhunderts in Sachsen in der Luft lagen, kommen im Jahre 1780 zu noch vollerer Entfaltung als 1772. Im Jahre 1780 vereinigten sich elf größere vogtländische Baumwollwarenhändler „ zu einer unter der Firma ,Haussner und Compagnie* zu errichtenden gemeinschaftlichen Niederlage in Plauen , welcher jeder Verleger seine Waren nach Belieben zum Verkauf übergeben konnte , so daß diese Niederlage ein Kommissions - oder Kon¬ signationslager darstellte . Ein Zwang für die Innungsmitglieder entstand dadurch ebensowenig als ein Monopol daraus hervorging. Diese Sozietätshandlung überhob vielmehr eine Zahl sonst selbst zum Verkauf ausstehender kleinerer Händler des Meßbesuches und gab die Möglichkeit der Einhaltung von allen Beteiligten zugute kommenden gleichen Preisen , auch gleichzeitig Gelegenheit , daß die weniger vermögenden Verleger auf diesem Wege eventuell schneller zu ihrem Gelde kommen konnten , weniger Risiko hatten und nötigenfalls sogar auf die niedergelegten Waren Vorschuß erhielten. 1 Louis Bein , Die Industrie des sächsischen Voigtlandcs. Wirtschal 'tsgeschichtliche Studie . II . Teil . Die Textilindustrie. Leipzig 1884 . S. 84. 2 Über den Wert der „ Meßbcrichte '' als Quellen der sächsischen Wirtschaftsgeschichte des 18. Jahrhunderts . Vgl . Bein a . a . O., S. 57. 3 Es wäre durchaus verfehlt , die Konvention von 1772 als „Zunftbestimmung “ aus dem Kreise der Kartellgeschichte ver¬ weisen zu wollen . Mit Recht betont Bein , daß es der Innungs¬ einigung nicht gelungen war , die Verkaufspreise in feste Normen zu bringen . Man mußte „ zur Erreichung dieses Zweckes zu einem auch modernen Hilfsmittel , zur Bildung von Koalitionen “ greifen. A . a . O., S. 84 . Vgl . auch S. 86, die Weigerung der Regierung , solch freiwillige Übereinkünfte der Unternehmer landesherrlich zu kon¬ firmieren. 14 * 212 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. Diese Sozietät erwies sich dann auch , da ihre Bestrebungen sich von jeglichem Zwang fernhielten , also, wie gesagt , ein monopolisti¬ sches Vorgehen damit nicht verbunden war , als den Erwartungen entsprechend und als günstig für die Manufaktur im allgemeinen 1.“ Drittes Kapitel, Monopole , Kartelle und Aktiengesellschaften im sächsischen Zinnhandel des 15, und 16.Jahr¬ hunderts. Im Verlaufe des 15. Jahrhunderts hatte sich im sächsischen Zinnbergbau eine Entwicklung vollzogen, die die meisten Gewerken immer mehr in Abhängigkeit von reichen Kapitalisten brachte 2. Die Zinngewerken ■—■im allgemeinen den ärmeren Schichten der Bevölkerung angehörig 3 vermochten — je länger um so weniger das nötige Geld aufzubringen , um die wachsenden Unkosten zu decken . Ganz allgemein bildete sich daher bei ihnen die Gewohnheit heraus , Verlag zu nehmen , d. h. gegen Vorschußempfang ihr Zinn¬ produkt an bestimmte Kapitalisten zu liefern 4. In der zweiten Hälfte des 15. Jahrhunderts und vielleicht schon früher war der Verlag der Zinngewerken („Zinnkauf “ genannt ) eine vielfach übliche 1 Bein a . a . O., S . 88/89. 2 Vgl. J . Falke, Geschichte der Bergstadt Geyer in Mit¬ teilungen des Kgl . Sachs . Vereins für Erforschung u . Erhaltung vaterländischer Geschichts - und Kunstdenkmale . 15. Heft . 1866. S . 25 ff. 3 Für das 16. Jahrhundert cfr . J . Falke, Die Geschichte des Kurfürsten August von Sachsen in volkswirtschaftlicher Beziehung. S. 175. 4 Für die schlesischen Zinnbergwerke läßt sieh dieselbe Ent¬ wicklung konstatieren . Steinbeck, Geschichte d. schlesischen Bergbaues . II . Bd ., S. 10. Ebenso für Böhmen . A. v . Sclieurl, Cristoff Scheurl . S. 30 . Für die Erscheinung in England vgl. Levy, Monopole , Kartelle und Trusts in ihren Beziehungen zur Organisation der kapitalistischen Industrie . Dargestellt an der Ent¬ wicklung in Großbritannien . S. 5, 24 ff ., 46 f. Dazu ferner G. B. Lewis, The stanneries , a study of the english tin miner . Harvard economic studies . Vol . III . Boston u . New York 1908 , besonders S. 213 ff. Drittes Kap. : Monopole, Kartelle u. Aktienges. im sächs. Zinnhandel usw. 213 Form der Kapitalanlagé . In aen letzten Jahrzehnten des Jahr¬ hunderts gediente sich selbst der Landesherr des „ Zinnkaufs “ zur Anlage überschüssiger Gelder . In einem „Hauptbuch “ der säch¬ sischen Finanzverwaltung unter Albrecht dem Beherzten (von 1489—1497 reichend ), das ich im Dresdener Hauptstaatsarchiv gefunden habe 1, ist von 1491 an der Herzog von Sachsen als Teil¬ haber einer Gesellschaft aufgeführt 2, die den „Zinnkauf “ in Sachsen im obengenannten Sinne betrieb. Schon um 1470 war übrigens von seiten eines herzoglichen Zehntners zu Geyer usw. der Vorschlag gemacht worden , der Herzog solle, um den Zinnzehnten „im guten Fortgang “ zu erhalten , selbst das Zinn in der Flöße (wo es fertig gemacht wurde ), aufkaufen. Dann würden die Zinngewerken „um so kühnlicher die Bergwerke bauen “ 3. Da es dem Herzog für eine solche Verstaatlichung des sächsischen Zinnverlags und damit des sächsischen Zinngroßhandels an dem nötigen Kapital fehlte , kam nur die oben angeführte Teil¬ haberschaft des Herzogs an der Zinnkaufsgesellschaft zustande. Näheres über diese ältere Zinnkaufsgesellschaft hat sich nicht beibringen lassen . Wir müssen ès also dahingestellt sein lassen , ob die jüngere „ Gesellschaft des Zinnkaufs “ resp . des „Zinnhandels “, die uns seit 1497/98 in ziemlich scharfer historischer Beleuchtung begegnet , mit ihr identisch ist bzw. als ihre Fortsetzung zu gelten hat . Im höchsten Grade wahrscheinlich ist ein solcher Zusammen¬ hang immerhin. 1 Alexander Puff hat , vornehmlich auf Grund dieses Haupt¬ buches , das sächsische Finanzwesen von 1488— 1497 eingehend dar¬ gestellt. 2 Haupt- Staatsarchiv Dresden . Loc . 8678 . Hof - u . Haus¬ haltungssachen Herzog Albrechts . Bl . 180 , zu den Jahren 1491— 1492: „2000 fl . in zcinlcauf gelegt uffs nau jar .“ Bl . 195, zu den Jahren 1492— 1493 : „ 200 fl . vom zcin -kauffe entpfangen .“ Bl . 226 , zu den Jahren 1493— 1494 : „190 fl . Austeilunge des zcinkaufs halben ; An 2 fassen zcines 8 4 fl . u . an 20 fl . müntz entpfangen uffs nau jar im 94 ,cn. Ist von 5 virtl jars .“ Bl . 261“, zu den Jahren 1494— 1495: „200 fl. Austeilunge des zcinkaufs halben .“ Bl . 291a, zu den Jahren 1495— 1496 : „200 fl . des zcinkaufs halben .“ Bl . 323 “, zu den Jahren 1496— 1497 : „200 fl . des zcinkaufs halben entpfangen .“ Aus dem Worte „Austeilung “ schließe ich , daß es sich um eine Gesellschaft handelt , deren Mitglied der Herzog im Jahre 1491 durch seine Ein¬ lage von 2000 fl. wurde. 3 Falke, Geschichte der Bergstadt Geyer , a . a . O., S. 26. 2 214 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. Die privilegierte ..Gesellschaft von des Zinnkaufs“ 1498— 1500. Hauptquellen : Ein ziemlich genaues Urteil über das Wesen der „Gesellschaft des Zinnhandels“ l , Uber ihre Stellung im öffentlichen Recht, ihre Organisation im Innern und ihre Geschäfts¬ führung ermöglichen uns u. a. die folgenden Hauptquellen: 1. Das Privileg, das ihr Herzog Georg von Sachsen am 6. März 1498 ausstellen ließ. Im Hauplstaatsarchiv zu Dresden Loc. 7414, den Zinnhandel betr . 1497— 1544 , Bl . 9. Im folgenden zitiert als Loc . 7414 , Nr . 2. Von mir im Anhang abgedruckt. 2. Zwei Gutachten über einen Prozeß , den der Leipziger Jurist Dr . Cristoff Kuppener mit der Gesellschaft führte . Handschrift¬ lich im Kgl , Preuß . Staatsarchiv zu Königsberg i. Pr ., Msc . A. 34 Fol ., Bl . 199 ff. und Bl . 204 ff . Das handelsgeschichtlich Wichtigste der Gutachten habe ich gleichfalls im Anhang wiedergegeben. Wenn man den Eingang des obengenannten Privilegs für sich betrachtet, so kann man auf den Gedanken kommen, die „ Gesell¬ schaft des Zinnhandels“ habe erst mit dem Ausstellungstermin dieser Urkunde ihren Anfang genommen. Das ist jedoch nicht der Fall. Die Urkunde stellt nur eine starke Privilegierung einer schon früher bestehenden Gesellschaft dar 3. 1 Das scheint der offizielle Name der Gesellschaft gewesen zu sein . Aber auch Zinnkaufsgesellschaft kommt als Bezeichnung für sie vor. 2 Über Dr . Cristoff Kuppener vgl . Th . Muther, Aus dem .Universitäts - und Gelehrtenleben im Zeitalter der Reformation. S. 13. 3 Das ergibt sich unzweifelhaft aus der folgenden Urkunde des Hauptstaatsarchivs Dresden, Loc. 7414, Georg u. Hans v. Salhausen contra die Gesellschafter des Zinnhandels 1501— 1516 ; Bl . 15 (im folgenden Loc. 7414 Nr. 3 zitiert) : 1497 Montag nach Misericordiae domini. Georg v. Salhausen beurkundet, daß Langhaus, Bürger von Lauenstein vor ihm erschienen ist mit der Bitte, ihm zu gestatten, die Hälfte seiner Flut an Bastiah Jobstei u. Martin Spengeler, Bürger zu Dresden, an Stelle ihrer Herren, der Gesellschafter des Zinnkaufs zu verpfänden. Wenn Langhans oder seine Erben nicht bis zu einem bestimmten Tage die Schuld an die Gesellschaft zahle, soll letztere die Hälfte der Flut nach Nutz und Frommen ge¬ brauchen und genießen bis Hauptsumma, Expensa und erlittene Schäden ganz und gar entrichtet und bezahlt seien. — In dem Gut¬ achten 1 des Kuppener-Prozcsses heißt es, daß Dr. Kuppeners Kapita. „longe ante principis confirmationem“ in die Gesellschaft inseriert worden sei. Drittes Kap . : Monopole, Kartelle u. Aktienges. im sächs. Ziiirihanclel usw. 215 Die „ Gesellschaft des Zinnhandels “ war in den neunziger Jahren , vielleicht sogar noch früher , von einer Anzahl zum Teil sehr angesehener Untertanen des Herzogs von Sachsen 1gegründet worden . Leider ist der Gesellschaftsvertrag , durch den das geschah, soviel ich sehe, nicht auf uns gekommen . Trotzdem können wir das Gesellschaftskapital unserer Firma mit Hilfe einer Abrechnung, die sie einem renitenten Mitgliede aufmachte , ziemlich sicher auf ca. 30 000 fl. einschätzen 2. Eine stattliche Summe für den Aus¬ gang des 15. Jahrhunderts , und unverächtlich , wenn man bedenkt, daß das Gesellschaftskapital der Gebrüder Ulrich , Georg und Jacob Fugger laut ihrem Gesellschaftsvertrag vom 18. August 1494 nur 54 385 fl. betrug 3. Die Namen der Teilnehmer an der Gesellschaft waren mit der Höhe ihrer Einlage in einem Buche verzeichnet . Dazu hatte jeder einzelne von ihnen noch eine besondere Quittung über die von ihm eingelegte Geldsumme erhalten . Die Geschäfte der Gesellschaft führten zwei von den Mitgliedern der Kompagnie eingesetzte Faktoren oder Diener . Es waren die Dresdener Bürger Sebastian (Bastian ) Jobstei (auch Jöstel usw. geschrieben ) und Martin (Merten) Spengler 4. Außerdem hatte die Gesellschaft noch ihre Verordneten , d. h. Mitglieder der Kompagnie , die sich in besonderer Weise um die Geschäftsführung kümmern mußten ; ohne ihre Crenehmigung durften die Faktoren nichts verborgen , also weder den Zinnern Verlag gewähren noch den Zinnkäufern Kredit eröffnen. Den Verordneten der Gesellschaft mußten die Faktoren viertel¬ jährlich oder so oft sie es verlangten , Rechenschaft von ihrer Ge1 Herzog Georg nennt die Gründer der Gesellschaft in dem Privileg von 1498 seine „ Lieben " und „ Getreuen “ . Daß sie an¬ gesehene Personen waren , geht aus den Eingängen der Gutachten zum Kuppener -Prozeß hervor. 2 Als Georg v . Salhausen im Jahre 1499 seine Einlage von 4000 fl . an Stefan Alnpeck „ verwies “ und dieser den Anteil herausforderte , belastete die Gesellschaft Salhausens ehemaligen Geschäfts¬ anteil mit etwas mehr als dem siebenten Teile aller uneinbringlichen und ungewissen Forderungen der Firma . Demnach wären also 4000 fl. nicht , ganz der siebente Teil des Gesellschaftskapitals , dieses also ca . 30 000 fl. (Loc . 7414 Nr . 3 Bl . 101 H .-St .-A . Dresden .) 3 M. Jansen, Die Anfänge der Fugger . S. 63. 1 Über sie cf. Otto Richter, Verfassungs - und Ver¬ waltungsgeschichte der Stadt Dresden . 3 Bde . Dresden 1885/91. Siehe Namenregister. 216 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. schäftsführung geben . Die Faktoren hatten ihre ganze kauf¬ männische Kraft dem Handel der Gesellschaft zu widmen . Eigene Geschäfte zu treiben , war ihnen streng untersagt . Dafür sollten sie ein gutes Gehalt bekommen 1, und wenn sie wollten , Einlagen in die Gesellschaft machen dürfen . Am Jahresschluß hatten die Faktoren die Bilanz zu ziehen und innerhalb 14 Tagen die Unter¬ lagen dafür in einer genügenden Anzahl von Abschriften der Ge¬ sellschaft zu übergeben . Die Mitglieder der Gesellschaft oder deren Bevollmächtigte nahmen dann auf der alljährlich in Dresden .stattfindenden Generalversammlung die Bilanz ab . Alljährlich fand auch die Ausschüttung der Gewinnanteile statt. Die eben genannten Bestimmungen über die Tätigkeit der Faktoren im Dienste der Gesellschaft sind in das Bestätigungs¬ privileg aufgenommen , das Herzog Georg der Gesellschaft aus¬ stellte . Schon damit wurde die Zinnhandelsgesellschaft in etwa aus der Sphäre des Privatrechts in die des öffentlichen Rechts gehoben . Mehr geschah das noch durch die folgenden Vorschriften des Privilegs : Die Faktoren der Gesellschaft hatten genau wie der herzogliche Wagmeister einen Schlüssel zu der Wage in Altenberg 2. Es konnte also niemandes Zinn verkaufsfrei erklärt werden , wenn der Betreffende noch der Gesellschaft aus Verlag usw. Geld schuldete. Die Räte und Amtleute des Herzogs waren ferner angewiesen , den Faktoren allzeit mit Rat und Tat zur Seite zu stehen . Mit Rat, wenn „ Händel und Geschäfte “ vorkamen , in denen sie sich nicht zu helfen wußten ; mit der Tat , wenn faule Schuldner nicht zahlen wollten . Die Zusicherungen sind nicht leere Formeln geblieben. Wirklich hat die Regierung die Gesellschaft des Zinnhandels tat¬ kräftig unterstützt . So wurden z. B. Bergmeister , Richter und Schöffen auf dem Geising angewiesen , dafür zu sorgen, daß die Gesellschaft immer pünktlich Zahlung erhalte 3. Und es zeugt von 1 Der Nachfolger des Jobstei und Spengler erhielt 50 fl. pro J ahr. 2 Aus den Schmelzhütten hatte alles Zinn in die herzogliche Wage zu gehen . Hier wurde es vom Wagemeister , einem herzog¬ lichen Beamten , zum Zwecke der Berechnung des herzoglichen Zehnten usw . gewogen . Zinn , das nicht die Wage passiert hatte, durfte nicht verkauft werden . Vgl . Christoph Meißner, Umständliche Nachricht von der Zien -Berg -Stadt Altenberg . Dresden u . Leipzig 1747 . S. 130. 3 Nach einem Konzept : Hauptstaatsarchiv Dresden , Loc . 7414 * Drittes Kap. : Monopole, Kartelle u. Aktienges. im sitchs. Zinnbandel usw. 217 einem scharfen Zugreifen der herzoglichen Regierung zugunsten der Gesellschaft , wenn ein Herr v. Colditz im Jahre 1499 die flehent¬ liche Bitte an Herzog Georg richtete „ der Schuld halben , so er der Gesellschaft [des Zinnkaufs ] vorhafft “, noch etwas Geduld mit ihm zu haben 1. Freilich scheint auch die kapitalkräftige Gesellschaft nicht verfehlt zu haben , ihren Privilegien durch Gefälligkeiten usw. an die Räte des Herzogs eine nachdrückliche Handhabung zu sichern 2. Wie schwer dabei die Privilegierung der Zinnhandelsgesellschaft auf den übrigen Einwohnern des Herzogtums lasten konnte , erkennt man aus einer Beschwerde des Rates von Freiberg i. S. 3. Als sich im Jahre 1499 Unregelmäßigkeiten in der Geschäftsführung der Gesellschaft des Zinnhandels einstellten , auf die wir noch zu sprechen kommen werden , erhielt der Rat von Freiberg vom Herzog den Befehl , das Zinn , das , „ der Gesellschaft zuständig “, in Freiberg vorhanden war , bis auf weiteres in Verwahrung zu nehmen . Der Rat bat sehr bald um Befreiung von dieser lästigen Pflicht . Er begründete sein Gesuch mit dem Hinweis , daß im Rathaus keine genügenden Räume für das Zinn vorhanden wären und besonders mit den Schwierigkeiten , die sich in der Abrechnung ergeben müßten, wenn Zinn davon verkauft würde . Es erübrigt sich für unsere Zwecke näher auf diese Dinge einzugehen . Es genügt uns zu sehen, wie hier die Gesellschaft des Zinnhandels fast wie eine öffentlich rechtliche Institution erscheint. Wenn wir nach einigen Worten über die Stellung der Zinn¬ handelsgesellschaft im öffentlichen Recht und über ihre Organi¬ sation im Innern zu einer Darstellung der Geschäftspraxis über¬ gehen wollen, so ist etwa das folgende als besonders wichtig hervor(Nr . 2), Bl . 46. Nicht datiert , aber wohl in das Jahr 1498 oder 1499 gehörig. 1 Hauptstaatsarchiv Dresden , Loc . 7414 (Nr . 2), Bl . 5. 2 Unter dem 23. September 1501 schrieb G. v . Widebach , einer der Hauptgesellschafter , an den Obermarschall Heinr . v . Schleinitz, er solle sich in einer Prozeßangelegenheit für die Gesellschaft ver¬ wenden : „ Und wullet uch mangfeldigs ansuchen nicht besweren lassen , wirt ane zweifei die gesellschaft sich vleissigçn zeu verdinen. Wumit ich uch zue willen u . gefallen werden sal , soldt ir mich meyns Vermögens willig finden . . . .“ Hauptstaatsarchiv Dresden , Loc . 7414 (Nr . 3), Bl . 17. 3 Hauptstaatsarchiv Dresden , Loc . 7414 (Nr . 2), Bl . 10. 218 Drittes Qucli: Monopole, Kartelle und Aktiengesellschaften im Mittelalter. zuheben . Die Geschäftstätigkeit der Gesellschaft bestand nament¬ lich aus zweierlei : Einmal erwarb sie von den Zinnern , d. h. den Zinngewerken , das Zinnprodukt und zweitens verkaufte sie das Zinn weiter . Der Erwerb des Zinns durch die Gesellschaft vollzog sich in zwei Formen . Den vermögenden Zinngewerken kaufte sie das Zinnprodukt ab . Bei den ärmeren brachte unsere Firma das Verlagssystem in Anwendung . Viele Zinner waren nämlich nicht kapitalkräftig genug , um auf eigene Kosten Zinn produzieren zu können . Diese mußten sich von unserer Gesellschaft verlegen lassen . Sie erhielten eine gewisse Geldsumme und hatten dafür eine entsprechende Menge Zinn zu liefern . Als Pfand wurde dann viel¬ fach der Zinnbergwerksanteil des Betreffenden gesetzt L In solchem 1Auf solche Weise geriet 1499 die Gesellschaft in den Pfand¬ besitz des halben Zinn -Berg - und Seiffwerks eines gewissen Lang¬ haus . ,,llem Langehansen bergwergk und sein seiffwergk haben sie zugleich angenohmen mit den herren des capittels zu Freiberg , den solch bergwerg und seyffenwerg auch die helffte zuständig gewest, sollen sie zugleich mit der geselschaft verlegen .“ Aus dem General¬ versammlungsprotokoll von 1499 . Hauptstaatsarchiv Dresden , Loc. 7414 (Nr . 2), Bl . 14. — Um ihr Geld aus dem Pfandbesitz herauszuwirtschaften , schloß die Gesellschaft mit Langhans den nach¬ folgenden Vertrag , der einen interessanten Beitrag zur Geschichte des Konkursrechtes darstellen dürfte : „ In irnisse und gebrechen sco sich zwusc.hen den hem der geselschaft des zcinkaufs an einem und Langehansen der scliuldt halben , sco er den hern obgemelt zeu bezcalen schuldig , andersteils gehalden , ist vorlossen und beredt wie hirnach folget : Nemliehen , daß die hern der geselschaft die halbe flut, sco inen vormals verhallt , ein jar langk Langehansen zeu sunderlichem gefallen mit irem gelde vorlegen sollen , nach irem gefallen zeu erbeten lassen , auch die erbeter wochenlichen zeu loen bestellen ; darzcu und auf dieselbige erbeit und knechte sal Langehans zeu forderunge der hern und sein seihest nuze ein vleißigk aufsehen haben und nichts minners , denn im die flut zeustendigk , getraulicli allenthalben fertigen, Umb deswillen sal im alle Wochen zeu seiner enthaldunge und nottorft ein swert sehogk gegeben werden . Sco man den auf die zeeit zcin machen wurde und der geselschaft obergantwort , was den unkost zeu voiiertigunge des zcins , Langehansen Ion und anders darauf¬ gegangen , das sal nach redelicher rechnunge an dem gemachten zcin ye zc.endehalben gülden rheinisch abgerechent werden und was obermase befindet , sale den hern an ire schulde , die in Lange¬ hanns verschrieben , abegezcogen werden und seine schulde domit minnern . Wu den nach des jars ausgange den hern nicht geliebet ader geliehen wurde , obgedaclite flut lenger ader meher zeu haben, 2* Drittes Kap. : Monopole,, Kartelle u. Aktienges. im sächs. Zinnhandei usw. 219 Verlag hatte im Jahre 1499 die Gesellschaft insgesamt 370 fl. un¬ einbringliche und 1580 fl. zweifelhafte Außenstände 1. Der Verkauf des Zinns an die Verbraucher brachte die Gesell¬ schaft mit Kannengießern und anderen Zinnwarenproduzenten in Verbindung . Aber auch Zinnzwischenhändler waren ihre Kunden. Das meiste Zinn scheint in Nürnberg abgesetzt worden zu sein. Aber auch nach Breslau lieferte die Firma , wie aus dem Debitoren¬ konto ersichtlich ist ä. Im allgemeinen wird die Gesellschaft auf Kredit verkauft haben . Häufige Verluste waren die Folge . Im Jahre 1499 standen 939 fl. an uneinbringliche n Außenständen , die zumeist aus Kreditgewährungen an Abnehmer entstanden waren , zu Buch. Dazu 3046 fl. an zweifelhaften Außenständen bei Zinn¬ konsumenten 3. Die Höhe der guten Außenstände der Firma bei Zinnabnehmern und Zinngewerken (für Verlag) zusammen gibt der Generalversammlungsbericht von 1499 auf 24 000 fl. an 4. Uber die „Dividende “, die die Gesellschaft in den Jahren 1498 und 1499 gezahlt hat , läßt sich leider nichts näheres sagen. Das Glück war jedenfalls dem Unternehmen nicht allzu günstig . Die zwei Faktoren der Gesellschaft Jobstei & Spengler wirtschafteten, mögen sie darvon treten und sich hinfurder Langehansen vorschreibunge und vorwilligunge halden und damit obgedachten contract nicht obergeben haben .“ Beurkundet durch Hans Bircke von der Daube und Sigmund von Miltitz, Statthalter 1499. Hauptstaatsarchiv Dresden , Loc. 7114 (Nr. 3), Bl. 16. 1 Hauptstaatsarchiv Dresden, Loc. 7414 (Nr. 3), Bl. 101. 2 Fritz Rußwunn in Breslau schuldete der Firma 145 fl. ; 1. c., Bl. 101. Fritz Rußwurm spielte im Breslauer Handelsleben wie im schlesischen Bergbau um die Wende des 15. und zu Beginn des 16. Jahr¬ hunderts eine hervorragende Rolle. Vgl. K. W u 11 k c , Schlesiens Bergbau u. Hüttenwesen ( = Cod. dipl. Silesiae XX u. XXI ), die Nummern 273, 285, 288, 292, 294 und A. Zycha, Iglaucr Schöffen¬ sprüche , Nr. 83, 84, 93, 95. Weitere Nachrichten über die Ruß¬ wurm zusammengestellt bei A. Zycha, Zur neuesten Literatur über die Wirtschafts - und Rechtsgeschichte des deutschen Bergbaus in Vierteljahrsschrift für Sozial- u. Wirtschaftsgeschichte VI (1908), S. 116 u. 126. 3 Hauptstaatsarchiv Dresden, Loc. 7414 (Nr. 3), Bl. 101. 4 Hauptstaatsarchiv Dresden, Loc. 7414 (Nr . 2), Bl. 14. „ Item das ist der besließ der herren von der gcsellscliafft des zcinkaufs: Erstlich so finden sie in ircr rechnunge 24 000 fl. an zcin u. gelt¬ schuld als sic verhoffen über ire schult als sich befinden sal.“ 220 Drittes Buch: Monopole , Kartelle lind Aktiengesellschaften im Mittelalter. wie es scheint , ziemlich willkürlich . So nahmen sie ohne Befehl und Erlaubnis der Gesellschaft 2000 fl. von Dr . Cristoff Kuppener als Gesellschaftskapital an , trugen ihn in das Gesellschaftsregister ein und zahlten ihm auch , wie den übrigen Teilhabern , die Dividende aus . Der betrügerische Vorteil , den sich die Faktoren dabei ver¬ schafften , bestand darin , daß sie die neue Einlage nicht im Interesse der Gesellschaft verwandten , sondern für sich behielten , um wahr -, scheinlich eigene Schulden damit zu decken . Erst als die zwei Faktoren — vielleicht infolge dieser Unregelmäßigkeiten in der Geschäftsführung — im Jahre 1499 die Flucht ergriffen , erfuhr die Gesellschaft von der Einlage Kuppeners . Natürlich bestritt sie dem ohne ihr Wissen und Willen Aufgenommenen die Teilhaber¬ schaft . Es kam zu langen prozessualen Auseinandersetzungen — die beiden Gutachten Dr . Cristoff Kuppeners und Dr . Tilemann Branders sind hierfür als Unterlagen entstanden —, und erst als Jobstei und Spengler in einem Vergleichsverfahren unter Pfandsetzung ihrer Güter die 2000 fl. zurückzuzahlen versprachen , wurde Dr . Kuppener in die Gesellschaft aufgenommen 1. Wahrscheinlich infolge der Unregelmäßigkeiten , die die Faktoren Jobstei und Spengler heraufgeführt hatten , mußte von seiten der Generalversammlung des Jahres 1499 den Gesellschaftern ein Kapitalnachschuß zugemutet werden . Für je 1000 fl. Einlage sollte eine Nachschußpflicht von 50 fl. bestehen 2. Die „Gesellschaft Jahres 1500 bis des Zinnhandels zur Aufhebung “ vom Ende des ihres Monopols. Haupt quellen: Das Monopolprivileg des Herzogs Georg von Sachsen für die Gesellschaft vom 14. September 1500 . Im Hauptstaatsarchiv zu Dresden , Loc . 4491 . Allerhand Privilegien und 1 Hauptstaatsarchiv Dresden , Loc . 7414 (Nr . 2), Bl . 7. „ Doruff ist Dr . Cristofferus Guppner mit 2000 gülden zu gewin u . Verlust in der geselschafft ." 2 Hauptstaatsarchiv Dresden , Loc . 7414 (Nr . 2), Bl . 14 . „ Item es ist angelegt , wer in der geselschafft 1000 fl. ligen hat , sal itzundt 50 fl. ufs 1000 fl. zu Freiberg bei dem Techant u . Dr . Schrencke in¬ legen nach laut wie in irer verschreibunge angezeeigt ist .“ Die zwei Genannten waren nach der Flucht der Faktoren zu Sachwaltern der Gesellschaft eingesetzt worden : „ Item zu nottorfft irs handeis haben sie verordent ir gelt u . czin inzunehmen u . auszugeben , den Techant von Freiberg u . Dr . Schrencken .“ A . a . O., Bl . 14. Drittes Kap. : Monopole, Kartelle u . Aktienges. im säclrs. Zinnhandel usw. 221 Befreiungen in Bergwerkssachen mir im Anhang abgedruckt. de anno 1500— 1681, Bl . 1— -2. Von Der Herbst des Jahres 1500 bezeichnet eine wichtige Epoche für die Geschichte der Zinnhandelsgesellschaft . Am 14. September stattete Herzog Georg die Kompagnie mit einem neuen großen Privileg aus 1. Das wesentlich Neue, das dieses Privileg enthielt, bestand darin , daß der Gesellschaft auf drei Jahre ein Monopol des Zinngroßhandels eingeräumt wurde 2. Der Herzog bestimmte, daß alles Zinn , das in seinen Landen gewonnen oder fertiggestellt wurde , drei Jahre lang der Gesellschaft zu übergeben sei,- und zwar zu einem festbestimmten Preise . Die Zinngewerken , die nicht auf den Verlag der Gesellschaft angewiesen waren, , erhielten für den Zentner fertiges Zinn 11 fl. rh . (halb meißnisches , halb böhmisches Geld). Wer auf ein ganzes Jahr Verlag nahm , erhielt für den Zentner Zinn 9% fl. Wer nur halbjährigen Verlag benötigte, 10 fl. Wer von letzteren Verlagsnehmern den Termin der Zinn¬ lieferung überschritt , wurde behandelt , wie die auf ein Jahr Ver¬ legten . Konnte ein Verlegter das versprochene Zinn nicht liefern, so sollte er zunächst das liefern, was er hatte . Die Gesellschaft ging dabei vor jeden etwaigen anderen Gläubiger des Betreffenden. Die Gesellschaft hatte das Zinn billiger zu kaufen gedacht. Sie hatte 10y4 fl . für „bares “ Zinn geboten und bei drcivierteljährigem Verlag 9% fl- 3 Aber darauf hatten sich die Gewerken nicht eingelassen . Sie betonten , daß es ihnen leicht möglich sei, 1 Ein entsprechender Eintrag in das Kopialbuch des Fürsten belehrt uns , daß es sich nur um eine neue Privilegierung der älteren Gesellschaft , nicht um eine neue Gesellschaft handelt . Hauptstaatsarchiv Dresden , Cop . 106 , Bl . 38 : „ Anno 1500 , Montag nach exalta¬ tionis crucis [14. Sept .] ist den herren der geselsehafft des zcinkauffs ein neue Verschreibung über iren handel des zcinnkaufs , wies hinfiir darinne soll gehalten werden , gegeben .“ 2 Vgl. das Monopol der Zinnkaufsgesellsehaft in England gegen Ende des 16. Jahrhunderts . Levy a . a . O., S. 25. 3 In dem Vorschlag der Gesellschaft heißt es : „ Zeuin ersten. Wer uffm Geussingc oder umbligenden anhengigen gebirgen bar zeen in die wage bringt , daß dass 'elbigc bare zeen der ctr . vor 10 11. 1 ort . . . zu lcauff gegeben u . also bezealt werde . Zcum andirn . Wer vorlegunge bedarff , adir haben wil , daß denselbigen der ctr . zeen vor 9 fl. 3 ort abgekauft u . bezealt u . auch dafür gegeben werde in 3 j jars nach dem kaufe solch zeen zeu obirantworten .“ Hauptstaatsarchiv Dresden , Loc . 7414 (Nr . 2), Bl . 47. 1 222 Drittes Buch: Monopole , Kartelle und Aktiengesellschalten im Mittelalter. ihr Zinn mit 11 fl. im Auslande unterzubringen . Georg und Hans Alnpeck, die reichsten und mächtigsten Zinngewerken Sachsens, führten aus, daß sie selbst, wenn sie noch Zinn kauften, 114 fl. bezahlten . Dagegen hatten sich die Zinngewerken, die auf den Verlag der Gesellschaft angewiesen waren, mit dem Voranschlag der Gesellschaft einverstunden erklärt 2. Es ist nicht recht aus den Akten ersichtlich , wem sie schließlich die kleine Verbesserung ihrer Bezahlung zu danken hatten , wie sie sich in dem endgültigen Privileg festgelegt vorfindet. Außer der genannten Monopolisierungenthält das Privileg vom 14. September 1500 noch folgende Veränderungen gegenüber dem von 1498. Die Generalversammlung wird von Dresden nach Leipzig verlegt. Die Faktoren der Gesellschaft haben die Bilanz auf den Sonntag Exaudi zu stellen. Im übrigen bleiben die Bestimmungen über die Generalversammlung bestehen, wie sie schon in der Be¬ stätigung von 1498 aufgestellt waren. Die Verlegung des General¬ versammlungstermins auf die Zeit der Leipziger Ostermesse hängt mit der Verlegung der Generalversammlung nach Leipzig zusammen. Mit dem Ausscheiden der Dresdener Jobstei und Spengler aus dem Dienste der Gesellschaft war wohl der Hauptgrund weggefallen , die Generalversammlung in Dresden abzuhalten. Auf Befehl des Obex-marschalls wurde auch eine Bestimmung über den Austritt aus der Gesellschaft in das Privileg aufgenommen 3. Wer sein Kapital ganz oder teilweise aus der Gesellschaft zu ziehen beabsichtigte, der sollte es ein ganzes Jahr vorher den Verordneten oder den Dienern der Gesellschaft kündigen. Nach Ablauf des Jahres würde ihm sein eingelegtes Kapital , dem augenblicklichen Geschäftsstande der Gesellschaft entsprechend , in barem Gelde, in vorhandenem Zinn und an Außenständen übergeben werden. 1 Aus einer Gegenschrift der Zinngewerken , a . a . O., Bl . 2, 2 Ebenda. 3 „Daß disscr arlikel in briff komme, hat der obermarschalgk bcfollcn wie hernach folget: Wer sein gelt wider aus der gesclschaffl nemen wil, der sal ein ganz jar zuvor abschreiben off daz naue jar den vorordenten oder dinern diser gcselschafft. Alzdann nach abschreibung dez jars sal im sein eingeleget gelt an zin u. an schulden mit gewinn u. Verlust, wi der handcl of die zeit stet und leid, ober¬ antwort werden alz getreulich u. ungeferlich.“ Hauptstaatsarchiv Dresden, I.oc. 7414 (Nr. 2), BI. 45. Wie dann die Stelle in den Text des Privilegs aufgenommen wurde , siehe Anhang. 1 * Drittes Kap . : Monopole, Kartelle u. Aktienges. im sächs. Zinnhandel usw. 223 Was die Geschäftsführung der Gesellschaft angeht , so wurde der rein kaufmännische Teil wieder zwei Faktoren übertragen , und zwar dem Leipziger Bürger Nicolaus Ketzler und Nickel Hennel L Dagegen wurden als Vertreter der Gesellschaft , vor allem in Rechts¬ streitigkeiten der Kompagnie mit Schuldnern usw., drei Verordnete, auch Anwälte genannt , aus dem Kreise der Gesellschafter gewählt. Die Wahl fiel auf Georg v. Widebach , auf Lorenz Mordeisen und Heinz Probst , Bürger zu Leipzig 2. Ausdrücklich war den drei Verordneten das Recht Vorbehalten worden , im Bedarfsfälle Be¬ vollmächtigte für sich zu ernennen . Schon sehr bald machten sie von diesem Rechte Gebrauch . Am 19. November 1501 erteilten sie vor dem Stadtgericht zu Leipzig den Faktoren Ketzler und Hennel mitsamt Daniel Staufmehl 3, Simon Schwertzel 4, Peter Weymann und Paul Roth Prozeßvollmacht und erklärten sie für berechtigt , für die Gesellschaft Schulden einzukassieren und zu quittieren 5. Ich vermag nicht zu sagen , ob die letzteren vier Bevollmächtigten der Gesellschaft selbst Mitgesellschafter waren . In der langen Reihe der Teilhaber ’, die die Urkunde vom 26. Mai 1501 aufführt — ohne freilich vollständig zu sein 8 sind —, sie nicht genannt . Dort sind nur folgende , offenbar besonders hervorragende Namen verzeichnet: 1 Urk. 26 . Mai 1501 . Hauptstaatsarchiv Dresden , Loc . 7414 (Nr . 3), Bl . 2. 2 Nach derselben Urkunde . — Wiederum erfreute sich die Ge¬ sellschaft der tatkräftigsten Unterstützung der Krone ; vgl . Haupt¬ staatsarchiv Dresden , Cop . 106 , Bl . 35 b zum 13. September 1500. Dem Heinz Probst „ ist vom Herzog ein offene helffbrieve gegeben mit anzeeigung nachdem er von der geselschafft wegen des zcinkaufs etlich hinderstallige schuldt einmanen solle , sei m . g. h . begere , daß ime die ampt .leute u . a . seiner gnaden Untertanen auf sein oder seines gewalthabers ansuclien über bekentlieh und geständige schuldt gebürliche hülffe gäben .“ 3 Es fragt sich , ob Daniel St . aus einem Zweige der Familie Kunze stammte , deren Mitglied Kunz (kurfürstl . Münzmeister ) den Beinamen Staufmehl führte . E . Kroker, Neujahrsblätter IV, S. 69 f. 4 Bergmeister auf dem Altenberg genannt. 5 Nach Urkunde des Hauptstaatsarchivs Dresden , Loc . 7114 (Nr . 3), Bl . 3. 8 Der Namenaufführung folgt die Anmerkung : „ sambt allen der geselschafft des zcinkauffs .“ * 224 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. Hans von Minekwitz , Heinrich und Georg vom Ende (Ritter ), Andreas Krell , Dechant zu Freiberg , Dr . Cristoff Kuppener , Dr. Georg Haller , Dr . Joh . Schrenck , Dr . Erasmus Stüber , Georg von Starschedel , Hans von Gunterode 1, Steffan Alnpeck , Weygsch Seydtwitz , Erhärt Karnploff , Georg Adam , Hans und Georg Himmel¬ reich , Hans von Taubenhain 2, Ängsten und Hans von Nitzschwitz, Jacoff Wallwitz , Ewald Hessler , Simon Hilbrant , Merten Sparsbrot, Heinrich von Salhausen , Melchior Thiele 3, Michael Petzolt , Nicolaus Ulrich und der Küchenmeister von Waldenburg. Es sind klangvolle Namen der sächsischen politischen , Kultur* und Wirtschaftsgeschichte , die uns hier begegnen . Alten Adels¬ geschlechtern reihen sich Vertreter der Wissenschaft in der Teil¬ haberliste der Zinnhandelsgesellschaft an . Dazu erscheint im Vordergründe die kaufmännische Welt des Herzogtums : Stefan Alnpeck , der bedeutende Freiberger Gewerke und Bürgermeister dieser Stadt . Im Vorstande dann drei der größten Vertreter des Leipziger Handels um die Wende des 15. Jahrhunderts , Georg von Widebach , Lorenz Mordeisen und Heinrich Probst . Georg von Widebach , den bekannten Leipziger Amtmann und Finanz¬ beamten Albrechts des Beherzten , hat unlängst Alexander Puff in helles historisches Licht gerückt 4. Über die beiden anderen Führer der Zinnkaufsgesellschaft möge hier ein Wort erlaubt sein. Lorenz Mordeisen, der spätere Leipziger Ratsherr , war in der zweiten Hälfte des 15. Jahrhunderts aus der oberfränkischen Stadt Hof nach Leipzig eingewandert 6. Hier hatte er es zu einem be¬ deutenden Vermögen und zu einer geachteten Stellung gebracht. Seine Tochter Katharina war mit Hieronymus Walter jr ., dem be¬ kannten Leipziger Vertreter von Bart . Welser & Co., vermählt; sein Sohn Ulrich — Lorenz Mordeisen starb 1510 — wird uns auf den folgenden Blättern noch öfters begegnen. 1Spielt in der zentralen Finanzverwaltung der Ernestiner seit 1477 eine bedeutende Rolle . Über ihn auch Puff a . a . O., S. 24 bis 31, 60, 108. * Wohl der spätere kursächsische Landrentmeister. 3 Vielleicht verwandt mit dem bekannten Chemnitzer Bürger und Oeyerer Gewerken Nickel Thiele ; Krokcr a . a . O., S. 102. 4 A. Puff a . a. O., besonders S. 70— 86. 6 Um 1483 kommt ein Lorenz Mordeisen als Dresdener Rats¬ herr vor . Cf. O. Richter, Verfassungs - und Verwaltungsgeschichtc der Stadt Dresden . Siehe Register. 31 Dritte ? Kap . : Monopole, Kartelle u. Aktien,ses . im säclis. Zinnhandel usw. 225 Lorenz Mordeisens Andenken lebt in einer Anzahl größerer Stiftungen fort . Die deutsche Handelsgeschichte der beginnenden Neuzeit aber wird ihn immer als einen der bedeutendsten Leipziger Handelsherrn jener Zeit ansehen mü,ssen. Heinz Probst , mit vollem Namen Heinrich Wiederkehrei ' gen. Probst , stammte aus Willanzheim in Unterfranken h Unter die Leipziger Bürgerschaft trat er in den achtziger Jahren des 15. Jahr¬ hunderts . ïm Handelsleben seiner neuen Heimatstadt begegnet er uns zumeist in Gesellschaft mit seinem Landsmann Lorenz Mord¬ eisen. H . Probst hat sein im Handel und in Geldgeschäften er¬ worbenes Vermögen als Gewerke, insbesondere des Schneebergs, noch bedeutend zu vermehren gewußt . Einen außergewöhnlich großen Teil davon legte er am Abend seines I .ebens 2 in milden Stiftungen an : In Legaten für Studierende aus seiner Heimat , für das St . Georgs-Hospital , zur Verteilung an Arme, auch für kirch¬ liche Zwecke usw. Seine große Mildtätigkeit hat den Kaufherrn nicht davor bewahrt , daß ihn Luther einen Wucherer nannte und ihm einen schrecklichen Tod nachsagte 8. Bekanntlich hat auch Georg von Sachsen das Schicksal gehabt , von Luther als Begünstiger des Wuchers gebrandmarkt zu werden . Wir stehen heute solchen Verdikten sehr kritisch gegenüber . Wir wissen, daß Luther in der Beurteilung der neuen kapitalistischen Geistesrichtung noch völlig ein Kind des Mittelalters war . Eine objektive Würdigung eines erfolgreichen kaufmännischen Lebens war ihm ebenso unmöglich, wie die gerechte Wertung eines seiner Lehre abholden Fürsten , wie es Herzog Georg war . „Ein Begünstiger der Wucherer “ in unserem Sinne ist Georg nicht gewesen, wohl aber ein Förderer der säch¬ sischen Volkswirtschaft und insbesondere des Leipziger Handels wie kein Wettiner vor ihm und wie vielleicht nur Kurfürst August nach ihm . Was Sachsen diesem viel verkannten Fürsten dankt, wird erst dann ans Licht kommen , wenn ihm die Wirtschafts¬ geschichte ein biographisches Denkmal gesetzt haben wird . Dann wird sich ergeben, daß Kurfürst Augusts großes Werk der Erhebung der sächsischen Volkswirtschaft zur ersten in Deutschland nur die 1 Das folgende nach E . Kroker, Neujahrsblätter IV, S. 58 ff . Uber Mordeisen dort S. 71. 2 Wahrscheinlich starb Heinrich Probst am 17. Juli 1515. 3 Kroker a. a. O., S. 63. Strieder , Studien z. Gesch . kapitalist . Organisationsfonnen . 15 1 226 Drittes Buch: Monopole, Kartelle und Aktiengesellschaften im Mittelalter. konsequente Fortsetzung von dem war , was Georg begonnen und in die Wege geleitet hatte. * * Die Monopolisierung des Zirmhandels in der Hand unserer Gesellschaft muß für die Entwicklung des sächsischen Zinnberg¬ baues durchaus förderlich gewesen sein. Der Herzog konnte es im Jahre 1508 aussprechen , daß unter diesem System der Zinn¬ bergbau sich gedeihlich entwickelt hätte K Zweifellos war den kleinen Zinngewerken die kapitalkräftige Gesellschaft , die allezeit zur Vorlage bereit war , nützlich und notwendig gewesen. Besonders aber die Arbeiter im Zinnbergbau hatten die Wirksamkeit der Kompagnie angenehm empfunden . Periodischer Arbeitsmangel, wie er früher beim Versagen der Kapilalkraft der Zinner nicht selten war , existierte nicht mehr , seit hinter den Zinnern die geld¬ mächtige Gesellschaft des Zinnhandels stand . Vielfach waren die Zinngruben oder Zinnseifen der Zinngewerken an die Verlag spendende Gesellschaft verpfändet . Ging der Zinner bankerott, so hörte nicht , wie früher vielfach , der Betrieb teilweise oder gänz¬ lich auf und machte mehr oder weniger Arbeiter brotlos , sondern die Gesellschaft sprang in die Lücke. Auf der anderen Seite standen die kapitalkräftigen Gewerken des Zinnbergbaus einem Monopol der Gesellschaft durchaus ab¬ lehnend gegenüber . Sie zogen es vor , ihr Zinn freihändig zu ver¬ kaufen . Kaufleute , wie sie größtenteils waren , boten sich ihnen selbst genügend Absatzmöglichkeiten für ihr erbautes Metall . Ohne das Monopol hätten sie die Konjunktur nach jeder Richtung hin ausnutzen können . Und gerade beim Zinn gingen ja die Preise oft stark in die Höhe . So erschienen denn Vertreter dieser Zinngewerken vor dem Herzog und baten ihn , die bisherige Monopolisierung des Zinnkaufs aufzuheben und einem jeden Zinngewerken zu gestatten, sein Zinn nach freiem Willen zu verkaufen , an wen er wolle 2. 1 „Nachdem wir in vergangener zeit, gemeldtem unserm bergwergk zu gute ein geselschafft bestalt und verordnet , daß alles zin bar ump berait geld bezahlt ist und auch die es bedorft zimblich und leidlich weise sein vorlegt worden, daraus dann gemeldtes unsere bergwergkes gedeihen schcinbarlicli befunden . Hauptstaatsarchiv Dresden , Loc. 7414 (Nr. 2), Bl. 21. 2 Hauptstaatsarchiv Dresden, Loc. 7414 (Nr. 2), Bl. 21. Die besonderen Beschwerden der Zinner ergeben sich aus folgendem Drittes Kap.: Monopole , Kartelleu. Aktiengcs.Im säelis. Zinnhandel usw. 227 Vergeblich versuchte der Herzog die Gewerken umzustimmen. Seine Räte verhandelten in Freiberg und anderwärts mit ihren Vertretern und dén Bevollmächtigten der Gesellschaft des Zinn¬ kaufs . Die Mehrzahl der Zinngewerken behauptete , ein Monopol der Gesellschaft sei ihnen zum Schaden . So mußte sich denn Georg zur Aufhebung des Monopols entschließen . Er tat es mit dem ausdrücklichen Verbot , daß die Zinngewerken , die Verlag brauchten, diesen bei ausländischen Kapitalisten nähmen . Der Gesellschaft, teilte Herzog Georg diesen Entschluß in einem verbindlich ge¬ haltenen Schreiben mit . Er berichtete darin von seinen Be¬ mühungen , dte Ordnung des Zinnkaufs in der bisherigen Form zu erhalten und sprach die Hoffnung aus , daß die Gesellschaft bei¬ sammen bleiben und wenn nicht in der bisherigen Form , so in einer anderen dem Lande nützlich werden möchte . Tatsächlich entschloß sich die Gesellschaft , dem Herzog „zu gefallen und den bergwerken zu gut “ bis Exaudi 1504 zusammen zu bleiben . Wer wolle, könne auch fürderhin Verlag von der Kompagnie erhalten. Dafür erbat sich die Gesellschaft des Zinnhandels auch für die Zukunft die Unterstützung der Regierung bei der Eintreibung ihrer Schulden , Herzog Georg ist dieser Pflicht des Dankes gegen die Gesellschaft nachgekommen \ Schriftstück : Hauptstaatsarchiv Dresden, Loc. 4493, Bergsachen den Altenberg und Gießhübel bei. 1509—1697, Bl. 39 : „ Und als auch von den czinhern an uns gelangt ist., wi unser ordenunge des czinkaufs nicht gehalden werde, indem daß sie mit vorlegunge nicht gefurdert , auch einczeln czin nicht augenomen und beczalt, auch den ieutten , die vorstandt sein, ir czin vor der czeit der beczalunge liemen und ufhalden und vor ein gülden nicht mehr den 23 bemische groschen geben, derhalben wir mit geinelter geselschafft gehandelt und sie vormocht , wiewol sie sich derhalben merglicher beswerungen beclagen, daß sie bewilliget, in der ordenunge bis auf Laurenti schirstkunftig als sich der vortragk endet , bleiben und dem genugk thun, auch hinfurder vierundezwengisten halben bemische groschen vor ein gülden geben, auch niemaiide sein czine ehr der termins hemen ader annehemen ader das einczeln czin auch annehmen und beezalen wollen; aber das bare czin wollen sie noch Laurenti , so sich der vortragk endet , nicht im vorigen kauf behalten , sundern uf die czeit ein saczunge machen, wie sie das bare czin annehmen wollen und sal doch darumb czu geben ader an andere ende yderman frei sein zu vorkaufen . Gegeben czu Dresten am montage nach dem sontage quasimodogeniti anno domini 1500 tercio .“ 1Er gab ihr „einen gemeinen Befehlbrief, ihr in ihren aus15* * 228 Neue gestat Drittes Buch : Monopole, Kartelle und Aktiengesellschaften im Mittelalter. Versuche , eine große tete Zinnliandelskompagnie , mit Monopol zu gründen aus(1518). Hauptquelle : „Ein bedencken wie ein zinkauf auf dem Altenberg wiederum angerichtet werden könnte 1518 .“ Kleines Papierheft im Hauptstaatsarchiv Dresden in Loc . 7414 (Nr . 2), Bl . 27 ff. Das sehr interessante Schriftstück ist von mir im Anhang dieses Buches zum Abdruck gebracht. Es entzieht sich unserer Kenntnis , wie lange die im Jahre 1503 ihres Monopols beraubte Zinnhandelsgesellschaft in der nicht mit einem Monopol privilegierten Form bestand . Jedenfalls begegnen uns 1515/17 einzelne Verleger der Zinngewerken 1 neben einer Zinnkaufsgesellschaft 2. Aber eine Gesellschaft wie die im Jahre 1500 privilegierte war das nicht . Vielmehr muß Bartolome stendigen Schulden gebürliche Hilfe zu leisten “ . Hauptstaatsarchiv Dresden , Loc . 7414 (Nr . 2), Bl . 18. 1 Hauptstaatsarchiv Dresden , Loc . 7414 (Nr . 3), Bl . 209 , zum Jahre 1516 : Georg , Herzog von Sachsen , tut kund , daß Hans Friedr. und Wolf v . Salhausen Gebrüder ihm angezeigt haben , daß sie Michael Puffler und Urban Ulrich , Bürger zu Leipzig , etliche Zentner Zinn zu liefern versprochen haben . Und zwar Puffler für 4000, Urban Ulrich für 2000 fl. Wenn die Ablieferung nicht in diesem Jahre (1516 ) geschieht , sollen die genannten Leipziger Bürger die Güter der Salhausen zu Lauenstein , bei Altenberg gelegen , mit allem Zubehör als Unterpfand erhalten . Urban Ulrich war Leipziger Kauf¬ mann und Ratsherr ; auf Michael Puffler kommen wir noch häufiger zu sprechen . Auch Martin Leubel , der in der zweiten Hälfte des 15. Jahrhunderts aus Nürnberg nach Leipzig einwanderte und es hier zu einem bedeutenden Vermögen brachte , erscheint um 1510 als Zinngroßhändler . Er hatte dem Buchdrucker Nickel Keßler in Basel und dessen Sohn Bernhard für 346 fl . Zinn geliefert , worauf sie 1514 noch 142 fl . schuldig waren . Kroker, Neujahrsblätter, S. 75. — Vgl . auch für die Tätigkeit einzelner Leipziger Kaufleutc als Zinnverlegcr Hauptstaatsarchiv Dresden , Loc . 9826 . Altenberger Statuten od . Ordnung der Stadt . 1515 . Bl . 32 ff. Barthel Buchfürer hat durch seinen Faktor Altenberger Zinner verlegt . Die Verlegten blieben ihm ca . 420 fl. schuldig . Dafür wollte B . die Bergteile , die ihm „ ins bergkbuch ypotecirt , verpfandt und eingesatzt " , an sich nehmen . Aber ein anderer Verleger machte ältere Rechte geltend. 2 Hauptstaatsarchiv Dresden , Loc . 7414 (Nr . 2), Bl . 4, zum Jahre 1517 : Die Zinner auf dem Geising hatten mit dem Diener der Gesellschaft des Zinnkaufs eine Unterredung „ umbdas lehen , auch der zceyt und bar zcin zu betzallen .“ 1 Drittes Kap. : Monopole, Kartelle u. Aktienges. im sächs. Zinnliandel usw. 229 Welsers Gesellschaft und M. Puffler und Gesellschaft entweder vereint oder jede der Firmen für sich den Zinnkauf in der Hand gehabt haben x. Im ganzen hatte die Auflösung der alten Monopolgesellschaft der sächsischen Volkswirtschaft keinen Nutzen gebracht . Der Zinnbergbau lag am Ende des ersten Jahrzehntes des 16. Jahr¬ hunderts in Sachsen offenbar darnieder 2. Man dachte in Regierungskreisen und auch sonst damals eifrig darüber nach , wie der Not abgeholfen werden könne . Es war naheliegend dabei auch wieder auf die Errichtung einer großen privilegierten Zinnhandels¬ gesellschaft zu verfallen . Aus dein Jahre 1518 ist uns ein inter¬ essanter Vorschlag überliefert , wie eine neue Gesellschaft des Zinnkaufs errichtet werden könne 3. Der Vorschlag enthält nicht nur wichtiges Material zur Geschichte der Kapitalassoziations¬ formen im 16. Jahrhundert , sondern zugleich einen Beitrag zur Geschichte der internationalen Kartelle. An den Anfang seiner Denkschrift setzt unser Autor die Forde¬ rung eines sächsischen Zinngroßhandelsmonopols für die neu zu errichtende Kompanie . Alles Zinn , das im Fürstentum gewonnen wird , soll an die Gesellschaft abgeliefert werden . Diese hat es ent¬ weder bar zu bezahlen oder sie hat es sogar schon vor der Lieferung zu bezahlen . In letzterem Falle verlegt sie die betreffenden Zinner oder Zinngewerken . Den Zinngewerkeri ist der freihändige Ver¬ kauf ihres Produktes an andere Personen als an die Gesellschaft völlig untersagt. Aber auch bezüglich der Menge der Produktion soll ihrem freien wirtschaftlichen Verfügungsrecht eine Beschränkung auferlegt werden . Wenn anders bei den gegenwärtigen schlechten Zeiten und bei dem tiefen Stand des Zinnpreises dieses Metall mit Nutzen solle vertrieben werden , so müsse eine Produktionseinschränkung eintreten . Man dürfe im Jahre nicht über 2000—3000 Zentner Zinn hersteilen und müsse die ausländische Einfuhr zurückhalten . Auf welche Weise das letztere geschehen solle, werden wir noch zu be¬ sprechen haben . Zunächst möge ein anderer Einwurf behandelt werden , der in jener Zeit gegen ein Monopol wie das geforderte 1 Vgl . das Gutachten vom Jahre 1518. 2 Vgl . das Gutachten vom Jahre 1518, Einleitung. 3 Hauptstaatsarchiv Dresden, Loc. 7414 (Nr. 2), Bl. 23 ff. 1 230 Drittes Buch : Monopole, Kartelle und Aktiengesellschaften im Mittelalter. geltend gemacht werden mußte . Die moralische Erlaubtheit des vorgeschlagenen Monopols und „einer Sperrung und Steigerung der Ware “ — eine bedeutsame Frage in einem Zeitalter , wo Kirche und Staat jede Art von Monopol als wucherische Kontrakte theo¬ retisch verboten — folgert unser Autor aus der Tatsache , daß die vorgeschlagene Ordnung des Zirmkaufs um des „ Enthaltes der Armen “ willen geschehe . Dadurch , daß in dem Monopol „ vor¬ nehmlich ein gemeiner “, d . h. ein allgemeiner , und nicht eigner Nutzen gesucht würde , sei der Handel „ auf eine christ¬ liche Ursache gebaut “. Er solle und könne „ ohne alle Beschwerung des Gewissens betrieben werden “ . Und tatsächlich waren ja die Arbeiter im Zinnbergbau noch immer in eine sehr üble Lage ge¬ kommen , wenn nicht kapitalkräftige Verleger genug vorhanden waren , die die ärmeren Zinner und Zinngewerken verlegten . Schon in dem Privileg von 1498 war übrigens betont worden , daß das Geschäftsgebahren der Gesellschaft „ er bar“ sei . Wie streng es der Aussteller des Privilegs , Herzog Georg, mit dem Wucherverbot nahm , zeigt ein Brief aus dem Jahre 1499, in dem er einen un¬ genannten Rat auffordert , in der Frage der Zinnkaufsgesellschaft auch einige Leipziger Doktoren der Theologie heranzuziehen und wegen der ethischen Erlaubtheit dieser Dinge zu konsultieren \ Das war eine Art der Gewissensberuhigung , wie wir sie auch bei strenggläubigen Kaufleuten noch des 16. Jahrhunderts öfters finden 2. Die Frage , wie sind die nötigen Kapitalisten für eine Gesell¬ schaft wie die oben erwähnte aufzubringen , wird in unserem Gut¬ achten folgendermaßen beantwortet , Der Herzog möge „ als ein Beschützer des Handels “ 4000 fl. zeichnen . Eine Reihe teils ge¬ nannter , teils ungenannter reicher sächsischer Adliger insgesamt 16 000 fl. Die Leipziger Bürger , hofft unser Autor , würden den größten Teil der Anteilscheine übernehmen : Andreas Pflug , der Amtmann , etwa 2000 fl., M. Puffler und Gesellschaft 4000 fl., Heinz Scherl 2000 fl., Straub & Co. 2000 fl., Bartolome Welsers Hütten¬ gesellschaft 3 2000 fl., die Bräutigam 1000 fl., die Pucher -Gesell 1 Hauptstaatsarchiv Dresden, Loc. 7414 (Nr. 2), Bl. 15. 2 R. Ehrenberg, Das Zeitalter der Fugger. I. Bd., S. 32. 3 Bart . Welser war in den zwanziger und dreißiger Jahren hervor¬ ragendes Mitglied der Schützengesellschaft, einer Hüttengesellschaft zu Chemnitz, die besonders von der Familie Schütz gebildet war. * Drittes Kap. : Monopole, Kartelle u, Aktiengcs. im säclis. Zinnhandel usw. 231 schaft 2000 fl., Kilian Reihvieser 2000 fl., die Preußer 12000 fl., Dr . Breitenbach , der bekannte Rat Herzog Georgs und Ordinarius der juristischen Fakultät zu Leipzig, der auch sonst starken kapita¬ listischen Sinn bekundete 2, 2000 fl. Dazu einige andere Doktoren und Bürger in Summa 4000 fl. Im ganzen erwartete man aus Leipziger Kapitalistenkreisen etwa für 25 000 fl. Beteiligung an dem Unternehmen. Tn Freiberg gedachte der Verfasser der Denkschrift vom Jahre 1518 etwa 5000 fl. aufzubringen ; nämlich 2000 fl. von Merten Manwitz , 1000 fl. bei G. vom Steyg , 2000 fl. bei den Alnpecken 3. ln Chemnitz 3000 fl., und zwar 2000 fl. beim Gleitzmann und 1000 fl, bei „sunst zween bürgern “ . In Salza endlich 2000 fl. bei vier Bürgern . Man hoffte überdies , die Hauptgesellschaft des Bartolome Welser zu Augsburg zu einer Anteilszeichnuug in der Höhe von 1000—2000 fl. veranlassen zu können . Bart . Welsers Hauptgesellschaft würde dem Faktor der Zinnkaufsgesellschaft besonders in den Niederlanden nützlich sein können 4. Die Gesellschafter der neuen Zinnhandelskompagnie sollten gehalten sein, ihr eingeschossenes Kapital zu Gewinn und Verlust „dieweil der Kauf stehen würde “, beieinander liegen zu lassen. Wie man wohl aus den in Chemnitz und Salza erhofften Beteili¬ gungen schließen darf (s. o.), war der Anteilschein auf 500 fl. an¬ gesetzt. Um 1527 war Bart . Welser schon das Haupt dieser Gesellschaft. Um 1530 zeichnet sie Bart . Welser und Gesellschaft des Hütten¬ handels zu Chemnitz. Kroker, Neujahrsblätter IV, S. 102/103; Möllenberg a. a. O., S. 33. Trotzdem scheint es mir fraglich, ob mit der in unserer Denkschrift genannten Bart . Welserschen Hüttengesellschaft die alte Schützengesellschaft gemeint ist. 1 Alte, vornehme Leipziger Familie . Der bekannte , noch oft zu nennende Hieronymus Walter hatte eine Preußer zur Frau. 2 Vgl . W. Piickert, Das Münzwesen Sachsens von 1518 bis 1545. Nach handschriftlichen Quellen. I. Abt . Die Zeit von 1518—1525 umfassend . Leipzig 1862. S. 75. 3 Die drei genannten Freiberger Familien waren als Gewerken an dem Zinnbergbau führend beteiligt . Aus einer Urkunde des Hauptstaatsarchivs Dresden, Loc. 7249. Blei-, Zinn- und Kupfer¬ handel etc. 1524—32. Bl. 92. (Ohne Datum , aber sicher zu 1528 gehörig.) 4 Antwerpen, W'o die Welser eine wichtige Faktorei besaßen, war der bedeutendste Welthandelsplatz für Metalle. 2 232 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. Für den Fall , daß es wider Erwarten nicht gelingen sollte, bei den sächsischen Kapitalbesitzern das Grundkapital für die Gesellschaft des Zinnkaufs aufzubringen , schlägt unser Autor eine sonderbare , an die Blütezeit des Merkantilismus um die Wende des 17. Jahrhunderts erinnernde Lösung der Schwierigkeit vor. Man möge in dem Falle „feierndes Geld“, das bei Kirchen und Hospitälern niedergelegt sei und auch sonst auf Zinsen ausgeliehen würde , in die Gesellschaft des Zinnhandels stecken . Dort sei es vielleicht sicherer und „göttlicher “ aufgehoben und genützt als vorher . Also eine Kapitalsäkularisation großen Stiles 11 Wenn wir zunächst einen Blick auf die Personen bzw. Personen¬ gruppen werfen wollen, die der ungenannte Verfasser unserer Denk¬ schrift voraussichtlich als Anteilszeichner der neuen Zinnhandels¬ gesellschaft annahm , so dürfen wir uns nicht über den starken Prozentsatz von nichtkaufmännischen , besonders gelehrten und adligen Elementen darunter , wundern . Längst war auch in die Gelehrten - und Adelskreise der starke Erwerbstrieb eingedrungen, der die hervorstechendste soziale Signatur jener Zeit bildete . Längst begnügten sich auch diese und andere , dem Erwerbsleben ferner stehende Kreise , nicht mehr damit , ihre Kapitalien in Renten und Gülten anzulegen . Auch die vielfach geübte darlehnsweise In¬ vestierung in Handelsgesellschaften zu festem , meist fünfprozentigen, Zinssatz , erschien vielen als zu wenig gewinnbringend . Vielfach suchte vielmehr das private Kapital zu „ Gewinn und Verlust “ An¬ lage in Erwerbsgesellschaften . Wir sahen im Vorausgehenden Beispiele dafür . Aus der Geschichte , insbesondere der Saiger¬ handelsgesellschaften und der anderen Hüttengesellschaften des 16. Jahrhunderts ließen sich die Beispiele beliebig vermehren. Dabei geschah die Beteiligung privater Kreise entweder direkt oder indirekt durch Afterbeteiligung bei den sog. Prinzipalgesell¬ schaftern 1 Sollte es sich hierbei auch mit um Pupillengelder handeln , so dürfte daran erinnert werden , daß die Stadtverwaltungen öfters solche Gelder unter mißbräuchlicher Ausnutzung zu allen möglichen Zwecken verwendet haben . Vgl. Lehmann, Die geschichtliche Entwicklung des Aktienrechts , S. 13 Anm. 1. 2 Vgl . Möllenbcrga. a. O., S. 18 u. 27/28. Manche Gesell¬ schaften erlaubten den Räten usw. der Fürsten Beteiligung zu Gewinn und Verlust , um sie günstig für sich zu stimmen. 6 Drittes Kap. : Monopole, Kartelle u. Aktienges. im säch?. Zinnhandel usw. 233 Demnach durfte unser Autor mit Recht auf eine Beteiligung des sächsischen Adels und der Leipziger Gelehrtemvelt bei der neu zu gründenden Zinnhandelsgesellschaft hoffen. Von den Kaufleuten , die er als Gesellschafter in Aussicht stellte , war Michael Puffler seit längerer Zeit schon Ziunhändler und Verleger von Altenberger Zinngewerken l ; andere der vor¬ gesehenen Zeichner von Anteilscheinen der neuen Zinnhandels¬ kompagnie waren an anderen Erzhandelsgesellschaften beteiligt und galten als erfahrene Erzhändler . So Moritz Bücher , das damalige Haupt der Bücher -Gesellschaft , der in Eisleben als Hütten¬ meister emporgekommen war , dann nach Leipzig übersiedelte (um 1506) und bald darauf mit seinem Eislebener Bruder Wolfgang und mit Sigmund Fürer von Nürnberg zusammen die Saiger¬ handelsgesellschaft der Hütte Gräfenthal gründete 2. So die Bräutigam , deren Sproß Hermann schon 1464 die erste thüringische Saigerhütte mit finanziert hatte 3. So die Straub und Michael Puffler 4, auf die wir später noch des längeren zu sprechen kommen werden . Auch Heinz Scherl, der zu Anfang des 16. Jahrhunderts arm aus Nürnberg nach Leipzig eingewandert war und es hier bald zu einem stattlichen Vermögen brachte , spielte seit den zwanziger Jahren eine so bedeutende Rolle in den Mansfeldischen Saiger¬ handelsgesellschaften 5, daß wir auch schon vorher eine Tätigkeit dieses Mannes im Erzhandel annehmen müssen. So erweist sich der unbekannte Autor unserer Denkschrift von 1518 in seinen Teilhabervorschlägen als ein guter Kenner der per¬ sönlichen Verhältnisse in der sächsischen Kapitalistenwelt . Wahr¬ scheinlich gehörte er selbst zu ihrem Kreise und hatte möglicher1 Siehe oben. • Vgl. Möllen berg a. a. O., S. 32. Weitere Schicksale der Bücher-Gesellschaft, S. 120—121. Kroker, Neujahrsblätter IV, S. 12. 3 Mö 11 e n b e r g a. a. O., S. 6. 4 Die Puffler scheinen auch aus Süddeutschland, vielleicht aus Nürnberg, in Leipzig' eingewandert zu sein. Cod. dipl. Silesiae XX, S. 178/79. 6 Vgl . darüber jetzt vor allem Möllenberg a. a. O., siehe Register. Heinz Scherl war geschäftlich und freundschaftlich eng verbunden mit Jacob Welser d. Ä. zu Nürnberg. Uber Heinz Scherls .Lebensgang cfr. Kroker, Neujahrsblätter IV, S. 81 ff. 234 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. weise schon bei ihnen das Terrain für eine Gesellschaft , wie die von ihm vorgeschlagene , sondiert. Über die Ordnung der Geschäftsführung durch die Gesell¬ schaft ist in dem Vorschlag nicht viel Detail gebracht . Als Organe sind nur zwei Leiter genannt , die, aus der Zahl der Teilhaber ge¬ wählt , dem Handel vornehmlich vorzustehen haben (ihn „ allent¬ halben regieren “). Sie haben den Faktoren und Dienern der Gesell¬ schaft Anweisungen zu geben . Im Bedürfnisfalle haben sie sich von den Räten des Herzogs Rat und Hilfe zu erbitten . Zum Zwecke, dieser Auskunfts - und Hilfserteilung sollen von den herzoglichen Räten , die Gesellschafter sind , zwei nominiert und beauftragt werden . Die übrigen bürgerlichen Mitglièder der Gesellschaft sollen „ ein jeder in der Stadt , in der er gesessen oder die ihm am gelegensten ist , bei den Kannengießern und den anderen Kunden der Gesellschaft die Schulden kassieren helfen und darüber den Vorstehern Rechnung legen.“ Im übrigen gibt unser Autor einige allgemeine Vorschläge für ein Statut der Gesellschaft . Er rät , daß man eine gute Ordnung und eine kräftige Verschreibung darüber aufzeichnen müsse, „wer Rechnung halten , wer dieselbige verantworten , wann und vor wem man dies tun solle.“ Auch darüber müsse sich das Statut auslassen , „wie man es mit Austeilung des Handels , wann der Kauf ein Ende hätte , halten solle“. Die größte Schwierigkeit für eine Hebung des sächsischen Zinnhandels , wie sie der Autor unseres Gutachtens vorschlägt , be¬ stand unstreitig in der Frage , wie würde auf die ausländische Kon¬ kurrenz ein solches Vorgehen wirken . Denn das war doch selbst¬ verständlich : wenn durch Zölle u. a. den außersächsischen Zinn¬ produzenten — die schärfsten Konkurrenten waren die zu Schlacken¬ wald in Böhmen — die Einfuhr in die Albertinischen Lande ge¬ sperrt wurde , so mußten diese sich um so mächtiger auf die übrigen Absatzgebiete werfen , auf denen sich bisher schon böhmisches und sächsisches Zinn gegenübergestanden hatten . Nun war ja die böhmische Konkurrenz — durch Sperrung des Durchgangsverkehrs durch das Herzogtum und Kurfürstentum Sachsen 1 von — dem bequemsten Wege nach Norddeutschland und von dort zur See 1 Man hoffte hierzu den Kurfürsten zu bewegen. Drittes Kap.: Monopole, Kartelle u. Aktienges. im sächs. Zinnbandei usw. 235 in die Niederlande abgeschnitten . Es war auch nicht anzunehmen, daß das böhmische Zinn über Nürnberg auf Main und Rhein in den deutschen Westen bis nach Köln transportiert würde . Der lange Landtransport und die hohen Rheinzölle verteuerten es zu sehr. Aber in Schlesien und überhaupt in Osteuropa konnte doch eine preisdrückende Konkurrenz des Schlackenwalder Zinns äußerst un¬ angenehm werden. So schlägt denn unser Autor ein Produk¬ tionskartell mit den Zinngewerken zu Schlackenw a 1d v o r. Die Schlackenwalder müßten sich verpflichten , im Jahre nicht mehr als 2—3000 Ztr . zu produzieren ; die Altenberger Zinngewerken würden dementsprechend auf eine Jahresgewinnung von iy 2 bis 2000 Ztr ., die Ehrenfriedersdorf er auf eine solche von 1000 Ztr, festgelegt werden müssen . Dann könnte man wohl darauf rechnen, 2000 Ztr . Schlackenwalder Zinn in Nürnberg abzusetzen und 2000 Ztr . sächsisches in den Niederlanden , den Rest der beider¬ seitigen Produktion im Lande. Bestünde das Kartell , so wäre es nicht nötig , die sächsischen Grenzen gegen ausländisches Zinn zu sperren , damit das herzog¬ liche Geleitgeld zu schmälern und den Leipziger Messen Abbruch zu tun . Den Nutzen , den die Schlackenwalder durch das Kartell hätten , führt unser Autor darauf hin , daß es besser sei, alljährlich 3000 Ztr . Zinn mit gutem Profit zu verkaufen , als 6000 Ztr . ohne Profit , ja vielleicht mit. Verlust. Das hier vorgeschlagene böhmisch -sächsische Zinnkartell ist nicht zur Ausführung gekommen . Immerhin bietet schon das Projekt einen interessanten Beitrag zur Geschichte der Unter¬ nehmerverbände . Den Beweis aber , daß wir es in dem Projekt nicht mit dem chimärischen Hirngespinst eines die Realität der Dinge verkennenden Pläneschmieds zu tun haben , dessen Vorschläge die betreffenden Unternehmer niemals annehmen bzw. die dabei maßgebenden Regierungen nicht zulassen würden , den Beweis dafür haben wir in einem anderen Kartellprojekt jener Jahre . Im Jahre 1524 schlug der bekannte Nürnberger Kaufmann Christoph Fürer vor, die an der Silberproduktion beteiligten deutschen Fürsten sollten sich zu einem großen Silbersyndikat zusammenschließen, den Silberpreis hinauftreiben bzw. das Silber zu einem höheren 4 236 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. Nennwerte als bisher vermünzen l. Der Plan hat die Billigung der Grafen von Mansfeld, des sächsischen Kurfürsten und zunächst auch Herzog Georgs von Sachsen gefunden . Er ist dann im wesent¬ lichen daran gescheitert , daß Georg die Gefährlichkeit einer aus dem Plane resultierenden Münzverschlechterung für seine Lande einsah 2 und seine Beihilfe zurückzog. Auch die früher in einem anderen Zusammenhänge erwähnte internationale Verabredung dürfte zeigen, wie wenig den Zeit¬ genossen ein Kartell der sächsischen und böhmischen Zinnprodu¬ zenten , wie es in dem Projekt von 1518 vorgeschlagen worden war, als phantastisches , unausführbares Gebilde erscheinen konnte . Im Jahre 1520 vereinigten sich die Herzoge von Sachsen , die Schlick als Herren von Joachimstal und die Pflug als Herrschaftsbesitzer in dem böhmischen Schlackenwald 3 zu einem Abkommen , das im Grunde durchaus wirtschaftlicher Natur war und dazu dienen sollte , Angebot und Nachfrage von Arbeitskräften in einer den Kontrahenten günstigen Weise zu regeln “. Die Zinnhandelsgesellschaft von 1521 — 1527. Hauptquellen : 1. Privileg des Herzogs Georg von Sachsen für die Gesellschaft des Zinnkaufs . Datum Dresden , 20 . De¬ zember 1520 . Hauptstaatsarchiv Dresden , Loc . 9826 , Altenberger Statuten oder Ordnung der Stadt 1515 , it . Bergk -Sachen etc . Im folgenden Loc . 9826 zitiert . Das Privileg ist im Anhang ab¬ gedruckt. 2. Herzogliche Bestätigung des Dienstvertrages , durch den die Gesellschaft des Zinnkaufs den Hans Alnpeck zum Faktor der Ge¬ sellschaft annimmt . Datiert : 4. März 1521 . Hauptstaatsarchiv Dresden , Loc . 4500 . Bergwerkssachen zu Freiberg bel . Vol . I, 1453 — 1543 , Bl . 25—26 . Im Anhang abgedruckt. 1 W. L o t z , Die drei Flugschriften über den Münzstreit der sächsischen Albertiner und Ernestiner um 1530 , S. V . Nach W . Pückert, Das Münzwesen Sachsens 1518 — 1545 , S. 65 ff ., besonders S. 73, 75, 100 ff. 2 Beraten durch die Leipziger Kaufleute Urban Ulrich , Andreas Matstedt , Faktor der Fugger , Hieronymus Walter , Kunz Krott, Mich . Puffler und Wolf Wiedemann . Ihre Gutachten besprochen bei Pückert a . a . O., S. 111 ff. 3 Alle drei Bergherren hatten bedeutende eigene Bergwerke und Bergwerksanteile. 4 Siehe oben S. 44. Drittes Kap . : Monopole. Kartelle u. Aktienges. im sächs. Zinnhandel usw. 237 Die Vorschläge von 1518, so gesund sie in mancher Beziehung waren , sind nicht zur Ausführung gelangt . Erst im Verlauf des Jahres 1520 scheinen die . Verhandlungen über die Errichtung einer neuen Zinnhandelsgescllschaft — auf wesentlich anderer Grundlage freilich , wie das Projekt von 1518 vorschlug — zum Abschluß ge¬ kommen zu sein. An der Spitze des Unternehmens stand Hierony¬ mus Walter , der Leipziger Faktor der Bartolome Welserschen Ge¬ sellschaft , Michael Puffler und Ulrich Mordeisen 1. Also drei Leip¬ ziger Kaufleute von sehr gutem Ruf und bedeutender Kapitalkraft. Michael Puffler überdies seit langem im Zinnverlagsgeschäft und Zinnhandel erfahren 2. Zunächst hatte sich Andreas Madtstedt „ mit seinen Anhängern“ für den Zinnkauf stark interessiert . Er war auch einem Abschluß mit den Zinngewerken auf einem Verhandlungstage in Leipzig (29. Oktober 1520) nahe gewesen 3. Es entzieht sich meiner Be¬ urteilung , was Madtstedt im letzten Augenblick bewogen hat , von dem Abschluß des Zinnkaufs abzusehen . Bekanntlich war Andreas Madtstedt Faktor der Fugger in Leipzig 4. Wir haben somit das interessante Bild vor uns, daß schließlich die Welser — wenigstens mitführend — ein Unternehmen in die Hand nahmen , das vorher die Fugger in ihre Hände zu bekommen beabsichtigten. Die neue Gesellschaft des Zinnkaufs wurde für die Zeit von Oculi 1521 bis Oculi 1524 vom Staate privilegiert . Ihr Wesen — wenigstens nach der Seite ihres öffentlich -rechtlichen Charakters hin — ist uns aus dem Privileg Herzog Georgs vom 20. Dezember 1520 in seinen Grundzügen erkennbar . Das wichtigste war, daß die Gesellschaft wieder, wie ihre älteren Schwestern am Anfang des Jahrhunderts , für den sächsischen Zinngroßhandel ein Monop o 1 besaß . So war es mit Hilfe der herzoglichen Räte zwischen den sächsischen Zinngewerken und den Kaufleuten , die die Gesell¬ schaft bildeten , für drei Jahre verabredet worden . Die Zinn1 Hauptstaatsarchiv Dresden , Copiai 127 , Bl . 126 b. 2 Uber Hieronymus Walter vgl . K r o k e r , Neujahrsblätter IV, (1908 ), S. 93 ff. Ulrich Mordeisen wird der Sohn des obengenannten frühverstorbenen Lorenz Mordeisen gewesen sein . Krokera . a . O., S. 71. 3 Vgl. dafür die Urkunde im Ernestin . Gesamtarchiv zu Weimar, Reg . T , Bl . 273/74 , Nrn . 3— 6. 1 Als solcher 1519 noch genannt und wohl auch später. M. Jansen, Jakob Fugger der Reiche , S. 143. 238 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. gewerken des Altenbergs waren gehalten , all ihr erbautes Zinn an die dortige herzogliche Bergwage abzuliefern , wo es die Gesell¬ schaft gegen Barzahlung zum Preise von 11 fl. 1übernahm 2. Die Wage auf dem Altenberge sollte mit zwei Schlössern verwahrt und verschlossen werden . Zu dem einen Schloß erhielt der herzogliche Zehntner , zu dem anderen der Altenberger Faktor der Zinnkaufs¬ gesellschaft den Schlüssel. Die Schmelzer und die Hüttenmeister auf dem Altenberge waren der Gesellschaft gegenüber durch Eid gebunden , dafür zu sorgen, daß alles Zinn an die Wage kam und daß nicht schlechtes Zinn dem guten beigemischt wurde. Das Zinn , das zu Ehrenfriedersdorf usw. gewonnen wurde, brauchte nicht auf die Altenberger Wage gebracht zu werden. Die Gesellschaft nahm es an Ort und Stelle gegen Barzahlung und zu demselben Preise wie das Altenberger ab . Die Untertanen des Herzogs endlich , die auf dem Mückenberg Zinn erbauten , hatten sich bereit erklärt , unter denselben Bedingungen wie die Altenberger Zinngewerken , ihr Zinnprodukt in die Altenberger Wage zu liefern. Dagegen durften die Mückenberger , Graupenschen und andere Ge¬ werken , die nicht Untertanen Georgs von Sachsen waren , ihre Zinn¬ produkte außerhalb des Herzogtums verkaufen . Zogen sie den Verkauf im Lande vor , so unterstanden sie denselben Bedingungen wie die herzoglichen Untertanen , d. h . sie mußten das Zinn gegen fest bestimmten Preis (s. o.) der Gesellschaft des Zinnkaufs auf die Altenberger Wage liefern. Bis Oculi 1521 mußten alle etwa bestehenden anderweitigen Zinnlieferungsverträge abgewickelt sein. Wer nach diesem Termin — von den Untertanen des Herzogs — Zinn an jemanden anderen 1 D. h . 11 fl. in Münze , halb sächsisch (je 21 Zinsgroschen für 1 fl .) und halb böhmisch (je 24 böhmische Groschen für 1 fl .). An den Orten , wo man kein böhmisches Geld nahm , und die Gesellschaft mit „ eitel “ sächsischem Gelde zahlen mußte , brauchte sie nur 11 fl. minus 1 Ort pro Altenberger Bergzentner zu geben. 2 Die Zinner von Thum , Ehrenfriedersdorf und Geyer forderten einen höheren Preis , weil ihr Produkt besser sei als das altenbergische. Dieses Verlangen scheint , trotz der Weigerung der Gesellschaft ihm nachzukommen , nicht unbillig gewesen zu sein , denn auch die vom Herzog bestellten Vermittler schlugen vor , es sollte für jenes Zinn Y2fl . pro Zentner mehr gezahlt werden. J. Falke, der Bergstadt Geyer in Mitteilungen vereins , Heft 15, S. 108. Geschichte des Kgl . Sachs . Altertums¬ 2 Drittes Kap. : Monopole, Kartelle u. Akticnges. im sächs. Zinnhandel usw. 239 als an die Gesellschaft des Zinnkaufs lieferte , ging dieser seiner Ware verlustig . Sie fiel zur Hälfte der herzoglichen Kammer , zur Hälfte der Kirche in Altenberg zu. War eine der Parteien (die Zinngewerken oder die Zinnkäufer, d. h. die Gesellschaft des Zinnkaufs ) nicht geneigt , nach Ablauf dreier Jahre den Kontrakt zu erneuern , so mußte von der be¬ treffenden Partei ein halbes Jahr zuvor schriftlich gekündigt werden. Die Zinnkaufgeschäfte der Gesellschaft auf dem Altenberg usw. besorgte Hans Alnpeck jr . aus Freiberg , der seinen Wohnsitz in Altenberg nehmen mußte und 200 fl. Jahresgehalt erhielt . Einzel¬ heiten seiner Aufgabe sind aus dem Mietskontrakte zu ersehen , den die Zinnkaufsgesellschaft mit ihm abschloß 1. Es ist aus dem spärlichen Archivmaterial , das uns vorliegt, nicht genau zu ermitteln , ob die 1520 zwischen den sächsischen Zinngewerken und der Gesellschaft des Zinnkaufs vereinbarte Kündigungsfrist genau eingehalten worden ist ; jedenfalls hatten schon vor Oculi 1524 die Zinner zu Geyer, Ehrenfriedersdorf und Thum den herzoglichen Räten erklärt , daß sie mit der Zinngesell¬ schaft nicht über einen neuen Kontrakt einig geworden wären. Darauf erlaubte Herzog Georg den Zinnern der genannten Orte, ihr Zinn nach freiem Ermessen zu verkaufen 3. Es scheint , als hätten nun zunächst die Orte Geyer, Thum und Ehrenfrieders¬ dorf selbst den Verlag ihrer ärmeren Zinner übernommen . Dabei konnten sie freilich ■ — ohne Erfahrung wie sie im Absatz des so erworbenen Zinnes waren und auch mangels genügenden Verlags¬ kapitals — die Leipziger Kapitalisten nicht entbehren . Schon am 1 Ich habe das für die Geschichte der Faktoren (Handlungs¬ diener) interessante Dokument im Anhang abgedruckt. 2 Herzog Georg schreibt an die Richter , Schöffen und Zinner zu Geyer, Ehrenfriedersdorf und Thum : „ Lieben getreuen ! Als ir uns ytzt auf das schreiben, so nechst unsere abwesens unsere verordente rete zu I.eiptzk auf ansuchen der geselschaffter des zinkaufs an euch gethan , antwort gegeben, haben wir vernomen . Nachdem wir denn daraus befinden, das ir euch mit inen nit verainen und ver¬ gleichen könnt , deshalb ir auch beschwerdt seit, den neuen vertrage einzegehn, so ist uns nit entgegen, daß ir mit euerm zin freisteet und damit nach euerm gefallen gebäret und handelt . Wolten wir euch darnach ze richten nit verhalten . Datum Sandt Annaperg dornstags nach Oculi anno etc. 24 Kgl. Hauptstaatsarchiv zu Dresden, Copiai 127, Bl. 156 a. 240 Drittes Buch: Monopole . Kartelle und Aktiengesellschaften im Mittelalter. 24. April 1525 wurde ein “Vergleich wegen des Zinnverlags zwischen den Abgeordneten von Geyer, Ehrenfriedersdorf und Tlmin einer¬ seits und den Leipziger Zirmkaufleuten Michael Puffler und Ulrich Mordeisen andererseits auf drei Jahre abgeschlossen . Die genannten Kapitalisten versprachen darin , jeden der drei Orte zunächst mit je 200 fl. rheinisch pro Jahr zu verlegen . Die Verlagssumme sollte „mit fürstlicher Gunst “ genügend versichert und nach drei Jahren ohne Säumnis zurückgezahlt werden . Wenn eine der Städte die Vorlegung nicht brauchte und eine andere wollte dieselbe für sich nehmen , so hatte auch die nehmende Stadt den Verlag sicher¬ zustellen . Alles Zinn , das gewonnen wurde , war an die genannten Zinnkäufer zum Preise von 11y2 fl . für blankes Zinn und von liy 4 fl . für weißes Zinn abzugeben . Es wurde bar , halb in säch¬ sischer , halb in böhmischer Münze bezahlt . Wenn ein Zinner oder Gewerke bis zu einem Zentner Zinn für seine „Hausingesessen“ bedürfe , so solle ihm die Abgabe dieses Quantums erlaubt sein. Doch müßte solches den Faktoren der Zinnkäufer angezeigt werden. Der hier in seinen wichtigsten Bestimmungen wiedergegebene Vertrag wurde „ mit Gunst und Vermittlung “ Herzog Heinrichs, mit Vorbehalt der Genehmigung von Herzog Georg abgeschlossen 1. Die Genehmigung scheint erteilt worden zu sein. Anders als in Geyer, Ehrenfriedersdorf und Thum gestaltete sich das Verhältnis zwischen den Zinnern und der Zinnkaufs¬ gesellschaft in Altenberg . Noch vor Ablauf des Vertrags , der Oculi 1524 endete , richtete Herzog Georg zwischen Hieronymus Walter 2, Michael Puffler , Ulrich Mordeisen 3 samt der Gesellschaft des Zinnkäufs einerseits und den Zinnern und Gewerken von Altenberg 1 Hauptstaatsarchiv Dresden, Loc. 4500. Das Bergwerk zu Ehrenfriedersdorf bei. 1377—1538. BL 13/14. Vgl. J . Falke, Mitteilungen des Kgl. Sächs. Altertumsvereins , Heft 15, S. 108. Bei mir im Anhang abgedruckt . — Herzog Heinrich , Georgs schwacher Bruder , hatte 1505 zur Entschädigung für das unhaltbare , 1515 an die Habsburger zurückgegebene Friesland die Ämter Freiberg und Wolkenstein erhalten . O. Kaemmel, Sächsische Geschichte. Leipzig (Göschen) 1905. S. 71. 2 HieronymusWalter d. J . war seit 1528 oder 1529 mit Kath. Mordeisen, der Tochter Lorenz Mordeisens, verheiratet. 3 Ulrich Mordeisen hatte eine Tochter (Margarete) Michael Pufflers zur Frau . Kroker, Neujahrsblätter IV, S. 103. Drittes Kap . : Monopole, Kartelle u. Aktienges. im sächs. Ziimhandel usw. 241 andererseits einen neuen Vertrag auf 1. In diesem Vertrag war in bedeutsamer , für die Entwicklung charakteristischer Weise den Altenberger Zinnern und Gewerken ein ziemlich großes Freiverkaufs¬ kontingent zugestanden . Sie durften ein Drittel ihrer Zinnausbeute frei absetzen an wen und wohin sie wollten . Nur die übrigen zwei Drittel hatten sie an die Kompagnie zu verkaufen . Lag hierin schon eine starke Benachteiligung der Gesellschaft des Zinnhandels, so wurden dieser nun auch nocli Beschränkungen einem Teil der Konsumenten gegenüber zugernutet . Die Gesellschaft , heißt es in einer Ordnung und Satzung des Herzogs Georg vom 11. Februar 1524, solle einen „gemeinen , freien zinnkauf bestellen und halten, also daß sie den kandelgießern 2 oder anderen , so das zinn wollen verarbeiten lassen , bei einzelnen zentner zinn kaufweis zukommen lassen“ 3. Und zwar zu festgesetztem Preise . In Leipzig den Zentner für l2j/ 4 fl . und in Altenberg , wo keine Transportspesen zuzurechnen waren , für 11/4 fl . netto Kasse . Dafür sollten die Kannegießer von niemand anderem als von der Gesellschaft das Zinn kaufen . Wenn , was wir wohl an nehmen dürfen , die Gesell¬ schaft im Jahre 1524 noch 11 fl. pro Zentner an die Zinnproduzenten zahlen mußte — wie es im Jahre 1520 vereinbart war —, so wäre 4 fl. pro Zentner ein sehr bescheidener Gewinn für die Gesellschaft gewesen. Auch 1% fl. Bruttoverdienst pro Zentner in Leipzig war wenig . Sicherlich hat die Gesellschaft weitaus das meiste Zinn in außersächsischen Landen abgesetzt , wo ihrem Profit keine obrigkeit¬ lichen Schranken gesetzt waren . Die Beschränkungen , die dem freien Walten der Zinnmonopolisten im „ Inland “ gesetzt waren, sind ein interessanter Beleg für die Handwerkerpolitik des be¬ ginnenden Merkantilismus . Um so mehr , als wir auch sonst dieselbe Richtung eines Eingreifens des Staates für seine Gewerbetätigen 1 Hauptstaatsarchiv Dresden , Loc . 7414 (Nr . 2), Bl . 32 f. Der Vertrag selbst ist -— soviel ich sehe — nicht erhalten . Wir wissen nur davon durch die gleich zu besprechende herzogliche Schutz¬ verordnung , die die sächsischen Zinngießer vor einer Überteuerung durch die Gesellschaft des Zinnhandels schützen sollte. 2 Kannegießern. 3 Eine besondere Rücksichtnahme auf die Verbraucher im eigenen Land bedeutete wohl auch später die Maßnahme des Kur¬ fürsten August , die J . Falke, Die Geschichte des Kurfürsten August von Sachsen in volkswirtschaftlicher Beziehung , S. 294, erwähnt. Strieder , Studien z. Gesoh . kupitaliat . OrganiBationsformen . 16 1 242 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. und eine Abwälzung der „Monopolpreise“ auf das „ Ausland “ be¬ obachten werden. Der Vertrag zwischen den Altenberger Zinnproduzenten und der Gesellschaft des Zinnhandels , den Herzog Georg im Frühjahr 1524 aufgerichtet hatte , sollte sich keiner langen Lebensdauer er¬ freuen . Schon im Herbst dieses Jahres sendeten die Zinngewerken des Altenbergs zwei Abgesandte an Georg von Sachsen mit der Erklärung , sie könnten das Zinn nicht länger zu dem verabredeten Preise an die Gesellschaft des Zinnhandels abgeben . Sie erhielten daraufhin vom Herzog die Erlaubnis , mit der Gesellschaft des Zinnhandels , oder wenn diese nicht wolle, mit anderen Zinnkäufern sich zu einem neuen Zinnkaufskontrakt zu einigen. Natürlich be¬ dürfe der abgeschlossene Vertrag der Bestätigung der Regierung Als kluge Geschäftsleute müssen sich die Zinnkaufsgesellschafter zunächst den Anschein gegeben haben , als liege ihnen selbst nicht allzuviel an der Erneuerung des Kontraktes . Dann freilich be¬ trieben sie diese Erneuerung wieder eifrig. Sie baten den Herzog um eine Tagsatzung mit den Zinngewerken ; nur so sei ein Zustande¬ kommen des Kontraktes noch möglich. Herzog Georg willfahrte ihrem Wunsche und setzte für den 27. Januar 1525 eine Zusammen¬ kunft der Zinngew'erken und der Herren der Zinnhandelsgesell¬ schaft fest 2. Es entzieht sich meiner Kenntnis , welches der Erfolg dieser Bemühungen war. Das große Leipziger Monopolprojekt von 1527. Hauptqucllc: Anonyme Denkschrift vom Jahre 1527. Hauptstaatsarchiv Dresden , Loc . 10 532 , Leipziger Händel etc. 1525— 51, Bl . 131 ff . Abgedruckt im Anhang dieses Buches. Der mächtige , unbestrittene Aufschwung , den Leipzig in dem ersten Dritte ! des 16. Jahrhunderts erlebte 3 und der nur im 19. Jahr¬ hundert eine Parallele hat , kann vielleicht durch nichts besser 1 Hauptstaatsarchiv Dresden , Cop . 127 , Bl . 124 b, 125 a. Schreiben des Herzogs vom 25. Oktober 1524 datiert. 2 Hauptstaatsarchiv Dresden , Cop . 127, Bl . 126 b . Schreiben des Herzogs vom 16. November 1524 an Hieronymus Walter , Michael Puffler und Ulrich Mordeisen. 3 Zeitgenössische Stimmen über diesen Aufschwung bei E . K r o k e r , Leipzig und die säebs . Bergwerke , Schriften d. Vereins f. die Geschichte Leipzigs , IX . Bd ., S. 5 des Separatabdruckes. 2 Drittes Kap. : Monopole, Kartelle u. Aktienges. im sachs. Ziimhandcl usw. 243 illustriert werden , als durch die großartigen Monopol- und Kartell- Projekte im Handel mit Bergbauprodukten, die in den zwanziger Jahren aus der Mitte der Leipziger Kaufmannschaft auftauchten. Man kennt die Bemühungen 1 der Leipziger , die Ausbeute der reichen Kuttenberger Kupfergruben monopolistisch zu beherrschen, die bis dahin den Nürnberger Kapitalisten zugefallen war . Noch großartiger zeigt sich die wirtschaftliche Energie der Leipziger Kaufmannswelt . in einem Projekt aus dem Jahre 1527, auf das hier aufmerksam gemacht werden soll. Das Projekt war ein Gegen¬ schlag gegen die Bemühungen der Nürnberger Kaufleute und der mit ihnen verbündeten Grafen von Mansfeld , die niedersächsischen Kaufleute , also die Hansen , aus dem Zwischenhandel mit Mansfeldischem Kupfer zu drängen . Im August 1527 hatte Jakob Welser d. À. von Nürnberg an den Grafen Albrecht von Mansfeld berichtet , daß das mansfeldische Kupfer auf dem Nürnberger Pfingstmarkt von 30 Schock Groschen auf 28 gefallen sei, „ darumb es die Sachsen “, d. h, die hansischen Kaufleute , „geben haben “ -. Daraufhin verhandelten die Grafen Albrecht und Gebhard von Mansfeld mit den auf der Michaelismesse zu Leipzig versammelten Kapitalisten , die den mansfeldischen Saigerhandelsgesellschaften angehörten , über den Abschluß einer Konvention , laut der sie sich alle verpflichten sollten , „ den Sachsen hinfür kein kupfer zu ver¬ kaufen “ 3. Die meisten Leipziger Kapitalisten , die an mansfeldischen Saigerhandelsgesellschaften beteiligt waren — unsere Denkschrift führt Kuntz Keller, Heinrich Scherl und Moritz Bücher nament¬ lich auf —, traten der Konvention bei. Eis sind die von Nürnberg 1 Ygl. Kroker, Neujahrsblätter IY , S. 104 ff. Aus der Krokerschen Darstellung erhellt dasselbe , was sich auch aus den hier folgenden Ausführungen ergibt : ein heftiger Kampf Nürnbergs gegen das aufkonunende Leipziger Wirtschaftsleben und gegen das Leipziger Großkapital , das an allen Enden dem nürnbergischen, besonders im Handel mit Bci'gwerksproduklen , die gefährlichste Konkurrenz machte. 2 Für das folgende Möllcnbcrg a . a . O., S. 5t f., der aller¬ dings fälschlich unter „ Sachsen “ die Leipziger versteht . Daß hier die niedersächsischen Kaiifleute , also die Hansen , gemeint sind , er¬ gibt sich unzweifelhaft aus unserer Denkschrift von 1527 . Vgl . dort das Verzeichnis der Waren , die die „ Sachsen “ auf die Leipziger Märkte bringen. 3 Nach der oben genannten Denkschrift. IG 244 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. abhängigen und geschäftlich eng besonders mit Jakob Welser d. Ä. und anderen Nürnbergern verbundenen Kaufleute . Nur Wolfgang Wiedemann und Lucas Straub weigerten sich energisch , in die Vereinigung einzutreten . Sie machten mit Recht darauf auf¬ merksam , wie stark der Leipziger Markt durch das Fernbleiben der Niedersachsen leiden würde . Besonders auf diese Vorstellungen hin versuchten der Rat der Stadt Leipzig und Herzog Georg die Konvention zu sprengen . Sie verhandelten eingehend mit den Leipziger Kapitalisten , die der Konvention beigetreten waren , über ihren Austritt . Die Bemühungen waren vergeblich . Der geschäft¬ liche Vorteil wies die Scherl, die Bücher und Konsorten zu stark auf die Seite der Nürnberger und besonders der Mansfelder Grafen, als daß sie zum Austritt aus der Konvention hätten vermocht werden können. In diese Situation fällt das obengenannte Projekt . Es sollte den unabhängig von Nürnberg und den Mansfeldern gebliebenen Leipziger Kaufleuten eine Monopolstellung in dem von Mansfeld nicht beherrschten mitteleuropäischen Metallhandel sichern . Die Denkschrift , in der dieser Plan entwickelt ist , schlug dem Herzog von Sachsen vor , seine Hilfe dazu zu bieten , daß 1. die Blei¬ produktion zu Goslar, daß 2. die böhmische Kupfer - und Silber¬ produktion , daß 3. und 4. auch die gesamte böhmische und säch¬ sische Zinnausbeute in „ eine Hand und an die Einwohner der Stadt Leipzig gebracht würden “ 1. Der Vorschlag bedeutete nichts weniger und nichts mehr als eine großartige Monopolisierung des bedeutendsten Teiles des mitteleuropäischen Metallhandels in der Hand weniger Leipziger Kaufleute und Kapitalisten . Ich stehe nicht an, in dem Vorschlag eines der interessantesten Monopol¬ projekte des 16. Jahrhunderts zu sehen. Mit dem Versuch , die gesamte bedeutende Bleiausbeute der Goslarer Gruben in die Hände zu bekommen , sollte nach dem Autor unserer Denkschrift die Ausführung seines Planes beginnen. Hätte man erst einmal die Goslarer Bleiproduktion in der Hand, dann seien die Böhmen leicht zum Anschluß an das Projekt zu be¬ wegen . Zur Gewinnung von Silber aus den stark silberhaltigen Kupfererzen Böhmens konnte man dort das Goslarer Blei nur 1 An einer andern Stelle der Denkschrift heißt es : „ Diese händel sollen alle zusammen gebracht werden .“ * 8 Drittes Kap . : Monopole, Kartelle u. Aktienges. im säclis. Zinnliandel usw. 245 schwer entbehren 1. Wer also den Goslarer Bleihandel beherrschte, gebot auch bis zu einem gewissen Grade zugleich über den böhmi¬ schen Kupfer - und Silberhandel 2. Der Vorschlag mit der Monopolisierung des Goslarer Blei¬ handels die Ausführung des obengenannten großen Projektes zu eginnen , führt uns in dieselbe Interessentengruppe Leipziger Kapitalisten , die wir bereits als die schärfsten Gegner der Mansfelder Konvention kennen lernten . In der Zeit , als das Projekt an Herzog Georg gebracht wurde , war der Leipziger Bürgermeister Wolfgang Wiedemann und seine Gesellschaft sowie Lucas Straub seit einiger Zeit schon mit dem Goslarer Rat 3 wegen eines mono¬ polistischen Blei- und Vitriolabnahmekontraktes in Unterhandlung 4. Man war einem Abschluß tiahe gewesen 6 und hatte sich schließ1 Die Beschreibung des umständlichen Saigerprozesses des silberhaltigen Kupfererzes , zu dem man viel Blei brauchte , siehe bei Möllenberg a . a. O., S. 5. Aber auch zu der Silbergewinnung aus den Silbererzen brauchte man viel Blei. 2 Schon 1492 berichtete der oberste Burggraf zu Prag und Haupt¬ mann des Königreiches Böhmen an Herzog Georg von Sachsen: die Amtleute von Kuttenberg hätten gemeldet , daß Bleimangel in Böhmen herrsche und daß es den Böhmen nicht gestattet werde , in Sachsen Blei zu „ kaufen und durchzubringen “ . Das sei früher nicht geschehen 'und man bäte im Namen des böhmischen Königs das Ausfuhr - und Durchfuhrverbot aufzuheben . Nach einer Urkunde im Hauptstaatsarchiv Dresden , Loc . 7250 . Schreiben Herzog Georgs zu Sachsen , den Bleyhandel in St . Joachimstal und Goslar betr. Anno 1533 . Bl . 3. 3 Im Verlauf des 14. und 15. Jahrhunderts hatte es der Rat verstanden , die Verpflichtung durchzusetzen , daß alles aus den Rammeisberger Bergwerken gewonnene Erz nach seiner Verhüttung an ihn verkauft werden mußte . Im 16 . Jahrhundert wußten dann die Herzoge von Braunschweig dieses Regal an sich zu bringen. 4 Schon 1524— 27 hatten die zwei Genannten den Bleiabnahme¬ kontrakt , den sog . „Bleikauf “ , mit Goslar gehabt , d . h . sie schossen dem Rat der Stadt größere Geldsummen vor und übernahmen dafür die gesamte Ausbeute zu festbestimmtem Preise. 8 Das folgende nach Hauptstaatsarchiv Dresden , Loc . 10532. Leipziger Händel 1525— 31. Bl . 127 ff . Besonders aus der Urkunde, in der Wiedemann und Straub dem Herzog die fehlgeschlagcnen Verhandlungen mitteilen und ihn bitten , er möge den Rat von Goslar zum Abschluß des schon zugesagten Vertrags zwingen . Sie hätten 15 000 fl . längere Zeit für das Objekt bereitgehalten und starken Zinsverlust erlitten . — In der obengenannten Denkschrift ist auf 246 Drittes Bucli: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. lieh auf folgende Bedingungen geeinigt . Die Leipziger Kapitalisten sollten eine Vorauszahlung von 6000 fl. leisten und dafür und für weitere Vorschüsse (Verlag) die Bleiproduktion des Rammeis¬ berges zum Preise von 33 gr . 4 Pfg. pro Zentner drei Jahre lang übernehmen . Nur über die Lieferung des Vitriols war man noch nicht einig gewesen, als der Rat von Goslar die Unterhandlungen abbrach . Er teilte der Gesellschaft mit , daß augenblicklich viel zu wenig Blei gewonnen würde , um einen Bleikontrakt ab¬ zuschließen . Wenn das sich ändere , sollte die Gesellschaft an erster Stelle Berücksichtigung finden. In unserer Denkschrift wurde nun die sächsische Regierung aufgefordert , dafür zu sorgen, daß die Goslarer den Kontrakt ab¬ schlössen. Wer anders konnte ein Jnteresse daran haben , diese Aufforderung an die sächsische Regierung zu richten , als die Kauf¬ leute Wiedemann und Straub , die damit als die geistigen Urheber des Projektes von 1527 deutlich gekennzeichnet sind. Freilich ging das Projekt , wie angedeutet , noch bedeutend weiter als auf die Beherrschung der Goslarer Bleiproduktion . Der sächsischen Regierung wurde darin gleichzeitig nahegelegt , darauf hinzuwirken , daß es zwischen den Schlackenwalder Zinnproduzenten 1und den Leipziger Kaufleuten zu einem Zinnabnahme¬ kontrakt käme . Nur so könnten die Kontraktverhandlungen zwischen den sächsischen Zinngewerken und den Leipziger Kauf¬ leuten , die augenblicklich sclrwebten und die auf ein sächsisches Zinnhandelsmonopol hinausliefen , zu gedeihlichem Ende geführt werden. Was endlich die. böhmische Kupferproduktion angeht , so war der Verfasser unserer Denkschrift davon unterrichtet , daß Hierony¬ mus Walter , der Leipziger Vertreter der Augsburger Welser — die Augsburger hielten gegen die Nürnberger zu Leipzig —, wegen der Übernahme der gesamten Kuttenberger Kupferproduktion in Unter¬ handlung stand . Im Falle Walter den Vertrag allein nicht zum Abschluß brächte , möge der Herzog sofort Hans von Schönburg zu König Ferdinand senden 2. Soweit die Denkschrift von 1527. die Supplikation an den Herzog hingewiesen. Der Autor ist also wohl in den Kreisen um Wiedemann, Straub & Co. zu suchen. 1 Schlackenwald war der Hauptgewinnungsort des böhmischen Zinns. 2 Zum Verständnis dieses Teiles der Denkschrift vgl. Krokcr, Neujahrsblätter IV, S. 107. 1 Drittes Kap . : Monopole , Kartelle u . Aktienges . im sächs . Zinnhandel usw . 247 Die sächsische Regierung wies das Leipziger Projekt durchaus nicht von sich. Im Gegenteil , sie bemühte sich redlich , die darin geforderten Abschlüsse mit den fremden ßergwerksmächten zu¬ stande zu bringen . An den Goslarer Rat gingen mehrfach Schreiben ab, in denen die Stadt dringend gemahnt wurde , den mit Lucas. Straub und Wolfgang Wiedemann verabredeten Kontrakt durch¬ zuführen h Desgleichen an den Herzog von Braunschweig 2. Ebenso machte Herzog Georg die eifrigsten Anstrengungen , um den Kutten¬ berger Kupferkauf in die Hände des Hieronymus Walter und der Schützgesellschaft zu Chemnitz zu bringen 3. Besonders aber be¬ mühte sich Herzog Georg von Sachsen , einen monopolistischen Abnahmekontrakt der Leipziger Erzbändler mit den Schlackenwaldcr Zinnproduzenten zustande zu bringen . Seine Räte , Rudolf von Bünau und Anton von Kospott (Amtmann in St . Annaberg ), traten in lange Unterhandlungen mit Hans Pflug , dem Herrn von 1 Hauptstaatsareliiv Dresden , Loc . 7249 . Blei -, Zinn - und Kupfcrhandel und -kauf . Goslarisches Blei anno 1524—32. Bl . 8; Loc . 7250 . Den Bleihandel belangend etc . Anno 1534—42 . BI. 35. Auch daselbst Bl . 79 eine Antwort des Rates folgenden Inhaltes auf die Mahnungen Herzog Georgs (1528 , 5. Januar ) : Mit Wolfg . Wiede¬ mann und Lucas Straub hätten sie den Blei - und Vitriolkauf noch nicht fest abgeschlossen gehabt ; sie wollten ihn den eigenen Bürgern Vorbehalten. 2 Weil die Herzoge von Braunschweig dem Rate von Goslar das Bezugsrecht auf das Rammeisberger Blei streitig machten , so hatte Wolfg . Wiedemann und Lucas Straub & Co . auch mit Herzog Heinrich dem Jüngeren von Braunschweig einen Bieiabnahmekontrakt abgeschlossen . Als dann Herzog Heinrich Mitte 1527 den Leipziger Kaufleuten ihren Vertrag zurückgab , suchte Herzog Georg auch hier das Bleibezugsmonopol seinen Leipziger Untertanen zu retten, wenigstens prinzipiell und für die Zukunft . Denn zunächst war die Bleiproduktion in den vielfach von Pierzog Heinrich verwüsteten Goslarer Hütten zu unbedeutend , um sofortige größere Bleilieierungen zu ermöglichen . Erst 1533 ist es zu festen Bleikontraktcn Wolfg . Wiedemanns , Lucas Straubs & Co. mit dem Goslarer Rat und dem Herzog von Braunschweig gekommen . Vgl . für das Be¬ richtete Hauptstaatsarchiv Dresden , Loc . 7250 . Bleihandel Anno 1543—46 . Bl , 16 ff. ; Loc . 7250 . Schreiben Herzog Georgs zu Sachsen , den Bleihandel in St . Joachhnstai und Goslar betr ., Anno 1533 . Bl . 81, 85 ff . und Loc . 7250 . Den Bleihandel betr . etc ., Anno 1534— 42 . Bl . 34. 3 Kroker, Neujahrsblätter IV , S. 107. 1 248 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. Schlackenwald , ein ]. Hans Pflug versprach auch , daß die Leipziger Händler den Schlackenwalder Zinnkauf „vor anderen “ erhalten sollten , d. h. vor den Nürnbergern , deren Abnahmekontrakt noch bis zum 13. Juli 1529 lief. Aber die näheren Verhandlungen scheiterten an der Unvereinbarkeit der gegenseitigen Forderungen. Die Leipziger Zinnhändler forderten eine beträchtliche Preisreduk¬ tion ; sie würden , versicherte Rünau , höchstens 12— 13 fl. pro Schlackenwalder Zentner geben . Ferner waren die Leipziger Kauf¬ leute nicht gesonnen , den ärmeren Zinngewerken Verlag (Vor¬ streckung ) zu bewilligen, sie wollten vielmehr das Zinnprodukt „bar um bar “ kaufen . Für die ärmeren Zinngewerken , die ohne Verlag nicht arbeiten könnten , sollten von den Zinnhändlern bis 1500 fl. gegen Sicherheit und 5 % jährliche Zinsen bereitgestellt werden . Außerdem sollte die Produktion beschränkt werden . Die Leipziger Kapitalisten wollten sich nur zur jährlichen Abnahme einer gewissen Summe Zinns, im höchsten Falle 4000 Zentner, verstehen . Dazu wünschten sie den Zinnabnahmekontrakt nur auf zwei Jahre abzusehließen. Demgegenüber forderten die Schlackenwalder Zimier einen dreijährigen Kontrakt mit der Verpflichtung von seiten der Kapita¬ listen , alles Zinn abzunehmen , das gewonnen wurde . Im äußersten Falle könne die Jahresproduktion auf 5500 Zentner be¬ schränkt werden . An eine Preisreduzierung des Zinn sei nicht zu denken und ebensowenig an eine Lieferung nur gegen bare Zahlung. Ohne Verlag könnten eine große Anzahl Zinner nicht existieren 2. Es versteht sich leicht , daß bei derartig verschiedenen Interessen¬ richtungen der Schlackenwalder Zinner einerseits und der Leipziger Kaufleute anderseits der Abschluß eines Kontraktes nicht zustande kam . Hieronymus Walter , Michael Puffler und Ulrich Mordeisen, 1 Für das folgende: Hauptstaatsarchiv Dresden, Loc. 7249. Blei-, Zinn- und Kupferhandel und -kauf etc. anno 1524—1532. Bl. 77—81 und Bl. 101. 2 Als die. Schlackenwalder Zinner merkten, daß kein Zinnkauf zustande kam, baten sie Hans v. Pflug, ihnen zu gestatten, ein- bis zweijährige Verträge zu schließen, mit wem sie wollten. „Dann ir weren vil die uif 1000—1500 Ctr. . . . verhohlen zu kaufen und vertragen zu können, dass also einer dem andern sein zeyn mechte helfen vertreiben.“ Aus diesem Anträge sprach das Interesse der reichen Zinngewerken, die ihre ärmeren Genossen gern selbst verlegt hätten. Drittes Kap . : Monopole , Kartelle u . Aktien ;,'es. im siiclis. Zinnhandel usw. 249 die kapitalistischen Träger der Unterhandlungen mit den böhmischen Zinnern , ließen , trotz eifrigster Bemühungen Herzog Georgs, im Grunde schon Anfang 1528 keinen Zweifel mehr über ihre Un¬ geneigtheit , sich in das gefahrvolle Unternehmen einzulassen l. Wie die sächsische Regierung ihr möglichstes tat , um , den Bürgern von Leipzig den Goslarer Bleikauf , den Kuttenberger Kupfer - und den Schlackenwalder Zinnkauf zu sichern , so mühte sie sich auch , den Leipziger Kaufleuten den sächsischen Zinngroß¬ handel als Monopol auszuliefern . Die sächsische Regierung hatte dabei , außer dem Wunsche , die Leipziger Kaufmannschaft im Sinne des obengenannten großen Projektes zu fördern , auch den Nutzen der ärmeren sächsischen Zinngewerken und namentlich der Zinnbergleute im Auge. Je länger je mehr hatte es sieh gezeigt, daß die ärmeren Zinner nicht ohne kapitalistischen Verlag auskommen konnten . Fehlte dieser, so waren Betriebseinstellungen mit anschließender Arbeitslosigkeit unter den Bergarbeitern die häufige Folge. Zu den schon früher in sächsischem Zinnkauf tätigen Unter¬ nehmern Michael Puffler und Ulrich Mordeisen erklärte sich um 1527 noch Hieronymus Walter bereit , den sächsischen Zinnkauf zu übernehmen 2. Über die Bedingungen wurde zwischen den Zinnkäufern und Zinngewerken unter Vermittlung Herzog Georgs eifrig hin und her verhandelt , ohne daß es zu einer Einigung gekommen wäre . Außer an dem niedrigen Preis , den die Kaufleute nur zahlen wollten , stießen sich die Gewerken auch daran , daß die Kapitalisten den Kauf nur immer für ein Jahr zu übernehmen gedachten . Was man freilich den Unternehmern bei der starken Stimmung , die im Reiche gegen „die Monopole“ herrschte —r wir kommen sogleich noch hierauf zurück —, nicht verargen konnte. So ist schließlich mit seinen einzelnen Teilen auch das Ganze des großen Projekts von 1527 im Sande verlaufen . Die Nürn¬ berger behielten , wie es scheint , in Kuttenberg die Oberhand 3. Die Goslarer Bleiproduktion wurde durch die Kämpfe der Stadt 1 Ersichtlich aus einem ermunternden Schreiben Georgs an die Genannten vom 28. Januar 1528. Hauptstaatsarchiv Dresden, Cop. 151, Bl. 7 b, 9 a. 2 Für das folgende vgl. Hauptstaatsarchiv Dresden, Loc. 7249. Blei-, Zinn- und Kupferhandlung und -kauf 1524—1532. Bl. 91 ff. 3 Kroker, Neujahrsblätter IV, S. 109 Anm. 1. 250 Drittes Buch : Monopole, Kartelle und Aktiengesellschaften iin Mittelalter. mit Heinrich von Braunschweig einige Jahre arg hintangehalten. Englisches und polnisches Blei ersetzte in der nächsten Zeit viel¬ fach das Goslarer , und wenn auch 1533 ff. Wolfg. AViedemann und Lucas Straub mit Heinrich von Braunschweig zum Abschluß längerer Bleilieferungskontrakte gelangten , so war damals das Pro¬ jekt längst in Vergessenheit geraten. Die Leipziger Kaufleute und Herzog Georg erkannten , daß sie namentlich in Böhmen die älteren Rechte und Geschäftsverbin¬ dungen der Nürnberger nicht einfach beiseite schieben konnten. Es ist charakteristisch und dokumentiert bis zu einem gewissen Grade den Sieg der Nürnberger in dem Kampfe um die Vormacht¬ stellung im mitteleuropäischen Erzhandel , wenn sich Herzog Georg im Jahre 1530 daran begab , mit den Kaufleuten und Händlern von Leipzig und Nürnberg AVege „ , Maß und Mittel “ zu vereinbaren , daß sie alles Zinn , das in Sachsen und Böhmen gemacht wurde , „in eine liandt “ annähmen . „Damit “, so heißt es in einem Schreiben Plans Pflugs an Herzog Georg von Sachsen vom 11. No¬ vember 1530, „ein teil dem andern in den zcinkauffen kein fal ader schaden mache “ 1. Man wird aus diesen kurzen Bemerkungen nicht sicher schließen können , ob es sich hier um ein Kartellprojekt oder gar urn den Vorschlag einer Fusion der zu gründenden Gesell¬ schaften des sächsischen und des böhmischen Zirinkaufs handelt. Jedenfalls war die Ausschaltung der gegenseitigen Konkurrenz der Beherrscher der sächsischen und der böhmischen Zinnproduktion der klar ausgesprochene Zweck der Organisation . Aber auch dieses Kartell oder gar Fusionsprojekt kam nicht zur Ausführung . Die Leipziger und Nürnberger Kaufleute , an die Georg von Sachsen herantrat , waren nicht dafür zu haben , angeblich , „ die weil der entliehe abschied 2 yetzigs reichstags noch nicht geben und aus¬ gekündigt , dardurch sie vergewiss, ab sie auch ire gewerbe und kaufman -handlung im rych möchten fridlich gewarten “. Die Er¬ klärung der Kaufleute spielt auf die Reichstagsverhandlungen über 1 Hauptstaatsarchiv Dresden, Loc. 7249. Blei-, Zinn- und Kupferhandel und -kauf anno 1524—1532. Bl. 81 und für das folgende Bl. 101. 2 Gemeint ist der Reichstag zu Augsburg. Am 22. September 1530 war dort den Ständen der Entwurf eines Reichstagsabschiedes vorgelegt worden. Der Abschied selbst wurde erst am 19. November publiziert. Drittes Kap. : Monopole, Kartelle u. Aktienges. im siichs. Zinnhandel usw. 251 die Monopole an , die allerdings nicht sehr günstig für die Kauf¬ leute waren und die sich auch direkt gegen Monopolisierungen im Zinnhandel ’ richteten 1. Zeiten Erneutes des freien Zinn Verkaufs (bis 1538 ). Monopol (1538 ff .) und dessen Ende ( 1541 ). Hauptquellen: Die folgenden Urkunden : 1. Herzog Georg nimmt auf drei Jahre den Zinnkauf in Alten¬ berg , Lauenstein und Bernstein in seine Verfügungsgewalt . 27 . Mai 1538 . Hauptstaatsarchiv Dresden , Loc . 7414 . Den Zinnhandel betr. 1497—-1544 . Bl . 33 resp . 35 und 38 . Bei mir im Anhang abgedruckt. 2. Herzog Georg übergibt dem Leipziger Kaufmann Michael Puffler den Zinnkauf zu Altenberg , Lauenstein und Bernstein auf drei Jahre (nur die Gewerken Alnpcck von Freiberg ausgenommen ). 26 . Mai 1538 . Hauptstaatsarchiv Dresden , Cop . 103 , Bl . 30 , auch Loc . 7414 , den Zinnhandel betr . 1497— 1544 . Bl . 36 . Im Anhang abgedruckt. 3. Herzog Georg macht seinen Bergbeamten von der Verleihung des Zinnkaufs an M. Puffler Mitteilung , befiehlt den öffentlichen Anschlag der Verordnung und den Schutz und die Unterstützung Pufflers bei der Ausübung seines Privilegs . 28 . Mai 1538 . Haupt¬ staatsarchiv Dresden , Loc . 7414 , den Zinnhandel betr . 1497— 1544. Bl . 34 . Im Anhang abgedruckt. 4. Herzog Georg vermittelt zwischen Michael Puffler und den Gewerken von Ehrenfriedersdorf , Geyer und Thum einen dreijährigen Zinnkauf . 29. Mai 1538 . Hauptstaatsarchiv Dresden , Cop . 103, Bl . 33, auch Loc . 7414 , den Zinnhandel betr . 1497 —1544 . Bl . 37. Im Anhang abgedruckt. 5. Verlagskontrakt M. Pufflers mit einigen Gewerken zu Alten¬ berg . 21. September 1539 . Hauptstaatsarchiv Dresden , Loc . 4493. Bergsachen , den Altenberg und Gießhübel bfetr. 1509—1697 . Bl . 73 bis 74. Im Anhang abgedruckt. 6. Michael Puffler beklagt sich hei Herzog Heinrich von Sachsen, daß ihm einige Altenberger Zinngewerken , die er verlegt hat , ihr Zinnprodukt nicht übergeben wollen . Leipzig , 2. April 1541 . Haupt¬ staatsarchiv Dresden , Loc . 4493 . Bergsachen , den Altenberg und Gießhübel betr . 1509— 1697 . Bl . 72. Im Anhang abgedruckt. 1 Der bedeutsame „ Ratschlag der Monopolien halben , so sich auf dem Nurnbergischen des 24 . Jhars referirt , verlesen zu Augsburg 1530 “ , ein Schriftstück , dem man deutlich die Mitautorschaft klein¬ kaufmännischer Gegner der großen Handelsgesellschaften anmerkt, ist abgedruckt bei K . E . Förste mann, Urkundenbuch zu der Geschichte des Reichstages zu Augsburg im Jahre 1530 . 2 Bde. Halle 1833/35 . II , S. 191 ff. 252 Driltcs Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. An Versuchen , wenigstens die sächsische Zinnproduktion in eine Hand zu bringen , hat es die sächsische Regierung , hat es namentlich Herzog Georg in der Folgezeit nicht fehlen lassen . Im Verfolg dieser Bemühungen konnte er im Frühjahr 1530 Michael Puffler und Ulrich Mordeisen benachrichtigen , daß „ etliche Leute vorhanden seien, die den Zinukauf in Altenberg anzunehmen ge¬ sonnen wären “. Er , Georg, halte es aber für seine Pflicht , den alten Zinnkäufern Michael Puffler und Genossen vorher den Kauf noch einmal anzubieten . Es entzieht sich unserer Kenntnis , inwie¬ weit es sich bei den Kaufleuten , von denen Georg berichtete , um ernsthafte Reflektanten bandelt . Jedenfalls sah sich Michael Puffler nicht durch ihre Konkurrenz veranlaßt , seinerseits den Zinn¬ kauf zuzuschlagen . Er teilte vielmehr dem Herzog mit , daß er gern eine Zinnkaufsgesellschaft gegründet hätte , bisher aber noch keine Kompagnons für das Unternehmen gefunden habe . Allein könne er den Zinnkauf nicht auf sich nehmen „aus Ursaeh , daß er sein Armut “, soll heißen seine geringen Kapitalien , „in andere Händel gewendet “ habe . Dagegen sei er noch immer gern bereit, sich an einer von anderer Seite gegründeten Zinnkaufsgesellschaf L kapitalistisch zu beteiligen 1. Während dann Georg auf dem Augsburger Reichstag weilte, suchte sein Sohn Johann einen Zinnkauf zum Abschluß zu bringen. Es war verlorene Arbeit . Der junge Herzog mußte schon bald, unter dem 4. Juni 1530, dem Vater berichten , daß alle Bemühungen vergeblich seien, die divergierenden Interessen der Zinngewerken und der Zinnhändler zu vereinigen . Die Kaufleute zu Leipzig, Walter u. a., die sich in einen Zinnkauf einzulassen beabsichtigten, wollten nicht alles Zinn abnehmen , sondern nur ein bestimmtes Quantum , etwa 4000 Ztr . jährlich . Sie forderten zugleich, daß die Gewerken nicht mehr als dieses Quantum produzierten 2. Einen Sinn hatte die geforderte Produktionsbeschränkung nur , wenn die Einfuhr auswärtigen Zinns unterbunden war . Nur durch Aus¬ schaltung der böhmischen Konkurrenz wäre ein Steigen des Zinn1 Hauptstaatsarchiv Dresden , Loc . 7249 . Blei -, Zinn - und Kupferhandel und -kauf 1524— 1532 . Bi . 62 . Der Brief Pufflers ist datiert : Freiberg , 11. März 1530. 2 Hauptstaatsarchiv Dresden , Loc . 9810 . Münz - u . a. Händel amio 1490— 1530 . Bl , 387 . Der Brief Johanns ist datiert : Dresden, 4. Juni 1530. 1 4 6 Drittes Kap . : Monopole , Kartelle u. Aktionärs , im säclis . Zinnhaiulel usw . 253 preises die Folge der verminderten Zimierzeugung gewesen. Es müssen Verhandlungen zwischen der sächsischen Regierung und den Zinnhändlern über diesen Punkt gepflogen worden sein, denn Prinz Johann bat seinen Vater um Instruktion , „wie und welcher ge¬ stalt sie sich mit dem fremden zinn zuvor bieten halten sollten “ b Die Verhandlungen mit Walter und Gen. haben sich zer¬ schlagen . Im Frühjahr 1531 suchte Georg von Sachsen die be¬ deutende Firma Erasmus Schetz zu Antwerpen 2, die in Hans Randerott zu Leipzig einen tüchtigen Vertreter hatte 3, für einen längeren Zinnabnahme - und Verlagskontrakt mit den Gewerken von Altenberg zu gewännen b Auch Michael Puffler wandte sich brieflich nach Antwerpen an die Schetzgesellschaft mit der Anfrage, ob sie bereit wäre , „sich neben anderen in Zinnkauf einzulassen “ 5. Das in den beiden , hauptsächlich für diese Dinge in Frage kommenden Archiven zu Dresden und Weimar nur sehr spärlich erhaltene arehivalische Material gestattet nicht mit Bestimmtheit zu sagen, ob in den nächsten Jahren ein Zinnkauf zustande gekommen ist . Wir haben Grund , daran zu zweifeln. Erst 1538 wurde dem Zustande des freien Zinnverkaufs durch die Gewerken ein Ende gemacht . Wie es noch immer gewesen war, drängten die kleineren , in den Bergstädten und Städtchen heimischen Zinnproduzenten , im Gegensatz zu den großen , auswärtigen Ge¬ werken , zu einer Ordnung im Verkauf ihres Zinnproduktes . Nament¬ lich die kleineren Altenberger Gewerken arbeiteten darauf hin. In ihrem Interesse besonders und gegen die Wünsche der reichen auswärtigen Gewerken erließ Georg am 27. Mai eine fürstliche Verordnung , mit der er der freien Verfügungsgewalt der Gewerken über ihre gewonnenen Erzprodukte ein Ende machte . Alle Ge¬ werken in Altenberg , Bernstein und Lauenstein — gleichgültig , ob 1 In demselben Briefe. 2 Die Schetz waren wohl das bedeutendste niederländische Handelshaus des 16. Jahrhunderts . Näheres über sie bei R . Ehren¬ berg, Zeitalter der Fugger , siehe Register . Vgl . dazu für die Be¬ deutung der Firma Schetz besonders im Metallhandel Möllenb e r g a . a . O. an verschiedenen Stellen , siehe Register. 3 Für diesen Faktor vgl . Möllen ber g a. a . O., S. 156. 4 Hauptstaatsarchiv Dresden , Loe . 724Ö. Blei -, Zinn - und Kupferhandel und -kauf 1524-—1532 . Bl . 63 ff. 6 Daselbst. 31 254 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. sie daselbst oder anderswo angesessen waren — sollten von Johanni¬ sonnenwende an gehalten sein, ihr Zinnprodukt für 11 fl. den Zentner in die Altenberger Wage zu liefern und zu verkaufen h An wen ist in der Verordnung nicht gesagt . Es erhellt aber aus einem Privileg , das Georg am 26. Mai bereits für Michael Puffler hatte ausfertigen lassen . Durch dieses Privileg wurde dem genannten Leipziger Kaufmann für drei Jahre von Johanni an der Zinnkauf in Altenberg , Lauenstein und Bernstein als Monopol übertragen 2. Nur die reichen Freiberger Gewerken Alnpeck hatten sich der all¬ gemeinen Produktionsablieferung an Michael Puffler zu entziehen gewußt . Sie sind ausdrücklich in der Urkunde vom 26. Mai aus¬ genommen . Das gesamte übrige Zinnprodukt der Gewerken der genannten Orte mußte der Leipziger Kaufmann gegen Barzahlung zu 11 fl. pro Zentner abnehmen , sobald es an der herzoglichen Wage zu Altenberg für gut befunden worden war . Die zuständigen herzoglichen Bergbeamten wurden sofort von dem Privileg in Kennt¬ nis gesetzt . Gleichzeitig wurden sie angewiesen , den betreffenden Erlaß öffentlich anzuschlagen und dafür zu sorgen , daß er strikte durchgeführt würde a. War in Altenberg , Lauenstein und Bernstein der Zinnkauf auf die Weise in die Hände Michael Pufflers gelangt , daß der Herzog zunächst — auf Grund seines Bergregals —■den Zinnkauf für sich „verordnete “ und ihn sodann dem Leipziger Kaufmann als Privileg überwies , so gestaltete sich der rechtliche Übergang des Zinnkaufs in Ehrenfriedersdorf , Geyer und Thum auf ihn in einer anderen Weise. Hier vereinbarten die herzoglichen Räte Georg von Karlowitz , Ernst von Miltitz und Heinrich von Gersdorf mitsamt dem bekannten Leipziger Juristen Dr . Fachs den Zinnkauf direkt zwischen den faktischen Kontrahenten , d. h. zwischen Michael Puffler und den Zinngewerken 4. Auch der Geyrer , Ehrenfriedersdorfer und Thumer Zinnkauf lief auf drei Jahre , von Johanni angefangen. 1 Hauptstaatsarchiv Dresden , Loc . 7414 . Den Zinnhandel betr. 1497— 1544 . Bl . 33, 35, 38 (letzteres Abschriften ). Vgl . Anhang. 2 Hauptstaatsarchiv Dresden , Cop . 103, Bl . 30 ; auch Loc . 7414. Den Zinnhandel betr . 1497— 1544 . Bl . 36. Vgl . Anhang. 3 Hauptstaatsarchiv Dresden , Loc . 7414 . Den Zinnhandel betr. 1497— 1544 . Bl . 34. Vgl . Anhang. 4 Hauptstaatsarchiv Dresden , Cop . 103, Bl . 33 ; auch Loc . 7414. Den Zinnhandel betr . 1497— 1544 . Bl . 37. Vgl . Anhang. Drittes Kap .: Monopole, Kartelle u. Akticngcs. im säclis. Zinnhaudcl usw. 255 Der Preis , den der Leipziger Kaufmann für das Zinnprodukt zahlte, war — für die bessere Sorte Zinn wenigstens — % fl- höher als in Altenberg . Die übrigen Abmachungen deckten sich. Auch die Kündigungsfrist von einem Jahre vor Ablauf des Kaufs findet sich an beiden Stellen. Es ist aus den genannten , 1538 erlassenen Verordnungen und Privilegien nicht recht ersichtlich , wie es mit dem Verlag der ärmeren Zinngewerken gehalten werden sollte . Aber wir wissen aus anderen Quellen, daß Michael Puffler nicht ohne Verlag der kleineren und auch der geldbediirftigeri größeren Zinngewerken davongekommen ist . So mußte noch 1538 der Geyerer Gewerke Hans Glatz vor dem Bürgermeister und den Geschworenen des Altenbergs und in Gegenwart des Matthias Roth , des Faktors von Michael Puffler , bekennen , daß er 2000 fl. von Puffler empfangen habe 1, und zwar „zuvolge der verainigung umb den czinkauf “ und zum Zwecke des Verlags des Glatzschcn Anteils am Altenberger Zinnbergwerk . Das Geld war mit 5 % jährlich zu verzinsen . Als Sicherheit dienten die Bergteile , Hütten , Molen u. a. Immobilien des Glatz 2. Es ist charakteristisch , daß selbst ein Gewerke wie Hans Glatz, ohne Hilfe auswärtiger Kapitalisten und ohne deren Verlag seine Zinngruben nicht abbauen konnte . Hans Glatz war einer der bedeutendsten Zinngewerken zu Altenberg . Wir kennen die Höhe der Altenberger Zinnproduktion und die Namen und die Beteiligungs¬ ziffern der einzelnen Gewerken für 29 Wochen der Jahre 1545/46. Eine Abrechnung des Zehntners Franz Raupennest vom 14. Septem¬ ber 1545 bis 23. April 1546 ist uns erhalten geblieben 3. Danach betrug in der genannten Zeit die Zinnausbeute: des Hans Glatz vom Aldenhof ...... 311 Ztr. ,, Martin Manwitz ........... 228 ,, von Georg Raupennests Erben ...... 184 „ ,, Dr . Bernsteins Erben ........ 180 ,, der Stollenhcrren ufn Aldeuberg ..... 121 „ 1 Hauptstaatsarchiv Dresden , Loc . 7414 . Acta in Sachen Michael Puffler . Bl . 8. 2 Der Ausdruck „ hipoteciren “ kommt schon für diese Pfandund Sicherheitssetzung in der betr . Urkunde vor. 3 Friedr . Aug. S c h in i d , Diplomatische Beiträge zur sächsischen Geschichte . Dresden u . Leipzig 1839 . S. 29. 1 256 Drittes Buch: Monopole , Kartelle und Aktiengesellschalten im Mittelalter. des Hans Glenigk in Dresden ...... 114 Ztr. „ Mattes Morgenstern ......... 112 „ von Georg am Steigs Erben ....... 109 „ des Franz Schwertzel zu Altenberg . . . . 101 „ der Alnpeclc zu Freiberg ......... 100 ,, des Vallin Buchführer zu Freiberg . . . . 50 „ „ Stefan Herklotz ........... 9 ,, ,, Philipp Engelhardt ......... 7 „ Man sieht , Hans Glatz stand 1545 an der Spitze der Alten¬ berger Zinnproduktion . Er war , was die Höhe der Produktion angeht , der erste Gewerke der wichtigsten sächsischen Zinngewin¬ nungsstätte . Wenn er schon Verlag brauchte , wie sollten dann die kleinen Zinngewinner in Geyer, Ehrenfriedersdorf usw. ohne die reichen Leipziger Kapitalisten auskommen? Ausgezeichnet sind wir über die Formen unterrichtet , in denen sich der Verlag der kleineren Zinngewerken durch Michael Puffler vollzog . In einem Verlagskontrakt , den der Leipziger Handelsherr mit einer Anzahl Altenberger Zinngewerken am 21. September 1539 schloß 2, wurde festgestellt , daß die Rückzahlung des vorgeschossenen Geldes von seiten der Verlegten in der Form von Zinn sofort zu beginnen habe . Damit sollte die Möglichkeit unterbunden werden, daß einige der Gewerken ihr Zinnprodukt zur Abzahlung älterer Schulden usw. verwerteten und der Verleger das Nach¬ sehen hatte . Auch das Eingehen von Verlagsverträgen mit anderen Kapitalisten war den Verpflichteten M. Pufflers verboten , es sei denn , daß sie vorher ihren Verlagsverbindlichkeiten ihm gegenüber völlig nachgekommen wären 3. Falls einer oder der andere der Verlegten auf eine der ge1 Im Jahre 1516 betrug in Geyer , Ehrenfriedersdorf und den J)en ach barten Orten die höchste Ausbeute der einzelnen Gruben 5 Ztr ., die niedrigste y2 Ztr . In Summa 221 Ztr . Hauptstaatsarchiv Dresden , Loc . 4500 . Unterschiedene Register über den Zinn¬ zehnten auf der Zinnflöße zu Ehrenfriedersdorf 1490— 1522. 2 Hauptstaatsarchiv Dresden , Loc . 4493 . Bergsachen , den Allenberg und Gießhübel betr . 1509— 1697 . Bl . 73/74 . Ich habe das für die Geschichte des Verlagssystems wichtige Stück im Anhang abgedruckt. 3 Die betreffenden Verleger hatten natürlich solange M. Pui'fIcrs Zinnkauf dauerte , das mit Hilfe solcher Verlagsvcrträge Zinn an den Monopolinhaber Puffler zu verkaufen. erworbene 1 Drittes Kap,: Monopole, Kartelle u. Aktienges. im siichs. Zinnhandel usvv. 257 nannten Arten kontraktbrüchig würde , sollte es Puffler und den Seinen zustehen , sofort „ ohne alle weitere Ersuchung und Er¬ laubnis “ das Zinn des Übertreters mit Arrest zu belegen . Nur der Arbeiterlohn für den Abbau des verlegten Zinns ging vor die An¬ sprüche des Kapitaldarreichers und mußte auch von den verlegten Gewerken zunächst gezahlt werden , ehe sie noch — mit Hilfe von Zinnlieferungen — an die Rückzahlung des Verlags gingen. Lange hat die 1538 aufgerichtete Ordnung des sächsischen Zinnkaufs nicht bestanden . Schon am 7. Juni 1540 kündigte Michael Puffler in einem Schreiben an den Herzog den Zinnkauf zu Altenberg , Bernstein , Lauenstein , Ehrenfriedersdorf , Geyer und Thum . Puffler gab als Gründe für seine Kündigung einmal die „schlechten Zeiten “ an . Dann aber konnte er sich auch darauf berufen , daß einige Zinngewerken geäußert hatten , es sei für die Bergwerke nützlicher , wenn es jedermann zustände , sein Zinn¬ produkt frei zu verkaufen , an wen er wolle In den ersten Regierungsjahren des Herzog Moritz hat kein Zinnkaufsmonopol die freie wirtschaftliche Verfügungsgewalt der sächsischen Zinngewerken beschränkt . Aber wiederum zeigte es sich, daß dieser Zustand undurchführbar und dem sächsischen Zinnbergbau schädlich war . Schon 1544 fand Herzog Moritz, daß es „ die hohe nothturft erfordern thut , einen zcinkauf im fürstenthumb aufzurichten “ 2. Wohl wurden für den 24. Januar 1544 alle Zinngewerken des Landes zu einer Beratung über den Abschluß eines Zinnkaufs nach Dresden gefordert , aber der Abschluß scheint nicht erfolgt zu sein. Erst gegen Ende des Jahrzehnts sind dann wieder erneute energische Versuche gemacht worden , das sächsische Zinn in eine Hand zu bringen . Die Versuche um 1549 hängen aufs innigste mil gleichzeitigen Monopolprojekten im böhmischen Zinnhandel zu¬ sammen . Wir müssen daher , um hier ganz klar zu sehen, zunächst unsere Blicke nach Böhmen richten. 1 Hauptstaatsarchiv Dresden , Loc . 7414 . Den Zinnhandel bclr, 1497— 1544 . Bl . 43. 2 Aus einem Schreiben des Herzogs an den Bergmeislcr zu Altenberg , Ehrenfriedersdorf und Geyer, in welchem er ihn dazu auffordert , alle Zinngewerken im Lande für den ‘24. Januar nach Dresden zur Beratung über einen Zinnkauf zu laden . Hauptstaats¬ archiv Dresden , Loc. 7414 . Den Zinnhandel belr . 1497— 1544 . Bl . 48. StrieWcr , ShuUen z. Gesch . kapitalist . Organiaationefoninon . 17 258 Drittes Buch: Monopole , Knrlclle und Aktiengesellschaften im Mittelalter, Viertes Kapitel. Monopol-- und Kartellbestrebungen im böhmi¬ schen und sächsischen Zinngroßhandel seit¬ her Mitte des 16. Jahrhunderts. E r ster Absehnit t. Die Monopolisierung der böhmischen Zinnproduktion in der Hund des Augsburgers Conrad Mayr. Hauptquellen: t . Zinnkaufvertrag zwischen König Ferdi¬ nand und den Gewerken zu Schlaekenwald und Schönfeld . 22 . Ok¬ tober 1549 . Vgl . F . A . Schmidt, Chronologisch -systematische Sammlung der Berggesetze der österreichischen Monarchie . I. Ab¬ teilung (Berggesetze des Königreichs Böhmen , der Markgrafschaft Mähren und der Herrschaft Schlesien ). 2. Bd . (1548 — 1561 ). Wien 1832 . S. 310 . . 2. Zinnkaufvertrag zwischen König Ferdinand und den Ge¬ werken am Hengst , Lauterpaeh , Peringer , Lichtenstadt , Platten, Gotsgab , Kaff , MUckcnbcrg u . a . 22. Oktober 1549 . Vgl . Schmidt а . a . O., S. 312. • 3. Zinnkaufkonlrakt zwischen König Ferdinand und den Ge¬ werken zu Graupen . 23 . Oktober 1549 . Vgl. Schmidt a . a . O,, S. 315. 4 . König Ferdinand überträgt dem Conrad Mayr aus Augsburg den böhmischen Zinnkauf auf drei Jahre . Privileg , datiert : Prag, б. Dezember 1549 . K . K . gemeinsames Finanzarchiv in Wien . Ge¬ denkbuch , Böhmen 1549— 50. Nr . 305 , Fol . 112— 115. Vgl . Anhang. 5. Conrad Mayrs Einwilligungserklärung . Daselbst Gedenkbuch, S. 116—117. Es wäre interessant zu wissen, wie die Organisation des Zinn¬ großhandels , die wir für Sachsen seit dem Ausgang des 15. Jahr¬ hunderts im vorigen Kapitel verfolgen konnten , sich in derselben Zeit in den benachbarten bedeutenden böhmischen Zinnproduktioiisstädten , besonders in Schlackenwald und Schönfeld , entwickelte. Hier kann eine Darstellung dieser Entwicklung nur für diejenigen Epochen gegeben werden , wo durch Kartellbestrebungen usw. eine direkte Verbindung der zwei Wirtschaftsgebiete angestrebt wurde. 1 Im Vierten Kapitel bedeutet : F .A . Wien = K . u . K . gemein¬ sames Finanzarchiv zu Wien , Böhmen , M. u . B . = Fase . Böhmen, Münz - und Bergwesen. Viertes Kapitel : Böhm.-sächs. Monopol- und Kartellbestrcbungeh . 259 Noch kurz vor der Mitte des 16. Jahrhunderts war der Zinn¬ kauf in Böhmen allen Kapitalbesitzern offen, d. h . es existierte kein Monopol, das die Gewerken zwang , ihr Zinnprodukt gegen festbestimmten Preis usw. einer Zinnkaufsgesellschaft zu über¬ geben . Die Schlackenwalder Zinnordnung , die 1548 im Druck er¬ schien , bestimmte in Abteilung 16 das folgende : „ Von sonderlicher fürderung des zinbergkwercks wollen wir hiemit gnedigst allen denienigen so dasselbig bauen , zulassen , dass ein jeder gewerck sein z i n seines gefallen s und n a c li seinem nutz und {romen wem , wo und wenn er w i 1 zu vorkaufe n macht haben soi 1.“ Und auch bet den übrigen böhmischen Zinnbergwerken unterlag um diese Zeit der Zinnverkauf der Ge¬ werken keiner monopolistischen Beschränkung . In Artikel 29 der Zinnbergwerksordnung 1 für die übrigen böhmischen Zinnproduk¬ tionsstätten heißt es unter dem Titel „Von dem vorlag , Vorlegern und denen , so gelt auf zin entleheu “ : ' „Zu sonderlicher fürderung des zinbergwercks wollen wir genedigst allen deiiieiiigen, so dasselbig bauen , zulassen , dass ein Jeder gewerk sein zin seines ge¬ fall e n s u n d nach seinem nutz und [ romen wem u nd wo er w i 11 z u v o rkauf en macht haben soll. Es soll auch das vorlegen auf disert unsern zinbergkwercken idermann frei sein und obgleich sieb der rnehrer teil gewercken des bergwercks mit unser Zulassung sich in einen zinkauf bewilligen und einlassen 2, so sollen doch diejenigen , die es nicht bedürfen oder den es nicht gelegen sein wil, damit nicht verbunden , sondern frei sein.“ Das Verhältnis zwischen Verlegern und den verlegten Zinngewerken wurde in derselben Ordnung wie folgt geregelt : „ Alle diejenigen, so von Vorlegern gell; auf zin aufheben und entlehen. die sollen auf bestimpte und versprochene fristen unverzüglichen zalen . Da aber die vorleger derhalben gegen unsernt bergkmeister klaghaftig würden , alsdann soi er, ungeachtet irer ungegründeten ausflucht und behelf, zu ihrem leib und guL schleunig vorhelfen. Werden gewercken mehr denn von einem vorleger , auch mehr denn 1 Aus: „ Rom. auch Hungarn u. Beliem etc. kn. Mai. zinbergkwercks-ordnung auf die bergkwerclc Hengst , Perniger , Liehtenstadt, Platten , Gotsgab, Kaff, Mückcnbcrgk u. a. derselben orten und enden. Gegeben Prag , 1. Jan . 1548. Gedr. zu Zwickau 1548. 2 Soll wohl heißen : „ in Zukunft einlassen würden“ . 17* 260 1) rittcs ßucli: Monopole , Kartelle und Aktiengesellschaften irn Mittelalter. auf einer zechen gelt entpfahen und entlehen und darnach in der zalung sich der ausflucht und behelf gebrauchen und sagen das zin wer nicht mit des, sondern mit eins andern gelt erbauet und gemacht , oder der vorlegcr hett ihm nicht auf diese, sondern auf ein andere zech geliehen , an solche und dergleichen behelf soi sich unser bergkmeister nicht keren , sondern atwege den ersten und eitern Vorlegern mit des hülfe für die andern gehen lassen , ungeacht aller vorschreibung , so sie gegen einander eingangen und aufgericht haben. Es soi auch keinem Vorleger zin gefolgen, es seindt dann zuvorn die arbeiter , so die Zwitter gewunnen , fiirgefürt und ausbereitet ihres lidlobns und darnach die mühlherrn , scbmeltzer und hüttenherren ihres lohns und zins gantz und gar entricht. Damit aber auch die vorleger nicht bevorteilt , und abscheuhig gemacht , so sollen bergkmeister und gesclrworne vleissigk achtung haben und diejenigen , so von Vorlegern gelt auf zyn nehmen , dahin halten , dass sie in bergk und mühlen treulich arbeiten und dass sie auch wöchentlich anschneydcn bei straf . Und so der bergk¬ meister und gesclrworne befinden , dass mehr auf berck und mühlen gerechnet , dann gebürlicher weis darauf gangen , das soll unser hauptmann , Verwalter und bergkmeister mit ernst strafen. Wir wollen auch , dass alle dieienig'en, so von Vorlegern gelt auf zin entpfahen , desgl. auch alle andere den arbeitern an ihrem lohn nicht aufschlahen , sondern par lohnen sollen und mit kainerlai wahr ; dass auch keinem hendler gestatt werde , Schichtmeister zu sein.“ Nach allem , was wir wissen, waren es in erster Linie Nürn¬ berger Kaufleute , die in Schlackenwald und in den übrigen böhmi¬ schen Zinnproduktionsgebieten das Zinnprodukt aufkauften und den Verlag der ärmeren Gewerken in der Hand hatten . Ihre Ge¬ schäfte erlitten im Jalire 1549 eine ziemlich unerwartete Be¬ schränkung . Am 22. Oktober dieses Jahres kam es zwischen dem Landesherrn , König Ferdinand , und den böhmischen Zinngewerken zum Abschluß eines allgemeinen Zinnkaufkontraktes h Die Ge1Der König schloß am 22. Oktober drei Zinnkaulskontrakte ab, den einen mit den Gewerken von Schlackenwald und Schönfeld, den andern mit den Hengster , den Lauterbacher , den Peringer , den Lichtcnstädter , den Plattner , den Gottesgaber , den Kaffer , den Mückenberger Gewerken. Am 23. Oktober folgte der Abschluß des Vertrages mit den Gewerken zu Graupen. Die Verträge sind (aller- * Viertes Kapitel : Böhm.-sächs. Monopol- und Kai teilbestreb ungen. 261 werken sollten von Weihnachten 1549 bzw. für Graupen von Pfingsten 1550 an auf 20 Jahre ihr gesamtes Zinnprodukt dem König oder seinen Erben übergeben, und zwar zum festbestimmten Preise von 18% fl. 1pro Schlackenwalder Zentner für solches Zinn, das ohne Verlag erbaut ist, von 18 fl. für solches, das mit Hilfe von Verlag gewonnen wird. Hartes Zinn soll nur mit 17% bzw. 17 fl. bezahlt werden. Für die Ablieferung des Zinns und die Be¬ zahlung werden bestimmte Orte verabredet. Die Bezahlung sollte in barem Gelde geschehen. Der König versprach auch dafür zu sorgen, daß es den ärmeren Gewerken, die den Vorschuß nicht entbehren könnten, an Verlags¬ geld nicht fehlen würde. Um diesen ärmeren Gewerken noch mehr entgegenzukommen, wurde ihnen gestattet , auch andere Kapitalisten als die vom König offerierten, als Verleger anzunehmen, falls sie bei ihnen den Verlag zu bequemeren Bedingungen erhielten als von den Monopolinhabern. Freilich geschah diese Erleichterung unter der Voraussetzung, daß das gewonnene Zinn dann von den betreffenden Verlegern dem König oder dessen Erben übergeben würde 2. Eine Reihe der Vertragsbestimmungen sieht für Fälle, daß etwa besonders gold- oder silberhaltige Zinnerze gewonnen würden, eine entsprechend höhere Bezahlung des Zinnproduktes vor. Mit keinem Worte ist in den Verträgen der Kapitalisten gedacht, die dem König das Zinnprodukt abnehmen und an seiner Statt das Monopol über das böhmische Zinn in ihre Hände bringen würden. Trotzdem dürfte es nur wenigen Zinngewerken in Böhmen zweifel¬ haft gewesen sein, daß die Krone nicht auf eigene Faust und für sich das Monopol erwirkte, sondern daß dahinter Kapitalisten standen, denen der König das fertige Abkommen — natürlich gegen entsprechendes Entgelt — zedierte 3. dings ziemlich lässig) gedruckt bei F . A. Schmidt, Chronologischsystematische Sammlung der Berggesetze der österreichischen Mon¬ archie . I. Abt . 2. Bd. als Nrn . 42, 43 u. 44. 1 Florin in guter Münze, wie sie im Königreich Böhmen „gangbar, gieb und gab“ , d. h. den Florin für 24 weiße Groschen, den Groschen zu 7 weißen Pfennigen(d.) gerechnet. 2 Genaue, interessante Bestimmungen , wie der Verlag geschehen soll, damit die Verleger nicht ihr Geld verlieren ; vgl. F . A. Schmidt, a . a. O., I. 2., besonders Nr . 51, S. 343 ff. 3 Wir haben früher einen analogen Fall vor uns gehabt . Auch 262 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. Wenn man den Darstellungen dieses großen böhmischen Zinn¬ monopols, wie sie in den sechziger Jahren des 16. Jahrhunderts in Sachsen von Kennern der Materie vorgetragen wurden, glauben dürfte, so wäre eine Reihe der größten Augsburger Handelsgesell¬ schaften an dem böhmischen Zinnhandelsmonopol beteiligt gewesen. Die Fugger, die Manlich und die Mayr x. Nun liegt es nahe für die F'ugger und die Manlich — beides Firmen, die im internationalen Metallhandel des 16. Jahrhunderts eine hervorragende Rolle spielten, die auch mit Ferdinand durch vielerlei Geldgeschäfte eng verbunden waren —, an eine Beteiligung irgendwelcher Art an dem böhmischen Zinngroßhandelsmonopol zu glauben. Tatsächlich wissen wir auch, daß Erasmus Heidenreich und Hans Metzschberger2, die Geschäfts¬ träger Ferdinands, mit den Manlich neben den Herwart, den Haug, den Neidhart, den. Herbst und den Paller (alles Augsburger Finnen) wegen der Übernahme des böhmischen „Zinnkaufs“ in Unter¬ handlung gestanden haben 3. Die Unterhandlungen mit den Manlich die sächsische Regierung nahm 1538 zunächst den Zinnkauf in eigene Hand , um ihn dann an die Kapitalisten weiterzugeben . Wie das zu geschehen pflegte , erkennt man gut aus einem Schreiben des Kurfürsten Moritz von Sachsen an seinen Bergamtmann zu Altenberg (datiert : 16. September 1550: Hauptstaatsarchiv Dresden, Loc. 36 080, Nr . 666, Bl. 25 bzw. 53 a). Moritz hatte mit den Alten¬ berger Zinngewerken einen Zinnkauf auf 20 Jahre abgeschlossen. Jetzt ließ er den Gewerken melden, sie sollten den Leipziger Kauf¬ leuten Hieronymus Lotter und Valtin Buchführer „den bewilligten kauf, in massen wir den jüngst mit inen geschlossen, nemiich den etr , urnb zwölf gülden balir umb bar uff liegst künftigk Michaelis ahne wegrunge bies uff unser weiter Verordnung zu kommen lassen und dagegen bahr betzaUung von inen gewarten .“ 1 In einer Denkschrift der kurfürstlich sächsischen Räte des Jahres 1569 (Hauptstaatsarcliiv Dresden, Loc. 36 080, Nr . 664, Bl. 12 a unten ) heißt es: „Dan chf. g. seindt sonder zweifei bericht, wie vor etzlichen jaren in der chron B eh ei mb auch ein zcinkauf aufgerichtet , welchen die zeit die furnembsten hendler im reich als die Fucker , die Manliche und die Meyerische und andere in henden und eben in dem furhaben die czien ires gefallens zu steigern.“ Dagegen nennt die gleichfalls 1569 entstandene Zinnkaufs-Denlcschrift des Hans Jenitz nur die Manlich als die Finanzmacht , die das böhmische Zinnmonopol kapitalisierte (Haupt¬ staatsarchiv Dresden, Loc. 36 080, Nr. 664, Bl. 19). 2 Letzterer hat auch sonst als Finanzagent Ferdinands viel rnit den Kaufleuten in Augsburg und Nürnberg zu tun gehabt. 8 F .A. Wien. Böhmen, M. u. B. (1540—1569) Nr. 16 406. Viertes Kapitel : Bühm .-säclis . Monopol - und Knrtcllbestrebungen . 263 wie mit den anderen genannten süddeutschen Firmen sind daran gescheitert , daß die betreffenden - Kaufleute wissen wollten , das böhmische Zinn vertrage einen Preisaufschlag , wie ihn das Monopol nötig mache , nicht . Die Gefahr bestände , daß im Falle einer Preis¬ erhöhung Italien , der bedeutendste Zinnabnehmer der Böhmen, englisches Zinn „per mare “ einführe. Die Manlich und eine andere Firma , deren Namen ich leider nicht feststellen kann , haben dann König Ferdinand vorgeschlagen, er selbst möge das böhmische Zinn „empfangen und verlegen “ 1. Das Kapital , das der König dazu brauche , wollten sie ihm gern leihen . Ferdinand solle jährlich 10 % Zinsen dafür zahlen . Die Firmen wollten auch den Vertrieb des königlichen Monopolzinns übernehmen . Sie verlangten eine Vergütung von 2 % und Ersatz der Unkosten dafür 2. Das Geld, das die Firmen beim Zinnverkauf lösten , solle zunächst „ an der schuld a rata der zeit nach alspaldt abgehalten werden “ . Der Vorschlag ist nicht verwirklicht worden. Die Manlich scheinen dann an dem böhmischen Zinngroßhandelsmonopol über¬ haupt keinen Anteil genommen zu haben . Vielleicht erklärt sich aus den geschilderten Ansätzen die Annahme der obengenannten sächsischen Denkschriften , die den Manlich neben den Fuggern und den Mayr eine Beteiligung an dem böhmischen Zinnmonopol vom Jahre 1549 ff. zuschreiben . Inwieweit die Nachricht bezüglich der Fugger richtig ist , werden wir noch sehen . Jedenfalls standen die Mayr aus Augsburg , besonders Conrad Mayr, für das große Publikum im Vordergründe der großzügigen Spekulationsunter¬ nehmung 3. 1 Leider ist das Schriftstück , nach welchem obiger Sachverhalt dargestellt ist , undatiert und ohne Namen . F .A. Wien. Böhmen, M. u . B, (1540—1569) Nr . 16 406. Der Verfasser spricht von den „Manlich und wir“. Es handelt sich wohl um eine süddeutsche Handelsfirma , die mit den Manlich in Konsortialbeteiligung das Ge¬ schäft machen sollte. 2 Die2 % verstehen sich, wie es scheint , von dem hergeliehenen Kapital . Wenigstens sagt das Schriftstück : „ Sie. wollen . . . fur ir mie über den uncost , so darauf gen wurdt , 2 % haben oder auf das hinausleihen 12 % rechnen .“ 3 Die Denkschrift Hans Bernsteins , des bekannten Rates des Kurfürsten August von Sachsen* aus dem Jahre 1585, in der ein historischer Rückblick auf frühere mißlungene Zinnmonopolprojekte 1 264 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. Wer war dieser Conrad Mayr ? War sein Vermögen groß genug, um eine kapitalistische Transaktion von Bedeutung durchzuführen ? Leider sind wir nicht sehr gut über die geschäftliche Tätigkeit Conrad Mayrs unterrichtet . Mehr wissen wir von seiner politischen Tätigkeit im Dienste seiner zweiten Heimatstadt Augsburg. Conrad Mayr stammte aus Memmingen , wo er als Sohn des Andreas Mayr und der Barbara geb. Loeklein geboren war . Nach¬ dem er im Jahre 1531 mit Euphrosina , der Tochter des Lukas Walter , das Augsburger Bürgerrecht erheiratet halte , siedelte er in die Heimatstadt seiner Frau über . Dort gehörte er seit 1538 dem Patriziat an und war seit 1545 Mitglied des Kleinen Rates „von den Herren “ . Weil Conrad Mayr dem alten Glauben treu geblieben war , gehörte er zu der Partei im Augsburger Rat , die sich mit großer Energie , wenn auch vergeblich , einem Kriege gegen Karl V. widersetzt halte . Als dann die reiche Stadt mit dem sieg¬ reichen Kaiser ihren Frieden machen mußte und es galt , möglichst leichte Bedingungen zu erreichen , da war Conrad Mayr mit Marx Pfister , Sebastian Seilz, Georg Hopfer und Dr . Peutinger unter der Gesandtschaft , die Anton Fugger zu dem berühmten Fußfall vor dem Kaiser nach Ulm führte 2. In den Jahren 1550—60 be¬ kleidete Conrad Mayr die Würde eines Bürgermeisters der Stadt Augsburg 3. In dieser Eigenschaft wurde er vielfach zu Missionen an den Kaiser , bei dem er in großem Ansehen stand , und an König Ferdinand als Fürsprecher seiner zweiten Vaterstadt verwendet. Aber auch die Sache des Kaisers hat Mayr verschiedentlich durch Geldvorschüsse u. a. vertreten . Als es z. B. im Frühjahr 1552 galt, so schnell als möglich Truppen anzuwerben , da war Mayr mit Anton Fugger und dem kaiserlichen Oberst Konrad von Haustein Tag gegeben ist, nennt nur die „M ayrischcn von Augsburg“ als diejenige Firma , die 1549 ff. das böhmische Monopol finanzierte. Hauptstaatsarciiiv Dresden, Loc. 7294, Bernstein , 4, Bl. 409 ff. 1 Nach gütigen Mitteilungen des Herrn Rechtsrat Werner in Augsburg und des Augsburger Stadtarchivs. 2 Fr. Roth, Augsburgs Religionsgeschichte. 3. Bd. München 1907. S. 469. 3 Seit der Regimentsänderung vom 3. August 1548 gab es neben den beiden Stadtpflegcrn — den Stadtoberhäuptern — sechs Bürger¬ meister , von denen immer je zwei vier Monate lang besonders als oberste Polizeibeamte tätig waren. 6 Viertes Kapitel : Böhm.-sächs. Monopol- und Kartellbestrebungen . 265 und Nacht daran, die Hauptleute, die die Söldner sammeln sollten, anzufeuern und mit dem nötigen Laufgeld zu versehen 1. Ist es nach dem Gesagten erklärlich, wie König Ferdinand auf Conrad Mayr kam, als er daran ging, das böhmische Zinnmonopol als Finanzquelle zu benutzen, so wird man sich wundern, daß Conrad Mayr mit einem vergleichsweisenicht sehr großen Ver¬ mögen ein so umfassendes Geschäft begann. Aus den Augsburger Steuerlisten läßt sich nämlich erkennen, daß Mayrs Vermögen durchaus nicht zu den größten der reichen Handelsstadt gehörte. Wenn er 1534: 40 fl., 1540: 65 fl., 1548: 70 fl. steuerte, eine Summe, die sich 1554- 1562 auf 115 fl. erhöhte, so gab es Dutzende von Augsburger Kapitalistenfamilien, die ihn an Reichtum übevtrafen 2. Wahrscheinlich wird aber Conrad Mayr nicht allein mit seinem eigenen Geld die böhmische Transaktion unternommen haben. Andere standen wohl hinter ihm. Schon oben wurde auf zeit¬ genössische Stimmen aufmerksam gemacht, die neben den Mayr, die Fugger und Manlich als Inhaber des böhmischen Zinnmonopols nannten. Es verdient in diesem Zusammenhang Beachtung, daß unser Conrad Mayr einmal der Faktor der Fugger genannt wird 3. Nun war, wie wir genau wissen, noch beim Tode Jakob Fuggers des Reichen (1527) ein Conrad Mayr in Fuggerschcn Diensten 4. Höchstwahrscheinlich war dieser Conrad Mayr mit unserem Monopo¬ listen identisch, wenn damit auch nicht bewiesen ist, daß er 1549 noch wirklich in Fuggerschen Diensten stand und die böhmische Zinnaktion im Aufträge der Fugger durchführte. Jedenfalls waren die Fugger ganz genau über die Sachlage orientiert. Das ergibt sich aus folgendem: In mehreren Exemplaren 3ist eine Art Lehrbuch der Buch1 Ro t h a . a. O., 4 . Bd ., S. 449 . Die Rechnungen Mayrs , be¬ ginnend mit dem 2. April , über seine Ausgaben „ für den Lauf der Knechte “ haben sich erhalten ; cfr . Roth, S . 546 , Anm . 80 und S . 547 , Anm . 91. 2 Vgl. J . Strieder, Zur Genesis des modernen Kapitalismus. 3 In einer Beschwerde und Bittschrift der Landschaft unter der Enns (1552 ). Msc . im K . K . gemeins . Finanzarchiv zu Wien, Loc . Böhmen . Schlackenwald 1552 . Letztes Blatt des nichtpaginierten Konvoluts. 1 M . Jansen, Jakob Fugger der Reiche , S. 66 f., 181, 332. 6 Stadtbibliothek Elbing , F . 30 . Stadtbibliothek Danzig, 266 Drilles Buch : Monopole, Kartelle und Aktiengesellschaften im Mittelalter. haltung, von dem Fuggerschen Hauptbuchhalter Matheus Schwarz verfaßt, auf uns gekommen1. Darin hat Schwarz auch zwei große Musterbuchführungen mitgeteilt. Die eine bringt eine Muster¬ abrechnung der venetianischen Faktorei der Firma Fugger und bietet zweifellos in ihren weitaus meisten Partien die Original¬ geschäftsführung der Fugger in Venedig aus dem Jahre 1516. Leider konnte die wertvolle Quelle von Jansen in seinem „ Jakob Fugger der Reiche“ nicht benutzt werden. Wir gedenken später darauf zurückzukommen. Hier ist es die andere von Matheus Schwarz gegebene Musterbuchführung, die uns interessiert. Sie wird von dem Fuggerschen Hauptbuchhalter wie folgt eingeleitet: „Auf 17. Aprilis im 1550 jar kam ein namhafftige herr on namen zu mir M. Schwartzen und sagt wie das- er gern wolt eine neue handlung mit, dem Schlaggenwalder z i n anfahen. Darzu muss er haben vill gelts, auch diener an mer orten und er woldt das haupt zu Augspurg bleiben. Darauf bat er mich, ich wolle ein formular eins buchhalten machen. . . . Darauf hab ich im wie hierinnen volgt ein kurtzen formular gestelt, des ich ime aus guter freundschaft halber nit liab konden abschlagen2.“ Der Inhalt des „Formulars“ ist nichts weniger . jnd nichts mehr als der effektive (nicht fingierte) Niederschlag der tatsächlichen Geschäftsführung des böhmischen Monopolunternehmens des Konrad Mayr, wenigstens in den ersten Monaten des Bestehens der Firma. Die au den Augsburger von den böhmischen Gewerken gelieferten Mengen Bleis werden in dem Formular des Matheus Schwarz mit den einzelnen uns auch sonst überlieferten Quanten genau auf¬ geführt. Die dafür gezahlten Summen stimmen mit den kontrakt¬ lich verabredeten Preisen überein. Auch sonst ist die Ausführung im einzelnen so detailliert, so individuell, daß die Annahme einer methodologischen Fiktion unmöglich erscheinen muß. Also: Wenn der Hauptbuchhalter der Fugger dem Konrad Mayr für das Schlackenwalder Geschäft die Buchführung machte, Manuskript 2297 . Außer diesen beiden Handschriften , auf die mich Herr Prof . Di'. B . Penndorf freundlich aufmerksam machte , konnte Ich eine dritte in der Wiener Hofbibliothek feststellen. 1 Die Elbinger Handschrift ist 1551 , die Danziger 1564 datiert. Vgl - jetzt B . Penndorf, Geschichte der Buchhaltung in Deutsch¬ land . Leipzig 1913 . S. 48. 2 Aus Matheus Schwarz , Bl . 48 der Danziger Handschrift. Viertes Kapitel: Rohm .-Sachs . Monopol - und KavtellbestrebLUige.11 . 267 wenn ferner — wie aus dieser Buchführung ersichtlich 1— die Fugger für den Schlaekenwalder Zinnkauf mit Mayr in lebhafter Geschäfts¬ verbindung standen , wenn endlich Konrad Mayr auch sonst eng mit den Fuggern verbunden erscheint , so darf man sich nicht wundern, wie das Gerücht entstehen , sich verbreiten und Jahrzehnte lang erhalten konnte , die Fugger seien die Inhaber des böhmischen Zinnmonopols von 1549 lf. gewesen. Erwähnt sei schließlich noch, daß die Fugger auch sonst in jenen Jahren besondere Unternehmungen unter dem Namen und unter der Firma von treuen , zuverlässigen Faktoren gehen ließen a. Wie dem auch sei, ob hinter Konrad Mayr die Fugger standen oder nicht , in den Kontrakten , die Mayr mit König Ferdinand über das böhmische Monopol abschloß , ist von dem großen Augsburger .Handelshaus nicht die Rede . Hier steht Konrad Mayr allein dem Könige gegenüber , dem er — angeblich zinslos — 30 000 fl. als Darlehen gewährte , um dafür das böhmische Zinnhandelsmonopol auf vier Jahre (vom 1. Januar 1550 bis zum 31. Dezember 1553) in' Empfang zu nehmen . In Wahrheit erhielt Konrad Mayr 5% % p. a. Es wurden ihm nämlich , so war es im Kontrakt ausgemacht, drei Jahre lang je 100 Zentner geschmeidiges Zinn gratis geliefert („frei on ainich bezalung “). Sonst hätte Mayr für den Zentner 23 fl. zahlen müssen — 6900 fl. für 300 Zentner . Er erhielt also jährlich von 30 000 fl. 1725 fl. Zinsen, d. h. 5% %. Die. genannten vier Jahre hindurch mußte König Ferdinand dem Konrad Mayr alles Zinn , das er seihst von den Zimigewerken zum Preise von 18 /, resp . 18 oder 173/2 resp . 17 fl. (s. o.) erhielt 3, für 23 fl. bei geschmeidigem Zinn , für 22 fl. bei ungeschmeidigem überlassen . Konrad Mayr anderseits war verpflichtet , alles Zinn, das ei’baut wurde , abzunehmen , es sei denn , daß Krieg in Böhmen ausbi'äche . Wenn er in diesem Falle das fertige Zinnprodukt „ mit Sicherheit nicht erheben noch vertreiben “, oder das Geld zur Be- 1 Ich gehe hier nicht auf die Buchführung ein; ich hoffe in der geplanten, großen Edition der süddeutschen Handelsbücher und ver¬ wandten Akten des IS. und 16. Jahrhunderts, deren Leitung mir die Historische Kommission bei der Königl. Bayrischen Akademie der Wissenschaften übertragen hat, später auf die Buchhaltung des Matheus Schwarz zurückkommenzu können. 2 Vgl . K . H ä b 1e r , Die Geschichte der Fuggerschen Handlung in Spanien, S. 109. 3 Diese Summe wurde das Losungsgeld genannt, während der Verdienst des Herrschers „Bevorstand“ hieß. 268 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. Zahlung des Zinns nicht gefahrlos gen Schlackenwald bringen könne, so wollte sich Ferdinand mit ihm in Güte einigen. Damit Konrad Mayr besser über Absatz und Bezahlung des Zinnes disponieren konnte , waren die königlichen Verwalter oder Amtleute zu Schlackenwalde gehalten , ihm oder seinen Leuten bei jeder Zinnlieferung schätzungsweise anzugeben , wieviel Zinn im nächsten ,, Quartal “ geliefert werden konnte . Wollte der König nach Ablauf der festgesetzten vier Jahre den Zinnkauf weiter ver¬ geben , so mußte er ihn dem Konrad Mayr überlassen , wenn dieser dasselbe bot wie ein anderer Reflektant (Vorkaufsrecht ). Die Rück¬ zahlung der vorgestreckten Summe sollte in dem letzten Halbjahr der Kontraktzeit durch Zinnlieferungen des Königs erfolgen. Durch den Kontrakt vom 6. Dezember 1549 rückte Mayr in den Mittelpunkt des böhmischen Zinnbergbaus . Seine Kapitalien dirigierten die Produktion . Er bezahlte die Gewerken , die den königlichen Amtleuten ihr Zinnprodukt überlieferten . Er verrechnete dann halbjährlich die dem König vorbehaltene Gewinn¬ quote . Wollte der König auf diese Einkünfte eine Anleihe machen, so mußte er Konrad Mayr davon benachrichtigen und zunächst mit ihm über die Anleihe verhandeln , ehe er sie bei anderen Kapita¬ listen aufnahm . Der Vertrieb des böhmischen Zinnes war somit ganz in die Hände des Augsburger Kapitalisten gelegt . Nur einige Verkaufsbeschränkungen waren in dem Kontrakte des 6. Dezember festgelegt worden . So sollte Mayr der Gesellschaft des Zinnblech¬ handels zu Amberg in der Pfalz 1 alljährlich 500 Zentner Zinn zu dem Einkaufspreis (23 resp . 22 fl. pro Zentner ) überlassen . Auch den Zinngießern in Schlackenwald und Graupen mußte er jährlich 2—300 Zentner zum Erstehungspreise abgeben . Endlich behielt sich der König vor, jährlich bis 500 Zentner „zu Notturft des Geschütz“ und „ zu anderen Gebrauch “ der Krone direkt von den Gewerken zu beziehen. Bei der Bevorzugung der Amberger Gesellschaft des Zinnblech¬ handels müssen wir noch einen Augenblick verweilen . Die Gesell¬ schaft hatte schon früher , als Schlackenwald usw. noch den Herren von Pflug gehörte , eine Ausnahmestellung als großer Zinnabnehmer innegehabt . Freilich drohte auch bereits in den dreißiger Jahren 1Näheres über die Gesellschaft des Zinnblechhandels siehe oben S. 145 ff. 2 Vieltes Kapitel : Böhm.-sächs, Monopol- und Kartellbestrebungen . 269 hierin eine Änderung . Hans Pflug schrieb am St . Ursulatag 1535 an die Gesellschaft des Zinnblechhandels in Amberg , daß er schwer¬ lich in Zukunft noch , wie sie bäten , die üblichen 400 Zentner Zinn an sie abgeben könne , da er beabsichtige , bei „ seinen Bergwerks¬ verwandten und Untertanen “ durchzusetzen , alles Zinn , welches auf seinem Bergwerk in Schlackenwalde gewonnen würde , „kaufs¬ weise in ein handt zu gebe n “ \ Pflug konnte auch in demselben Briefe mitteilen , daß er bereits mit einer Gesellschaft über die Vergebung des Schlackenwalder Zinnkaufsmonopols in Unterhandlung stehe. Kurze Zeit hat damals die Amberger Kompagnie daran gedacht, selbst das Monopol zu übernehmen . Aber schließlich fand man doch nicht den Mut für solch ein großzügiges Unternehmen . Um so eifriger versuchte die Amberger Gesellschaft sich auch trotz der drohenden Monopolisierung noch ein monopolfreies Zinn¬ kontingent in Böhmen zu sichern . Als Ferdinand am Ende der vierziger Jahre den Monopolgedanken der Pflug auszuführen begann (siehe oben), wandten sich die „ Geordneten “ der Gesellschaft des Amberger Zinnblechhandels in einem dringenden Schreiben an den Pfalzgrafen Friedrich 1. Sie hätten , führten sie darin aus , davon gehört , daß Ferdinand beschlossen habe , alles böhmische Zinn durch die Kammer aufzukaufen . Dann folgt in dem Brief die nach¬ folgende , beachtenswerte Stelle : „ Dieweiln nun , wie man den hof erkhennt , unsers vermutens solicher contract directe nit uf die kgl. camern sonder vermittls darunder lauffenden practickhen uf sonders andere personen sich erstregkhen wirdet , haben wir uns ferner sovil erkhundigt , dass etliche genachparte zu Nürnberg und Augspurg im handl steCkhen, denselben zinkhauf aus der k. Mt. camern fortan in i r handt und gewalt zu pringen . Es soll auch vasst mit inen dahin abgehandlt sein, daruss gut abzunemen ist, mit solichen zin (nachdem das englisch [Zinn ] in disem OberTeutschlandt nit so vertriblich ) einen monopolischen , aigennutzigen 1 An anderer Stelle des Briefes heißt es: Pflug wolle „alle zin durchaus, so auf meinen bergwerckh zu Schlackenwalde gemacht und berait werden, mit der Vorlage und kauf in einer gesellschaft hand stellen.“ Amberger Kreisarchiv. Amberg Stadt. Nr. 42. Fase. 326. 2 .3 Mai 1549. Kreisarchiv Amberg. Amberg Stadt. Nr. 115. Fase. 327. 1 270 Drittes Buch: Monopol «, Kartelle und Aktiengesellschaften im Mittelalter. handl , wie mit andern mer ufzurichten .“ Als sich daraufhin Pfalzgraf Friedrich an Ferdinand mit der Bitte wandte , er möge die Monopolinhaber des böhmischen Zinns verpflichten , während ihrer Kontraktperiode jährlich der Amberger Gesellschaft 450—550 Ztr. Zinn zu mäßigem , festen Preis zu liefern \ willfahrte der König diesem Wunsche , wie wir aus dem Kontrakt mit Konrad Mayr sahen. Den obengenannten Beschränkungen der Verfügungsgewalt Konrad Mayrs über das böhmische Zinn standen bedeutende Förde¬ rungen von seiten der Krone gegenüber . Im Königreich Böhmen war das Zinn Mayrs von allen Zöllen, Mauten usw. ausgenommen. In den übrigen Ländern Ferdinands durfte es über die gewöhn¬ lichen „Zölle, Mauten und Aufschläge “ nicht in Anspruch genommen weiden. Ferner genoß Mayr noch einen erhöhten königlichen Schutz gegenüber allen anderen Kaufleuten in Ferdinands Landen . Dieser Schutz bezog sich auch charakteristischerweise auf etwa vor¬ kommende Belästigungen Konrad Mayrs durch die gegen Monopole gerichtete Reichsgesetzgebung . Wenn Mayr „ diser zinhandlung halber , als ob er ain verbotene oder monopolische handlang damit getriben oder geuebt het , vor der röm . kais . Majestät , derselben camergericht oder kaiserlichen viscall 2, oder andern orten beclagt werden sollte “, so wolle ihm Ferdinand helfen und ihn in seinen Schutz nehmen 3. Der König war sich übrigens ganz klar darüber , daß Mayr 1 F.A. Wien . Böhmen . Schlackenwalde 1549. 2 Diese Rückendeckung durch den Kaiser resp . den deutschen König scheint damals allgemein üblich gewesen zu sein . So wurde 1524 in einem Quecksilberkontrakte Ferdinands I . und der Gewerken von Idria mit den Höchste Item von Augsburg der König zur Hilfe¬ leistung verpflichtet , falls die Kontrahenten „ ditz kaufs halb von des raichs regimen t oder desselben fiscalen “ angesprochen wurden. F . A . Schmidt a . a . O., III , 1, S. 113 ff . hat sinnlos „ Filialen “ . Der Fiskal ist der Anwalt des Reiches , der die Prozesse gegen die Monopolisten zu führen hatte . Siehe oben S. 71 ff ., besonders S. 80/1. 3 Erst auf heftiges Drängen Mayrs und auf dessen Hinweis, daß auch andere einen derartigen Schutz erhalten hätten , hatte sich Ferdinand zu dieser Zusage bereitfinden lassen . Vgl . das Schreiben an die Unterhändler Heidenreich und Metschberger 23 . Oktober 1549 . F .A . Wien . Böhmen , M. u . B . 1576— 1590 (16 407 ). 3 Viertes Kapitel : Böhm.-säclis. Monopol- und Kartellbcstrcbungen . 271 den Kontrakt benützen würde, um Monopolpreise zu erzielen. Er schreibt in der Instruktion für Heidenreich und Mctschberger1: „Wissentlich ist wo die zin in aines handt lchumen, dass dersclb die schier seines gefallens mit grosser staigerung und nutz hin¬ bringen thuet . Und ob er gleich ain halbs oder gantz jar etwas wenig sp..... 2so bringt er doch denselben Verzug in den nachvolgenden jaren topelt wider herein.“ Das wichtigste Versprechen aber, das König Ferdinand dem Konrad Mayr in dem Kontrakte vom 6. Dezember 1549 gab, bezog sich noch auf etwas anderes. Der Augsburger wäre ein schlechter Kaufmann gewesen, wenn er nicht gewußt hätte , daß sich das Zinnhandelsgeschäftnur durch Verabredungen mit den sächsischen Produzenten wirklich hochrentabel gestalten würde s. So ließ er sich denn in dem Kontrakte vom 8. Dezember 1549 von Ferdinand feierlich versprechen, daß dieser mit dem Kurfürsten von Sachsen in Unterhandlungen eintreten wolle, die ein Kartell der Beherrscher der sächsischen und der böhmischen Zinnproduktion herbeiführen sollten. Entweder ein Preiskartell, in welchem sich die sächsische Regierung und die Gewerken des sächsischen Zinnbergbaues ver¬ pflichteten, das Zinnprodukt ihrer Gruben nicht billiger au die Zinnhändler abzugeben, als es Konrad Mayr in Böhmen erhielt. Oder ein Gebietskartell, in welchem die Kaufleute, die mit säch¬ sischem Zinn handelten, versprachen, ihre Ware nur dorthin zu führen und zu verkaufen, wo damit Konrad Mayr keine Konkurrenz gemacht würde. Sollten sich unüberbrückbare Schwierigkeiten für das Zustande¬ kommen eines Kartells ergeben, so wurde Konrad Mayr ein Ab¬ schluß der Grenzen der habsburgischen Lande gegen sächsisches und überhaupt fremdes Zinn zugesichert. 1 F.A. Wien . Böhmen AI. u . B . (1570 —1590 ), Nr . Iß 407. 2 Unleserliches Wort , das soviel bedeutet wie „verdient “ . 3 „Do anno im 50 . johr die kaufleute von Augspurck mit denen zue Schlackenwahle uff 20 jahr einen zihnkauf geschlossen , dormeber brief und Siegel geben , auch Vorhabens gewesen die z i h n in E . c h f. G. landen an sich zue z i h e n . . .“ Aus einem Gutachten der gemeinen Zinngewerken und Vorleger von St . Annaberg , Buchholz , Geyer , EhrenEricdersdorf und Thum vom Jahre 1569 . Hauptstaatsarchiv Dresden , Loc . 36 080 , Nr . 660, Bl . 102 a. 272 Drittes Buch : Monopole , Kartelle und Aktiengesellschaften Zweiter im Mittelalter. Abschnitt. Vergebliche Versuche den böhmischen und sächsi¬ schen Zinngroßhctndel zu kartellieren (1549 ff.). Konrad Mayrs Fall. Als Ferdinand in dem Kontrakt vom 6. Dezember 1549 Konrad Mayr versprach , dafür zu sorgen , daß die sächsische Konkurrenz ihm nicht in den Rücken fiele, waren schon seit längerer Zeit dies¬ bezügliche Verhandlungen gepflogen worden . Schon in einem Bericht vom 5. August 1549, den Erzherzog Ferdinand dem König Ferdinand I. über die Verhandlungen gab , die er mit den Bevoll¬ mächtigten Konrad Mayrs gehabt hatte , erwähnte der Erzherzog die Forderung des Augsburgers , die sächsische Konkurrenz aus zuschalten 1. Er schlug vor , Ferdinand I. möge auf der bevor¬ stehenden Reise nach Sachsen mit Kurfürst Moritz darüber unter¬ handeln. Ferdinand zweifelte nicht , daß Moritz von Sachsen für die finanziellen Vorteile , die auch für ihn aus einem sächsisch -böhmischen Zinnmonopol resultierten , empfänglich sein würde . Er meinte charakteristisch in einem Schriftstück aus jenen Tagen : „ gedachter churfürst von Sachsen werde den genues und nutz so wenig als wir nit dahinden lassen , sondern ee noch ain merers als wir heraus bringen “ wollen 2. Tatsächlich hat Ferdinand I. von seinem säch¬ sischen Besuche im September 1549 das Versprechen Moritzens mit nach Hause gebracht , auch in Sachsen einen Zinnkauf einzuführen, der dem böhmischen „gleich und gemäss gestelt “ sei 3. Er konnte 1 K. K. Statthaltereiarchiv Prag, Kopialbuch-Nr. 45 (Hof¬ berichte 1549—1550), Fol. 14—15. Der Vertreter Mayrs hatte aus¬ geführt, daß „zu fürderung des handeis sowol Ew. Mt. als iine zue guten von nötcn sein weil, die zin, sovil deren in des churfürsten zu Saxon landen und gepieten gemacht, auch an Ew. Mt. zu bringen oder mit seinen churfürstlichen gnaden sovil zu handeln, damit sein churfürstlichegnaden bei derselben zin-pergkwerchen und gewercken verfliegen welle, die zin in dem khauf wie sich Ew. Mt. mit ime Maier oder andern vergleichen wurden, auch zu verkhaufen und hinzugeben." 2 Instruktion für Erasmus Heidenreich und Hans Metschberger. 5. September 1549. F.A. Wien. Böhmen, M. u. B. (1570—1590), Nr. 16 407. 3 Schon am 26. September drang Ferdinand I. in Moritz, sein Versprechen zu halten . F .A. Wien. Fase . Böhmen, M. u. B. (1570 Viertes Kapitel : Böhm.-säehs. Monopol- und Kartellbcstrebungen . 273 am 11. Oktober 1549 seinen Unterhändlern mit Konrad Mayr er¬ klären , daß Moritz seine „ zin nit geringer oder leichter als wir die unsrigen zu geben willens sei“ 1. Kurfürst Moritz von Sachsen hat es denn auch nicht an Ver¬ suchen fehlen lassen , die Zinngewerken seines Landes zu einem Zinn¬ monopol zu bewegen. Am 5. November 1549 ließ er den säch¬ sischen Zinnern mitteilen , erhalte es für nötig — um , über dem jetzt wieder wachsenden Export englischen Zinnes , den Zinnpreis nicht sinken zu lassen 2 daß „ zwischen den ziengewercken und etlichen kaufleuten auf eine antzal jhar ein gewisser zienkauf “ geschlossen werde 3. Moritz weiß auch schon, daß die Kaufleute nicht für einen bis 1590 ), Nr . 16 407 . Moritz antwortete unter dem 3. Oktober von Chemnitz aus , er möchte zuvor wissen , wie Ferdinand den Zinn¬ kauf in Böhmen kontrahiert habe , damit er sich danach richte und „sovil muglich gleichheit hielte “ . F .A . Wien . Böhmen , M. u . B. (1540 — 1569 ), Nr . 16 406. 1 A . a . O., Böhmen , M. u . B . 1549. 2 Interessant sind die bevölkerungspolitischen Begründungen , die, in rein merkantilistischer Art , den churfürstlich -sächsischen Denk¬ schriften jener Zeit beigegeben werden . Da wird stets auch die Ver¬ mehrung bzw . numerische Erhaltung der Bevölkerung — der Mann¬ schaft , wie man sich ausdrückt — als politischer Maxime gedacht. 3 Der folgende Entwurf eines Zinnkaufs von Dr . Fachs (Ende 1549 ) zeigt , wie man sich ungefähr die Sache in Dresden dachte. Hauptstaatsarchiv Dresden , Loc . 36 080 , Nr . 666 , Bl . 11: „1 . Der zienkauf sal N jhar stehen. 2. Der kaufman sal das zien auf seinem kosten besage des zienkaufs wie der mit den gewerken beschlossen , vorlegen , gewegen , an¬ nehmen , einschlahen und wegschaffen. 3. Der kaufman sal auf unsers gnedigsten hern gebure 20 000 fl. ungeverlich ane interesse hiraus geben und auf die letzten jhar an jeden centner die gebuer abrechnen und dadurch die 20 000 fl. wieder bekommen. 4. Sal der kaufman den gewerken ire zien alle sonnabent bahr um bahr betzalcn und unserm gnedigsten hem s. cf. gn . gebure alle Leipzigkcr merckte erlegen. 5. Der kaufman sal im lande von den handwergs -leuten über 1 fl. von einem centner zien nicht zu gewin nehmen. 6. Den Rulicken [Rholingkj ist im vertrauen zugesagt , sie mit etwas zu bedencken , davon man weiter sollte reden . [Dieser Para¬ graph ist durchgestrichen .] 7. Wie der zienkauf mit den ziengewercken beschlossen also solten es die hendler annehmen und unserm gn . h . von jedem centner 2 fl . entrichten ." Strieder . KUelicn z. Oeseli . k y.pitalist , Organisai ionsformen . J8 1 274 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften Im Mittelalter. Zinnkontrakt zu haben sein werden , wenn ihnen nicht ein Monopol gegeben würde . Sein Ausschreiben an die Gewerken enthält außer¬ dem bereits den Vorschlag des von den Monopolinhabern zu zahlenden Zinnpreises (1214 fl. in bar , bei Verlagsgewährung im höchsten Falle 9 Groschen pro Zentner weniger) . Waren die meisten Altenberger Gewerken und einige von Geyer für das Monopol, so erklärten sich die übrigen Geyrer Zinner sowie die Thumer und Ehrenfriedersdorfer dagegen und baten , sie mit dem monopolistischen Zinnkauf zu verschonen . In erster Linie natürlich die Großgewerken . Besonders lebhaft klang der Protest der reichen Familie Rholingk , deren Mitglieder zu den größten Gewerken im Lande gehörten und überdies hohe Stellen in der sächsischen Bergverwaltung bekleideten . Ihr Führer , Hans Rho¬ lingk , sprach seine Verwunderung darüber aus , daß man die Ge¬ werken von Altenberg , auch etliche von Geyer „zue einem zienkauf “ nach Dresden beschieden habe , ohne auch sie zu laden 2. Sie seien doch die größten Gewerken in Geyer und Thum . Ihrer Unternehmungslust und Kapitalkraft hätten die dortigen Berg¬ werke auch in schwierigen Zeiten das Meiste zu danken gehabt. Hans Rholingk wies auch darauf hin , daß ihm die Gewerken „ allent¬ halben in 2000 fl. vorleggelt schuldig “ seien. Wie sollte er zu diesen Vorschüssen kommen , wenn auswärtigen Kaufieuten das sächsische Zinngroßhandclsmonopol zugesprochen würde ? Wie ständen sie auch in diesem Falle vor ihren bisherigen Abnehmern? Sie hätten Abschlüsse gemacht , sich „auch zum theil gelt darauf geliehen “, jetzt möchten sie doch „glauben halden , auf dass sie ein andermal dorften wiederkommen “ 3. Zweifellos war es dem energischen Zugreifen der Rholingks in erster Linie mit zu danken , wenn sich die Gewerken der Zinnberg¬ werke in Annaberg , Geyer, Ehrenfriedersdorf , Buchholz , Thum, Eibenstock , Schwarzenberg usw. ebenfalls gegen ein Zinngroß¬ handelsmonopol aussprachen . Die Schriftstücke , die die genannten Zinngewerken nach Dresden expedierten 4, ähneln einander oft so 1 Hauptstaatsarchiv Dresden , Loc . 36 080 , Nr . 666 , Bl . 1 ff. a Das folgende alles nach einem Schreiben der Rholingk vom 2 . November 1549 . Hauptstaatsarchiv Dresden , Loc . 36 080 , Nr . 666, Bl . 19 ff. 3 Zu ihren Kunden , denen sie Zinn verkauft hatten. 4 A . a . O., Bl . 21 ff. Viertes Kapitel : Böhm .-sächs . Monopol- und Kartellbestrebungen . 275 stark und klingen in manchen Teilen so lebhaft an Rholingksche Gedankengänge an , daß man versucht ist , die starke Hand Hans Rholingks dahinter zu suchen . So betonten sie fast sämtlich , daß es den ärmeren Gewerken schwer fallen , ja unmöglich sein würde, die Verlagssummen den bisherigen Verlegern sofort zu bezahlen. Und schwerlich nur würden sich die Monopolinhaber bereit finden lassen , mit dem neuen Verlag auch c&e Testierenden Vorschüsse zu übernehmen . Lagen solche Erwägungen immer noch im Gesichts¬ felde der kleinen Gewerken der genannten sächsischen Gebirgsorte , so lassen gewisse bevölkerungspolitische Erwägungen ihrer Denkschriften , die auf die Regierung Eindruck machen sollten , den Rholingkschen Einfluß deutlich erkennen l. Dasselbe ist von einigen handelspolitischen 2 und historisch -statistischen Argumenten gegen eine Monopolisierung des sächsischen Zinngroßhandels zu sagen . Wenn z. B. in den Denkschriften ausgeführt wird : es würden schwerlich auf allen sächsischen Zinnbergwerken fünf Gewerken ge¬ funden werden , die ohne Verlag arbeiten könnten ; oder die Gesamt¬ summe des im sächsischen Zinnbergbau angelegten Verlags betrage ca. 10 000 fl. ; oder noch immer sei nach einem Zinngroßhandels¬ monopol ein Preissturz erfolgt , so sind das Erwägungen , die den genannten kleinen Gewerken fern lagen , die ihnen aber von Rholingk an die Hand gegeben sein mögen. Auf die fast allgemeine Absage der sächsischen Zinngewerken hin blieb Moritz kaum etwas anderes übrig , als von dem Projekte einer Monopolisierung des sächsischen Zinngroßhandels und eines Kartells mit den böhmischen Zinnmonopolinhabern abzusehen. Rholingk hatte dadurch dem Kurfürsten die bittere Pille schmack¬ hafter zu machen gewußt , daß er vorschlug , der Landesherr möge von jedem Zentner Zinn , der in seinem Herrschaftsgebiet (außer in 1 Da heißt es zum Beispiel : Durch den Zwang die bisherigen Verleger sofort zu bezahlen , würden viele Gewerken bankrott gehen und vom Bergbau lassen . Die Arbeiterschaft müßte dann brotlos werden und aus Sachsen auswandern. 2 Ein „ Zinnkauf “ schade dem Handel auch deshalb , „ weil das zin in eine handt , daraus ein monopolischer handel entsteht , ge¬ bracht " , nun nicht mehr vielen Leuten Verdienst gäbe . „ Denn wann under vil leuthen ein wahr vertrieben , so mag die bei wirden . . . erhalden werden . Denn mancher furth dem lande etwas zu , legt das gekaufte gelt an zihn , brengt dadurch sein gelt und Wechsel ane gefahr in sein gewarsam . Dadurch sein die zhin bisher in grosse staigerung kommen .“ 18 276 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. Altenberg ) gewonnen würde , 4 fl. über den bisherigen Zehnten hinaus nehmen . Moritz war auf die Anregung eingegangen , hatte aber — soviel ich sehen kann — 1 fl. gefordert und wohl auch zu erhalten gewußt . Nur für Altenberg hatte Moritz einen Zinnkauf auf 20 Jahre übernommen und zeitweise an Leipziger Kaufleute übergeben (s. o. S. 261 Anm . 3). Mit dem Scheitern des Versuches, in Sachsen ein allgemeines Zinngroßhandelsmonopol aufzurichten , war der kühne Plan einer Beherrschung des mitteleuropäischen Zinnbergbaues und Zinngroß¬ handels , der Konrad Mayr vor Augen gestanden hatte 1, vernichtet. Zwar verzagte der kühne Augsburger nicht . Wenigstens einem Teile Europas wollte er mit Hilfe seines böhmischen Monopols den Zinnpreis diktieren . Konrad Mayr hat es bei Ferdinand I. durch¬ zusetzen gewußt , daß dem sächsischen , wie überhaupt allem fremden Zinn der Weg in die habsburgischen Erblande durch eine umfassende Grenzsperre verlegt wurde . Schon am 20. September 1550 erging ein von Augsburg , dem Wohnsitz Konrad Mayrs, aus datiertes Generalmandat des Habsburgers an alle Landeshauptleute , Land¬ vögte , Bürgermeister und Räte der Städte , besonders aber an alle Zollstätten ab , in welchem die strikteste Schließung der Grenze vor fremdem Zinn angeordnet wurde 2. In dem Mandat war es aus¬ gesprochen , daß die Ausländer ihr Zinn in dem bisherigen böhmischen Absatzgebiete billiger — „ in nehern khauf und pretio “ — verkauft hätten als der böhmische Monopolinhaber . Jetzt wurde nicht nur die Einfuhr , sondern auch die Durchfuhr , der Transit dieses fremden Zinnes durch die habsburgischen Lande bei Strafe der Einziehung der Ware angeordnet. Auf solche Weise meinte Konrad Mayr unbehindert von der sächsischen Konkurrenz , wenigstens im Osten und Süden Europas 1 „Wie es weylendt dem Conradt Mayrn von Angspurg ergangen sei, als der sich aucli auf e r h a n d 1 u n g der Meisnischcn zin verlassen und hernach , da er dieselben nit gehaben mugen und an den verschleuss seiner datzumal ge¬ habten behaimbischen zin dardurch gesperrt und verhindert worden, in verderben gerathen muessen .“ Aus einem Schreiben Kaiser Maximilian II . an Kurfürst August von Sachsen (Prag , 8. Jan . 1570). Hauptstaatsarchiv Dresden , Loc . 36 080 , Nr . 664 , Bl . 32. 2 Das Mandat ist gedruckt bei F . A . Schmidt, Berggesetze Österreichs . I . Abt . 2. Bd ., Nr . 49, S. 339 ff. 1 Viertes Kapitel : Böhm.-sächs. Monopol- und Kartellbestrebungen. 277 seine Pläne verwirklichen zn können. Er hielt eine geraume Zeit mit dem Zinnabsatz zurück und hoffte dadurch den Preis seiner Ware in die Höhe treiben zu können. Das Gegenteil der erhofften Wirkung trat ein. Zwei Tatsachen besonders machten die Pläne des Augsburger Kapitalisten scheitern. Einmal ließen sich die Grenzen der habsburgischen Länder nicht vollständig gegen die Einfuhr und Durchfuhr sächsischen und wohl auch anderen, be¬ sonders englischen Zinnes sperren. Eine starke Kontrcbande setzte ein. König Ferdinand I. sah sich am 6. März 1551 in einem wiederum in Augsburg ausgestellten Mandat gezwungen, seine Amtleute an den Zoll- und Mautstellen auf die unbedingte Durch¬ führung der Grenzsperre aufmerksam machen zu lassen, „auf dass sovil muglich die contrabanden hierinnen verhuet werden“ L Die immer wieder notwendige Wiederholung des Mandats zeigt, daß sich der Schmuggel nicht unterbinden ließ 2. Bald mußte sich Mayr von neuem bei Ferdinand beschweren, „dass dieselbigen fremden zin allenthalben in Ferdinands khunigreich, fürstenthumb und lande gebraucht, darinnen kauft, verkauft und durch dieselben in andere land und nationen ungeacht der derhalben ausgangen generalverpot verfuert“ würden 3. Zweitens aber hatte Konrad Mayr bei seinem Projekte einer künstlichen Preissteigerung des Zinnes die zweite wichtige Vor¬ bedingung des Gelingens eines derartigen Planes nicht in Rücksicht gezogen, die relative Entbehrlichkeit dieses Handelsartikels. Es war durchaus richtig, wenn der sächsische Kammerrat Bernstein im Jahre 1585 in einem Gutachten über ein sächsisches Zinnmonopol schrieb: „ Zin ist ein wahre, der man so gar notwendig nicht bedarf.“ So kam, was kommen mußte. Konrad Mayr fehlte es schließlich 1 Mandat an die böhmischen Kammerräte, das Zinneinfuhr- und Durchfuhrverbot strikte durchzuführen. Augsburg, 6. März 1551. Abgedruckt bei F. A. S c h m i d t a. a. O., Nr. 50, S. 342. 2 Cfr . F.' A. S c h m i d t a. a. O., Nr. 55, S. 354. Zwei Zinneinfuhrverbote (vom 24. November 1550 resp. 12. April 1551), die Schmidt nicht aufführt, sind abgedruckt in den Mitteilungen des Vereins für Geschichte der Deutschen in Böhmen. 46. Jahrgg. (1908). S. 286 ff. Buchholtz, Geschichte Ferdinands I. 4. Bd., S. 520, nennt außerdem ein Zinneinfuhrverbotvom 1. Juli 1551. Vgl. auch J. Falke, Die Geschichte des Kurfürsten August von Sachsen in volkswirtschaftlicher Beziehung, S. 297 ff. 3 Anfang 1553. F.A. Wien, Gedenkbuch (Böhmen 1553—1554), Nr. 307. 278 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. an Kapitalien , um die gesamte Produktion des böhmischen Zinnes zu bezahlen und dann in seinen Faktoreien zurückzubehalten , bis die Hausse kam . Wohl ließ ihm Ferdinand an dem Zinn , das er von Mitte des Jahres 1552 bis zum Ende seines Kontraktes noch abzunehmen hatte , 2 fl. von seinem Profit (dem sog. Bevorstand) nach , wohl hob ferner Ferdinand 1. in all seinen Ländern für alles böhmische Zinn Konrad Mayrs , das er „darinnen verschleissen und verkaufen oder dardurch in ander land und nationen furen wirdet , alle dreissigist , aufschleg , meut , zoll und andere dergleichen gepurnus “ auf 1, aber alles das genügte nicht , um das drohende Unheil abzuwehren . Der Zentner sächsischen und anderen Zinnes wurde um 5 fl. billiger auf den Markt gebracht , als Mayr das böhmische Zinn erstand ; eine Änderung dieser Verhältnisse war nicht abzusehen . Die Gewerken waren nicht zu bewegen, eine Preisreduktion eintreten zu lassen 2. So mußte der Augsburger schließlich mit seinem Zinn losschlagen . Eine Masse des bis dahin etwas knappen Produktes kam dadurch auf einmal zu Markt . Ein ungewöhnlicher Preissturz war die natürliche Folge. Zinn , das noch 1549 etwa mit 14 rh . fl. allgemein bezahlt worden war , galt jetzt (1551—1553) kaum noch 10 fl. Man erzählte sich in Sachsen, daß die Augsburger eine Tonne Goldes bei dem Handel verloren hätten . Andere schätzten den Verlust des Konrad Mayr auf 100 000 fl. 3 1 Nach einem Schreiben König Ferdinands I. an Erzherzog Ferdinand. Graz, den 20. Januar 1553. F.A. Wien. Gedenkbuch (Böhmen 1553—1554), Nr. 307, Bl. 7—10. 2 Sternberg, Umrisse einer Geschichte der böhmischen Bergwerke I. 1, S. 290. 3 Vgl . die Denkschrift der kurfürstlichen Kammerräte vom 12. Oktober 1569 (Hauptstaatsarchiv Dresden, Loc. 36 080, Nr. 664, Bl. 12 a unten). „Es werete aber solcher kauf nicht allein nicht lange, sondern es volgete auch das doraus, dass entlieh aus mangcl des geldes sie domit losschlagen mussten und dordurch die czin nichts weniger in s. chf. gn. landen als in der chron B e h e i m b in solchen fall kommen, dass man den centner umb 10 fl. kaufte. Nicht mit geringem schaden und verterb etzlicher uf s. chf. gn. czienberckwergs bauenden gewerkhen. Und werete solcher fall etzliche jar bis solcher gesamleter vorrath, doch mit grossem schaden sonderlich der hendler, so uf etzliche hunderttausent fl. sich erstreckht, verhandelt.“ Die Kammerräte, die den Kurfürsten gegen ein sächsisches Zinnmonopol einnehmen wollten, mögen die Sache etwas übertrieben haben. Viertes Kapitel : Böhm.-säclis. Monopol- und Kartellbestrcbungen. -' ■J Dritter 279 Abschnitt. Erneuter Plan einer Monopolisierung oder wenigstens Kartellierung der böhmischen und sächsischen Zinn¬ produktion (1569). Etwa zwei Jahrzehnte nach dem Versuche Konrad Mayrs , ein mitteleuropäisches Zinnmonopol in die Hand zu bekommen , setzten neue Projekte dieser Art ein. Wenn wir es nun — infolge Mangels an genügendem Urkundenmaterial — für die Monopolisierung der böhmischen Zinnproduktion von 1549 ff. nicht unbedingt sicher behaupten konnten , ob die Anregung dazu von der Gewinnbegierde Augsburger Kapitalisten oder von dem Geldbedürfnis , der Finanz¬ not des Königs ausging 1, so sind wir für die Pläne von 1569 besser daran . Für sie läßt sich mit aller nur wünschenswerten Deutlich¬ keit nachweisen , daß es die schwere Finanznot des Kaisers war, die aus einer Monopolisierung der böhmischen und der sächsischen Zinnproduktion größere Geldmittel bzw. Anleihemöglichkeiten er¬ hoffte und deshalb den Plan eifrig betrieb und leitete . Schon am 13. Dezember 1568 fragte Kaiser Maximilian II ., der zugleich König von Böhmen war , bei Georg Ilsung , dem langjährigen Vertrauens¬ mann der Habsburger in Finanznöten , auf den wir noch des längeren zu sprechen kommen werden , an , ob in Nürnberg , Augsburg oder Ulm keine Kaufleute vorhanden seien, die den böhmischen „Zinn¬ kauf “ um 20, 19 oder doch 18)4 A- den Zentner anzunehmen bereit wären 2. In den weiteren vertraulichen Verhandlungen Maximilians II. mit Ilsung und anderen Beauftragten kommt es dann ganz deutlich zum Ausdruck , daß dem Kaiser der finanzielle Vorteil, den das Monopol der kaiserlichen Kammer abwerfen mußte , die Haupt¬ sache bei der ganzen Angelegenheit war . Da heißt es einmal : der Nutzen , den die kaiserliche Finanzverwaltung von dem Zinnkauf 1 Für den, der die Finanzpolitik Ferdinands I. einigermaßen kennt, wird es keinem Zweifel unterliegen, daß auch 1549 die könig¬ liche Finanznot noch bestimmender für die Entstehung des Monopols gewesen war, als die Spekulationsneigung der Augsburger Kaufleute. 2 Vgl . auch für das folgende: F. A. Schmidt, Berggesetze der Österreich. Monarchie I. 3, Nr. 103. Allerdings ist die Urkunde dort mit recht vielen Lesefehlern abgedruckt; z. B. ist aus Konrad Mayr Conradt Mugen geworden. 280 Drittes Buch : Monopole, Kartelle und Aktiengesellschaften im Mittelalter. haben würde, könnte erst präzisiert werden, wenn man mit dem¬ jenigen süddeutschen Kapitalisten handelseinig sei, der das Monopol übernähme. Immerhin könne Ilsung schon jetzt seinem kaiser: liehen Herrn versichern, daß „derselbe der Mühe wohl wert“ er¬ scheine, und „etwas darauf zu antizipieren sein werde“, d. h. daß man auf die aus dem Zinnkauf zu erhoffenden fiskalischen Ein¬ nahmen eine Anleihe werde fundieren können 1. Ein anderes Mal heißt es in einem Bericht der an Ilsungs Seite für die Monopol¬ angelegenheit stehenden Kommission: „Also verhoffen wir auch bei den handelsleuten zu Nürmberg oder Augspurg ain solche partita zue treffen, dass Eure röm. kais. Mt. iren gebürenden nutz auch darbei haben khünden“ 2. Maximilian hat bis zuletzt erwartet, von jedem an den Monopol¬ inhaber abgegebenen Ztr. Zinn allenvenigstens 1l/ 2 fl. „Arrenda“ zu erhalten. Außerdem natürlich erwartete er, dem Brauch der Zeit bei all solchen „Käufen“ gemäß3, die Gewährung eines Darlehens in der Weise etwa wieder, wie es auch Konrad Mayr in seinem Zinnkaufskontrakte vom 6. Dezember 1549 zugesagt hatte . Sehr charakteristisch betonte der Kaiser in verschiedenen Schriftstücken zu dieser Angelegenheit, er benötige das Geld, um die Grenzen gegen den Erbfeind der Christenheit, die Türken, zu schützen! Das war offenbar als Rückendeckung gegen wirtschaftsethische Vorwürfe gedacht. Wir brauchen hier nicht näher auszuführen, wie Maximilian II. durch eine besondere Kommission, der Georg Ilsung, Wenzel Berckha und der Hofzahlmeister David Haug angehörten, im Sommer 1569 mit den böhmischen Zinngewerken über das Zinnmonopol ver¬ handeln ließ 4, wie er zunächst wieder — vergleiche im Jahre 1549 1 F.A . Wien . Böhmen , M. u . B . (1540— 1569), Nr . 16 406 . Am 8 . Februar 1570 schrieb Maximilian II . an Ilsung : „ Und ehe inan ein solche angefangne stattliche handlung , deren wir jährlichumb was ansehnliches geniessen möchten . . .“ F .A . Wien . Böhmen , M. u . B . (1570— 1590 ), Nr . 16 407. 2 Konnnissionsberichte vom 8. August 1569 . F .A. Wien a . a , O. 3 Siehe oben S. 31 fl. und sonst. 4 Näheres darüber aus der Instruktion der kaiserlichen Kom¬ missarien und aus ihren Verhandlungsprotokollen mit den Gewerken. F .A. Wien . Böhmen , M. u . B . (1570— 1590 ), Nr . 16 407 , und Haupt¬ staatsarchiv Dresden , Loc . 36 080 , Nr . 664 , Bl . 5 ff . und Bl . 23 ff. Dort auch der Entwurf des Zinnkaufskontraktes . Ich hebe daraus nur den folgenden Artikel hervor , der versucht , den kleinen Hand- Viertes Kapitel : Böhm .-sächs . Monopol- und Kartcllbestrclningcn . 281 Ferdinand I. — sich selbst das Monopol pro forma übertragen ließ und inzwischen durch Ilsung mit verschiedenen süddeutschen Kapitalisten zwecks Übernahme desselben in Verbindung trat. Das alles entwickelte sich ungefähr in derselben Weise, wie wir es bereits früher kennen gelernt haben , und wie wir es für das Idrianer Quecksilbermonopol noch weiterhin sehen werden . Nur daß sich diesmal unter den Augsburger Großkaufleuten keiner fand , der Konrad Mayrs Schicksal zu teilen gewillt war 2. Wo immer auch Ilsung in Augsburg anklopfte , bei der Gesellschaft David Haug, Hans Langenauer & Co. 3, bei der Firma Manlichsche Erben , bei Melchior Manlich, bei Hans Paul Herwart usw., überall hörte er denselben Bescheid . Man könne nur dann an die Übernahme eines böhmischen Zinnmonopols denken , wenn der Kurfürst von Sachsen „sich in ain gleiche khaufhandiung mit seinen zinen einlassen würde“ 1. In Summa : ohne ein korrespondierendes sächsisches Zinnmonopol war ein Unternehmer für einen böhmischen „ Zinn¬ kauf “ nicht zu haben , selbst wenn der Kaiser „ denselben frei ohn allen gwin übergeben wolt “. werker des Inlandes (Böhmen ) vor Monopolpreisen zu schützen: „Also auch da die zingiesser zue Schlackenwaldt und Schönfeld in zeit des werenten neuen khaufs jerlichen ein zimbliclie anzall zin, als ungevarlich von zwei bis in dreihundert c.ennten , aber doch nit merers , zu irer handtarbait (aber sonst in khain andern weeg ) be¬ dürftig sein würden , so sollen inen dieselben die neuen kaufer in dem gelt , wie sic solche von gemeinen gewerckhen angenommen doch mit staigerung aines halben guldens volgen zu lassen schuldig sein .“ 1 „Zum andern so hat des Conrad Mairs alte handlung und aufstondt die leuth allhie dermassen ersehröckht , dass weder sie, die Haugischen , noch aiuicher liandelsmann alhie disen khauf annemen wirdet , es sei dann , dass der churfürst von Sachsen sich zuevor seines zin halben in gleichmessigen verstandt einlass .“ Aus einem Bericht Ilsungs an den Kaiser , Datum : Augsburg , 29. Dezember 1569 . Dorther auch das folgende. 2 Diese Firma hatte damals das Idrianer Quecksilbermonopol und wünschte sehr , es gegen das böhmische Zinnmonopol ein¬ zutauschen. 3 In seinem Bericht erwähnt Ilsung , daß Herr Hans Paul Her¬ wart früher „ auch nach disem khauf gestellt “ habe . Er sei 1549 ein starker Konkurrent Konrad Mayrs gewesen , habe aber den böhmischen Zinnkauf nicht ohne den sächsischen zugleich über¬ nehmen wollen und sei schließlich vor K . Mayr zurückgetreten. 282 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. Bei dieser Sachlage blieb nichts anderes übrig —- wenn anders man kaiserlicherseits nicht überhaupt auf ein Zinnmonopol und die daraus resultierenden Einnahmen verzichten wollte — als eine Ver¬ ständigung mit Sachsen zu suchen. Eine Verständigung mit den sächsischen Zinngewerken und in erster Linie eine solche mit dem Regalherrn, mit Kurfürst August. In Sachsen hatten sich die Aussichten auf eine Zustimmung der Gewerken1zu einem Zinnmonopol seit 1549 noch bedeutend verschlechtert. Waren 1549 wenigstens die Altenberger Gewerken — aus Sorge um einen regelmäßigen Verlag — für einen gesamt¬ sächsischen„Zinnkauf“ zu haben gewesen2, so hatten sieh inzwischen die Verlagsverhältnisse in Altenberg leidlich geregelt. Bereits im Jahre 1556 war Kurfürst August „darauf gedacht, das die zienvorlagk dises orts in eine handt gestalt, doch uf namhaftige, vormogende zienvorleger gedacht wurde, und dass mit denselben ein abrede bis uf unsern gefallen gemacht wurde, wie theur sie ein centner zien zalen auch wie lange solche zienvorlagk stehen solle“ 3. August hatte erwartet, daß sich die auch vorher schon im Zinngroßhandel und Verlag tätigen Freiberger Firmen Mathias Rot oder Valentin Buehführer bereitfinden würden, den Altenberger Zinnkauf zu übernehmen. Ein Finanzgeschäft war die Angelegenheit für den Kurfürsten nicht. Natürlich wünschte er auch deshalb den ruhigen Fortgang der Altenberger Zinnproduktion, weil sonst die Regaleinkünfte daraus (der sog. Zehnte) geschmälert wurden, in erster Linie aber war es offenbar August um die Bergarbeiterschaft bei seinen Altenberger Zinnkauf¬ bemühungen zu tun. Bisher hatte öfters der Verlag plötzlich aus¬ gesetzt — der betreffende Verleger war vielleicht nicht mehr zahlungsfähig oder zahlungswillig gewesen—, das bedeutete für die Bergarbeiterschaft zumeist die drückendste Arbeitslosigkeit. Dem sollte durch den Zinnkauf und die Verlagsübernahme durch kapital¬ kräftige Großkaufleute vorgebeugt werden 4. Es ließ sich mit dem 1 Natürlich konnte der Landesherr auch ohne diese ein Zinn¬ monopol festsetzen. 2 HauptstaatsarchivDresden, Loc. 36 080, Nr. 666, Bl. 3 ff.,. Bl. 53. Auch Loc. 4493, Bergsachen, den Altenberg und Gießhübel betr. 1509—1697, Bl. 204 b. 3 Hauptstaatsarchiv Dresden, Cop. 276, Bl. 219—22. 4 Vgl . Hauptstaatsarchiv Dresden, Cop. 276, Bl. 219 ff. Vieltes Kapitel : Böhm .-sächs . Monopol- und Kartellbestrebungen . 283 mir zur Verfügung stehenden Archivmaterial nicht feststellen , ob in den fünfziger Jahren des 16. Jahrhunderts in Altenberg ein allgemeiner Zinnkauf , wie ihn Kurfürst August wünschte , öfters bestand , jedenfalls ist das Anfang der sechziger Jahre für die wichtigsten Altenberger Zinngruben , den sog. Radeschacht , ge¬ schehen . Am 6. Mai 1562 schlossen die Leipziger Zinnhändler Georg Huttherr , Michael Jerniger , Hans Eibe , Dominicus Breun, Adrian von Hilsen und Christof Walburger unter Vermittlung Wolf von Schönbergs , Hauptmann des Erzgebirges , und Mathias Arnoits , Amtsverweser daselbst , einen Zinnkauf auf drei Jahre (von Exaudi 1562 bis Exaudi 1565) 1. Die Zinnhändler empfingen danach alles Zinn des Altenberger Radeschachts . Und zwar von Exaudi 1562— 1563 zum Preise von 12 fl., von Exaudi 1563—1565 zum Preise von 12% fl. Für das Vorrecht alleinigen 'Kaufs gewährte die Leipziger Ge¬ sellschaft den Gewerken sofort ein Geschenk von 200 fl., ferner ein Darlehen von 4600 fl. Die geliehene Summe sollte dadurch allmählich zurückerstattet werden , daß bis zu ihrer völligen Tilgung für jeden Zentner Zinn , den die Gesellschaft von den Gewerken erhielt , nur 11% fl- resp . 11% fl- (von Exaudi 1563 ab) gezahlt wurde . Außer dem einmaligen Darlehen übernahmen es aber die Leipziger Zinnhändler , dem Bergwerk allwöchentlich bis zu 700 fl. Verlag zuzuführen . Die Verlagsnehmer waren gehalten , die Vor¬ schüsse pünktlich zurückzuerstatten . Die sämtlichen Ge¬ werken des Radeschachts zu Altenberg hafteten dafür als Bürgen und Selbstschuldner . Nur die Großgewerken Mathias Roth und die Buchführergesellschaft , die auf Verlagnahme von vornherein verzichteten , waren der Bürgschaft enthoben . Für die Zeit von Exaudi 1565 an übertrug dann Kurfürst August schon im Sommer bzw. Herbst 1564 auf acht Jahre den Altenberger Zinnkauf einer Gesellschaft, die größtenteils aus kapitalkräftigen kurfürstlichen Be¬ amten bestand 2. Der Regalherr hatte bei der Übertragung die 1 Ich möchte nach dem Wortlaut des Kontraktes, dessen Original uns im Hauptstaatsarchiv Dresden (Loc. 36 080, Nr. 666, Bl. 75 ff.) erhalten ist, glauben, daß schon vor 1562 ein Zinnkauf für den Altenberger Radeschacht existierte. Siehe Anhang. 2 Dem Sekretär Hans Jenitz, dem KammermeisterHans Harrer, dem Zehntner zu Freiberg Wolf Prager, dem Münzmeister Hans Biener, dem KammerschreiberHieronymus Krahwider. 284 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. Bedingung gestellt , daß sein Leibarzt mitsamt dessen Eidam in die Gesellschaft des Altenberger Zinnkaufs aufgenommen würde 1. Die Gesellschaft sollte sich mit den Altenberger Gewerken dann immer von zwei zu zwei Jahren „ eines gewissen zinkaufs nach gelegenheit der leuffe vergleichen “, welche „ Vergleichung der churfiirst dann auch jedes mais gnedig confirmiren “ wollte 2. Durch die Bildung der genannten Altenberger Zinnkaufs¬ gesellschaft war nun — um zu unserem Ausgangspunkte zurück¬ zukehren — auch dort , wo 1549 noch Neigung für ein sächsisches Zinnmonopol bestanden hatte , das Interesse daran im wesentlichen erlahmt . Es fragt sich, ob die Gewerken und Verleger der übrigen sächsischen Zinnbergwerke im Verlaufe der letzten zwei Jahrzehnte ihre Ansicht geändert hatten . Die Frage muß verneint werden. Bei Gelegenheit einer Sondierung der Gewerken und Verleger der Zinnbergwerke zu Annaberg , Buchholz , Geyer, Ehrenfriedersdorf und Thum durch kurfürstliche Beamte 3 baten diese dringend , sie mit einem allgemeinen Zinnkauf zu verschonen 4. Die Zinn¬ produzenten sprachen dabei die Vermutung aus , daß „ etwa frembde und auslendische kaufleute , die sich zuesammen verbunden , bei seiner churfürstlichen gnaden in angebung eines gueten wercks, anhalten mögen, gedachten zihnkauf an sich zu bringen . Wie es dan durch die selbst und sonderlich von N. Schlüsselfelder von Augspurck neulicher weil uff S. Annenbergk geruhmet worden, der dan öffentlich ausgesagt , daß ehr wegen seiner lierren und principalen mit s. cf. gn . in handlung stehe , alle zihn in diesen und anderen landen zue behefften und in seiner lierren hende zu bringen,“ Die sächsischen Gewerken der genannten Orte erinnerten dem¬ gegenüber den Kurfürsten an Konrad Mayrs Geschick und an die 1 Hauptstaatsarchiv Dresden , Loc . 36 080 , Nr . 666 , Bl . 86 1. Urkunde vom 24 . August 1564 . Vgl . Anhang. 2 Der zweite Verlagskontrakt genannter Art (Datum : 27 . No¬ vember 1566 ) ist im Anhänge abgedruckt. 3 Wir werden sofort sehen , wie Kurfürst August zu dieser An¬ frage kam. 4 Hauptstaatsarchiv Dresden , Loc . 36 080 , Nr . 666 , Bl . 101 ff. Die interessante Denkschrift der Zinnproduzenten der genannten Orte gibt eine eingehende Begründung der Bitte unter genauer Dar¬ legung besonders der Kreditverhältnisse im sächsischen Zinnbergbau und -handel . Das Stück ist eine wertvolle Quelle zur Geschichte des Verlagssystems im Bergbau. Viertes Kapitel : Böhm.-säclis. Monopol- und Kartcllbestrebungen. 285 Baisse, die dessen fehîgeschlagene Monopolversuche im Zinnhandel hervorgerufen hatte . Außerdem wiesen sie, genau wie 1549, auf die Schwierigkeiten hin , die es machen würde , von den bisherigen Verlegern loszukommen . Es seien, führten sie aus , nur noch wenige Gewerken unter ihnen zu finden , „ die ire zihnpergkwerge selbst zu vorlegen und zu bauen “ imstande wären . Die meisten seien über dem Bergbauen in große Schulden geraten . Sie hätten ihren „gleubigern und Vorlegern all ir hab und guth , haus und hoff, zechen, riess und halden in die berclcbuch vorsichert und vorpfendet “. Sicherlich betrage der Gesamtverlag in den genannten Zinnbergwerken mehrere Tausend Gulden . Wie sollten solche Summen jetzt auf einmal aufgebracht werden ? Überdies aber sei es auch undankbar gegen die Verleger , wenn man ihnen jetzt den Laufpaß geben wollte , nachdem sie erst durch ihre Kapital¬ vorschüsse den armen Gewerken die Möglichkeit der Weiterarbeit verschafft und diese oft vor „ eusserster noth und verderben “ ge¬ rettet hätten. Wie die Gewerken, so sprachen sich auch die Verleger der genannten Zinnbergwerke gegen ein Monopol aus . Sie wiesen be¬ sonders darauf hin , daß sie selbst ja wieder oft von Kaufleuten in den großen Handelsstädten verlegt würden , denen sie gegen Vor¬ schüsse feste Zinnlieferungen zugesichert hätten h Wie könnten 1 So wurden die Verleger wiederum ihrerseits Verlegte (siehe oben S. 126). Der ganze Passus , der für die Geschichte des Verlagssystems wichtig ist , mag hier im Wortlaut folgen : „ Aber wir Vor¬ leger haben uns zue beforderung E . cf. gn . zehnten und gemeines bergkwergs mit kaufleuten und zihnhendlern — ausser - und inner¬ halb landes — nambaftige summen zins zue liefern vorschrieben und vorsprochen , auch stadlieh gcldt doruff entpfangen , dogegen genüg¬ same caution und vorsicherung mit vorpfendung unser hab und gueter , trauen und glaubcns aus bezwungener noth thuen müssen und forder dasselbige geldt zu beforderung E . cf . gn . zehenten unter die armen zihngewcrcken ausgespendet und vorlegt ; dass wir uns also von unsern gläubigem auch nicht entledigen können , es were dan sach , dass inen an unsere stelle alle schulden erlegt und bar bezalt wurden , welches uns vor unsere personell zue thuen unmöglich, konte auch ohne voranderung unserer hab und gueter nicht gesclieen. Im fall nhuen ein zihnkauf geschlossen werden solte , würden die kaufleute , unsere gläubiger , ire schulden wie billich von uns abmahnen und fodern , alsdann diejhenigen , so die armen vorlegt und bei dein bergwerge bis anhero mit noth erhalten sic zur zalung dringen und 280 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. sie ihr Versprechen halten , wenn man fremden Kaufleuten das Monopol in die Hände gäbe . Außerdem machten die Verleger darauf aufmerksam , daß der tiefste Stollen des Bergwerks zu Ehren¬ friedersdorf , der über 50 000 fl. gekostet habe 1, zumeist durch fremde , ausländische Gewerken getrieben wurde . Noch niemals sei ein Pfennig Ausbeute darauf gefallen . Wenn man jetzt den auswärtigen Gewerken die Selbstbestimmung über das erbeutete Zinn durch Einführung eines Monopols nähme , so sei Gefahr vor¬ handen , daß die Gewerken des tiefsten Stollens den Betrieb über¬ haupt aufgäben , was natürlich für den Bergbau Sachsens einen erheblichen Schaden bedeute. Dieselben Argumente , die wir in der hier kurz besprochenen Denkschrift erwähnt finden , besonders auch „ der Verlag der Ver¬ leger“ kehren in Bittschriften wieder , in denen die Gewerken und Verleger von Eibenstock ä, von Schwarzenberg usw. um dieselbe Zeit sich gegen die Einführung eines Zinnmonopoles wandten. So stand also alle Hoffnung auf ein sächsisches Zinnmonopol bei dem Kurfürsten , der als Regalherr natürlich auch gegen den Willen der Gewerken und Verleger einen allgemeinen sächsischen Zinnkauf hätte einführen können . War Kurfürst August des Willens ? Zweifellos hat der genannte Fürst um die Wende des Jahres 1568 — vielleicht auch schon früher — an die Einführung eines sächsischen Zinnmonopols gedacht . Schon in einem Schreiben Augusts vom 8. Januar 1569 an Wolf Schönberg , den Hauptmann des Erzgebirges , heißt es : „Wir haben bewegende Ursachen, dass wir den zinkauf in unsern landen gleich dem kupferkauf gerne in ein enge zusammen bringen wolten . Begeren derhalben Du wollest darauf bedacht sein und Dich bevleissigen ob Du solches also füglichen und ohne clage der zingewerken könntest zuwege bringen 3.“ Als darauf Schönberg nach Rücksprache mit den Zinngewerken von haus und hof treiben musten , was dan E . cf. gn . zchenten , gemeinem bergkwerge und auch den armen gewercken vor nachtheil und beschwerung erfolgen wurde , das bitten die gewercken unterthenigst E . cf. gn . genedigst behertzigen und zue bedencken .“ 1 Man hatte natürlich auf Silber , nicht auf Zinn gemutet . Aber die Zinnausbeute deckte doch wenigstens einen Teil der Kosten und ermöglichte den Weiterbetrieb in der Hoffnung , dann noch auf Silber zu stoßen. 2 Hauptstaatsarchiv Dresden a . a . O., Bl . 109 usw. 3 Hauptstaatsarchiv Dresden , Loc . 36 318 , Nr . 4311 , Bl . 2. Viertes Kapitel : Böhin.-sächs. Monopol- und Kartellbestrebungcn . 287 berichtete , daß den Gewerken der Vorschlag sehr bedenklich er¬ scheine, ließ der Kurfürst seinen Plan durchaus nicht fallen. Er antwortete vielmehr etwas gereizt , er begreife das Zaudern der Gewerken nicht , da er sich erboten habe , „ire zyn in dem werth und kauf , wie sie dieselbigen andern verkaufen und lassen müssen, zu bezahlen , sie auch dardurch einen stedten , gewissen kauf er¬ langten und die armen gewerken sich darzu eines zimblichen vorlags zu getrosten “ hätten l. Schönberg erhielt dementsprechend die kurfürstliche Weisung, nochmals mit den vornehmsten Ge¬ werken zu reden , „wie tlie /.in uff dem oberen gebirge alle in einen gewissen kauf bracht werden möchten “. Wir haben oben gesehen , daß die Gewerken — von ihren Ver¬ legern dabei kräftig geschoben — auf ihrem ablehnenden Stand¬ punkte beharrten . Es fragte sich demnach für das Projekt eines vereinigten sächsisch -böhmischen Zinnmonopols , würde es Maxi¬ milian II . gelingen , den Kurfürsten August zu bewegen, daß ei¬ sernen sächsischen Zinngewerken einen Zinnkauf oktroyierte . Die diesbezüglichen Bemühungen des Kaisers setzten Mitte 1569 ein. Damals wurde auf Anraten der obengenannten Monopolkommission (Georg Ilsung usw.) der kaiserliche Rat Christoph von Karlowitz an den Kurfürsten von Sachsen abgesandt . Er sollte , lautete seine Instruktion , entweder im Aufträge des Kaisers oder auch privatim, wie es ihm angemessen erscheine , den Kurfürsten zu gewinnen suchen , „ damit solche zin [die sächsischen ] sowol als die behmischen in ain oder zwo henndt gebracht und zwüschen kaiserl . mt . und seiner cf. gn . ain solcher verstandt gemacht werden rnöcht , dz dise handlung nit allain baiderseits gewerckhen in gernain , sonder auch röin . kais . mt . und seiner cf. gn . zu grossem nutz , wolfardt und guetem khomen würde “ 2. Den Gewerken sollte durch das Monopol ein Preisaufschlag von y2 fl . pro Zentner , dem Landesherrn eine Arrenda von 1 /, fl. Zuwachsen. Begründet wurde die dadurch notwendig gemachte künstliche Preissteigerung und der damit gegebene Widerspruch gegen die strenge Wirtschaftsethik der Zeit 3 aus dem Darnieder1 Daselbst, Bl. 3. 2 Aus dem Kommissionsbericht vom 8. August 1569. F.A. Wien. 3 In der vom 22. August 1569 datierten Denkschrift , die Georg Ilsung für Karlowitz verfaßte (Hauptstaatsarchiv Dresden, Loc. 36 080, Nr. 664, Bl. 1 ff.), heißt es charakteristisch : „Es khündtc 288 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. liegen des Zinnbergbaues . Die Ausbeute sei geringer , die Kosten (für Unscblitt , Stahl , Eisen , Fleisch , Brot usw.) seien größer ge¬ worden , trotzdem stünden die Zinnpreise so tief , daß „ etliche arme gewerckhen khaum das truckhen brodt von irer harten arbait“ hätten . Eine Änderung dieser mißlichen Verhältnisse sei nur da¬ durch möglich, daß „ der zinkhauf aus sovil underschidlichen henden genomen und allein in ain h a n d t gebracht , die mit der zeit die zin wider in iren alten preis und werdt bringen khünde “ 1. Nun bestanden zwei Möglichkeiten , ein sächsisch -böhmisches Zinnmonopol aufzurichten . Entweder wurde sowohl der böhmische wie der sächsische Zinnkauf in ein und desselben Kapitalisten Hand gegeben . Das war die Art , wie im 19. Jahrhundert die Rothschild durch Fusion des Idrianer und Almadener Monopols ein Welt¬ monopol in Quecksilber schufen 2. Oder aber der sächsische Zinn¬ kauf und der böhmische gelangten an zwei verschiedene Kapita¬ listen , die aber unter Vermittlung der beiderseitigen Regierungen ein Kartell miteinander eingingen . Zunächst mußte Maximilian II ., um nicht die sächsischen Kapitalisten 3 und mit ihnen den Kursicli auch solcher staigerung aller billichait nach niemandts be¬ schweren, dann die zin nit höher hingebracht würden, dann si vor vilen iharen gegolten . . . Und wicwol auch aus disem handcl, wo also zwüschcn der kais. mt. und sein cf. gn. ein nachbarlicher verstandt gemacht wurdt, ain gross monopolium nufgcricht werden möcht, angesehen dass sonst khainc andere zin in der gantzen chrislcnhait ausserhalb Engenland — welche zin aber auch in grossem abnemen — gemacht werden. So ist doch solches, weil es ein wahr, die meniglicli arm und reich brauchen mues, kainswegs stattlich noch göttlich. Aber obgcnielter staigerung, weil die perckbwereh arm und der saumbcost gross, klian sich niemandts beschweren.“ Das war natürlich Ilsung selbst nicht ernst. Er wußte ganz genau, daß die Kaufleule, denen das Monopol übertragen wurde, das Zinn im Preise steigern würden , soviel sie immer könnten! 1 Aus der Denkschrift Ilsungs vom 22. August 1569. 2 Handwörterbuch der Staatswissenschaften . 2. Auf], V. Bd„ S. 851 unten. 8 So schrieb einer der Ratgeber des Kurfürsten, Hans Jcnitz, der selbst ein großer sächsischer Kapitalbesitzer war, voll Eifersucht auf die süddeutschen Kaufleute : „Ob aber e. chf. g. umb des ver¬ trösteten, ungewissen génies willen denen von Nurenberg oder Augs¬ burg gestatten soltcn, sieh in ire lande und bergwerge einzuflechten, dieselben auszusaugen und zin und gewin ausser landes zu furen, das stehet in derselben gnedigstem bedencken.“ Hauptslaatsarchiv Dresden, Loc. 36 080, Nr. 664, Bl. 16 ff. 31 * Viertes Kapitel : Böhm.-sächs. Monopol- und Kartellbestrebungeu . 289 fürsten August vor den Kopf zu stoßen , den Vorschlag eines Kartells machen . Die kaiserliche Hofkammer ließ durch Karlowitz an den Kurfürsten gelangen , „ dass es nit die meinung hab , dass eben der churfurst an diejenigen handelsleut , mit denen ire k. mt . ires teils handeln wollen lassen , angebunden sein solle, sonder dass es principaliter darumb zu thun sei, ob gleich ire cf. g. selbs ire eigne zinkauf in denselben landen treiben oder sonst andern hinlassen wolten , dass doch der wert des kaufs einer jeden sort zin und des¬ selben gewichts und die arenda irer k. mt . und des churfursten gemess pro rata auf eine gleiche handlung und correspondentz ge¬ richtet würde . Also dass diejenigen , so irer k. mt . zin an sich bringen neben und mit denen , so mit denen Meissnischen zinen handeln, in gleichem wert , doch nach gestalt einer jeden sort underschiedlichen guete , inmassen auch mit den gewerken in Beheim beschehen, hinkommen und keiner dem andern disfalls, wie etwa zuvorn be¬ schehen , eintrag thun möge V * Diesen Ausführungen fügte frei¬ lich die Hofkammer sofort die weitere Aussicht hinzu , daß am Ende doch die Fusion beider Zinnmonopole — des sächsischen und des böhmischen — das Beste sei 2. Auch das hatte selbstverständlich Maximilian II . dem Kur¬ fürsten überlassen , ob er den sächsischen Zinnkauf einer Gesell¬ schaft von sächsischen Kapitalisten übergeben wollte oder einem auswärtigen Großkaufmann 8. In letzterem Falle war der Kaiser bereit und imstande , reiche süddeutsche Kapitalisten vorzuschlagen. Kurfürst August hat sich weder zu dem einen noch dem anderen entschlossen . Er hat sich überhaupt dem Projekt eines mitteleuropäischen Zinnmonopole versagt . Nachdem er schon im Herbst 1569 nach Wien geantwortet hatte , daß er vorläufig sich nicht entschließen könne 4, lehnte er, nach wiederholter Anfrage ’ 1 Schreiben vom 20 . Jan . 1570 . Hauptstaatsarchiv Dresden, hoc . 36 080 , Nr . 664, Bl . 29 ff. 2 „Konte rlan sowol irer k . mt . als seiner cf . g, zin in einerlei gesclschaft handen kommen , wie sich dau on zweivel statliche leule angeben , die solchen kauf mit gutem irer cf. g. überschus annemben wurden , mochte es villeicht umb sovil besser sein/' 3 Das und das folgende nach Akten des Hauptstaatsarchivs Dresden , Loe . 36 080 , Nr . 664 , Bl . 1 ft'. ; Loc . 36 131, Nr . 2034 , Bl . 1 ff. 4 Hauptstaatsarchiv Dresden , Loc . 36 080 , Nr . 664 . Bl . 21. 6 Daselbst Bl. 31 ff . Auch l’.A . Wien . Böhmen 51. u . B . ( 1570 bis 1590 ), Nr . 16 407. Striatior , Studien z. Gosch , kapitalst . Uvt:ani3r\ i i.Mistormen . 1!) 290 Drittes Buch: Monopole, Kartelle und Aktiengesellschaften im Mittelalter. Maximilians II . Ende Januar 1570 den Plan definitiv und be¬ stimmt ab 1. Fragt man nach den Gründen , die Kurfürst . August bei seiner Ablehnung bestimmten , so sind von wirtschaftspolitischen — auf andere gehe ich hier nicht ein — die folgenden etwa zu nennen . - Einmal hat sicherlich die einmütige Ablehnung eines sächsischen Zinnkaufs durch Gewerken und Verleger auf den Kur¬ fürsten Eindruck gemacht . Manchen Großgewerken war ja über¬ haupt ein freier Verkauf ihres Zinnes von seiten der Krone garantiert worden . Ferner mußte sich der Kurfürst , der die wirtschaftlichen Kräfte seines Landes genau kannte , sagen, daß in Sachsen kaum genügend reiche und genügend unternehmungslustige Kapitalisten zu finden waren , die den sächsischen Zinnkauf in ihre Hand nehmen würden 2. Aber den Zinnkauf in seinen Landen auswärtigen , etwa Augsburger Kaufleuten zu übertragen , dagegen sträubte sich sein Selbständigkeitsgefühl und sein Wunsch , die sächsische Wirtschaft in jeder Beziehung zu heben . Zweifellos hätte auch der Handel Leipzigs stark leiden müssen , wenn der Zinngroßhandel ganz nach Süddeutschland abgezogen worden wäre 3. Weniger ernst zu nehmen waren wohl die Bedenken des Kur¬ fürsten , die in der Richtung einer Handwerkerpolitik lagen . Wenn August meinte , „ die handwerger in seinem lande , die ir nahrung vom zin haben muessen , als kandelgiesser , rothgiesser , pfannenschmidt , schlosscr, gurtler “, würden nach Einführung des Monopols „zur notturft kein zin, es were dan umb ubermessige bezahlung von den Verlegern bekhommen können “, so ließ sich hierin leicht Abhilfe schaffen . In vielen Monopolkontrakten der Zeit ist den Handwerksleuten ein Materialkontingent zu bestimmtem , niedrigen 1 Hauptstaatsarc -hiv Dresden a. a . 0 ., Bl . 35 ff. 2 Vgl. die Denkschrift der kurfürstlichen Räte vom 12. Oktober 1569 . Hauptstaatsarchiv Dresden , Loc . 36 080 , Nr . 664 , Bl . 11 ff. Dazu Brief Augusts an Maximilian II . (29 . Januar 1570 ), a . a . O., Bl . 35 ff . „ Viel weniger habe ich Vermutung , dass in meinem lande hendeler oder andere leuth zu finden , die sich dis w' eitleuftigen wercks understelien werden .“ Wie mußte in Leipzig da die Unternehmungs¬ lust zurückgegangen sein ; man denke an die dortigen kühnen Kapita¬ listen in der ersten Hälfte des 16 . Jahrhunderts! 3 „Es ist aber solches wegen des handeis zu Leiptzigk (dass dadurch die niederlage und auch ander liendel , so des zins halben bis anher in dise lande gangen , geschwecht ) hochbedencklicb .“ Aus dem genannten Brief des Kurfürsten . Und ein andermal heißt es: * Viertes Kapitel : Bölim .-sächs . Monopol- und Kartellbestrebungen . 29! Preis Vorbehalten . August hätte das bei ernstem Willen auch für die sächsischen Handwerker durchsetzen können . Nein, der Haupt¬ grund , weshalb der Kurfürst von Sachsen nicht auf den Plan eines mitteleuropäischen Zinnmonopols einging , lag auf einem anderen Gebiete . Auf dem Gebiete der Finanzpolitik. Es kann , wie wir sahen , keinem Zweifel unterliegen , daß für Maximilian II . der innerste Beweggrund , das Projekt zu betreiben, in dem Wunsche lag, sich eine neue Geldquelle zu eröffnen . Eine solche Notwendigkeit bestand für den Kurfürsten August von Sachsen mit seinem wohlgeordneten Finanzsystem , mit seiner reichlich gefüllten Staatskasse nicht . Hierin ist zweifellos der letzte Grund der Weigerung des Sachsen zu suchen , dem Kaiser auf dem gefährlichen Monopolwege zu folgen. Damit war aber nun auch das Schicksal eines böhmischen Zinnmonopols besiegelt . Es fand sich kein Kapitalist , der Konrad Mayrs Geschick zu teilen gewillt war . Den böhmischen ärmeren Zinngewerken aber gereichten die verfehlten Projekte ihres Regal¬ herrn zu rechtem Unglück . Sie hatten ihren alten Verlegern kündigen müssen, teilweise hatten diese sich auch selbst zurück¬ gezogen und ihre Kapitalien anderswo angelegt . Woher nun die Kosten für die Grubenarbeit decken ? Wer sollte die unentbehr¬ lichen Kapitalvorschüsse leisten , „sonderlichen diser zeit, do der winter fur der thür , dass man allerlei berckwercks -notturft an unslet , eisen, zimmer -, schacht -, khol - und lachterholtz sambt andern mehr gezeug in vorrath schaffen und etwas mehrers dann sonsten zu wöchentlicher verlag haben soll und muss “ 1. Die Bergarbeiter¬ schaft geriet über diesen Verhältnissen in die höchste Not . In manchen Distrikten waren sie „in 19 wochen nit gelondt worden, welches under dem perckhgesindt auch iren weib und kindern ein solche hungersnot und ungeduldt verursacht , dergleichen nie zuvor gewesen“ 2. Unter solchen Umständen war es als ein Glück zu bezeichnen , daß der Kurfürst August von Sachsen im Januar 1570 „ . . . zu geschweigen, dass durch disen weg die niederlag und handtlung mit dem zin aus meiner handelsstadt Leiptzigk gar hinwegk khommen mechten . . . .“ 1 F.A. Wien. Böhmen M. u. B. 1540—1569, Nr . 16 406. 2 Maximilian II . an Georg Ilsung, 8. Februar 1570. F .A. Wien. Böhmen M. u. B. 1570—1590, Nr. 16 407. 19* 1 292 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. das Monopolprojekt rund ablehnte L Bald darauf wurde in Böhmen der freie Zinnverkauf wieder öffentlich verkündet und damit einer Besserung der Verhältnisse im Zinnbergbau die Wege geebnet 2. Fünftes Kapitel. Monopole und Kartelle im Idrianer Quecksilberhandel des 16. Jahrhunderts. Um das Jahr 1523— zu welcher Zeit Kartell- und Monopol¬ bestrebungen lebhaft in Idria einsetzten — waren drei Gewerk¬ schaften mit dem Abbau der bedeutenden Idrianer Quecksilber¬ gruben beschäftigt 3. Zwei davon waren Privatgesellschaften. Einmal die Gewerkschaft St. Katarinen, die erst im Jahre 1520 gegründet worden war 4, und zweitens die Gewerkschaft St. Achaci. Letzterer hatte Maximilian I. das im Jahre 1510 den Venetianern wieder abgenommene „Bergwerk Idria“ übertragen. Und zwar 1 Es bleibt zu untersuchen , inwieweit die sächsischen Zinnkaufs¬ projekte , die im Jahre 1583 auftauchten , sich auf eine Vereinigung mit Böhmen stützten . Vgl. Joh . Falke, Die Geschichte des Kurfürsten August von Sachsen in volkswirtschaftlicher Beziehung, S. 298 f. Dazu Hauptstaatsarchiv Dresden, Bernstein 4, Loc. 7294, Bl. 409—11. 2 Vgl . Urkunde vom 28. Aug. 1572, durch die Maximilian II . den Zinngewerken von Schlackenwald, Schönfeld und Lauterbach freien Zinnkauf erlaubt . F . A. Schmidt a. a. O., I. 3, Nr . 118, S. 173. 3 Die Geschichte des Idrianer Bergbaus liegt sehr im argen. Noch immer sind wir angewiesen auf das Werk von Peter Hit¬ zing e r, Das Quecksilberbergwerk Idria . Laibach 1860. Unsere folgenden Idrianer Monopol- und Kartellstudien stützen sich aus¬ schließlich auf Archivmaterial , besonders auf solches aus dem k. und k. gemeinsamen Finanzarchiv in Wien. Im folgenden als F . A. Wien abgekürzt . Die besonders benutzten großen Aktenfaszikelreihen: Österreich. Inneröstr . Quecksilberbergwerke (1523—1563) Fase. 22 Nr . 18 320 und Österreich . Inneröstr . Quecksilberbergwerke (1564 bis 1599) Fase . 23 Nr . 18 321 sind im folgenden als Inneröstr . Queck¬ silber 22 resp . 23 zitiert. 4 „Das k. k. Quccksilbcrbcrgwcrk zu Idria " . Wien 1881, S. 3. Nach Hitzing er gehörten Ihr folgende bedeutende Gewerken an : Gabriel, Graf zu Ortenburg , Hans von Auersberg, Christof Räuber, Sigmund von Dietrichstein , Sigmund von Lamberg u. a. Fünftes Kapitel : Monopole u. Kartelle im Idrianer Quecksilberhandel usw . 293 zum Lohne dafür, daß die Gewerken dem Kaiser „ mit Darstreckung von Leib und Gütern“ gedient hätten 1. Bedeutende Namen finden sich unter den Gewerken von St. Achaci genannt. Zum Beispiel Hans von Auersberg, Kaspar und Sigmund von Lamberg, Sigmund von Dietrichstein, Lienhart Räuber, Jörg von Egkh, Jakob Villinger, Blasi Höltzl, Gabriel Vogt, Paul Rasp, Ulrich Putsch, Kaspar Herbst, Math. Hofer, Hans Strigl von Luentz, Wilhelm Neumann u. a. In die Achacigewerkschaft hatten sich noch unter Maximilian „alle anderen alten“ Gewerkschaften aufgelöst 2, so daß vor der Grün¬ dung der Katarmengewerkschaft (1520) zeitweise nur eine Idrianer Gesamtgewerkschaft existierte. Aber auch die Gewerkschaften St. Açhaci und St. Katarinen standen sicli sehr nahe. Das ergab sich schon aus der teilweisen Identität der Mitglieder und aus der Zusammenlegung ihrer Schürfe in dem gleich zu besprechenden Monopol. In den gleichzeitigen Idrianer Quellen erscheinen sie zumeist als „die Idrianer Gewerkschaft“ zusammengefaßt. Kurz nach Maximilians Tod, im Jahre 1522 wohl, hatte so¬ dann Erzherzog Ferdinand als „regierender Herr und Landesfürst aus fürstlicher Obrigkeit“ eine neue Grube, „das furstenpau ge¬ nannt in dem perckhwerch Idria aufzuschlagen und von seinem chammergut zu pauen bevolhen“ 3. Die eigene Bergwerkstätigkeit des Landesfürsten in Idria hat jedoch nicht lange gewährt. Schon im Frühjahr 1523 übergab Erzherzog Ferdinand einer Gewerkschaft, in der besonders der bekannte Gabriel von Salamanca, Graf von Ortenburg und Johann Castro hervorragten 4, den Fürstenbau. Daß den neuen Eigentümern kein wertvolles Geschenk damit ge¬ macht war, erkennt man aus den Privilegierungen, die die Über¬ tragung begleiteten. Darin wurden die neuen Gewerken unter anderem auf vier Jahre von Fron und Wechsel befreit. „Damit sie,“ heißt es in der Uberlassungsurkunde, „die Gruben und Berg¬ werke desto besser erbauen und erheben mögen“. 1 Haus-, Hof- und Staatsarchiv Wien Rep. I Österreich. 2 F. A. Schmidt, Berggesetze Österreichs III, 1. S. 103. 3 Schmidt, a. a. O. S. 101, 107. Nach letzterer Stelle wäre es 1522 geschehen. 4 Die übrigen Gewerken sind in der Innsbruck, den 2. April 1523 datierten Urkunde genannt, in der Ferdinand den neuen Ge¬ werken des Fürstenbaues die oben erwähnten Erleichterungen ver¬ lieh. Vgl. F. A. Schmidt, III , 1. Nr. 49. 294 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. Mit dieser neuen Gewerkschaft des Fürstenhaus war der „Idrianer Gewerkschaft “ eine gewisse Konkurrenz gegeben . Er¬ wies sich die Arbeit der jungen Gründung ertragreich , so konnte leicht , namentlich bei Absatzstockungen , ein gegenseitiges Unter¬ bieten des geförderten Metalles Vorkommen. Um dem vorzubeugen, einigten sich die beiden Gewerkschaften zu einem Kartell, das insbesondere folgende Bestimmungen enthielt : Alles von den beiden Gewerkschaften gewonnene Quecksilber wurde zum Zweck des Verkaufes zusammengelegt . Kein Gewerke war befugt , das Quecksilber , das auf seine Kuxe entfiel — die Austeilung geschah damals noch in natura —, freihändig und auf eigene Faust an die Quecksilberhändler zu veräußern . Erforderte es die Not , daß er seine Ausbeute schnell verkaufte , so durfte das nur an einen seiner Gewerkschaftsgenossen geschehen . Der Verkauf des also „in eine Hand “ zusammengelegten Produktes an die Quecksilberhändler geschah dann „ mit Wissen und Willen “ des Bergherrn — der durch das ebenfalls in natura ausgezahlte Fron - und Wechsel¬ quecksilber interessiert war -— und der Gesamtheit der Gewerken. War es unmöglich , das ganze Produkt auf einmal abzusetzen , so sollte bei jedem Verkaufsabschluß jede der beiden Gewerkschaften im Verhältnis ihrer Förderung beteiligt wrerden 1. 1 Die betreffenden Stellen der Verabredung lauteten wörtlich: „Ir fürstl. durchleuchtigkait hat sich auch mit uns genedigclichen vergleicht und veraint, in craft berurts Vertrags deshalben aus¬ gangen, also dass ir f. dt. und derselben erben und nachkhomen, auch diejhenen, den ir f. dt. derselben aufgeslagen grueben zuegestellt oder noch zuestellen mochten, dass ir f. dt. und dieselben ir dt. begabten [gewerken] mit uns in ainem kauf des quegksilbers und alles gewannen guets beieiben sollen und wellen und solchen kauf alweg in ainer handt halten und die keuf mit ir f. dt. und aller gwerkhen wissen und willen beslissen und machen, wie die ir f. dt. und wir auf das höchst bekhenen mugen. Und soll khain gwerkh ausserhalben des gemainen kaufs nichts hingeben oder verkaufen. Wir sollen und mugen auch an einander in unsern notturften unser Silber, zinober, varb oder ander vorat, wie und wann es uns fuegt, unverhindert menigclichs wol verkaufen, wie das alles bisher auch beschehen ist. Doch mit dem undterschidt, dass die, so also kaufen, dasselb ausserhalb des gemainen kaufs, auch nit verkaufen sollen noch mugen. . . . Dieweil auch an baiden vorbenanten orten mer * Fünftes Kapitel : Monopoleu. Kartelle im Idrianer Quecksilberhandelusw. 295 Das Kartell der beiden Gewerkschaften ist, soviel ich sehe, von keiner Bedeutung geworden. Der Fürstenbau erwies sich als wenig förderungsreich. Um so ergiebiger zeigten sich die Gruben der „gemeinen Gewerkschaft“ von Idria, so daß sich bald Absatz¬ schwierigkeiten und Preisstürze ergaben und an Mittel, diesem Übel abzuhelfen, gedacht werden mußte. Zwei Umstände waren es, die mit diesen Absatzschwierigkeitenzusammen dahin führten, daß schon in den letzten Tagen des Jahres 1524 eine Handels¬ monopolisierung des idrianischen Quecksilbers ins Auge gefaßt und bald darnach verwirklicht wurde. Einmal dér Wunsch Erzherzog Ferdinands, Darlehen für seine kriegerischen Unternehmungen gegen die Türken in die Hand zu bekommen 1und zweitens die kühne Spekulationslust der Augsburger Kapitalisten Ambrosius und Hans Höchstetter. Ferdinand hatte schon Mitte des Jahres 1524 die Idrianer Gewerkschaft schärfer als bisher für seine Geldbedürfnisse heran¬ gezogen. Sie hatte 13 000 fl. rh. für eine Grenzhinausschiebung ihrer Gruben und 20 000 fl. rh. für nichtgezahlten Wechsel her¬ geben müssen2. Zu Anfang des Jahres 1525 machte der Erzgrueben gebaut werden sollen , darumben zuversichtlich ist , es werde mer Silber , zinober und färb gemacht , als bisher beschehen , des¬ halben sich im verkaufen aber irrung begeben möchte in dem , die¬ weil bisher nit alles , das gemacht ist auf ainmal gar , sonder nur ain tail hat inugen verkauft werden , da villeiclit furan auch also be¬ leihen und beschehen ; demnach ist entslossen : Wann furan die keuf beschehen , so soll von jeder grueben nach gebuerunder antzall ires gemachten Silbers , zinobers und färb in die kaufsumma genomen und verkauft werden .“ Schmidt , a , a . O. S. 110 f. 1 Für die außergewöhnlich großen Geldbedürfnisse des persönlich so anspruchslosen Ferdinand während seiner kriegerischen Regierung vgl . Karl Oberleitner, Österreichs Finanzen und Kriegs¬ wesen unter Ferdinand I . Archiv für Kunde österr . Geschichts¬ quellen Bd . 22 , S. 1 ff. Außerdem O. Thors ch, Materialien zu einer Geschichte der österreichischen Staatsschulden vor dem 18 . Jahr¬ hundert . Leipziger Dissertation 1891. 2 In dem Vertrage vom 28 . August 1524 , in dem diese Vergütungen an die Krone fcstgelegt wurden , wurde die Fron für die Zukunft auf ein Zehntel der Förderung und der Wechsel auf den vierten Pfennig oder Zentner festgelegt . Allerdings brutto ! Für Brenn¬ kosten , Verpackung in Fäßchen oder Lederschläuche mußte die Krone 10 % der Fron wiedererstatten . Auch die Verpackungs- 296 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. herzog dann ein größeres Anleihegescliäft mit den Idrianer Ge¬ werken, bei welcher Gelegenheit ein Großhandelsmono¬ pol für Quecksilber aufgerichtet wurde. Am 1. Januar 1525 schlossen nämlich die Gewerken von Idria mit Erzherzog Ferdinand einen Vertrag ab, demzufolge sie dem genannten Herr¬ scher die 300 000 fl. als Darlehen überließen, welche sie im Ver¬ laufe der nächsten vier Jahre für den alleinigen Verkauf ihres Queck¬ silbers und Zinnobers von Ambrosius und Hans Höchstetter & Co. erhielten l. Kurz vorher war der betreffende Monopolvertrag mit den Höchstettern aufgerichtet worden 2. Darin war be¬ stimmt : Alles Quecksilber und Zinnober, das augenblicklich vor¬ rätig war oder in den nächsten vier Jahren gewonnen wurde, sollte zum Preise von 30 fl. rh. für den Wiener Zentner Quecksilber und 35 fl. rh. für den Wiener Zentner Zinnober der Höchstetter -Ge¬ sellschaft überlassen werden. Ausgenommen wurde nicht einmal das Quecksilber, das sich schon in den Händen Hans Baumgartners aus Augsburg, Wilhelm Neumanns und Hans Pfluegels Erben 3 befand. Auch dieses sollte sofort den Höchstetter übergeben werden 4. Für den Fall, daß die Gewerken oder der Erzherzog während der vierjährigen Dauer des Kontraktes an jemand anders Unkosten des Wechsels fielen dem Erzherzog zur Last. Schmidt, a. a. O. III, 1, Nr. 50 bes. S. 105—106. 1 Oberleitner, a. a. O. S. 21. Der Vertrag ist im Wiener gemeinschaftlichenFinanzarchiv erhalten. Österr. Gedenkbuch(1. Ja¬ nuar 1525) Bl. 248. Das Darlehn erfolgte in einzelnen Raten. So übergaben die Höchstetter im Auftrag der Gewerken am 1. und 2. Jan. je 40 000 fl. (Österr. Gedenkbuch 1523—1525, Bl. 249 a, 250). Die Rückzahlung erfolgte durch die Überlassung der fürstlichen Fron und des Wechsels in der Höhe der Schuldsumme an die Gewerken. 2 Der Vertragsentwurf, den Johann Zott als Vertreter des Erz¬ herzogs, die Bevollmächtigten der Gewerken und Ambrosius Höch¬ stetter auf dem Verhandlungstage zu Ponte (19. Dezember 1524) aufgesetzt hatten, ist ■ — allerdings arg verballhornt — abgedruckt bei Schmidt, a. a. O. III, 1, Nr. 51. Der Originalvertrag ist von mir im Anhang wiedergegeben. 3 Das waren die Gewerken bzw. Kaufleute, die bisher den größten Teil des Idrianer Quecksilbers an sich gebracht und verkauft hatten. 4 Österr . Gedenkbuch S. 250 b. Willi. Neumann erhält Befehl, all sein Quecksilber, sei es zu Wasserneuburg, Villach, Venedig oder sonst lagernd (auch wenn er Verlag darauf gezahlt habe) sofort an die Höchstetter zu geben- (2. Jan. 1525). Fünftes Kapitel: Monopoleu. Kartelle im Idrianer Quecksilbcrlnmdtl usw. 297 als die Höchstetter Quecksilber oder Zinnober verkauften oder sonstwie abgaben, war eine Konventionalstrafe von 300 Zentner Quecksilber und 200 Zentner Zinnober festgesetzt. Demgegenüber verpflichteten sich die Höchstetter in der aus¬ gemachten vierjährigen Frist für 300 000 fl. Quecksilber oder Zinnober abzunehmen. Dabei verstanden sich die obengenannten Preise loco Idria. Für das bereits versendete Metall, das die Höchstetter -Kompagnie in Venedig, Laibach, Kronburg und in anderen Lagerstätten übernahm, hatte sie außerdem den Fuhrlohn und andere bereits von den Gewerken darauf verwendete Spesen zu zahlen. Dieses bereits versendete Quecksilber sollten die Höch¬ stetter übrigens noch im Laufe des Jahres 1525 abnehmen. Als Lieferfristen des noch zu gewinnenden Quecksilbers wurden die jeweiligen Quatemberzeiten festgesetzt. Auch die Zahlungsfristen des Monopolinhabers wurden genau in dem Vertrage festgelegt. Als Zahlungsorte nahm man Hall, Salzburg, Wien und Villach in Aussicht. Sollte das Bergwerk nicht für 300 000 fl. Quecksilber in der genannten Frist liefern können, so war natürlich auch die Zahlung der Höchstetter entsprechend herabzusetzen. Die üblichen Zölle und Mauten, die bisher bei der Verführung des Quecksilbers bzw. Zinnobers gezahlt worden waren, sollte auch die HöchstetterGesellschaft entrichten. Jedoch verpflichtete sich der Erzherzog für sich, seine Erben und Nachkommen diese Zölle während der Kontraktszeit nicht zu steigern. Eine wichtige und interessante Bestimmung des Monopol¬ kontraktes befaßte sich mit einem voraussichtlichen Einschreiten der Reichsregierung gegen das Quecksilberhandelsmonopol der Höchstetter. Im Falle der Gesellschaft auf Grund der Reichs¬ gesetzgebung gegen Monopole irgendwelche Schwierigkeiten er¬ wachsen sollten, verpflichtete sich der Erzherzog, für sie einzutreten und ihr „ Gewährschaft zu leisten“. Das konnte nicht wohl anders sein! Kam doch die Steigerung des Quecksilberpreises, die als oberster Zweck der Verabredung genannt war, am meisten dem Erzherzog zugute. Wenn, so lautete der betreffende Passus des Vertrags, die Höchstetter das Quecksilber und Zinnober „in höheren Wert und Kauf“ während der genannten vier Jahre zu bringen imstande wären 1, so solle das ihr Schade nicht sein. Für diesen 1 Auch von einem späteren Idrianer Quecksilbermonopolkontrakt 298 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. Fall, und wenn sie sich auch sonst „ehrlich und ersprießlich ge¬ halten hätten, “ verpflichteten sich der Erzherzog und die Ge¬ werken, den Höchstettern ein Vorrecht auf ein späteres Monopol einzuräumen. Das heißt, sie versprachen für den Fall, daß sie nach Ablauf des jetzigen vierjährigen Kontraktes das Quecksilber wiederum „in eine Hand“ verkaufen wollten, die Augsburger auch dann zu bevorzugen, wenn sie für den Zentner Quecksilber y2ü. weniger böten, als der höchstbietende sonstige Bewerber. Erzherzog Ferdinand hat den Schutz der Höchstetter gegen die Monopolgesetzgebungdes Reiches nicht übernommen, ohne sich an der höchsten Reichsgewalt, dem Kaiser, einen starken Rückhalt zu sichern. Kaiser Karl V. erließ am 13. Mai 1525 von Toledo aus, wie wir sahen, ein Mandat, in dem er bestimmte, daß die Kontrakte , die den Erzgroßhandel in die Hände weniger Kauf¬ leute auslieferten, nicht als monopolistisch im Sinne der Reichs¬ tagsverhandlungen über Monopole angesehen werden sollten und dürften 1. Vielmehr solle den Gewerken oder denen, die Metalle oder Erze zu verkaufen hätten , das Recht zustehen, ihre Pro¬ duktion „in eine Hand“ zu verkaufen und über diesen monopolisti¬ schen Verkauf „ Gedinge und Pacta “ (also Monopolkontrakte) mit den Käufern aufzurichten. Und anderseits natürlich den Kauf¬ leuten auf solche Monopole einzugehen. Entgegenstehende Erlasse oder Bestimmungen der Gesetzgebung, wie sie etwa „aus ungenüg¬ samem bericht oder verstand“ durch irgend jemanden verordnet wären oder würden, sollen durch kaiserliche Machtvollkommenheit cassiert sein 2. (der im Jahre 1539 mit Hans Baumgartner aus Augsburg abgeschlossen wurde ) sagt Ferdinand ausdrücklich , daß er ihn zu dem Zwecke ein¬ gegangen sei, „damit das quecksilber umb sovil höher gestaigert werden müge“ . Schmidt, a. a. O. III , 1, S. 253. 1 Übertreter dieser Auffassung hatten der kaiserlichen Kammer 50 Mark Goldes zu zahlen. 2 Der Kaiser erklärte direkt : „Ob wir ictz ainicherlei Ordnungen zu abstellung der monopolien aufgericht und gemacht lietten , oder hinfuro machen oder aufrichten würden , dass dieselben Ordnungen und fürsehungen in kaufen , verkaufen , handlungen , contracten , gedingen , handtierungen und verfuerungen der gemelten métal und ertzt , sunderlich der kupfer , Silber und quecksilber halben , nit statt haben , noch verstanden werden , sonder in denselben andern unsern Ordnungen und fürsehungen gentzlich und gar ausgeslossen sein sollen .“ Fünftes Kapitel : Monopoleu. Kartelle im Idrianer Quecksilberhandel usw. 299 Die Bedeutung des kaiserlichen Erlasses ist an einer anderen Stelle dieses Werkes bereits eingehend gewürdigt worden. Hier muß noch auf das folgende hingewiesen werden. Es kann keinem Zweifel unterliegen, daß das Mandat Karls V. vom 13. Mai 1525 mit seiner Verteidigung der Erzgroßhandelsmonopole in erster Linie das Idrianer Quecksilbermonopol der Höchstetter im Auge hatte . Wenn auch der Name der Augsburger Kapitalistenfamilie nicht darin vorkommt, so ergibt das sich doch daraus, daß unter den Metallen für die Großhandelsmonopole erlaubt sein sollen, Quecksilber— neben Kupfer — an mehreren Stellen des Privilegs besonders hervorgehoben ist. Freilich waren es die Höchstetter nicht allein, für die Erzherzog Ferdinand bei seinem kaiserlichen Bruder das Toledaner Mandat erwirkte x. Außer ihnen sollten besonders die Fugger in ihren Tiroler Kupfermonopolisierungsbestrebungen durch das Privileg geschützt und vor einer anti¬ monopolistischen Reichsgesetzgebung für die Vergangenheit und die Zukunft salviert werden. Schon unter Sigismund von Tirol, mehr dann noch unter Maximilian und Ferdinand hatte sich die geschäftliche Organi¬ sationsform in dem ganz bedeutenden Tiroler Kupfer- und Silber¬ großhandel herausgebildet, die man mit „ Silberkauf“ ’resp. „Kupfer¬ kauf“ bezeichnete und die wir bereits in einem anderen Zusammen¬ hang kennen gelernt haben. Von einem Monopol konnte dabei solange nicht die Rede sein, als neben den Fuggern eine ganze Anzahl anderer Kapitalisten „Kupfer- und Silberkäufe“ mit den Tiroler Landesherren ab¬ schlossen. Aber seit Jakob Fugger durch seine Verbindung mit der ungarischen Kapitalistenfamilie der Thurzo die ungarische Kupferproduktion in der Hand hielt, gingen die Gedanken des großen deutschen Kaufmannes immer deutlicher darauf hinaus, ein Fuggersches Weitgroßhandelsmonopolfür Kupfer zu schaffen. Besonders seit ein Syndikat der Fugger, der Baumgartner, der Herwart und der Gossembrot für den Verkauf von Ti oler Kupfer in Venedig (1498) nach kurzem Bestände sich — wif es scheint, 1 Daß Erzherzog Ferdinand der unmittelbare Veranlasser des Privilegs bei Karl V . war , ergibt sich daraus , daß das Originalexemplar, das jetzt im Fugger -Archiv ruht , die Rückennotiz der kaiserlichen Kanzlei trägt : „ Taxa nihil , quia pro archiduce “ . M. Jansen, Jakob Fugger der Reiche . S. 403 unten. 300 Drittes Buch: Monopole, Kartelle mul Aktiengesellschaften im Mittelalter. nicht ohne der Fugger Verschulden— aufgelöst hatte 1, ging Jakob Fugger raschen Schrittes auf sein Ziel los. Für einige Zeit ist dem kühnen Mann der Plan geglückt. Zeitweise ist Jakob Fugger in den ersten Jahrzehnten des 16. Jahrhunderts der Herr über die beiden größten Kupferproduktionsstätten der damaligen Welt, über Sclrwaz in Tirol und Neusohl in Ungarn gewesen. Für Ungarn blieb dieser Zustand bestehen. Versuche aber einer dauernden Beherrschung auch des Tiroler Kupferbergbaues hat Jakob Fugger eigentlich bis zu seinem Tode gemacht. Dabei scheint die monopolistische Absicht besonders bei zwei „Kupfer¬ käufen“ hervorgetreten zu sein. Bei dem Kontrakt , den Jakob Fugger am 7. November 1514 für die Zeit von Weihnachten 1516 bis Weihnachten 1520 mit Maximilian schloß und bei dem, den die Fugger und Höchstetter zusammen am 30. Oktober 1515 für die Zeit von 1521—1524 mit dem Kaiser verabredeten. Wenigstens hielt es Jakob Fugger noch einige Zeit nach Ablauf der Kontrakte für geraten, sich vom Kaiser bestätigen zu lassen, daß damit keine unziemliche oder sträfliche Teuerung in deutscher Nation oder sunst gemacht worden sei 2. Der Nachweis dieser Behauptung dürfte Jakob Fugger schwer geworden sein; verpflichtete ihn doch der Vertrag mit den Höchstettern und Kaiser Max, der den zwei Firmen auf vier Jahre alles Schwazer Kupfer in die Hände gab, während der Vertragsdauer kein fremdes (d. h. ungarisches) Kupfer nach Flochdeutschland oder Welschland zu führen, sondern sein ungarisches Kupfer bei Strafe der Konfiskation nur in Nieder¬ deutschland abzusetzen 3. Selbstverständlich war mit dieser Ver¬ abredung bezweckt, den Preis des Tiroler Kupfers, auf den das ungarische Kupfer von jeher niederdrückend gewirkt hatte , in die Höhe zu treiben. Man versteht es aus den Vorgängen des Jahres 1525 sehr wohl, weshalb sich Jakob Fugger nicht mit dem Mandat vom 13. Mai 1525 begnügte, das ja, wie wir sahen, die Monopole auf dem Gebiete des Erzhandels ganz allgemein für rechtlich und ethisch erlaubt erklärte. Sicherlich hielt er für die damals besonders 1 R . Ehrenberg, Zeitalter der Fugger I, S. 417 . M. Jansen, Jakob Fugger der Reiche , S. 52 ff. Letzterer ist auch für das folgende zu vergleichen. 2 Mandat Karl V . vom 26. Oktober 1525 (Toledo ). Teilweise abgedruckt bei M. Jansen, Jakob Fugger der Reiche 'S. 404 ff. 3 Jansen, a. a. O. S. 115. 4 Fünftes Kapitel : Monopoleu. Kartelle im Idriancr Quecksilberhande! usw. 301 gefährlichen politischen Verhältnisse in Tf«}l und Ungarn einen besonderen Schutz unter Bezugnahme auf die zwei genannten — für eine strenge Wirtschaftsethik der damaligen Zeit etwas anrüchigen ■ — Geschäfte für angebracht. Mehr als einmal ist in der sozialen Bewegung, die 1525 Tirol erschütterte >, ist auch in der nationalen ungarischen Erhebung, die den Fuggern so be¬ deutende Verluste in ihrer Ofener Faktorei brachte 2, das Argu¬ ment der „wucherischen Monopole“ gegen die Firma gehandhabt worden. Es entzieht sich vorläufig unserer Kenntnis, ob auch die Höchstetter sich von Karl V. für ihr Idrianer Quecksilbermonopol noch ein besonderes, auf ihren Namen lautendes Schutzprivileg ausfertigen ließen, oder ob sie sich mit dem allgemeinen Mandat vom 13. Mai 1525 begnügten. Die Habsburger, mit denen die Firma seit alters in den innigsten geschäftlichen Beziehungen stand 3, dürften ihnen kaum jede mögliche Unterstützung ver¬ weigert haben. Und Grund genug, sich auf alle mögliche Weise vorzusehen, hatten die Höchstetter. Die Firma —- aus kleinen Anfängen erwachsen1 stand — in den ersten Jahrzehnten des 16. Jahrhunderts neben den Fuggern und Weisem im Vorder¬ gründe der deutschen Kaufmannschaft. Dabei war die Organi¬ sationsform des Höchstetterschen Geschäftes noch besonders dazu angetan, vieler Augen auf die Firma zu ziehen. Während andere 1 Vgl. H . W opiner, Quellen zur Geschichte des Bauern¬ krieges in Deutschtirol . Acta Tirolensia III , 1. Vgl . das Register sub Fugger und Handelsgesellschaften. 2 Vgl. jetzt darüber ausführlich Jansen, a . a . O. S. 179 ff. Wie beunruhigend die Bewegung gegen die „ Monopolisten " auf den Handel des 16. Jahrhunderts wirkte , und wie beinahe jede eigen¬ nützige Wegnahme von Kaufmannsgut damit entschuldigt wurde, erkennt man auch aus H anse rezesse III . Abt . 6. Bd , Nr . 220. 3 R . Ehrenberg, Zeitalter der Fugger 1, S. 212 ff . Ich halte mit Ehrenberg die Nachricht des Augsburger Chronisten Clemens Sender , der zufolge Ambrosius Höclistettcr im Jahre 1489 den von den Bürgern Brügges gefangen gehaltenen Maximilian mit Geld versah , für richtig , über die geschäftlichen Beziehungen der Habsburger zu den Höchstettern vgl . außer Oberleitner und Thorsch jetzt besonders noch Jans e n , Jakob Fugger der Reiche , siehe Register sub HöehstcUrr. 4 Näheres bei Strieder, Zur Genesis des modernen Kapita¬ lismus , S. 166 ff. 302 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. große Augsburger Handelshäuser kleine, festverzinsliche Depositen nur gelegentlich von Verwandten, Freunden, Faktoren usw. annahmen und von Fremden überhaupt keine oder nur größere Be¬ träge zu festem Zinssatz (meist 5 %) zu ihrem sonstigen Betriebs¬ kapital liehen, nahmen die Höchstetter Depositen in jeder Höhe und woher sie konnten. Der gleichzeitige Augsburger Chronist Clemens Sender berichtet darüber : ,,Zu Ambrosius Höchstetter haben Fürsten und Grafen, Edelleute und Bürger, Bauern, Dienst¬ knechte und Dienstmägde ihr Geld, was sie gehabt haben, gelegt und vom Hundert 5 fl. genommen. Mancher Bauemknecht und Leute, die nicht mehr als 10 fl. in Besitz hatten , die haben es dem Höchstetter in seine Gesellschaft gegeben und haben gemeint, es sei ihnen ganz wohl dort aufgehoben und bringe jährliche Nutzung. Dieser Höchstetter hat eine Zeit lang in seiner Gesellschaft 1 000 000 fl. verzinst — die allgemeine Ansicht war, er lüge gern —, kein Mensch hat gewußt, daß er soviel Geld verzinst hat “ L Es versteht sich, daß ein Geschäft, auf das so viele blickten und an dem so viele Interesse hatten , der Aufmerksamkeit der den Kapi¬ talisten übel gesinnten Kreise besonders ausgesetzt war. Man würde es also schon aus diesem Grunde verstehen, wenn die Höch¬ stetter ihr idrianisches Quecksilberhandelsmonopolsich so dicht wie nur möglich mit obrigkeitlichen Schutzmaßregeln umgeben ließen. Aber ein anderes kam hinzu. Die Höchstetter hatten, schon ehe sie das Idrianer Quecksilberhandelsmonopol übernahmen, den Ruf als besonders gefährliche und schädliche Monopolisten. Clemens Sender erzählt, Ambrosius Höchstetter habe mit seiner Kaufmannschaft oft den gemeinen Nutzen und den armen Mann bedrückt, nicht allein mit großem, namhaften Gut und Waren, sondern auch mit billiger Ware. Er habe Fichtenholz aufgekauft und erst verkauft, als schlechtes Wetter die sonstige Zufuhr zum Markte hinderte. Desgleichen Wein und Korn und Saiten für die Lauten. Oft sei von Höchstetter ein ganzer Warenvorrat teurer, als er wert gewesen, aufgekauft worden, damit er die anderen Kaufleute, die das nicht vermochten, nach seinem Gefallen drücken konnte 3. Es wird sich heute kaum mehr feststellen lassen, was an diesen 1 Die. Chroniken der deutschen 2 a . a . O. S. 220. Städte , 23 . Bd ., S. 219. Fünftes Kapitel : Monopole u. Kartelle im Iclrianer Quecksilberhandel usw . 303 Aussagen des Chronisten, der darin wohl die Augsburger öffent¬ liche Meinung wiedergab, wahr ist und was erdichtet wurde. Das nur vermag ich festzustellen, daß Ambrosius Höchstetter das Idrianer Quecksilbermonopol mit rücksichtsloser Energie durchführte. Im deutschen Reiche war außer in Idria im Böhmer Wald ein Queck¬ silberbergwerk gelegen. Zwar war die böhmische Förderung nicht sehr bedeutend, immerhin vermochte sie preisdrückend auf das idrianische Produkt zu wirken. Um das zu verhüten, mußte Ferdinand schon am 11. April 1526 von Wien aus ein offenes Generalmandat erlassen, in dem alle Obrigkeiten und Zöllner der österreichischen Erblande streng angewiesen wurden, böhmisches Quecksilber nicht durch die österreichischen Lande nach Italien oder sonst wohin führen zu lassen, sondern sofort mit Beschlag zu belegen1. Das Mandat wurde am 27. Juli 1526 in verschärfter Fassung wiederholt 2. Als dann aber im Jahre 1527 Erzherzog Ferdinand König von Böhmen geworden war, ließen sich solche kräftige Maßregeln gegen die Ausfuhr böhmischen Quecksilbers nach Italien usw. nicht mehr gut treffen. Jetzt drang Ambrosius Höchstetter mit allen Mitteln in Ferdinand, er solle das böhmische Quecksilberbergwerk in seine Hände zu bekommen suchen 3. Der neue Böhmenkönig hat sich dem Wunsche des Augsburger Kapi¬ talisten nicht versagt. Bevor er aber noch energische Schritte auf dieses Ziel hin tat , hatte sich Ambrosius Höchstetter fürs erste schon mit dem Besitzer des böhmischen Werkes, einem gewissen Wigkell, durch eine private Abmachung geeinigt. Wigkell ver¬ pflichtete sich in dem Syndikatskontrakt, seine Produktion auf ein Maximum von jährlich 800 Zentner einzuschränken und alles von ihm gewonnene Quecksilber allein an die Höchstetter-Kompanie zu verkaufen 4 Aber mit der Zuriickdrängung der relativ unbedeutenden 1 Das Mandat ist abgedruckt bei F . A . Schmidt, III , 1, Nr . 53. 2 Daselbst Nr . 54. 3 Höchstetter weiß kein anderes Mittel , um Idria hoch zu halten, „dann dass das bechmisch berckwerckh dem pergkwerckh Idria mecht verwannt und zugestelt werden , damit Ew . ku . Mt . [Ferdinand] des hohen ansuchens von denen Pehainen umb ain pass die Silber durch Ew . ku . Mt . erbland auf Venedig zu furen absein mecht “ . F . A . Wien . Innerösterr . Quecksilber , Fase . 22 (1523— 1563). 4 Daselbst. 304 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. böhmischen Konkurrenz begnügte sich ein Mann wie Ambrosius Ilöchstetter nicht. Seine Gedanken schweiften weiter. Sein Ziel war die Errichtung eines Höchstetterschen Weltmonopols für Queck¬ silber. Nun war neben Idria das spanische Almaden zu jener Zeit die bedeutendste Fundstätte für Quecksilber. Glückte es, die dortige Produktion in die Hand zu bekommen, so konnte der Augsburger der Welt die Preise für Quecksilber nach seinem Gut¬ dünken vorschreiben. Die Almadener Quecksilberbergwerke gehörten zu den Be¬ sitzungen des Großmeisters des Calatravaordens. Die Einkünfte dieser wie der anderen beiden Großmeistereien der spanischen geistlichen Ritterorden 1St( . Jago und Alcantara) zog, seitdem Ferdinand von Aragonien die Großmeisterwürde an den König von Spanien gebracht, die Krone an sich 2. Schon seit dem 15. Jahr¬ hundert war es üblich, die Ordenseinkünfte im ganzen oder zumeist geteilt an Kaufleute gegen bedeutende Geldvorschüsse an die Krone zu verpachten. Im 16. Jahrhundert bildete sich dieser Brauch noch mehr aus. So hatte im Jahre 1516 Alfonso Gutierrez das Bergwerk von Almaden auf vier Jahre übernommen. Er durfte in den Gruben ganz nach Belieben schalten — Gewerken gab es nicht — und hatte nur die Verpflichtung, nach Ablauf seiner Pacht das Werk in gutem Zustande seinem Nachfolger zu überlassen. Der Pächter erhielt die Zusicherung des unbedingten Quecksilber¬ handelsmonopols für ganz Spanien; alles Quecksilber, das nicht von ihm stammte und in Spanien angetroffen wurde, verfiel der Konfiskation. Die Regierung durfte für kein zweites spanisches Quecksilberbergwerkdie Genehmigung erteilen. Auf der anderen Seite war der Quecksilberhandel des Pächters absolut unbeschränkt 3. Er durfte exportieren, wohin er wollte usw. Nach Ablauf der Pachtfrist von 1516 scheint Alfonso Gutierrez im Jahre 1520 noch einmal die Bergwerke von Almaden auf vier Jahre in Pacht genommen zu haben. Dann übernahmen mit der Pacht der gesamten Einkünfte der Großmeistereiender drei spani¬ schen Ritterorden die Fugger auch die Pacht der Almadener Queclc1 Die Einkünfte der Großmeistereien der drei spanischen geist¬ lichen Ritterorden wurden Maëstrazgos genannt (von mesa maestral ). 2 K. Hü bl er , Die Geschichte der Fuggersclien Handlung in Spanien . Kap . IV. Die Maëstrazgos. 3 H ii b 1e r a. a. O. S. 93. 4 Fünftes Kapitel : Monopoleu. Kartelle im Mrianer Quecksilbex-handcl usw. 305 silbergruben (1. Januar 1525)1auf drei Jahre . Die Fugger ver¬ kauften das Quecksilberprodukt, das sie in Almaden gewannen, zumeist nach Italien. Mit den großen Genueser Firmen Joh . Bapt. Grimaldi und Thomas Fornari liefen 1527 große Quecksilber¬ lieferungsverträge der Augsburger2. Gerade die Konkurrenz der Fugger in Italien war nun den Höchstettern sehr lästig. Denn auch das idrianische Quecksilberund Zinnoberprodukt ging größtenteils nach der Apenninhalbinsel3. Höchstwahrscheinlich hat Ambrosius Höchstetter schon bei der Übernahme des idrianischen Quecksilberhandelsmonopols daran ge¬ dacht, die spanische Quecksilberproduktion in seine Hände zu nehmen. Jedenfalls gehen alle diesbezüglichen Bemühungen Fer¬ dinands — auf die wir gleich zu sprechen kommen werden — von Höchstetter aus. Zunächst freilich trat der Kaufmann noch nicht hinter seinem hohen Beschützer hervor. Als sich die Fuggersche Pacht der spanischen Maëstrazgos zu Ende neigte, bewarb sich zunächst — der in Spanien erzogene— Ferdinand für seine Person 1 1 Hä bl er a. a. O. S. 75 und S. 94. 2 Strieder, Inventur der Firma Fugger aus dem Jahre 1527, S. 38 und 82. 3 Das Quecksilber scheint von Italien aus besonders in die Levante exportiert worden zu sein. So berichtet Hans Ulrich Krafft in seinen Denkwürdigkeiten (ed. K. D. Häßler unter dem Titel „Reisen und Gefangenschaft H . U. Kraffts “ als 61. Bd. der Bibliothek des literarischen Vereins in Stuttgart 1861) von einem Schiff der Augsburger Firma Melchior Manlich und Mitverwandte, das einen großen Vorrat von Quecksilber von Marseille aus nach Alexandria brachte. Schon 1248 wurde übrigens spanisches Quecksilber aus Marseille nach Syrien exportiert. Vgl. A. Schaube, Handels¬ geschichte der romanischen Völker, S. 206. Aus Genua schon im 12. Jahrhundert nach der griechischen Romania, a. a. O. S. 247; Mitte des 13. Jahrhunderts von Marseille nach Messina, a. a. O. S. 501. 4 Allerdings„previous to the appointment and acceptance of a mercantile.house to be responsible for the payment and to undertake the works etc.“. Calendar of letters, despatches and state papers relating to the negotiations between England and Spain prcserved in the archives at Simancas and elsewhere, Vol. III, Part. II (1527—1529), ed. Pascual de Gayangos. London 1877, S. 337. H ä b 1e r a. a. O. S. 95 f. Die Unterhandlungen mit Karl V. bzw. mit dem spanischen Finanzrat (Hacienda) führte für Ferdinand Martin de Salinas. Jetzt sind diese Unterhandlungen bequem zu erkennen in den Originalberichten Martin de Salinas an Ferdinand I. Strieder , Studien z. Gosch , kapitalist . Organisationsformen . 20 30(1 Drilles Duell; Monopole , Knrlelle lind Alilieiif'ese.llseluiflen im Millelnllev. um die Neupacht der Almadener Queeksilbe .rborgwcrkc. Bald aber stellte cs sich heraus, daß Ferdinand die Unterhandlungen für die 1löchsLctler führte. In dem Bericht, den Martin de Salinas, der spanische Geschäftsträger des Erzherzogs, an seinen Auftraggeber sandte, ist die Firma, für die Ferdinand sieh bemühte, zwar Ostrctes genannt, aber wir sind mit II ä bl er der Ansicht, daß hier eine Entstellung vorliegt, wie. sie für deutsche Namen in ausländischen Urkunden gang und gäbe ist. Den Beweis dafür, daß mit den Oslrel.es wirklieh die Augsburger 1löchstettcr gemeint sind, dürfte damit gegeben sein, daß tatsächlich die Gewerken von tdria an Erzherzog Ferdinand mit der Bitte herangetreten sind, er möchte die an ihn gebrachte Aufforderung der Gebrüder Höchsletter be¬ rücksichtigen und ihnen beim Kaiser die Pacht der spanischen Quecksilberbergwerke erbitten k Die Vereinigung der maßgebenden Quecksilberbergwerkein der Hand des Ambrosius Hoch sie lier ist trotz der Bemühungen Ferdinands nicht zusLande gekommen. Ais der Faktor der Höch¬ stetter in Spanien erfuhr, daß die Fugger noch zirka 2000 Zentner Vgl. Ant . Rodriguez Villa , El cinperador Carlos V y su cortc. Scgtiti las eartas de Don Marlin de Salinas, em baj ador del infante Don Fernando (1522—1539), Madrid 1903/05, S. 368, 370 bis 373. Nach Villas Publikation ist es nun ganz sicher, daß die Ostrctes die llöchstctter sind; ist doch direkt dort von Ambrosio Osterl aus Augsburg die Rede. 1 F. A. Wien. Inncrösterr. Quecksilber 22. „Ew. Mt. well gnedigist fürschung tlnm, damit das qwegsilbcrpcrgwerchin Hispania disem E. K. Ml. pergwcrch Idria nit zu schaden und abfall gebaut, gcarwait oder in frembder person band und gewaltsam gebracht werdt. Und weil dann die röm. kais. Mt. dasseib pergwcrch den Fuggern von Augspurg eilieh jar uinb nin jerlich zins, jeds jar umb 2000 ducaten verüben hat, so aber nun jetzund dieselben jar vcrschin und vergangen sein und das gcmell pergwcrch der röm. kais. Mt. widcruml) haimgefallcn ist, so langt an Ew. ku. Mt. unser underthanigst bit, die welle auf suplicircn und anpringen Ambrosi und Hans Gebrüder der Hochslctcr gnedigist furschung thun und bei der röm. kais. Mt. verhelfen, damit dasseib pergwcrch in Hispania in Ew. Ml., der Hochslctcr und unser band gcpracht werdt. So mag alsdan dasseib pergwcrch im bau und arwait dem pergwcrch Idria o» schaden gehalten werden." Sonderbarerweise stammt diese Bittschrift der Gewerken erst vom 12. Februar 1528, während sich doch schon 1527 spätestens Ferdinand entschlossen hatte, den Höclislettern die Almadener Pacht zu verschaffen. Fünftes Kapitel : Monopoleu. Kartelle im Idripner Quecksilberhandel usw. 307 Quecksilber im Vorrat hätten — eine Summe, die bei dem damals noch nicht sehr erheblichen Weltverbrauch genügte, um eine be¬ deutende Preiserhöhung des Inhabers der vereinigten Idrianer und Almadener Produktion auf viele Monate hinaus zu verhindern —, als ferner eine spanische und eine genuesische Firma die bisher von den Fuggern gezahlte Pacht bei weitem überboten, da traten die Höchstetter von der Bewerbung um Almaden zurück. Es müssen sich aber ziemlich bedeutende Massen von Quecksilber für das Projekt eines Weltmonopols schon in den Lagern der Firma angesammelt haben, die nun schwer verkäuflich waren. Sonst hätte sich Ambrosius Höchstetter schwerlich dazu verstanden, im August 1528 mit dem Brüsseler Hofe, der für die kaiserlichen Heeresvölker in Geldern unbedingt Geld brauchte, folgendes Ge¬ schäft abzuschließen1: Der Augsburger lieh dem Brüsseler Hofe 200 000 Carolusgulden, aber er zahlte die Summe nicht in bar aus, sondern stellte 350 700 Pfund Quecksilber und 60 760 Pfund Zinnober zur Verfügung, welche die Regierung selbst verkaufen lassen mußte. Die niederländische Regierung hat bei dein Geschäft 74 000 Carolusgulden eingebüßt. Ihr Beauftragter, Lazarus Tücher, erhielt für das von Höchstetter gelieferte Quantum Quecksilber und Zinnober statt 200 000 nur 126 000 Carolusgulden2. Die Höchstetter aber sind des gewaltsamen Verkaufes eines Teiles ihrer großen Quecksilbervorräte nicht froh geworden. Bald darauf ist die Firma Ambrosius und Hans Höchstetter und Mit¬ verwandte in einem Bankrott , der die Kaufmannschaft von ganz Europa in Aufregung versetzte, unrühmlich zugrunde gegangen3. Die Augsburger allgemeine Ansicht, die der Chronist Clemens Sender wiedergibt, nannte als Hauptursache des Falliments die verfehlte Quecksilberspekulationder Firma. Ambrosi Höchstetter, heißt es dort, hat in allen Königreichen und Landen das Quecksilber auf¬ gekauft, teurer als der allgemeine Kauf war um 8 fl., damit er durch diese Listigkeit die andern Kaufleute drücke. Da er nun das Quecksilber ganz in seine Hand gebracht hatte , verkaufte er den Zentner für 14 fl. Da gab aber Gott zu, daß der Kaiser in Spanien und der König in Ungarn große Mengen Quecksilber 1 Ehrenberg , Zeitalter der Fugger I, S. 215. 2 Näheres bei Ehrenberg a. a. O. 3 Ehrenberg a . a. O. I, S. 214 tf. und 398. 20* 308 Drittes Blich: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. fanden. Und der Höchstetter hatte für 200 000 fl. Quecksilber aufgekauft, daran mußte er den dritten Teil verlieren 1. Aus unserer vorangehenden, auf sicherem Aktenmaterial beruhenden Darstellung ist zu erkennen, daß die Nachricht in der Weise, wie sie Sender gibt, nicht wahr sein kann. Immerhin hatte die all¬ gemeine Anschauung im damaligen Augsburg wenigstens darin recht, daß verfehlte Versuche, ein Weltmonopol für Quecksilber zu schaffen, den Sturz der Höchstetter mit herbeiführen halfen. Die Almadener Quecksilberbergwerkesind mit den übrigen Maëstrazgos bei der Neupacht von 1528 in die Hände der Fugger und Welser gekommen. Das Konsortium der beiden deutschen Bankhäuser mußte dabei die Pachtsumme zahlen, wie sie von den spanischen und genuesischen Konkurrenten geboten worden war 2. Erst 1538 übernahmen die Fugger die Maëstrazgos und damit auch die Almadener Quecksilbergruben wieder als alleinige Pächter. Sie hatten seitdem wiederum die alleinige Verfügungsgewalt über die nicht unbedeutende spanische Quecksilberproduktion, bis 1550 ein großer Brand das Bergwerk vernichtete und auf längere Zeit zum Stillstand brachte 3. Für die Zeit von 1547—1550 sind wir durch einige glück¬ licherweise erhaltene Abrechnungen in der Lage, einen Einblick in die Größe des Almadener Monopolunternehmens zu gewinnen4. In der genannten Epoche war verkauft worden: an Quecksilber und Zinnober 3761 Zentner 21% Pfund, an Sublimat 652 Zentner 98% Pfund. Am Schlüsse der Pachtperiode waren noch vorrätig: 2549 Zentner Quecksilber, 691 Zentner Zinnober und 450 Zentner Sublimat. Häbler dürfte Recht haben, wenn er davor warnt, aus der Addition dieser Zahlen etwa die Höhe der Förderung fest¬ stellen zu wollen. Offenbar waren von früheren Pachtungen größere Summen des wertvollen Metalls in den Händen der Fugger ver¬ blieben, die in den genannten Zahlen mit enthalten sind. Diese Annahme wird bestätigt durch einen Rechenschaftsbericht des Faktors Hans von Schüren, in welchem die Summe von 3240 Zentner Quecksilber und Zinnober sowie von 450 Zentner Sublimat wohl als Ausbeute für die Zeit vom 1. Januar 1547 bis 31. Dezember 1 Chroniken der deutschen Städte, Bd. 23, S. 220. 2 Hä b 1e r a. a. O. S. 96 und 77. 3 Hä b 1e r a. a. O. S. 103. 4 Hä b 1e r a. a. O. S. 102. Fünftes Kapitel : Monopoleu. Kartelle im Idrianer Quecksilberhandel usw. 309 1551 ifnztisehen ist. Demnach würde die jährliche Förderung durchschnittlich 650 Zentner Quecksilber und Zinnober und 90 Zentner Sublimat betragen haben. Die Fuggersehe Abrechnung von 1547—1551 gibt uns auch einen Anhaltspunkt für die Frage, wohin während dieser Zeit die Quecksilberausbeute von Almaden verkauft wurde. Wenn dar¬ nach ein Posten von 2188 Zentner Quecksilber und 4051/2 Zentner Zinnober nach Antwerpen, Venedig und Marseille unterwegs war, so erkennt man neben dem großen Meßplatz Antwerpen, auf dem in der Mitte des 16. Jahrhunderts schließlich jede Ware gehandelt wurde, noch immer die Mittelmeerhäfen, von denen aus das Queck¬ silber und Zinnober in die Levante 1und nach Indien 2ging , als Hauptabnahmestellen der genannten Metalle. * * Während so bis zur Milte des 16. Jahrhunderts das spanische Quecksilbermonopol, in die Hände der Fugger gelegt, eine relativ einfache Entwicklung durchlief, kam es in Idria nach dem Bankrott der Höchstetter zunächst nicht wieder zu einem längeren Monopol im Quecksilberhandel; Es müssen um 1529 schwere Zeiten für die dortigen Gewerken angebrochen sein. Die Produktion ließ, wie aus der untenstehenden Tabelle ersichtlich ist 8, außerordentlich 1 Von Marseille aus versendete die Augsburger Firma Melchior Manlich & Co. mit eigenen Schiffen Quecksilber nach Alexandria. Siehe oben S. 305 Anm. 3. 2 Daß die Venetianer Quecksilber nach Indien führten, ergibt sich aus gelegentlichen Bemerkungen in den Akten des Wiener F. A. 3 Höhe der Produktion in Idria in Wiener Zentnern mit Hin¬ weglassung der Bruchteile. (Nach Msc. im Wiener F. A. Innerösterr. Quecksilber 22, zum Jahre 1536.) Jahr Zinnober Quecksilber 1525 65 524 1526 103 118 1527 621 143 1528 902 180 1529 536 30 1530 20 24 1531 54 6 1532 85 41 1533 94 11 1534 _ 14 1535 — 4 * 310 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. nach , um zeitweise fast ganz einzugehen . Noch 1539, als schon eine wesentliche Besserung der Dinge bevorstand , klagten die Gewerken in einer Bittschrift , sie hätten „ seit der Höchstetter Verderben die meiste Zeit das Bergwerk aus ihrem eigenen Säckel verlegen müssen “ '. Nur zeitweise gelang es den Gewerken, Lieferungsverträge mit Großhandelsfirmen abzuschließen , bei denen dann der Verlag durch Anleihen auf diese abgewälzt werden konnte. So muß Hans Baumgartner , der selbst ein bedeutender Gewerke zu Idria war , verschiedentlich in den dreißiger Jahren solch kleinere „Quecksilberkäufe “ abgeschlossen haben 2. Wir haben Kunde von einem solchen für 10 000 fl, und einem anderen von 32 000 ÎL 3. Aber diese Erleichterungen der Lage der Idrianer Gewerken fallen doch erst in das Ende der dreißiger Jahre . Zunächst , am Anfang des vierten Jahrzehnts des 16. Jahrhunderts fand sich kein kapital¬ kräftiger Helfer, der in der Weise, wie es die Höchstetter getan hatten und wie es dann Hans Baumgartner tat , den Gewerken die Sorge um den Absatz des Quecksilbers und um die Beschaffung genügender Verlagssummeu abnahrn . Es war doch nur ein Ausweg, den man in Ermangelung von etwas besserem traf , wenn die Ge¬ werken 1532 mit dem venetianischen Kapitalisten Nicola Venier einen — wirtschaftsgeschichtich interessanten — Tauscbkontrakt verabredeten , in. welchem sich der Venetianer verpflichtete , inner¬ halb sieben Jahren für 106 000 Dukaten Quecksilber und Zinnober (zum Preise von 12,5 Dukaten pro Zentner Quecksilber resp. 16 Dukaten pro Zentner Zinnober ) gegen entsprechende GewürzWien . F . A . Innerösterr . Quecksilber 22. Es wird also als das Natürliche hingestellt , daß ein Kaufmann den Gewerken diese Leistung durch Darlehen abnahm . Ygl . hierfür auch den interes¬ santen Text des Vertrages von 1539 , auf den wir noch zu sprechen kommen. 2 Wird doch in dem Monopolvertrag mit diesem reichen Kauf¬ mann , zu dem es, wie wir noch sehen werden , 1539 kam , betont, daß die Idrianer Gewerken Baumgartner zu Dank verpflichtet seien, „neben anderem der practicen halben , so der Paumgartner in vorigen kheufen diser handlung zu guetem geen Alcheuro und Constantinopl mit etwas grossen ehosten , wie er glaubhaft dargetan , gefuert hat “ . Daß es sich dabei nur um I .ieferungsabschlüsse eines Teiles der Idrianer Produktion handelte , erkennt man aus dem weiteren Wortlaut des Kontraktes , wo im Gegensatz zu diesen „ Käufen “ von einem „ großen Kauf “ die Rede ist. 3 Abgeschlossen auf 1 % Jahr am 14. Februar 1536. 4 Fünftes Kapitel : Monopole u . Kartelle im Idrianer Quecksilberhandel usw . 311 lieferungen abzunelimeu Bald aber weigerte sich der Vcnetianer, den Kauf zu halten 2, und auch Vorstellungen der Gewerken beim Rat von Venedig und ihr Ansuchen , Nicola Venier zum Einhalten des Kontraktes oder zur „Bezahlung des Profites “ anzuhalten, dürfte vergebliche Mühe gewesen sein 3. So kam im Verlaufe der dreißiger Jahre eine ziemlich resignierte Stimmung über die Gewerken von Idria . Die Kartellverhandlungen, die sie um 1534 mit fremden Quecksilberproduzenten zur Behebung der Absatzkrisis führten , sind durchaus aus dem Drange der Not geboren i. Zunächst handelt es sich dabei um Verabredungen mit der Nürnberger Firma Steher (oder Staber ), die die böhmischen Quecksilbergruben 5 beherrschte , und böhmisches Quecksilber und 1 Der Entwurf des Kontraktes Wien. F . A. Innerösterr . Queck¬ silber 22. 2 Er wollte einen geringeren Kaufpreis zahlen als vereinbart worden war, angeblich, weil spanisches und böhmisches Quecksilber in Venedig den Preis drücke. F. A. Wien. a. a. O. 3 Aus einer Resolution König Ferdinands. Wien, 2. Juli 1534. Abgedruckt bei F. A. Schmidt, III, 1, S. 147. 4 Bekanntlich sind sich die Nationalökonomen nicht darüber einig, ob die Erkenntnis der proiitdrückenden Wirkung des Kon¬ kurrenzkampfes die Unternehmer mehr in ungünstigen oder in günstigen Marktlagen zum Abschluß von Kartellen veranlaßt habe. Klein wüchter, der wissenschaftliche Entdecker der Kartelle, nimmt das erstere an. Ihm sind die Kartelle Notstandskinder. R. Lief mann, Die Unternehmerverbände, S. 53 ff., kennt zwar demgegenüber auch eine ganze Reihe von Syndikaten, die in Zeiten günstiger Konjunktur aus dem Wunsche heraus entstanden sind, unbelästigt von der Konkurrenz, die gute Wirtschaftslage durch Preissteigerungauszunützen, aber auch ihm sind die meisten Kartelle aus der Not schlechter Zeiten heraus geboren. Auf der anderen Seite steht u. a. Sombart Die ( deutsche Volkswirtschaft des 19. Jahrhunderts . Berlin 1903. S. 368). Er meint , es seien nicht die Zeiten schwerster Depression, in denen sich die Unternehmer eines Gewcrbszweigcs zu Kartellen zusammenschlössen— in solcher Zeit hoffe jeder sich noch eher auf eigene Faust durchzuschlagen— sondern vielmehr die Zeiten des Aufschwungs, die Zeiten, wo ein Absatz zu höheren Preisen, als sie den Produktionskosten ent¬ sprächen, gesichert erscheint. Die geschichtliche Erfahrung lehrt, daß die Ansicht Liefmanns im ganzen das richtige trifft. 5 Die Gruben lagen bei Schönbach und gehörten den Schlick. Die Firma Steher aus Nürnberg hatte sich um 1534 für drei Jahre verpflichtet , jährlich 50 Meiler = 500 Zentner abzunehinen . Dem * 3 312 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. Zinnober nach Venedig brachte. Die Verabredungen, die die Idrianer Gewerken mit Steber in Villach trafen, sind, soviel ich sehe, nur in einigen Unterbandlungsbruchstücken auf die Nach¬ welt gekommen, immerhin erkennt man auch schon daraus deutlich genug, daß es sich urn Kartellbestrebungen bandelt. Da ist von Verhandlungen die Rede, wieviel Quecksilber Steber in Venedig auf den Markt bringen dürfe; auch von anderen Beschränkungen der gegenseitigen Konkurrenz wird gesprochen, „damit baider quecksilber und Zinnober gleich als durch a i n band verkauft würde“. Ferdinand hat auf das Verhandlungsprotokollzunächst geschrieben: „Placet, dass sie solch Quecksilber und Zinnober miteinander ver¬ treiben“. Dann wurde dieses Placet in folgende königliche Re¬ solution umgewandelt, die noch deutlicher zeigt, daß wir es hier mit einem Kartell zu tun haben : „Des queksiiber halber so aus Beheim durch den Steber zu Nurenberg hinein gen Venedig verfuert wurdet, lasst ir die k. Mt. genedigclich gefallen, dass si, die gewerkhen, irem anzaigen nach mit gedachtem Staber bandien, also damit das quecksilber in ainem khauf beleih und mit einander vertriben und ains durch das ander nicht verhindert werde“ l. Auch die Bemühungen der Idrianer Gewerken, das spanische Quecksilber von dem venetianischen Markt auszusehließen, zeigt die damalige geringe Nachfrage nach dem Produkt . Bei diesen Bemühungen handelt cs sich nicht um Verabredungen der Idrianer Gewerken mit den Beherrschern der spanischen Produktion, mit den Fuggern 2, sondern um eine Vereinbarung der beiderseitigen Landesfürsten. Die Gewerken von Idria baten Ferdinand, bei seinem Bruder Karl V. durchzusetzen, „ dass das spanische Queck¬ silber und Zinnober hinfüran nicht mehr gen Venedig geführt Nürnberger lag viel an einem Kartell mit den Beherrschern der Idrianer Produktion; nur dann erlaubte Ferdinand eine Durchfuhr des böhmischen Quecksilbers durch seine Erblande nach Venedig. 1Kgl . Resolution. Wien, 2. Juli 1534. Vgl. F. A. Schmidt, III, 1, S. 147. 3 Es ist wohl nur aus dem damaligen schlechten Absatz von Quecksilber und Zinnober zu erklären, daß die Fugger — die Be¬ herrscher der spanischen Quecksilberproduktion— nicht ihrerseits versuchten, die Idrianer und böhmische Produktion an sich zu bringen und auf diese Weise ein Wcltmonopol in Quecksilber zu schaffen. Die Vorbedingungen waren günstig. 12 Fünftes Kapitel : Monopoleu. Kartelle im Idrianer Quecksilberliandel usw. 313 werde“ x. Karl V. — so begründen die Gewerken ihre Wünsche — habe aus den spanischen Quecksilberbergwerkennur sehr wenig Gefälle, dagegen schade es Ferdinands Budget außerordentlich, wenn das idrianische Quecksilber geringen Absatz fände. Ferdinand versprach, mit Karl V. über den Wunsch der Idrianer Gewerken zu verhandeln, wenn der Kaiser demnächst nach Deutschland komme. Soviel ich sehe, ist aber die Angelegenheit vorläufig liegen geblieben. Auch die Kartellverabredungen mit Steher scheinen die Lage der idrianischen Gewerken nicht sehr gebessert zu haben. Ihre gedrückte Stimmung hielt an. Die Mehrzahl der Gewerken hätte sich am Ende der dreißiger Jahre am liebsten ganz von einem Risiko zurückgezogen und die Nutznießung ihrer Kuxe wie eine feste Rente genossen. Es taucht unter ihnen der Plan auf, „daß Hans Baumgartner das Bergwerk auf etliche Jahre in Pacht nehmen und auf seine Kosten verlegen solle, damit sie, die Gewerken, des schweren Darlegens zur Unterhaltung der Gruben überhoben blie¬ ben“ . Zwar weigerte sich Hans Baumgartner, diesen Vorschlag anzunehmen, aber gegen die Übernahme eines fünfjährigen Idrianer Quecksilberhandelsmonopols hat er schließlich nichts einzuwenden gehabt. Am 1. August 1539 gelangte der Kontrakt zwischen König Ferdinand, den Geweiften von Idria und Hans Baumgartner von Baumgarten zu Hohenschwangau und Erbach 3zum Abschluß 4. Er band die Kontrahenten auf fünf Jahre , vom 1. Januar 1540 an. Hans Baumgartner verpflichtete sich, den Wiener Zentner Queck1 Wenn das geschehen und das Kartell mit Steher abgeschlossen sei, „alsdann ist niemand, der weiter quecksilber oder zinnober gen Venedig fuere". 2 Wien . F. A. Innerösterr. Quecksilber 22. Also wie es in Almaden war. 3 Hans Baumgartner war 1539 in den Adelstand erhoben worden. Ehrenberga, a. O., I, S. 193. Für den Reichtum Hans Baum¬ gartners vgl. Strieder, Zur Genesis des modernen Kapitalismus, S. 48 ff. Für seine zahlreichen Geldgeschäfte mit Ferdinand E h r e n b e r g a. a. O., I, S. 193. Dazu neuerdings Jansen, Jakob Fugger der Reiche, s. Register. Jansens Forschungen haben ergeben, daß der reiche Hans Baumgartner von Kufstein nicht mit dem Augsburger identisch ist ; a. a. O., S. 13 Anm. 3. 4 Der Kontrakt ist nach dem Manuskript des Wiener F. A. (Innerösterr. Quecksilber 22) im Anhänge dieses Buches abgedruckt. 314 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. siîber mit 26 und den Wiener Zentner Zinnober mit 30 fl. rh. locoidria zu bezahlen und innerhalb der genannten Zeit für 100 000 fi. rh. abzunehmen. Mit Rücksicht auf die gefährlichen Zeitläufe und auf die schätzbaren Dienste, die der Augsburger Kapitalist den Ge¬ werken schon früher geleistet hatte \ erhielt Hans Baumgartner als „Auf-“ und „Eingabe“ 687 Zentner Quecksilber und 166% Ztr. Zinnober „frei ohne Bezahlung“. Auf seinen Wunsch war ihm alles Quecksilber und Zinnober, das die Gewerken in Triest, Villach oder sonstwo lagern hatten , gegen Bezahlung von 26 resp. 30 fl. zuzüglich der von den Gewerken ausgelegten Transportkosten aus¬ zufolgen. Hans Baumgartner stand das absolute Großhandelsmonopol für Idrianer Quecksilber und Zinnober während der fünfjährigen Dauer seines Kontraktes zu. Weder durfte der König etwas aus seinem Fron- und Wechselquecksilber an irgend jemanden anderen abgeben als an den Monopolinhaber, noch durften es die Gewerken aus den ihnen zukommenden Teilen der Förderung. Außerdem ver¬ pflichtete sich Ferdinand für sich und seine Erben während der Vertragsdauer kein fremdes Quecksilber und Zinnober durch seine Erblande passieren zu lassen. Übertreter des Transitverbotes sollten strenge bestraft und ihre Ware konfisziert werden. Dagegen durfte Baumgartner das Quecksilber und Zinnober gegen Erlegung der üblichen Zoll- und Mautgebühren hinführen, wohin er immer wollte. Von neuen Zöllen und Mauten, die etwa eingeführt wurden, war er im voraus befreit. Ein Wclthandelsmonopol für Quecksilber dem Baumgartner in die Hand zu geben, dazu fehlte es König Ferdinand an Macht. Immerhin versprach er dem Augsburger Kapitalisten, soviel er vermöchte dafür zu sorgen, daß „das spanische und böhmische Quecksilber dem idrianisclien keinen Nachteil bringe“. Besonders wollte Ferdinand sich an den Kaiser mit der Bitte wenden, er möchte gebieten, daß das Almadener Werk nicht „dermaßen wie eine Zeit her, überbaut, sondern daß, wie von altersher üblich, Maß gehalten und bedacht werde, daß beide Bergwerke — das 1Siehe oben S. 310 Anm . 2. Darnach scheint Baumgartner Quecksilber und Zinnober in die Levante geführt zu haben ; wir hätten demnach in den Baumgartner eine zweite deutsche Firma zu sehen , die im 16. Jahrhundert direkten Levantehandel trieb . Die andere waren die Manlich . Vgl . Ehrenberg a . a . O., I , S. 224 i. Fünftes Kapitei : Monopole u. Kartelle im Idrianer Quccksilberhandel usw . 315 Idrianer und das Almadener — wie hievor nebeneinander ohne Nachteil noch lange bestehen möchten .“ Außer den genannten verdienen noch folgende Bestimmungen des Kontraktes vom 1. August 1.539 besondere Hervorhebung : Sollte Hans Baumgartner in der Zeit des Vertrags mehr als für 100 000 fl. Quecksilber abnehmen und absetzen können — und er versprach es nach Kräften anzustreben —, so sollten die Ge¬ werken und König Ferdinand dieses Mehr zum obengenannten Preise liefern . Auch für diese Mehrabnahme solle eine entsprechende „Ein -“ und „Aufgabe “ dem Augsburger gegeben werden 1. Ver¬ möchte die Baumgartner -Gesellschaft die. über die ursprünglich ausgemachte Quantität (für 100 000 fl.) gelieferten Mineralien nicht ganz zu vertreiben , so sollte sie gehalten sein, nach Ablauf ihres Kontrakts den Rest gegen entsprechende Rückvergütung abzu’iefeni . Ohne diese Maßregel wäre es dem König und den Ge¬ werken nur schwer möglich gewesen, nach Ablauf des Baumgartner¬ sehen Quecksilberkontrakts einen andern Kapitalisten für einen neuen Abschluß zu finden . Denn selbstverständlich hätte Baum¬ gartner mit der ihm Testierenden Quecksilbermenge die Preise des neuen Kontrahenten empfindlich drücken können . Von einem Handelsmonopol in idrianischem Quecksilber wäre so lange nicht mehr die Rede gewesen, bis Baumgartner all sein Quecksilber ver¬ kauft gehabt hätte. Die zuletzt genannten Paragraphen des Kontraktes vom 1. August 1539 waren für den Fall vorgesehen , daß Baumgartner nach Ablauf seines Monopols bei einem neuen Abschluß nicht in Frage kam . Man hoffte , daß dieser Fall nicht eintreten würde und machte dem Augsburger sogar starke Hoffnungen auf einen Neuabschluß , Wenn sich Baumgartner während seiner Monopol¬ inhaberschaft dem Bergwerk nützlich erzeigen und das Quecksilber und Zinnober auf einen hohem Marktpreis als den jetzigen bringen würde , so wollte der König und die Gewerken ihn anderen Be¬ werbern vorziehen . Auf keinen Fall war es dem König und den Gewerken erlaubt , mit jemandem während des laufenden Kon¬ trakts einen neuen Abschluß zu verabreden oder abzuschließen. 1 Tatsächlich hat Hans Baumgartner für 10 000 fl. Quecksilber und Zinnober mehr abgenommen. Vgl. den Eingang des zweiten Vertrags Hans Baumgartners vom 14. Februar 1544. Wien. F. A. Innerösterr. Quecksilber 22. 1 316 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. Eine wichtige Bestimmung , die der Höchstettersche Monopol¬ vertrag nicht enthielt , gestattete dem Hans Baumgartner die ganze oder teilweise Abtretung seines Kontraktes an einen anderen Kapi¬ talisten . Das Versprechen des Königs , den Monopolinhaber gegen jegliche Belästigung von seiten des kaiserlichen Fiseals zu schützen, findet sich wie im Höchstetterschen Vertrag so auch im Bauingartnerschen . Wie überhaupt die beiden Verträge sehr viele — auch textliche — Übereinstimmungen zeigen. Wie es in dem Kontrakte vorn 1. August 1539 bestimmt war, ist fünf Jahre hindurch das Idrianer Quecksilbermonopol gehandhabt worden . An Versuchen , das Monopol zu durchbrechen , hat es dabei natürlich nicht gefehlt . Manche Gewerken versuchten entgegen den Kontraktbestimmungen heimlich Quecksilber und Zinnober auf eigene Rechnung zu verkaufen . So mußte sich am 20. November 1541 auf eine Klage Baumgartners hin , daß in Venedig und Villach Idrianer Quecksilber und Zinnober von Fremden verkauft würde , König Ferdinand mit einem Schreiben voll heftiger Vorwürfe an die Idrianer Gewerken wenden 2. Er verbot darin auf das strengste , wider den Kontrakt von 1539 heimlich Quecksilber und Zinnober zu verkaufen . Ganz abgesehen, daß er bei solchen „ Partikularverkäufen “ um Fron und Wechsel betrogen werde , gingen sie gegen die Absichten , die ihm bei der Monopolisierung des Quecksilberhandels in der Hand des einen Augsburger Kapitalisten vorgeschwebt hätten . Nur wenn das Quecksilber „in einer Hand “ bliebe, könne die Ware „ umb sovil höher gesteigert “ werden . Verhinderten die Gewerken das , so schadeten sie nicht zuletzt sich selbst. Es entzieht sich unserer Beurteilung , ob die energischen Vor¬ stellungen Ferdinands dauernd ihren Zweck erreichten . Jedenfalls hat Baumgartner von seinem Rechte , den Vertrag — wenn er nicht redlich gehalten würde — zu lösen, keinen Gebrauch gemacht. 1 In dem Kontrakt, den Hans Baumgartner 1544 abschloß, — wir kommen sofort im Text darauf zu sprechen —, ist als Be¬ dingung hinzugefügt, daß der betreffende ein ehrlicher, vermögender Mann ist, der „disen Sachen gemäss zu thuen guet macht hat“. 2 Abgedruckt bei F. A. Schmidt, III , 1, S. 252 f. Schmidt setzt als Adresse „An die Statthalter und an die Regierung und Kammer in Österreich", was sicherlich, wie sich aus dem Text ergibt, falsch ist. 1 fünftes Kapitel : Monopoleu. Kartelle im Idrianer Quecksilberhandel usw. 317 Im Gegenteil , Hans Baumgartner sollte länger als fünf Jahre der Inhaber des Großhandelsmonopols für idrianisches Quecksilber bleiben . Als man 1544 an eine Erneuerung des Monopols gehen mußte , versuchten die Gewerken zunächst wiederum , Hans Baum¬ gartner zu einer Pacht der idrianischen Gruben , diesmal auf acht Jahre , zu überreden . Aber auch dieses Mal glaubte sich der Augs¬ burger einem solchen Ansinnen versagen zu müssen . Dagegen übernahm er noch einmal unter ganz ähnlichen Bedingungen wie 1539 das Handelsmonopol in Idrianer Quecksilber und Zinnober h Es war das letzte Mall Mit dem Jahre 1548 brachten neue Männer das Monopol für den Handel mit den idrianischen Berg¬ werksprodukten an sich. Wiederum ist es eine bedeutende Augs¬ burger Kaufmannsfamilie , eine Firma , die seit Generationen schon mit den Habsburgern in regem geschäftlichen Verkehr gestanden hatte : die Herwart 2. Von diesem berühmten Handelshause schlossen im Jahre 1548 Hans Paul und Hans Heinrich Gebrüder den ersten Herwartischen Kontrakt eines idrianischen Queck¬ silberhandelsmonopols mit König Ferdinand und den Gewerken ab . Der Vertrag 3 lief vom 26. April 1548 bis zum 26. April 1553. Die Herwart verpflichteten sich, in der genannten Zeit für 140 059 fl. rheinisch 30 Kr . Quecksilber und Zinnober (den Zentner Queck1 F. A. Wien. Innerösten-. Quecksilber 22. Der Kontrakt lief vom 14. Februar 1544 bis 14. Februar 1548. Wiederum erklärte sich Baumgartner bereit, für 100 000 fl. Quecksilber und Zinnober abzunehmen. Zum Preise von 26 fl. rh. pro Wiener Zentner Queck¬ silber und 30 fl. für Zinnober. Wiederum betrug die „Auf- und Eingab" 687 Zentner Quecksilber und 166% Zentner Zinnober. Der Wortlaut des Kontraktes für diese zweite Monopolperiode Hans Baumgartners stimmt fast wörtlich mit dem vom 1. August 1539 überein. 2 Für die Bedeutung dieser Familie und ihren Reichtum vgl. Strieder, Zur Genesis des modernen Kapitalismus, S. 115 ff. Für ihre zahlreichen Geldgeschäfte Ehrenberga , a. Ü.,I,S . 218 ff. Am 22. Oktober 1548 wurden die Brüder Hans Paul, Hans Heinrich und Hans Jakob Herwart von Karl V. geadelt. In der Nobilitierungsurkunde heißt es : für die „getreuen willigen dienst, so ire voreitern weilendt unsern vorfarn und si uns und dem reiche getan haben und hinfüran ze thun sich guetwillig erpieten“. 3 Original mit der Unterschrift der Gebrüder Herwart und ihrem Siegel (mit der berühmten Eule) im F. A. Wien. Innerösterr. Queck¬ silber 22. 318 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. siiber für 29 fl. 15 Kr ., den Zentner Zinnober für 33 fl. 15 Kr., alles Wiener Gewicht) abzunehmen. Als „Ein- und Aufgab“ er¬ hielten die Brüder 860 Zentner Quecksilber und 210 Zentner Zinnober. In den beiderseitigen Verpflichtungen und Berechtigungen stimmt der Vertrag im wesentlichen mit den früheren Baumgartnerschen Kontrakten überein. Wie dort findet sich das Schutz¬ versprechen König Ferdinands gegen ein Eingreifen des Reichs¬ regiments oder des kaiserlichen Fiskals. Wie dort das Versprechen, eine allzu heftige „unlautere“ Konkurrenz der spanischen Queck¬ silberwerke nach Möglichkeit zu unterdrücken. In letzterer Be¬ ziehung kamen Naturgewalten den am idrianischen Bergbau Inter¬ essierten zu Hilfe. 1550 zerstörte ein furchtbarer Brand die Almadener Gruben vollständig. Bis auf weiteres hatten die Fugger nun nur noch mit dem Rest ihrer bisherigen Produktion Einfluß auf den Quecksilber- und Zinnobermarkt. Bei dieser Sachlage ist es sehr verwunderlich und läßt auf ein starkes Nachlassen des Weltbedarfs in Quecksilber und Zinnober oder auf die Eröffnung sonstiger neuer Gruben schließen, wenn die Herwart in ihrem zweiten idrianischen Monopolkontrakte1für den Wiener Zentner Quecksilber nur 20 fl. rh. und für Zinnober nur 24 fl. zahlen mußten. Namentlich in der letzten Zeit ihres zweiten Kontraktes haben die Herwart allerdings bei solch niedrigem Einkaufspreis und bei einem infolge der veränderten Marktlage — wir kommen sogleich darauf zurück — starken Steigen der Quecksilberpreise ein glänzendes Geschäft gemacht. Für die Geschichte des Quecksilberhandels beginnt in der Mitte der fünfziger Jahre des 16. Jahrhunderts eine neue Epoche, eine Zeit der Hochkonjunktur. Man hatte — wahrscheinlich in Deutschland 2ein — Verfahren erfunden, um Silber und Gold auf 1 Der sie für die Zeit vom 8. Oktober 1554 bis zum 8. Oktober 1559 zur Abnahme von einem 100 000 fl . rh . entsprechenden Quantum Quecksilber und Zinnober verpflichtete . Wien . F . A. Innerösterr. Quecksilber 22. 2 Hä b 1 e r hat mit guten Gründen darauf hingewiesen , daß nicht Bartolome de Medina aus Sevilla der Erfinder des Amalgamationsverfahrens ist — wie bisher angenommen wurde — sondern nur derjenige , der das neue Verfahren in den neuspanischen Berg¬ werken einführte , a . a. O., S. 138. 1 Fünftes Kapitel : Monopoleu. Kartelle im Idrianer Quecksilberhandei usw. 319 dem Wege der Amalgamierung bequem aus den silber- und gold- haltigen Erzen auszuscheiden L Nun eignete sieh zwar die neue Technik auf ihrem damaligen Standpunkte nicht für alle Silber¬ erze 2, aber für die Erze in den amerikanischen Kolonien Spaniens war die denkbar beste Edelmetallgewinnungsweise damit gegeben* Seit den Jahren 1556/57 entsteht so in Neuspanien eine ungemein starke Nachfrage nach Quecksilber 3. Die Preise des Produktes schnellten dadurch so merklich in die Höhe , daß sich die spanische Regierung sofort veranlaßt sah , die Einfuhr von Quecksilber in die Kolonien für ein Reservatrecht , ein Regalrecht der Krone zu erklären , eine Maßnahme , durch die den spanischen Finanzen in den nächsten Jahrzehnten schon viele Millionen Dukaten zuflossen. Es fragte sich, ob das Angebot der spanischen Quecksilber¬ gruben genügen würde , um die starke koloniale Nachfrage zu decken . Das Almadener Bergwerk , das einzige von Bedeutung in Spanien, war nach dem Brande von 1550 noch kaum wieder aufgenommen worden . Die Fugger hatten sehr wenig Eile be¬ wiesen, das zerstörte Werk wieder aufzurichten 4, um so weniger, als sie sich durch eine kluge Wirtschaftspolitik große Vorräte gerade 1 Das Verfahren ist in Brockhaus ’ Konversationslexikon unter dem Stichwort . Amalgamierung näher beschrieben. 2 Hä b 1 e r a . a. O., S. 139. Zum Beispiel kam es für die um 1555 entdeckten außerordentlich ergiebigen Silbergruben in dem spanischen Guadalcanal nicht in Frage. 3 „Copia, was Cristoff Baiser auf 5. Junio 1558 aus Sevilla an Spanischen hof geschriben hat : Auf datum sein zeitung herkhoinen von 5 scheffen , so in Sandt Lucar aus Nueva Spania einkhomen . Sein 3 monat , dass die ausgefarn ; das landt in fridt ver¬ lassen ; die gueter wenig schleiss , ausgenomen queckhsilber , thet 150 ducaten das quintal gelten und sonder daselbig wenig Silber in den perckhwerchen machen. Das guet zeitung fur die , so das perckhwerch draussen arrendirt haben . Und die 500 quintal , in den liulckhen jetzundt khumen , auch mit aufschlagen werden . Dan sie es angefangn zu 60 ducaten halb par , rest ain jar zeit verkhauft haben, nummer darumb finden und dise zeittung das queckhsilber draussen auch machen aufschlagen . Doch wan der khonig dessen vil zu Almaden umb das hoh verkhaufen möchte , verhielten lassen, khains von aussen khumen , alsdann ir vil ! ain plossen legen .“ Wien. F . A . Innerösterr . Quecksilber 22. 4 Hä b 1 e r a . a . O. S. 104 ff. 320 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. der reichhaltigsten Quecksilbererze aufgespart hatten 1, und wei sie durch Zuwarten hofften, die Kosten des Wiederaufbaues des Bergwerks zum wenigsten durch eine verminderte Pachtsumme wettzumachen. Als jetzt infolge der erhöhten amerikanischen Nachfrage der Quecksilberbedarfaußerordentlich stieg, machte die spanische Regierung wahr, was sie gedroht hatte , seit die Fugger Almadcn zu vernachlässigen anfingen: sie begann das Bergwerk auf Staatskosten wieder in Betrieb zu setzen 2. Die Fugger aber und ihre Unterpächter, d. h. diejenigen Spanier, denen die Augs¬ burger Firma die Aufarbeitung von Halden und Schlacken usw. unterverpachtet hatte , wurden gezwungen, ihr Quecksilberprodukt an die Regierung zu verkaufen 3, die es dann selbst zu ungeheuer erhöhtem Preise an spanische Kaufleute abgab. Erst die letzteren setzten das durch soviele Hände gegangene Produkt in die neu¬ spanischen Bergwerke ab 4. Aber die geringen Mengen des auf diese Weise erworbenen Quecksilbers und die noch geringeren, die die Regierung in dem verstaatlichten Betriebe von Almadcn selbst gewann, genügten bei weitem nicht, um den veränderten Marktverhältnissen Rechnung zu tragen. Die Regierung sah bald ein, daß nur die Fugger imstande waren, Almaden wieder auf die Höhe der früheren Leistungsfähigkeit oder womöglich noch darüber hinaus zu führen. Da auch die Fugger ihrerseits jetzt wieder gern geneigt w'aren, die Pacht von Almaden anzunehmen, so kam der folgende Vertrag (geltend vom 1. Januar 1563) zustande. Die Fugger stellten das Bergwerk wieder her und erhielten dafür die alten Privilegien (Freiheit des Holzfällens für die Stollenanlagen, Weidegerechtigkeit auf den Almaden benachbarten Wiesen für 1 Nach dem Brande ging der Fuggersche Quecksilberhandelin unverminderter Größe weiter, und selbst 1558, als der Betrieb von Almaden unter königlicher Verwaltung wieder in Angriff genommen war, hatten die Fugger noch immer 800 Zentner in Vorrat. H ä b 1er, a. a. O. S. 106. 2 Hä b 1e r a. a. O. S. 139. 3 Zunächst zum Preise von 16 Dukaten pro Zentner. 4 „Der Kunig schafft ainen grossen nutz mit disem quecksilber, dann er hat ainen spanischen handelsman gefunden, welcher ir Mt. zalt für ainen jeden centner, damit, er denselben in Indias frei fieren mege, 120 ducaten“. Martin Guzmann an Kaiser Ferdinand I. Barcelona, 1. März 1564. F. A. Wien. Innerösterr. Quecksilber 23. (1564—1599) Nr. 18 321. Fünftes Kapitel : Monopole u. Kartelle im Idriancr Quecksilberliandcl usw . 321 ihre Transportiere usw.)- Eine eigentliche Pachtsumme zahlte das Augsburger Handelshaus nicht, dafür wurde aber die alte Freiheit der Pachtinhaber, ihr Produkt frei zu verkaufen, beseitigt. Alles geförderte Quecksilber und Zinnober mußte gegen festgesetzten Preis — während der ersten fünf Jahre für 25 1Dukaten , wahrend der nächsten Jahre für 20 Dukaten —- an die Regierung abgcliefevL werden. Außerdem verpflichteten sich die Fugger zu einer jähr¬ lichen Mindestproduktion von 1000 Zentner 2. Diese Bestimmungen erfuhren insofern in den nächsten Jahren eine Abänderung, als von 1567 an die Regierung für den Zentner 26 fl. zu zahlen ver¬ sprach und die Fugger ihr Mindestlieferungskontingent auf jährlich 1200 Zentner erhöhten. Ja die Geschäfte, die die spanische Re¬ gierung in den nächsten Jahren bei dem Quecksilberverkauf nach Amerika machte, waren so glänzend3, daß sie schon 1569 den Fuggern 29 Dukaten pro Zentner bot, wenn sie imstande wären, 1500 Zentner jährlich zu liefern. Die erste Fuggerschc Pacht¬ periode nach dem neuen Stil lief nach zehnjähriger Dauer 1572 ab. Sie endete mit einem sehr guten finanziellen Ergebnis für die Firma. Im ganzen waren 13 100 Zentner Quecksilber an die Krone ab¬ gegeben worden, teilweise zu 25, teilweise zu 26 und 29 Dukaten. 1 Wegen der größeren Betriebsunkosten. 2 Leider ist uns das Original des Vertrags nicht erhalten . Obige Darstellung , die auf H ä b 1 e r s Untersuchungen beruht , wird etwas anders gegeben in dem Berichte des kaiserlichen Orators Martin Guzinann (Barcelona , 1. März 1564). F . A. Wien . Innerösterr . Queck¬ silber 23 . „Wie der quccksilberhandlung zu Almaden diser zeit allenthalben steet , habe ich mich mit allen fleis erkundiget und befinds wie volget : erstlich dass die Fuggcren von kuning dise qucck¬ silberhandlung auf 10 jar angenomen haben , welch bestanndt sich den 1. Januarii verschincn 63. jars angefangen . Die ersten 3 jaren soll bemelte Fuggcren dem kunig liveren wie volgl : das erst jar 600 ctr , das ander 800 , das drit 1000 etr . Für einen jeden zalt inen der kunig 25 ducaten bis zu ausgang der dreien jaren von wegen der uncost , so inen auf die widererbauung des bergwercks gangen ist . Die iberige 7 jar ist inen der kunig nit mer schuldig für ainen ctr. dan 20 ducaten zu geben . Und ist schuldig ain jedes jar von 1000 bis in die 1500 ctr. von inen zu nevnen. Was si iber diselbig antzal machen werden , dorten sis nindert ain verhandeln noch ver¬ kaufen .“ 3 Über die Preise , die für Quecksilber in Neuspanien gezahlt wurden, vgl . Ifä hier a. a. O., S. 143 Anna. 1. StrirdiT , Sin. lion Gnd«:li. kupit alint . Organisatiousforinoii . 1 322 Drittes Buch : Monopole, Kartelle und Aktiengescl' sclinflcn im Mittelalter. Die Unkosten der Fugger beliefen sich im Durchschnitt auf 144/s Du¬ katen pro Zentner , so daß ein Durchschnittsgewinn von 127/ 30 Du¬ katen pro Zentner , also zirka 85 % bei dem Unternehmen heraus¬ sprang Sehen wir zu, wie die veränderte Lage des Weltmarktes für Quecksilber , die dieses schöne Geschäftsergebnis für die Fugger verursacht hatte , auf die Idrianer Quecksilberproduktion und ihre Beherrscher wirkte . Da müssen wir konstatieren , daß besonders in der ersten Zeit des amerikanischen Massenbedarfs , als Almaden noch wenig liefern konnte , Idria gewaltigen Vorteil von der ge¬ steigerten Kauflust des Marktes zog. Einmal fiel für Idria , seit Almaden nicht einmal die amerikanische Nachfrage befriedigen konnte , die spanische Konkurrenz in Venedig, der Levante usw. weg. Aber auch in die spanischen Kolonien Amerikas gingen jetzt bedeutende Mengen idrianischen Quecksilbers . Die LIerwart und auch ihre Abnehmer verkauften in Antwerpen bedeutende Massen Quecksilber an spanische Kaufleute . Die Spanier , die ihrem König zu hohem Preise das Almadeuer Quecksilber für den Export in die amerikanischen Bergwerke abuahmen 2, kauften gern , wenn sie konnten , idrianisehes Produkt auf und verführten es als Almadener nach Amerika . Ihr Gewinn an dem österreichischen Produkt , das sic viel billiger als das spanische kauften , war enorm. Aber auch die Beherrscher der idrianischen Quecksilbergruben machten kein schlechtes Geschäft bei diesem Handel . Die Herwart, deren zweiter Kontrakt bis zum 8. Oktober 1559 lief, waren so zu¬ frieden mit dem Erfolg dieser Kampagne , daß sie sich beeilten, die Herrschaft über die Idrianer Produktion auch weiterhin in ihrer Hand zu behalten . Noch viele Monate vor Ablauf ihres Monopolkontraktes verabredeten sie prit den Vertretern des Kaisers und der Gewerkschaft zunächst einen Zusatzvertrag , der ihnen für 32 000 fl. Quecksilber zu sofortiger Lieferung zum alten Preise von 20 fl. rh . pro Zentner in die Hand geben sollte . Gleichzeitig vereinbarten die zwei Augsburger Kapitalisten mit dem Kaiser und den Gewerken von Idria für die Zeit vom 1. Oktober 1559 bis zum 1. Oktober 1564 einen neuen Kontrakt 3. Die Gebrüder 1Nach H ä b 1 e r a. a. O. S. 144. 3 Siehe oben. 3 Original (Augsburg , 23. Februar 1559 datiert ) im Wiener F . A. Inneröstcrr . Quecksilber 22. Fünftes Kapitel : Monopoleu. Kartelle im Idrianer Quecksilbcrhandelusw. 323 Herwart verpflichteten sich darin , in der genannten Zeit für 120 000 fl. rh . Quecksilber und Zinnober abzunehmen . Dabei wollten sie für den Wiener Zentner Quecksilber 24 fl., für den Wiener Zentner Zinnober 28 fl. zahlen . Ferner behielten sich die Herwart vor , daß ihnen auf Wunsch im letzten Jahre des Kon¬ traktes noch für 20 000 fl. Quecksilber zum alten Preise geliefert werden müsse. Doch sollte die Firma bis spätestens Oktober 1563 darum ersuchen . Schließlich erklärten sich die Augsburger bereit, die Fron des Kaisers aus den 32 000 fl. und den 120 000 fl. „ ihrer kaiserlichen Mt. alsbald auszuzahlen , zu erlegen und gut zu machen “. Wenn die klugen Augsburger Handelsherren das letztere Ver¬ sprechen als eine besondere Wohltat hinstcllten , die sie dem Kaiser erwiesen , so mochten sie mit Rücksicht auf die. furchtbare Finanz¬ not , in der Ferdinand sich befand , einigermaßen Recht haben. Aber Ferdinand war doch viel zu gut über die Handelskonjunkturen des Weltmarktes unterrichtet , um nicht zu wissen, daß der Gewinn, den er durch die Bestätigung der zwei genannten Verträge den Herwart ermöglichte , die vorausgezahlte Fron - und Wechsel¬ summe um ein mehrfaches überstieg . Schon spätestens Mitte des Jahres 1558 hatte die Wiener Regierung genaue Kenntnis der obengeschilderten Veränderungen im Welthandel mit Quecksilber. Der Schwazer Fuggersche Faktor Sebastian Kurz 1, der als Idrianer Gewerke ein starkes Interesse an diesen Dingen nahm , berichtete unter dem 30. August 1558 an den Kaiser wie folgt : Er (Kurz) habe , als er kürzlich am spanischen und englischen Hofe war , aus Sevilla von der jüngst allda angekommenen Armada aus India (Amerika ) Nachricht empfangen , daß in India das Quecksilber pro Quintal (etwas weniger als ein Wiener Zentner ) bis auf 150 Du¬ katen aufgeschlagen sei. Weil man aber jetzt nicht allein in India, sondern auch in Spanien das Silber schmelze n ganz abgestellt und sich ganz und gar auf das Verquicken (d. h. das Atnalgamicrungsverfahren ) eingerichtet habe , weil ferner das spanische Queck¬ silberbergwerk zurzeit sehr im argen liege, so sei zu hoffen, daß das idrianische Quecksilber einen hohen Preisaufschlag erführe . Wohl stehe zu befürchten , daß der König von Spanien die Zufuhr von nichtspanischem Quecksilber in die Kolonien verbieten werde, aber 1 Ehrenberg, Zeitalter der Fugger , I, S. 344. 21 * 3 324 Drittes Bueli: Monopole , Kartelle und Aktiengesellschaftenim Mittelalter. ein solches Verbot könne auf die Dauer nicht durchgeführt werden. Das spanische Quecksilber genüge kaum zur Anwendung des Amalgamationsverfahrevis in den alten und jüngst neuentdeckten (Guadalcanal ) spanischen Silberbergwerken . So bleibe dem idrianischen Quecksilber in Neuspanien ein weites Feld , das die spanische Regierung schon aus eigenem Interesse nicht sperren würde 1. Enthielt der Bericht des Sebastian Kurz eine an die Adresse des Kaisers gerichtete Warnung , sich auf einen zu „ billigen Queck¬ silberkauf “ mit den Herwart oder anderen Kapitalisten einzulassen, so traten bald noch andere Berater an Ferdinand mit Fingerzeigen heran , wie man die günstige Quecksilberkonjunktur ausnützen könne . In einem lateinischen Briefe vom 1. Januar 1559, dessen Unterschrift leider herausgeschnitten ist, wird dem Kaiser nahe gelegt , er möge anstatt der Kaufleute das Q u e c k s i 1b c'r handelsmonopol selbst übernehmen 2. Bei der jetzigen starken Nachfrage und der augenblicklich so minimalen spanischen Förderung sei ein großes Stück Geld damit zu ver¬ dienen und der kaiserliche Kredit zu erweitern . Der Kaiser könne das Quecksilber und Zinnober durch seine „ Amtleute “ oder andere Mittelspersonen den ausländischen Kaufleuten zu einem bestimmten Preise verkaufen . Weil aber das meiste Quecksilber nach der Pyrenäenhalbinsel abgesetzt würde , von wo aus die Könige von Spanien und Portugal auf ihren Schiffen das Metall nach India brächten , so sei anzuraten , mit den genannten Königen einen Quecksilberlieferungsvertrag (conventionem ) zu schließen 11 . Das¬ selbe könne bei den Venetiancrn versucht werden , die Quecksilber nach der Levante verführten. 1 Original des Schreiben des Sebastian Kurz im F . A. Wien. Inneröstcrr . Quecksilber 22. 2 Der Schreiber des Briefes fügt entschuldigend und um die elbische Erlaublheit eines solchen kaiserlichen Monopols zu erweisen hinzu : „ 1-loc non esset indecorum ! Nam honestissimum est principi fructus fundi sui vendere , meialla censentur ipso iure fructus fundi " . F . A . Wien . Inneröstcrr . Quecksilber 22. 3 Der Verfasser des Briefes weiß von früheren Versuchen des Kaisers für seine Feldzüge gegen die Türken vom König von Portugal Geld zu erhalten . Fr meint die Verstaatlichung des Quccksilberhandclstnonopols böte das beste Mittel bei der jetzigen Türkengefahr ein großes Darlehn vom König von Portugal zu erlangen . Das Dar¬ lehen könne in Quecksilber zurückgezahlt werden. Fünftes Kapitel : Monopole u. Kartelle im Idriancr QuecUsilbcrliandel usw. 325 Eine dritte Aufforderung an den Kaiser , sich die. günstige Konjunktur für eine Änderung der idrianischen Quecksilberhandels¬ organisation zunutze zu machen , ging —- wie mir scheint im Jahre 1560 — von einem kleinen, in Wien wohnenden idrianischen Ge¬ werken aus x, der von der Übernahme des Handelsmonopols durch den Kaiser und die Gewerken eine Besserung der Lage der von den großen Gewerken angeblich übervorteilten und überstimmten kleineren Gewerken erhoffte 2. Der Verfasser der Denkschrift machte über den Verdienst der Herwart folgende Rechnung auf: Die Augsburger Firma habe sich durch den letzten Kontrakt (der im Oktober 1564 zu Ende gehe) 5000 Zentner Quecksilber, den Zentner zu 24 fl., gesichert . Nun sei bekannt , daß „ hier in Wien“ und in andern Handelsstädten der Zentner mit 80— 100 fl. bezahlt werde 3. In andern Gegenden, wohin der Transport weit und schwierig sei, sei der Preis noch bedeutend höher. So habe der 1 Die Schrift enthält auch eine interessante Notiz zur sozialen Geschichte der Bergarbeiterschaft (Trucksystem ), die hier mitgeteilt werden soll . Der Verfasser betont : „ dass auch sonst die gemainen gewercken und arbeiter bei dem perckwerch von denselben fürnemisten gewercken mit allerlai aigennützigkait und vorthin , also wo si, die gemainen gewercken , ir pargelt auf die ablonung geben, dass dieselben gwalttrager [d . h . der Vorstand der gemeinen Ge¬ werkschaft ) inen , den arbaitern dafür allerlai provanndt ines gefallens eindringen und allen notdurften , die zum perekhwerch ge¬ hören , zum höchsten anschlagen und verraiten . Und verlassen sich darauf , dass inen , als den merern niemand einreden darf , also dass si, die gemainen gewercken , die das perckwerch mit irer schwären darlag , ehe es noch zu disen wirden körnen ist , haben erhalten helfen, ungeacht diser reichen augenscheinlichen gotsgab , wenig gewinns bisher empfunden haben . Darumben dan zu abstellung solicher unordenlichn , aigennützigen liaushaltung , sollen anderst die ge¬ wercken bei guctem willen und lust erhalten werden und der allmechtig seinen segen geben , einsehends hoch vonnöten .“ 2 „Ain verzaichnus und bericht die perekhwerch Idria betreffend, wie dasselb etlichen Privatpersonen , so iren gwinn allain darin suechen und daselb regiern , widerumb mecht aus handen gebracht und mit merern nutz dem landtsfuersten zugetailt werden .“ Es handelt sich in der Denkschrift nicht um die Verstaatlichung des Bergwerks, sondern nur darum , den Vertrieb des Produkts in die Hände des Landesherrn und der Gewerken zu bringen. 3 „Nachdem die queckhsilberperckhwerch in Hispania und an¬ derer orten vasst zu grün dt gangen und man nun die notdurft daraus nit mer haben kan . . . 326 Drittes Huch: Monopole, Kartelle und Aktiengesellschaften im Mittelalter. Verfasser durch den Faktor der Fugger , Kurz , der in Spanien „mit dem Quecksilberhandel umgegangen “, erfahren , daß das Quecksilber, das nach der Türkei verfrachtet würde , dort mit 160 Dukaten bezahlt würde . Aber selbst, wenn man nur 80 fl. als Durchschnittspreis für den Zentner Quecksilber ansetze und für „Fuhrlohn , Unkosten , Haltung der Diener , Wagnus und für ihren ziemlichen Gewinn“ 14 fl. rechne , so bleibe noch ein Über¬ schuß von 42 fl. pro Zentner als „Übergewinn “ . Insgesamt ge¬ wannen die Herwart an dem letzten Kauf mindestens 210 000 fl. Daß dieses Ergebnis nicht übertrieben ist, erkennt man aus der folgenden Gewinnberechnung , die Georg Ilsung , der Augs¬ burger Finanzagent Ferdinands I. und seiner Nachfolger , im Früh¬ jahr 1561 aufstellte . Ilsung gibt auf Grund genauer Erkundigungen an Ort und Stelle folgende Kalkulation: ln A u g s b u r g verkaufen die Herwart , den Krämern den Augsburger Zentner um bare fl. 70. Das würde Wiener Gewicht (pro Zentner 15 Pfund mehr) 80 fl. 30 kr . betragen. Den Zentner (wieniscb) von Kärnten nach Augsburg zu führen kostet ungefähr 2 fl. Da die Herwart den Zentner für 24 fl. von den Gewerken erhalten , so gewinnen sie in Augsburg pro Zentner 54 fl. 30 kr. Weil der Preis des Quecksilbers aber in den Niederlanden viel höher ist und deshalb naturgemäß dorthin das meiste Produkt abfließt , so ist in Augsburg Quecksilber nur schwer erhältlich. Die Krämer , die es den Goldschmieden pfundweise verkaufen, lassen sich pro Pfund 1 fl., zeitweise sogar 16—17 Batzen geben. Zu Nürnberg gilt der nürnbergischc Zentner (der um 4 Pfund größer ist als der augsburgische ) 77 fl. 40 kr . Rechnet man von Augsburg nach Nürnberg den Fuhrlohn pro Zentner 1 Gulden, so verdienen die Herwart in Nürnberg am Zentner Quecksilber 55 fl. 30 kr. In Antwerpen gilt der Zentner Quecksilber (Antwerpener Gewicht) jetzt fl. 96 kr . 40. Also der Wiener Zentner 115 fl. Rechnet man 6 fl. Unkosten pro Zentner von Kärnten bis Ant¬ werpen , so verdienen die Herwart pro Zentner in Antwerpen 85 fl. 40 kr . b 1Ilsungs „ Erkundigung per Verkauf des Quecksilbers . Actum Augsburg , den 1. April 1561 . Verzeichnis , was das Quecksilber Fünftes Kapitel : Monopoleu. Kartelle im Idrianer Quecksilberhandelusw. 327 Diesen gewaltigen Verdienst könnten — nach Vorschlag des obengenannten Gewerken —- der Kaiser und die Gewerken durch „Selbstführung und Handlung des Kaufes “ in ihre eigenen Kassen leiten . Im einzelnen gibt die Denkschrift noch folgende Ratschläge für die Ausführung des Planes . Man solle feststellen , wo am meisten Quecksilber und Zinnober gekauft würde und dort Faktoreien eröffnen . Das brauche nicht im Namen des Kaisers zu geschehen, sondern könne durch tüchtige und vermögende , in den betreffenden Städten eingesessene Kaufleute besorgt werden . Die betreffenden Kommissionäre müßten alljährlich über Verkauf und Restbestände Rechnung legen. Vielleicht genüge eine Faktorei in einer Stadt nahe bei Idria und eine zweite in Venedig oder Wien. Eine weitere Frage sei die, ob der Kaiser allein die Unternehmung auf sich nehmen wolle (durch einen beauftragten Kaufmann natürlich ) oder ob er den Kauf „mit den Gewerken in gesamter Gesellschaft haben wolle“. Auf alle Fälle dürfe kein neuer Kontrakt mit den Herwart oder anderen Kaufleuten Zustandekommen . Wenn diese auch jetzt bereit seien, einige Gulden mehr für den Zentner zu zahlen , so würde „ doch soliche staigerung bei weitem nit sovil austragen als sonst die kais . Mt. und die gewerckhen selbs des kaufs gemessen mechten “. Leider verhinderte den Kaiser die furchtbare Finanznot jener Jahre die günstige Konjunktur voll auszunutzen . Er mußte froh sein, daß die Herwart seinem Rate Gg. Ilsung schou am 23. November 1560 einen neuen Quecksilberkauf für 80 000 fl. abnahmen und Fron und Wechsel im voraus zu zahlen versprachen . Zwar ver¬ sagten die Gewerken infolge von Differenzen mit dem Kaiser dem verabredeten Kontrakt ihre Zustimmung b aber um die Mitte des jetzo gilt." Wien. F. A. Inneröstcrr. Quecksilber 23. Uber Gg. Il¬ sung, der in den 60er Jahren und zu Anfang der 70er Jahre noch im Mittelpunkt der kaiserlichen Finanzpolitik stand, vgl. „Allgemeine deutsche Biographie". Als kaufmännischer Beirat erscheint er auch bei Hirn, a. a. O., I, S. 401. Namentlich vermittelte Ilsung die großen Anleihen des Kaisers in Augsburg usw. Auch seinen per¬ sönlichen Kredit stellte er dabei den Habsburgern zur Ver¬ fügung. So mußte Ilsung mit einigen Augsburger Kaufleuten zu¬ sammen Bürgschaft leisten, als Maximilian II. im Jahre 1564 bei den Städten Ulm und Augsburg 100 000 fl. „zur befurderung des Kriegswesens in Zips" aufnahm. F. A. Wien a. a. O. 1 Die Idrianer Gewerken hatten Differenzen mit dem Kaiser, 328 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. Jahres 1561 — nacli Schlichtung der Streitsache •— wurde ein neuer Vertrag verabredet . Die Herwart sollten darnach wiederum auf fünf Jahre das Idrianer Quecksilberhandelsmonopol in die Hand nehmen . Im ganzen sollten ihnen bis zum 8. Oktober 1566 für 140 000 fl. Quecksilber bzw. Zinnober zum Preise von 28 bzw. 32 fl. übergeben werden . Die kaiserliche Fron und der Wechsel in Summa 29 750 fl. versprachen die Herwart zwei Monate nach der kaiserlichen Bestätigung des Kontraktes in Augsburg zu zahlen .. Diesmal war es der Kaiser , der von dem verabredeten Kontrakt zurücktrat . Der Vorschlag der spanischen Krone , die idrianischo Quecksilberproduktion zu festem Preise für den Export in die amerikanischen Silber- und Goldbergwerke zu übernehmen , änderte die Situation völlig. Es läßt sich mit dem zur Verfügung stehenden Aktenmatcrial unbedingt sicher entscheiden , daß die Initiative zu einem festen Lieferungsvertrag des idrianischen Quecksilbers an die Krone Spanien von Philipp II . ausging KDas war damals ja auch das natürliche , da Spanien die glänzende Konjunktur am besten kannte und während der nächsten Jahre in Almaden nicht genug Queck¬ silber erzeugen konnte , um die amerikanische Nachfrage zu decken. Im Frühjahre 1561 waren die Verhandlungen bereits im Gange. Am 24. April schrieb König Ferdinand an Martin Guzmann , der für ihn in Madrid die Unterhandlungen führte , er sei imstande , in Jahresfrist 1000—1500 Zentner und dann jährlich ebensoviel zu liefern. Er fordere als Preis dafür loco Antwerpen 115 fl. rh . für den Wiener , 96 fl. rh . für den Antwerpener Zentner 2. Guzmann der vom Jahre 1554 an wieder den vierten Zentner als Wechsel forderte , einen Prozentsatz , der seit 1524 üblich gewesen war und den die Krone 1539 nur der schlechten Zeiten wegen auf den achten Zentner herabgesetzt hatte. 1 Im „ Jahre 1785 schloß die österreichische Regierung mit Spanien einen Vertrag auf sechs Jahre ab, in welchem sie sich ver¬ pflichtete , jährlich mindestens 4480 metr . Zentner Quecksilber an Spanien abzuliefern . Dieser Vertrag wurde im Jahre 1791 auf weitere sechs Jahre für Lieferung von jährlich bis zu 5600 metr. Zentner erneuert .“ Das k. k. Quecksilberbergwerk zu Adria , S. 37. 2 „Essemus propterea contenti ut nobis per dictum serenissimum regem pro quovis centenario Viennensi praemissi 115, pro Antverpiensi vero 96 floreni et 40 crucigeri illic Antverpiae , quo nos argentum vivum sumptibus et periculo nostro pervehi tradique curaremus exolveretur .“ F . A. Wien . Innerösterr . Quecksilber 22. Fünftes Kapitel: Monopoleu. Kartelle im Idrianer Quecksilberhandel usw, 329 bekam aber Weisung, daß er eventuell bis auf 100 fl. rh . für den Wiener Zentner heruntergehen dürfe 1. Auch zu diesem Preise noch dürfe er den Kontrakt auf vier bis fünf Jahre in Ferdinands Namen abschließen . Der Kaiser zweifelte nicht , daß der Kauf zum Abschluß käme , lieferte er doch dem König von Spanien das absoluteste Weltmonopol für Quecksilber in die Hände . Dieser konnte dann besonders für Amerika den Quecksilberpreis nach eigenem Gutdünken bestimmen 2. Man versteht es, daß Ferdinand seinem Beauftragten Martin Guzmann befahl , die Unterhandlungen in tiefstem Geheimnis zu führen , damit besonders die Herwart vor dem völligen Abschluß nichts erführen . Käme der Abschluß zustande , so sollte das Augs¬ burger Handelshaus , das kontraktlich ihm zugesicherte Quecksilber sofort erhalten und abnehmen müssen. Wie Ferdinand sich im übrigen mit den Herwart auseinandersetzen wollte, die bis zum 1. Oktober 1564 das Handelsmonopol in idrianischem Quecksilber vom Kaiser zugesichert erhalten hatten , ist in dem lateinischen Schreiben an Martin Guzmann nicht gesagt. Wenn Ferdinand geglaubt hatte , den Wiener Zentner Queck¬ silber zu 115 oder doch wenigstens zu 100 fl. nach Spanien ab¬ setzen zu können , so sollte er bald eines anderen belehrt werden. Die spanische Regierung bot 80 Carolusgulden für den Zentner loco Antwerpen , was 64 fl. rh . gleichkam . Wie enttäuscht man in Wien über dieses niedrige Angebot war, erkennt man aus einem Briefe König Maximilians (II .) an seinen kaiserlichen Vater vom 13. Oktober 1561 3. Maximilian betont darin ausdrücklich , daß auch er gehofft hätte , „mit Hilfe des kaiserlichen Orators (Martin Guzmanns ) fleissiger handlung “ einen annehmbaren Vorschlag aus Madrid zu vernehmen . Wie jetzt die Dinge lägen, müsse man durch erneute Unterhandlungen ein höheres Gebot zu erreichen 1 „ . . . damus tibi potestatem atque facultatem precium singu¬ lorum centenariorum Viennensium ad centum usque florenos remittendi dummodo ultra omnem tuam positam operam et dili¬ gentiam maioris eam emptionem contrahi non posse deprehenderis ." a. a. O. 2 „Nam quando ipse praelibatum metallum a nobis emptum penes se habuerit , illud tunc pro suo arbitrio et commodo vel in Hispania vel India divendere poterit .“ a. a. O. 3 F . A . Wien . Innerösterr . Quecksilber 22. 2 330 Drittes Buch: Monopole , Kartelle und Aktiengesellschaftenim Mittelalter. suchen . Wenigstens solle Spanien sich damit einverstanden er¬ klären , daß Idria für 80 Carolusgulden den leichteren Antwerpener und nicht den schwereren Wiener Zentner liefere. Mehr freilich als einen Versuch , mehr als 80 Gulden zu erhalten , riet Maximilian seinem Vater nicht an . Der Kaiser dürfe diesen Quecksilber¬ kontrakt mit Spanien nicht am Geldpunkte scheitern lassen , dafür sei der kluge Francisco Erasso 1der Leiter der Verhandlungen auf der spanischen Seite — schon zu sehr in die Idrianer Verhält¬ nisse eingeweiht worden . Tatsächlich blieb ja auch dem Kaiser, wenn er das Handelsmonopol von Idria selbst in die Hand nahm und den Zentner Quecksilber für 61 fl. rli. an die spanische Krone nach Antwerpen lieferte , ein nicht unbedeutender Verdienst . Von den Gewerken erhielt der Kaiser einen Wiener Zentner für 28 fl. Laut Ilsungs Bericht betrugen die Unkosten pro Zentner bis nach Antwerpen 6 fl., gibt zusammen 34 fl. Der König von Spanien hot für den Zentner loco Antwerpen 64 fl. Der Kaiser würde demnach noch an jedem Zentner 30 fl. Verdienst haben , bei einem Jahresumsatz von 1200 Zentner also 36 000 fl. 3. Rechnet man 1000 fl. als jährliches Gehalt für einige Faktoren ab , — was reichlich hoch angesetzt ist —, so würde ein jährlicher Reingewinn von 35 000 fl. bleiben. Erwägt man demgegenüber freilich, was die spanische Krone an dem ihr überlassenen idrianischcn Quecksilber verdienen konnte, so mußte der Gewinn des deutschen Kaisers dagegen erblassen. Nach Erkundigungen , die Ilsung eingezogen hatte , wurde in Spanien der kleine spanische Zentner (= 89 Pfund des Wiener Zentners) mit 85—88 spanischen Dukaten verkauft . Das käme bei dem oben¬ genannten Einkaufspreise (80 Carolusgulden pro Wiener Zentner) auf einen Verdienst von mehr als 100 % hinaus 3. Verkaufte aber die spanische Krone das idrianisclie Quecksilber nach Amerika, so war der Profit nach Ilsungs Schätzung mindestens 200 % (dortiger Preis zirka 140 Dukaten ). Es war nicht sehr ernst zu nehmen und wurde auch von Ilsung 1 Über ihn siehe Ehrenberg, Zeitalter der Fugger , vgl. Register. 2 Hierzu natürlich noch Fron und Wechsel. 3 „dergestalt , daß die kgl . Würde [von Spanien ] oder derselben räth iner als ain gülden am andern nur von Antorff bis in Hispanien daran gewinnen würden .“ Fünftes Kapitel : Monopoleu. Kartelle ira Idriancr Quecksilberliandelusw. 331 durchaus als ein Scheinmanöver erkannt , wenn Spanien den Kaiser dadurch zum schnellen Abschluß des Kontraktes drängen wollte, daß es für einen voraussichtlichen späteren Kontrakt einen höheren Preis in Aussicht stellte . Die Annahme lag nahe , daß in zwei bis drei Jahren die spanischen Quecksilberbergwerke von Almaden soweit wieder hergestellt waren , daß der König von Spanien die Idrianer Produktion überhaupt entbehren konnte 1. Ilsung meinte, Spanien müsse und könne mindestens für den Antwerpener Zentner 94 fl. rli. zahlen 2. Wahrscheinlich infolge, des niedrigen spanischen Angebots ist der geplante Quecksilberlieferungskontrakt zwischen der spanischen Krone und dem Kaiser nicht zustande gekommen 3. Damit war aber auch das Schicksal eines kaiserlichen Handelsmonopols mit Idrianer Quecksilber besiegelt. Schon am 26. Mai 1562 traf Ilsung mit Hans Paul und Hans Heinrich Herwart — auf kaiser¬ liche Ratifikation bin — eine Verabredung , laut welcher der Kaiser auf die Herwart den Kontrakt übertrug , den er am 24. Oktober mit den Idrianer Gewerken abgeschlossen hatte 4. Die Herwart sollten darnach von den Idrianer Gewerken das Quecksilber und Zinnober zu dem mit dem Kaiser verabredeten Preise erhalten. 1 Aus Bericht Ilsungs . Dat . Augsburg 19. Oktober 1561 . Wien. F . A . Innerösterr . Quecksilber 22. 2 ,, . . . aller billigkhait nach weniger nit darumben geben kinden, dan wie die queckhsilber anjetz zu Antorff gelten und durch die Herwart teglich verkauft werden , nämblich das lib . per 50 und 52 Groschen , kliomt der Antorffer centner , wie gemainlich die wixl gonndt , auf 94 gülden rh . in muntzs .“ 3 Im Jahre 1564 führt Kaiser Ferdinand allerdings als Haupt¬ grund , weshalb der Plan eines Weltmonopols der spanischen Krone in Quecksilber nicht zustande kam , die Rechtsansprüche der Herwart an , die auf Grund der älteren Kontrakte bestanden. 4 Ilsung versprach im Namen des Kaisers den Herwart : ,,ain genuegsame ubergab und bevelch an die gewerckhen des inhalts fertigen zu lassen , dass sein röm . kais . Mt . iren getroffenen kauf aus beweglichen Ursachen und gemainem perckhwerch [ !] zu nutz und guetem , obgedachten Herwarten auf - und ubergeben hab und dass dargegen die Herwart schuldig sein sollen , die bestimbt antzall queckhsilber von inen , den gewerckhen antzunemen , die betzallung auf die erneute termin und sonst alles das zu thuen und zu vol¬ tziehen , so beruerte kaufsabredt mit sich bringt und also sein röm. kais . Mt . gegen den gewerckhen gäntzlich zu vertreten .“ 1 332 Drittes 13uc .li: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. Sie traten in die verabredeten Waren - und Geldliererungsfristen usw. ein. Aber natürlich übergab der Kaiser den Herwart das Idrianer Quecksilberhandelsmonopol nicht ohne bedeutenden Gewinn für die kaiserliche Kasse . Als „Fron , Wechsl, Zehendt , Achtel und sonst all ander ir Mt. Recht und Gerechtigkeit , wie die Namen haben möchten “ versprachen die Herwart dem Kaiser für jeden Zentner 49 4 fl. rh . in Müntz Augsburger Währung „als Ge¬ winnung“ zu geben . Das machte für den verabredeten Kon¬ trakt in Summa 277 250 fl. Sic waren bis zum t . November 1564 in fünf genau festgesetzten Terminen in Augsburg zu zahlen . Dem¬ gegenüber sollte den Herwart der neue Zoll „an der Krcmbspruggen“ erlassen werden . Von dem guten Geschäft , das der Kaiser bei der Übergabe des Vertrags an die Herwart machte , sollten die Idrianer Gewerken nichts erfahren . Sie erhielten nur von der Tatsache der Übergabe selbst Mitteilung h Der Kaiser hat die Abmachung Ilsungs mit den Herwart im wesentlichen bestätigt 2. Die Herwart erhielten überdies die Zu¬ sicherung , wenn sie nach Ausgang des fünfjährigen Kontraktes noch für 20 000 fl. Quecksilber von den Idrianer Gewerken beziehen wollten , so solle ihnen diese Quantität zum alten Preise geliefert werden . Für künftige Zeiten wird dazu verabredet : Wenn der Kaiser nach Ablauf des jetzigen Kontrakts wiederum mit den Idrianer Gewerken abschließen würde , so wollte er „ die Herwardten vor andern zu solichen kheufen khomen lassen “. Wenn aber der Kaiser nicht wieder mit den Gewerken kontrahieren wollte , so versprach er den Herwart das Monopol „vor andern zu vergönnen “ . Man hätte meinen sollen, bei dem glänzenden Entgelt , das die Herwart Ferdinand für die Überlassung des Idrianer Quecksilber¬ handelsmonopols leisteten , habe der Kaiser das Ende des laufenden Kontraktes ruhig abgewartet , dann seinerseits einen neuen Kontrakt mit den Idrianer Gewerken abgeschlossen und diesen wiederum 1 Der betreffende wichtige Passus der Verabredung Ilsungs mit den Herwart lautet : „Es sollen auch obgemelte ubergab und bevelch allain in genere gestellt werden und der Herwarten bewilligter staigerung khain nieldung darin beschechen , damit die gewerckhen der¬ selben nit gewahr werden .“ 2 Original der Verschreibung der Herwart , in der sie den Kon¬ trakt mit den Idrianer Gewerken vom Kaiser übernehmen . 2. Juni 1562 . Wien . F . A. Innerösterr . Quecksilber 22. 2 Fünftes Kapitel : Monopoleu. Kartelle irn Idrianer Quecksilberliandelusw. 333 gegen einen entsprechenden Aufschlag den Gebrüdern Herwart ab¬ getreten . Aber offenbar erlaubten die schlechten kaiserlichen Finanzen ein derartiges ruhiges Zuwarten der Dinge nicht . In seinen Finanznöten mußte Kaiser Ferdinand schon bald wieder daran denken , aus einem neuen Quecksilberkontrakte Kapital zu schlagen , d. h. Geld auf die ihm aus dem Quecksilbermonopol zu¬ fallende Gewinnquote zu antizipieren . An Georg Ilsung erging um die Wende des Jahres 1563 die Anfrage , ob und wie er glaube, daß sofort oder in einiger Zeit ein neuer Quecksilberkauf getroffen werden könne , mit dessen Hilfe eine ansehnliche Summe Geldes in die leeren kaiserlichen Kassen geleitet werden möchte l . Ilsung mußte in der Notwendigkeit , eine neue kaiserliche Anleihe unterzubringen , Grund genug sehen, einen neuen Queck¬ silberkauf cinzuleit.en, aber anderes kam hinzu , um ihn für den Abschluß eines neuen Kontraktes einzunehmen . Wozu die Not der kaiserlichen Krone riet , dazu ermahnte auch die wirtschaftliche Lage auf dem internationalen Quecksilbermarkte . Aus Spanien kam die Kunde , daß die Quecksilberbergwerke von Almaden von Tag zu Tag an Ergiebigkeit Zunahmen. Die Fugger müssen eine fieberhafte Tätigkeit entfaltet haben , um die günstige Konjunktur des Quecksilbermarktes so schnell wie möglich für sich ausnutzen zu können . Ilsung erhielt Nachricht aus Spanien , daß die Fugger in Almaden einen wahren Raubbau trieben , um Idria den Absatz zu nehmen ä. Auch sonst weckte im Anfang der sechziger Jahre der so außer¬ ordentlich erhöhte Weltbedarf an Quecksilber die Bergbaulust auf dieses Metall. In Böhmen erwiesen sich die neuentdeckten Queck¬ silberfundstätten als ziemlich förderungsreich . Und schließlich war in dem Herrschaftsgebiete des Pfalzgrafen Wolfgang von PfalzZwcibrücken ein neues Quecksilberbergwerk eröffnet worden . Das Bergwerk war dem reichen Augsburger Kaufmann Thomas Frei1 Schreiben Georg Ilsungs vom I . Februar 1564 . Wien . F . A. Innerösterr . Quecksilber 23 .- Dem Kaiser war die Hauptsache , bares Geld „ auf den kauf zu leihen “ . 2 ,, . . . bericht , dass die lierren Fugger im queckhsilberberckhwerckh in Spanien allein auf den raub arbeiten und guglen lassen , damit sic in eil ein grosse anzal qneckhsilber machen . . .“ Aus Ilsungs Bericht an den Kaiser vom 11. September 1564. Wien. F . A. Innerösterr . Quecksilber 23. 334 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. hainer und seiner Gesellschaft überlassen worden . Freihainer er¬ klärte sich fähig , jährlich 600 Zentner zu liefern und bot das Produkt der neuen Gruben bereits in Augsburg an. War aus diesen und ähnlichen Gründen mit einiger Sicherheit anzunehmen , daß der hohe Stand des Quecksilberpreises sich in der nächsten Zeit in absteigender Kurve, bewegen würde , war also dem Kaiser (wie auch den Gewerken) zu raten , durch baldigen Abschluß eines Quecksilberkontraktes sich einen Abnehmer und einen möglichst hohen Preis des Quecksilberproduktes zu sichern, so erhoben sich doch auch bedeutende Schwierigkeiten gegen einen baldigen neuen Abschluß . Einmal hatten die bisherigen Kon¬ trahenten , die Herwart , kaum die Hälfte des verabredeten Queck¬ silberquantums erhalten . Sie hatten auch gerade jetzt die Ab¬ nahme des fertig gestellten Produktes mit der Begründung hinaus¬ gezögert , daß der Markt augenblicklich für größere Quecksilberquanlen nicht genügend aufnahmefähig sei. Es war demnach, wenn sich die Konjunktur nicht stark änderte , kaum anzunehmen, daß der Hcrwartsche Kontrakt bald sein Ende, erreichte . Das um so weniger, als die genannte Firma sich nach Ablauf ihres Vertrages noch die Lieferung von Quecksilber für 20 000 fl. rh. zum alten Preise ausbedungen hatte. Unter diesen Umständen war es das naheliegendste , wenn man sich kaiserlicherseils mit dem Wunsche eines baldigen Neuabschlusscs des klrianer Quecksilbermonopols und mit den daran hängenden Anleihen an die bisherigen Inhaber , die Herwart , wandte. Freilich verschloß sich lisung der Einsicht nicht , daß es schwer halten müsse, die Herwart dazu zu überreden . Er hielt es für an¬ gebracht , sich erst genau über die Quecksilberkonjunktur in Venedig und Antwerpen zu erkundigen , ehe er an die Ilerwart herantrat 1. Den Kaiser bat er, in der Zwischenzeit durch seinen Botschafter in Madrid anfragen zu lassen, „ ob der könig von Spanien in die 1ln einem Berichte Ilsungs an den Kaiser (Augsburg , 1. Februar 15(54. Wien . F . A . lniioröslerr . Quecksilber 23) heißt es : „ Die weil die handlslcul alhic lieslig , gcscliwindt , unthreu und vorthaillig und der mit inen handle » will , wol) getast sein und inen mit dem grundl under äugen geen muess , so hat ) ich in gehaim gen Venedig und Antilorl 'l' uinb bericht geschahen , wieviel queckhsilber der enden unvertriben lige , was nachfrag cs anjetz hab und wie theur es verkhnuft werde , mich in alweg desto hass haben darnach zu richten .“ Fünftes Kapitel: Monopole u. Kartelle im Idrianer Quecksilbciinmdel usw. 335 handlung wie hievor gebracht werden möchte “ . Die Begründung dieser Maßnahme macht dem psychologischen Scharfblick listings als Unterhändler und Kaufmann alle Ehre . Wenn man , meint der geriebene Geschäftsmann , auch mit dem König von Spanien zu keinem Kontraktsabschluß käme , so würden doch schon die Unterhandlungen mit ihm bei den Interessenten , also bei den Herwart , Aufsehen erregen und sie oder andere süddeutsche Kauf¬ leute zu einem schnellen und für den Kaiser und die Gewerken günstigen Abschluß veranlassen h Sorgenvoll schließt Ilsurtg seinen Bericht an den Kaiser , er fürchte , wenn man die Herwart mit solcherlei Mittel nicht anreize , so sei wohl wenig bei ihnen zu er¬ reichen . Der eigene Vorteil weise die Augsburger zu deutlich darauf hin , erst einmal das bisher contrahierte Produkt zu ver¬ kaufen , ehe man sich auf neue , weitsichtige Abschlüsse einließe. Was aber die Herwart , die langjährigen Monopolinhaber des idrianisclien Quecksilberproduktes nicht täten , das würden andere Kauf¬ leute erst recht unterlassen. Dagegen wird es Ilsung nicht allzu schwer geworden sein, die Bevollmächtigten der Idrianer Gewerken zum Abschluß eines neuen Quecksilberkaufs mit dem Kaiser zu bewegen. Ein Hinweis auf den spanischen Wettbewerb und auf die wachsende Konkurrenz der neuen böhmischen und pfälzischen Gruben bei einem späteren Abschlüsse dürfte genügt haben . Tatsächlich kam es zu einem neuen Abschluß zwischen Kaiser und Gewerken (Frühjahr 1564). Ferdinand verpflichtete sich, 5000 Zentner Quecksilber und Zinnober zum Preise von 31 resp . 35 fl. abzunehmen . Als Ilsung dann auch genügende Aufschlüsse über die Lage des Quecksilbermarktes er¬ halten hatte , trat er an Hans Paul und Hans Heinrich Herwart heran . Er cröffnete ihnen die für den Kaiser bestehende Not¬ wendigkeit , einen neuen Quecksilberkontrakt abzuschließen . Er machte ihnen auch davon Mitteilung , daß die Gcwaltträgcr des Bergwerks von Idria dem Kaiser einen neuen Kauf bereits bewilligt 1 Ilsung faßt seine Erfahrung in diesen Dingen in die charakte¬ ristischen Worte zusammen: „Will man nun solche abgeribne leut [wie die süddeutschen Kaufleute] ad terminos equitatis bringen, so hab ich aus erfarung, dass man sie mil nichten bass treiben khan, dann so man inen ander an die seitten setzet. Alsdann khommeu sie selbst und tinrent das [was] man sonst mit khainer lumst von inen bringen klian.“ 336 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. hätten . Ferdinand — so fuhr Ilsung in seinen Eröffnungen fort — würde am liebsten den Gebrüdern Herwart das Monopol wieder übertragen . Sie seien selbst Gewerken in Idria , sie hätten auch bisher den Kauf zur Zufriedenheit des Kaisers geführt usw. Die Herwart waren von dem Antrag des Kaisers überrascht. Sie sprachen zunächst ihre Mißbilligung darüber aus , daß die Gewaltträger in Idria dem Kaiser einen neuen Kauf bewilligt hätten , ohne die übrigen Gewerken zu Rate zu ziehen. Mit ihrem Wissen und Willen, fuhren dann die zwei Augsburger Handels¬ herren fort , wäre jetzt kein neuer Kontrakt abgeschlossen worden. Sic hätten ja noch über 2000 Zentner von dem alten Abschluß ab¬ zunehmen , auch in ihren Faktoreien zu Antwerpen , Venedig und Lyon noch große Vorräte lagern . Vor Ablauf der in ihrem letzten Kontrakt festgesetzten fünf Jahre sei an keine Erneuerung des Kaufs zu denken . Man müsse vom Kaiser erwarten , daß er diese Zeit verstreichen ließe, ehe er mit einem neuen Kontraktanerbieten an sie, die Herwart , oder sonst wen heranträte . Als dann Ilsung trotz dieser deutlichen Absage fortfuhr , von den Herwart eine bestimmte Antwort zu fordern , ob sie den neuen Kauf anzimehmen gedächten oder nicht , da geschah etwas Unerwartetes . Die Firma Herwart erklärte , daß sie nicht nur den Neuabschluß ablehne, sondern auch durchaus damit einverstanden sei, wenn der Kaiser den Kontrakt mit einer anderen Firma abschlösse. Um diesen anderweitigen Abschluß zu erleichtern , erboten sich die Gebrüder Herwart , ihre Rechte am Idrianer Monopol in die Verfügungs¬ gewalt des Kaisers zurückzugeben . Das heißt , die Augsburger Handelsherren erklärten sich bereit , alles Quecksilber, das sie schon in Händen oder noch auf Grund ihres Kontraktes zu fordern hätten, dem Kaiser zu zedieren . Unter folgenden Bedingungen : Für Queck¬ silber, das in den Faktoreien der Herwart zu Antwerpen , Lyon, Venedig oder sonstwo lagerte , sollte der Kaiser pro Wiener Zentner 100 fl. zahlen . Für die 2000 Zentner Quecksilber, die den Augs¬ burger Kaufleuten noch von den Gewerken zu liefern waren , solle der Kaiser den Herwart zu Idria pro Wiener Zentner 77 4 fl. rh. geben. Das war der Preis , den auch die Herwart dem Kaiser zu zahlen sich verpflichtet hatten . Ferner sollten die gegenseitigen sonstigen Geldverbindlichkeiten der Herwart und Gewerken einer¬ seits und der Herwart und des Kaisers andererseits endgültig ge¬ regelt werden . Auf der einen oder der anderen Seite sich ergebende Fünttes Kapitel : Monopoleu. Kartelle im Idrianer Quecksilberhandelusw. 337 Schulden waren sofort zu tilgen . Endlich verzichteten die Herwart zugunsten des Kaisers auf die Verpflichtung , die die Gewerken in dem Kontrakt von 1562 übernommen hatten , ihnen nach Ablauf des laufenden Vertrags noch für 20 000 fl. Quecksilber zum alten Preise 1 zu liefern. Ilsung war erstaunt , daß die Herwart so leichten Herzens auf ihr Monopol verzichteten . Er war aber klug genug , den Verzicht der Herwart nicht sofort anzunehmen . Er müsse, so bedeutete er die Firma zunächst die Einwilligung des Kaisers einholen . In Wahrheit zweifelte Ilsung nicht daran , diese zu erhalten . Vielmehr wollte er erst noch mit einer anderen Augsburger Firma , mit der .er bereits seit einiger Zeit über den neuen Quecksilberkontrakt unter¬ handelte , zum endgültigen Abschluß kommen , ehe er die Herwart von ihrer kontraktlichen Verpflichtung entband. Die Firma , mit der Ilsung seit einiger Zeit wegen Übernahme eines neuen Quecksilberkaufs in Unterhandlung stand , war die des Melchior Manlich. Melchior Manlich hatte sich Ilsung gegenüber bereit erklärt , in einem neuen Kontrakt 100 fl. rh . für den Zentner Quecksilber zu zahlen unter der Bedingung , daß die Herwart ver¬ hindert werden könnten , ihm Konkurrenz zu machen . Ilsung war bereits am 23. Februar 1564 zu einem provisorischen Abschluß mit Melchior Manlich & Co. gelangt . Darin war — das Zurücktreten der Herwart vorausgesetzt — folgendes verabredet worden . Melchior Manlich übernimmt in den nächsten fünf Jahren 5000 Zentner Quecksilber und Zinnober von den Gewerken zum Preise von 100 fl. rh . Davon sind 28 fl. rh . resp . 32 fl. rh . (für Zinnober ), in Summa (unter Abzug der regalis tischen Gefälle) für 5000 Zentner 110 250 rheinische Gulden den Gewerken zu zahlen , die übrigen 389 750 fl. fallen dem Kaiser für Fron , Wechsel, „Verehrung und all ander Gerechtigkeit “ zu. Die letztgenannte Summe ist an die kaiserliche Kammer in 6 Terminen zu Augsburg zu entrichten. Die Gewerken erhalten ihr Geld pro rata der Quecksilberlieferung zu Villach in dort gangbarem Gelde ausgezahlt . Die Manlich sind gehalten , mit der Abnahme des fertiggesteilten Quecksilberproduktes nicht zu säumen , wie auch umgekehrt die Gewerken dafür zu sorgen 1 Quecksilber pro Wiener Zentner für 24 fl. rh. und Zinnober für 28 fl. rh. Strieder , Studien z. Gesch . kupitiüist . Organisafcionaformen . 22 12 338 Drittes Buch : Monopole , Kartelle und Aktiengesellschaften im Mittelalter. haben , daß genügend Quecksilber und bei rechtzeitiger Bestellung auch Zinnober fertiggestellt wird. Bis zur völligen Lieferung der 5000 Zentner dürfen weder die Gewerken noch der Kaiser jemand anderem als unserer Firma Quecksilber „verkaufen , verschreiben oder sonstwie vergeben “ . Auch die Durchfuhr fremden Quecksilbers durch seine Länder will der Kaiser nicht gestatten . Ebenso macht sich Ferdinand an¬ heischig, soweit es in seiner Macht liegt , dafür zu sorgen , daß das Quecksilbermonopol der Gesellschaft des Melchior Manlich ein wirkliches Monopol wenigstens für Mittel - und Osteuropa bleibt. Solange der Kontrakt währt , will der Kaiser kein anderes Queck¬ silberbergwerk in seinen Ländern dulden , noch aufkommen lassen 3. Bezüglich der Lieferung im einzelnen , der Verzollung , des Ge¬ wichtes , der Verpackung , soll cs gehalten werden , wie das alles in dem Vertrag mit den Herwart festgelegt worden war . Auch die Vorbehalte , die dort für Kriegsfälle vorgesehen sind , sollen in dem Kauf der Manlich-Gesellschaft platzgreifen . Bemerkenswert art dem Entwurf ist die Tatsache , daß sich der Kaiser nicht auf fünf Jahre band , sondern nur für die Zeit, deren man zur Lieferung der 5000 Zentner Quecksilber bedurfte . Ausdrücklich heißt es; „Und so bald obgemeltc 5000 Centner oder nach en dt dabei gelifert worden , alsdann soll diser kauf (die 5 vorbebaltn« j a r sein verstrichen oder n i t) ain volligs endt haben V So lebhaft hatte Melchior Manlich gewünscht , das Österreich! sehe Quecksilbermonopol in die Hände zu bekommen , daß Ilsung zeitweise hoffen konnte , für den Zentner von ihm 100 Taler zu erhalten , statt 100 rheinische Gulden . Um so mehr war der kaiser¬ liche Finanzagent erstaunt und entrüstet , als ihm plötzlich Melchior Manlich eröfi'nete , es sei ihm unmöglich , das Quecksilbermonopol zu übernehmen . Wie war der plötzliche Umschwung in der Unter 1 Iti dem Kontraktentwurf (F. A. Wien. Inneröstcrr . Queck¬ silber 23) heißt es mit einer Spitze namentlich gegen den böhmischen Quecksilberbergbau : „ Desgleichen soll auch sein kais. Mt. gcmelL» 5 jar oder so lang diser kauf werdt , in der chron Behaim und ander seiner rönt. kais. Mt. Königreichen und erblanden khain ander frembdt queclchsilherperckhwerch noch die jetzigen erheben lassen, noch ge¬ dulden in khainen weeg.“ 2 An einer anderen Stelle heißt es nochmals : „Dergleichen soll auch sein röm. kais. Mt. gemelte 5 jar oder so lang diser kauf werdt d [ . h. währt ] . . . Fünftes Kapitel : Monopoleu. Kartelle im Idrianer Quecksilberhandel usw. 339 nehmungslust des Augsburger Kapitalisten entstanden ? Einmal spielen bei der Absage der Manlich —- wie auch bei der oben ge¬ schilderten Geschäftsunlust der Herwart -— allgemeine Gründe eine hervorragende Rolle. Infolge der Zahlungseinstellungen der spani¬ schen , der französischen und der portugiesischen Krone war eine ernste Krisis im süddeutschen Wirtschaftsleben eingetreten . Von 1561 an hatten besonders in Augsburg eine Anzahl einst bedeutender Firmen rasch hintereinander den Bankrott anmelden müssen l. In¬ direkt wurde davon auch Melchior Manlich-schwer getroffen . Auch sein Vetter Cristoff Manlich hatte in jenen für Augsburg so schweren Jahren „Pancarotta gespilt “ und nicht allein die Firma Manlichs Erben , sondern auch die Handelsgesellschaft Melchior Manlich & Co. bedeutend in ihrem Kredit erschüttert 2. Melchior Manlich hatte alle Hände voll zu tun , um seine Waren zu Geld zu machen , damit er das Geld, das ihm täglich gekündigt wurde , auszahlen und „Treu und Glauben “ halten , konnte . Innerhalb acht Tagen hatte er so nicht weniger als 80 000 fl., auszahlen müssen und noch immer ließ der Sturm auf seine Kassen nicht nach . „Weder Vater noch Sohn noch Bruder und Vettern trauten einander in jener schlimmen Zeit , noch wollten sie miteinander Geduld haben, “ so berichtete listing an den Kaiser nach Wien . Und die ganze miserable Stim¬ mung der damaligen Augsburger Großhandels weit faßt der Fiuanzagent in die folgenden charakteristischen Worte zusammen : „ In Summa da ist kein Geld und wollt ich auch 20 % Interesse zahlen, sondern nur Verzweiflung, daß man bei dem jetzigen Geschrei und Aufkündigung des Gelds von Treu und Glauben kommen würde “ . Es nutzte lisung nichts , wenn er Melchior Manlich Vorhal¬ tungen machte , daß er sich aus einem fast schon abgeschlossenen Geschäft zurückzöge ; es half auch nichts , daß er dem Handels¬ herrn im Preise entgegenkam . Melchior Manlich konnte und wollte sich nicht in ein neues und so zweifelhaftes Engagement wagen, bevor nicht die Krisis auf dem Geldmärkte zu Ende war. War die Baisse im süddeutschen Handelslebeu ein allgemeiner Grund für die Manlich, wie für die Herwart , sich der Übernahme 1 R. Ehrenberg , Zeitalter der Fugger , I. Bd . S. 77. 2 Das folgende nach den Berichten Ilsungs an den Kaiserhof aus dem Sommer 1564 . Wien . F . A . Innerösterr . Quecksilber 23. Interessantes Material zu diesem Bankrott des Manlich in Innsbruck, k . k . Statthalterei -Archiv Abt . Ferdinandeum : Stritt . Part . M. •22 * 1 340 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften ini Mittelalter. eines neuen riskanten Idrianer Quecksilberhandelsmonopols zu ver¬ sagen, so kam noch ein spezieller Grund für diese Absage hinzu. Es wird sich leider bei der klugen Art , wie Geschäftsleute ihre Geheimnisse zu wahren wissen, niemals mit voller Sicherheit mehr feststellen lassen , auf welchen indirekten Wegen das Idrianer Quecksilber in die Bergwerke der spanischen Kolonien gelangte. Aus Erkundigungen , die Ilsung einzog, läßt sich darüber nur etwa das folgende feststellen , wobei freilich zu beachten ist , daß sich Ilsung offenbar selbst nicht recht klar über diese Dinge war . Auf keinen Fall exportierten die Herwart das Quecksilber selbst in die spanischen Kolonien . Dagegen werden einzelne spanische Kauf¬ leute , die von den Herwart Quecksilber in Antwerpen kauften, das Metall nach Neuspanien geschmuggelt haben J. Vielleicht be¬ teiligten sich an diesem Quecksilberschmuggel auch Portugiesen und Franzosen , „ die eine besondere Anfahrt in Brasilien haben “ 2. Auch die spanischen Kaufleute , die dem König das Almadencr Quecksilberprodukt abkauften , müssen sich direkt oder indirekt idrianisches Quecksilber zu verschaffen gewußt und als Almadencr in die Kolonien exportiert haben . Sie kauften das österreichische Produkt von den Herwart natürlich erheblich billiger als das spanische vom König . Demgemäß war ihr Profit bei der Ausfuhr von Idrianer Quecksilber sehr bedeutend. Mit spanischen Kaufleuten , die Idrianer Quecksilber auf die eine oder die andere Art in die amerikanischen Kolonien zu schmug¬ geln gedachten , hatte nun Melchior Manlich offenbar vorbereitende 1 Die spanische Krone hatte das Recht des Exportes von Queck¬ silber in die Kolonien für sich und die von ihr beauftragten Kauf¬ leute Vorbehalten. Siehe oben. 2 Die ganze etwas unklare Stelle in dem Bericht Ilsungs vom 19. Oktober 1561 (F. A. Wien. Innerösterr. Quecksilber 22) lautet: „Mich will aber, allcrgencdigisterkaiser, gentzlich bedünckhen, dass die Herwart haimlichc leuth haben, die inen das queckhsilber in Niderland abnemen und cs hievor unangeseclien des bcscliechnen Verbots und anjetz unangeacht des Zolls in Spanien dannocht unangezaigt fur und in Indien gebracht haben. Ob si aber nun solches durch die Portugaleser oder aber die Frantzosen, so ain soliderer anfart in Presill [= Brasilien] haben zuwegen bringen oder aber andere kaufleuth, so die queckhsilber von den Herwarten kaufen, solche rennckli oder contrabanda brauchen, das khan ich bis anher, weil es die Herwart gar in gehaim halten, nit erfaren.“ Fünftes Kapitel : Monopoleu. Kartelle im Idrianer Quecksilberhandcl «sw. 341 Quecksilberkaufsverhandlungen in Antwerpen gepflogen, als er sich Ilsung gegenüber zur Annahme eines idrianischen Quecksilber¬ handelsmonopols bereit erklärte . Ilsung hält in seinen Berichten an den Kaiserhof dafür , daß die Spanier aus irgendeinem Grunde in dem Schmuggel behindert wurden und daraufhin Manlich ab¬ sagten . Vielleicht boten die spanischen Zollbehörden eine erhöhte Wachsamkeit auf , seit Almaden genügend Quecksilber liefern konnte. * * * Nach der Absage der Manlich hat Ilsung bei verschiedenen Augsburger Großkaufleuten noch das Idrianer Quecksilberhandels¬ monopol unterzubringen versucht . Vergeblich ! An der Handels¬ krisis jener Zeit scheiterte jedes Bemühen . Die sechziger Jahre des 16. Jahrhunderts waren keine Zeit , in der sich die Augsburger Handelsherren auf große riskante Unternehmungen einlassen konn¬ ten . So mußte Ilsung denn den Herwart ankündigen , daß er sie vorläufig nicht aus ihrem Kontrakt entlassen könne . Den Kaiser aber mußte er auf andere Geldquellen hinweisen und auf eine andere Art , das Idrianer Quecksilber mit Vorteil für die Krone abzusetzen . In letzterer Beziehung dachte Ilsung zunächst an die Fugger . Der kaiserliche Finanzagent ging dabei von der sicherlich richtigen Annahme aus , daß die Übernahme eines Idrianer Queck¬ silberhandelsmonopols niemandem nutzbringender sein könne , als der Firma Fugger . „Dann die Fugger vielleicht die subtilen Wege finden möchten , das idrianische Quecksilber mit oder ohne Licenz nach Spanien zu bringen , es dort unter ihr spanisches zu mischen und alsdann eins mit dem andern zu vertreiben .“ Georg Ilsung hat im Herbst 1564 Markus Fugger , dem da¬ maligen Führer der Firma , entsprechende Vorschläge unterbreitet l. Aber auch hier erfuhr der kaiserliche Finanzagent eine Absage. Selbst die Fugger hatten unter der süddeutschen Handelskrisis jener Jahre zuviel zu leiden , um sich auf weitgreifende Projekte einzulassen . Persönlich hatten sie überdies gerade im Herbst 1564 mit den Schulden des leichtsinnigen Hans Jakob Fugger genug zu schaffen , um ihren Kredit voll aufrecht zu erhalten. 1 Bericht Ilsungs vom 11. September 1564. F. A. Wien. Innerösterr . Quecksilber 23. 342 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. Nac'n diesem vergeblichen Versuch bei den Fuggern ein neues idrianisches Quecksilberhaudelsmonopoi unterzubringen, blieb Ilsung nichts anderes übrig, als dem Kaiser ein ruhiges Abwarten zu emp¬ fehlen, bis sich die süddeutsche Handelswelt etwas beruhigt und neuen Mut zu großzügigen Unternehmungen gefunden hätte . Um nichts unversucht zu lassen, wurde aber auf Ilsungs Rat der kaiser¬ liche Gesandte von Dietrichstein angewiesen,mit den spanischen Kaufleuten, die dem König von Spanien das Älmadener Queck¬ silber abkauften und nach Amerika führten, in Verbindung zu treten ]. Vielleicht, daß sie sich verpflichteten, jährlich „eine anzahl der idrianischen quecksilber anznnehmen und ihrer gelegenheit nach auszuführen“. Ein etwaiges Bedenken, ob man durch eine solche Verbindung nicht dem König von Spanien schade, wies Ilsung mit dem — in seiner Naivität natürlich nicht ernst gemeinten—- Ausspruch zurück: Da die spanische Krone alljährlich alles Älmadener Quecksilber verschleiße, so habe sie „nit ursaeh einem anderen zu wehren“, der neben ihr „ Indien der noldurft nach mit quecksilber versehe“. Inzwischen erwuchs Ilsung die schwierigere Aufgabe, die Idrianer Gewerken zu beruhigen, die auf schnellere Abnahme ihres fertigen Quecksilbers durch die Herwart drängten. In Idria lag 1564 ein bedeutendes Quantum Quecksilber vorrätig. Je schneller die Herwart abnahmen, um so schneller war ihr Kontrakt zu Ende und um so eher konnte der neue, zu höheren Preisen mit Ilsung verabredete, beginnen. Den Gewerken lag aber noch aus einem anderen Grunde am flotten Absatz. Nur bei schnellem und genügendem Verkauf konnten sie den „Verlag“ von jährlich zirka 12 000 fl. ohne Mühe aufbringen. Von seiten der Herwart gesehen, sah das Bild nun freilich anders aus. Der Zwang für sie, sofort bedeutende Mengen Quecksilber abzunehmen und auf den Markt zu bringen, hätte ohne Zweifel einen sofortigen Preissturz des Artikels auf dem Weltmärkte Antwerpen bedeutet. Ilsung schlug vor, um den Gewerken Verlagsgelder zu beschaffen, die Herwart sollten den Gewerken ein Darlehen gewähren, das aus dem künftigen Kontrakt gedeckt werden könne. Würden die Herwart aber den nächsten Kontrakt nicht übernehmen, so sollten 1 29 . September 1564 . Kaiser Max . II . an Dietrichstein . F . A. Wien . Innerösterr . Quecksilber 23. Fünftes Kapitel : Monopoleu. Kartelle im Idrianer Querksilbcrluindel usw. 343 die Kaufleute, die ihn übernahmen, gehalten sein, die Schuld¬ summe den Herwart sofort in bar auszuzahlen. Die Herwart ließen sich freilich weder auf ein solches Darlehn ein, noch machten sie Ilsung Hoffnung, daß sie den nächsten Kon¬ trakt übernehmen würden. Die Firma verfolgte vielmehr eine Politik des Hinhaltens. Sie berief sich dabei auf einen Paragraphen des Vertrags von 1544, laut dem erst acht Monate vor Ablauf des alten Kaufs über einen neuen verhandelt werden dürfe. Ich weiß nicht, ob von Dietrichstein wirklich Verhandlungen der ebengenannten Art mit spanischen Kaufleuten eingeleitet liât. Die wären dann bald dadurch unterbrochen worden, daß Ilsung den älteren Gedanken wieder aufnahm, das Idrianer Quecksilber ganz oder doch zum größten Teil an die spanische Krone selbst zu liefern. Es ist ein Beweis für das Vertrauen, das Georg Ilsung beim Kaiser genoß, wenn Ferdinand sich beeilte, die spanischen Pläne seines Rates ganz und gar in dessen Sinne und nach dessen Anweisungen zu verwirklichen. Man ging dabei österreichischcrseits mit guten Hoffnungen an die Krone Spanien heran, hatte doch noch kurz vorher (Februar und Anfang März 1564) Martin Guzmann aus Barcelona berichtet, daß die Fugger so gut Avie keinen Vorrat mehr an Quecksilber hätten , daß sie im Jahre 1563 nur zirka 800 Quintal Quecksilber in Almaden erbaut und kaum über jährlich 1500 Zentner erbauen könnten, während der ameri¬ kanische Bedarf jährlich 3000 Zentner übersteige. Guzmann ver¬ ließ um jene Zeit Spanien; an seiner Stelle war von Dietrichstein berufen, die spanische Krone zu einer kontraktlichen Abnahme idriauischen Quecksilbers zu bewegen. An ihn richtete der Kaiser Mitte 1564 eine genaue, von Ilsung entworfene Instruktion. Dietrich¬ stein solle, heißt es darin, König Philipp erklären, daß Spanien am meisten Interesse an dem Abschluß eines idrianischen Queck¬ silberlieferungsvertrageshabe, wie ja auch der spanische König vor zwei Jahren „derohalben bei der kaiserlichen Mt. umb handlung ansuechen lassen“. Philipp dürfe und könne nicht verkennen, daß Almaden nicht imstande sei, genügend Quecksilber für Spanien und besonders für Spaniens amerikanische Kolonien zu liefern. Bisher hätten die Fugger nie mehr als 800—1000 Quintal in Al- 344 Drittes Bucli: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. maden gewonnen , im laufenden Jahre (1564) würde die Produktion vielleicht auf 1200 Quintal höchstens steigen . Mehr aber als 1400 Quintal könne Almaden nicht liefern . Demgegenüber betrage der Bedarf der amerikanischen Kolonien Spaniens , Spaniens selbst und Portugals an Quecksilber und Sublimat 1jährlich 3000 Quintal. Wenn jetzt die neu aufgefundenen Inseln erobert würden , werde die amerikanische Nachfrage sich noch um ein bedeutendes stei¬ gern 2. Auf Grund der genannten Tatsachen bekam von Dietrich stein die Weisung , dem König von Spanien vorzuschlagen , er möge sich mit Ferdinand über die jährliche Abnahme einer bestimmten Menge idrianischen Quecksilbers einigen . Die spanische Krone könne dann dieses idrianische Produkt in Sevilla an diejenigen spanischen , nach Amerika exportierenden Kaufleute absetzen , an die sie auch das Almadener Produkt verkaufe . Auf diese Weise behalte der König von Spanien „ den Gewinn selbst , anstatt daß andere Leute mit oder ohne Licenz das Quecksilber heimlich nach Amerika brächten und den Profit einsteckten “. Ferdinand machte sich anheischig, während der nächsten drei bis vier Jahre alljährlich 800—1000 Zentner Quecksilber Wiener Gewicht, eventuell auch mèhr, loco Antwerpen für 105 fl. rh . oder loco Sevilla für 80 Dukaten zu liefern . Es würde nicht schwer fallen, gute , vermögende Kauf¬ leute zu finden , die das Quecksilber in Idria annähmen und gegen eine entsprechende Frachtvergütung auf ihre Gefahr nach Ant¬ werpen bzw. Sevilla brächten . Der Verdienst des Königs von Spanien würde sich, selbst wenn er nur 110 Dukaten für den Quintal in Sevilla erhalte , an 3000 Quintal auf zirka 150 000 Dukaten belaufen. Könne sich der König von Spanien nicht zu einem festen Abnahmekontrakt entschließen , so wurde von Dietrichstein an¬ gewiesen, darauf hinzuarbeiten , daß Ferdinand wenigstens für die 1 Aus dem Quecksilber werde Mercurium sublimatum gemacht und nach Indien zur Scheidung des Silbers von Gold geführt , „bei welchem vil ain grössrer nutz als mit dem quecksilber ist “. Martin de Guzmann an den Kaiser . Barcelona, 22. Februar 1564. In F. A. Wien. Inncröstcrr . Quecksilber 23. 2 Über das Phantom der Gold- und Silberinseln, das gerade in jenen Jahren wieder lebhaft die Gemüter bewegte, vgl. Sophus Rüge, Geschichte des Zeitalters der Entdeckungen . Berlin 1881, S. 207 ff., bes. 211. 1 Fünftes Kapitel : Monopoleu. Kartelle im Idrianer Quecksilberhandel usw. 345 nächsten fünf Jahre die Licenz einer Einfuhr von 4—5000 Quintal gegen Zollgebühr (24 Dukaten pro Quintal , aber womöglich weniger) erhielte. Etwaige Bedenken Philipps II ., daß „unter dem Scheine“ des spanischen Abnalimekontraktes oder der kaiserlichen Licenz mehr idrianisches Quecksilber als verabredet , nach Spanien im¬ portiert und so der Preis gedrückt würde riet Ilsung durch die Zusicherung folgender Maßnahmen zu zerstreuen . Der Kaiser wolle die feste Zusicherung abgeben, daß über die. verabredete Menge hinaus nicht ein Pfund idrianischen Quecksilbers nach Spanien eingeführt werde. Für die übrige, nicht an Spanien abgegebene idrianische Quecksilberproduktion schlug Ilsung ein Preiskartell zwischen der Krone Spanien und dem Kaiser vor. Der Kaiser solle sein Testierendes Quecksilber nicht wohlfeiler verkaufen als der König von Spanien das Almadener an die spanischen, nach den Kolonien exportierenden Kaufleute lieferte. So würde das spanische und das idrianische Quecksilber „ ain weg wie den andern in iren würden erhalten , beide nebenainander vertriben , die betrüglichen handelsleute ausgeschlossen werden und der ansechlich nutz aus diser handlung obgedachten beiden potentaten selbst bleiben“. Von Dietrichstein war gehalten , mit der größten Vorsicht bei seinem Auftrag zu Werke zu gehen, damit der spanische Faktor der Fugger, Cristoff Hörmann , keinen Wind davon bekäme, und den König von Spanien gegen das Projekt einnähme. Auch die spanischen Kaufleute , die bisher Einfuhrlicenzen nach Spanien auf auswärtiges Quecksilber erhalten hätten , durften natürlich aus leicht ersichtlichen Gründen nichts von dem Vorschlag des Kaisers wissen. Die Verhaltungsmaßregeln , die Ilsung für den spanischen Bot¬ schafter des Kaisers ausgearbeitet hatte , gelangten am 11. Juli in die Hände des Herrn von Dietrichstein . Bald darauf schon hatte der kaiserliche Beauftragte Gelegenheit, seine Sache Philipp II. vorzutragen . Zunächst bat sich der König Überlegungszeit aus 2. Bald aber ließ er dem kaiserlichen Orator durch Francisco Erasso folgenden Bescheid geben : von Dietrichstein möge dem Kaiser 1 Was natürlich einen Rückgang der Einnahmen der spanischen Krone bedeutete , die das Quecksilber an die nach Amerika expor¬ tierenden Kaufleute sehr teuer verkaufte. 2 Das folgende nach dem Bericht von Dietrichsteins vom 29. Juli 1564 . F . A . Wien . Innerösterr. Quecksilber 23. 346 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. Philipps Dank für seinen Vorschlag melden. Der König von Spanien wisse wohl, daß er sich im Jahre 1561 um einen festen Lieferungskontrakt idrianischen Quecksilbers bemüht habe. Da¬ mals sei Almadert „noch nit in Schwung“ gewesen. Jetzt seien die dortigen Quecksilbergrubengenügend leistungsfällig. Das um so mehr, als der amerikanische Bedarf nachzulassen beginne. Wohl sei noch im vorigen Jahre in Neuspanien Mangel an Quecksilber gewesen, wirklich habe man dort im vergangenen Jahre bis zu 300 Dukaten für den Zentner gezahlt, aber die Zeiten seien vorbei. Der Vizekönig habe mitgeteilt, daß der Bedarf an Quecksilber falle. Man wisse jetzt in Neuspanien besser mit dem wertvollen Produkt umzugehen als früher. Von Dietrichstein hat sich nicht mit der Antwort Francisco Erassos begnügt, er hat sich dann eine direkte Ablehnung der kaiserlichen Vorschläge von Philipp II. selbst geholt. So waren die deutschen Habsburger — von ihren spanischen Vettern im Stich gelassen — wiederum darauf angewiesen, das Idrianer Quecksilberhandelsmonopoleiner deutschen Käpitalistenfamilie zu übergeben. Die Herwart kamen nicht mehr in Frage, sie weigerten sich aufs neue, ihr Geld in das schlechte und gewagte Unternehmen zu stecken. So schlecht freilich, wie sie es hinstellten, muß aber das Quecksilbergeschäftauch 1565 noch nicht gewesen sein, sonst würde man es nur schwer verstehen, daß die Firma von ihrem Recht, nach Abnahme der kontraktlichen 4000 Zentner noch für 20000 fl. zum alten Preise fordern zu können, Gebrauch machte. Nach langem Suchen hat endlich llsung für Erzherzog Karl 1 mit der Firma David Haug, Hans Langenauer und Mitverwandte am 8. Oktober 1566 einen neuen Kontrakt abgeschlossen. Die bekannte Augsburger Firma 2 übernahm den Vertrag, den seiner¬ zeit (Villach, 29. März 1564) llsung mit den Gewerken abgeschlossen hatte 3, d. h. sie verpflichtete sich, vom 8. Oktober 1566 bis zum 1 Dieser hatte nach Ferdinand, seines Vaters, Tod in der brüder¬ lichen Teilung den Vertrag übernommen. 2 Näheres über die Firma besonders bei R. Ehrenberg, Das Zeitalter der Fugger, I. Bd. S. 227 ff., ferner hei J. Hartung, Aus dem Geheimbuche eines deutschen Handelshauses im 16. Jahr¬ hundert. Zeitschr. f. Sozial- und Wirtschaftsgeschichte, Bd. 6 (1898) S. 36 ff. 3 Revers der Firma. F. A. Wien. Innerösterr. Quecksilber 23. „Doch ausserhalb fies articklils der khonftigen kaufshandlungen . . .“ Fünftes Kapitel : Monopoleu. Kartelle Im Idrianer Quecksilberhandel usw. 347 8. Oktober 157t 5000 Wiener Zentner idrianischen Quecksilbers und Zinnobers abzunehmen. Dafür hatte sie 350 000 fl. zu zahlen 1 und zwar den Gewerken in bestimmten Raten 122 062 fl. 30 kr. und dein Erzherzog 227 937 fl. 30 kr. 2. Die Augsburger Handels¬ herren setzten als Pfand der rechtzeitigen und ordentlichen Zahlung alle ihre und ihrer Erben „ligende gegenwürtige und khunftige samer.t- und sonderliche hab und gueter wie die namen haben und genanndt werden mögen, keine davon aus¬ genommen“. Daß auch die Haug, Langenauer & Co. nicht leicht zur Über¬ nahme des idrianischen Quecksiiber-Großhandelsmonopols vermocht wurden, erkennt man aus den vielfach neuen Bestimmungen, die die Augsburger Firma zu ihrer Sicherung in den Kontrakt zu bringen wußte. Selbstverständlich versprach der Erzherzog, wie auch Ferdinand in den früheren Kontrakten getan hatte, während der Kontraktszeit „khain Idrianisclies noch ander queckhsilber oder zinober an khainem ort noch end verkhaufen, hingeben, verschenckhen noch verfüren zu lassen, noch auch jemands andern solches zu thuen zu gestatten “. Der Erzherzog verpflichtete sich aucli, kein Quecksilber oder Zinnober bei Strafe der Konfiskation durch sein Land und Gebiet passieren zu lassen. An die Amtleute sollte ein entsprechender Befehl ergehen. Endlich sagte Erzherzog Karl der Augsburger Firma zu, in der Zeit ihres Kontraktes Quecksilberbergwerke„in Behaim oder andern seiner kais. Mt. königreichen und erblanden, so anjetzo vor äugen oder khünftig aufkhomen möchten nit erheben oder in gang Idiomen zu lassen, damit das Idrianisch queckhsilber desto hass verschlissen werden mög“. Karl hat seine Versprechungen gehalten; so sistierte er Ende 1566 ein Quecksilberbergwerk , das ein Leobener Bürger und ein Radmeister aus Vordernberg eröffnet hatten 3. Auch die Schutzversicherung der Monopolinhaber vor einer 1 Also 70 fl. rh . in Müntz zu 15 Batzen = 60 Kr . für den Wiener Zentner „ Quecksilber und Zinnober durchainander gérait“ . 2 Die älteren Historiker (Hurter, Geschichte Ferdinand IL, Hitzinger a. a. O. S. 21) haben aus dieser Gewinnverteilung ganz falsche Schlüsse auf die Größe des landesfürstlichen Anteils an dem Bergwerk gezogen. 3 .7 Dezember 1566. F. A. Wien. Loc. Idria. 348 Drittes Buch : Monopole , Kartelle und Aktiengesellschaften im Mittelalter Belästigung durch den Reichsanwalt, den kaiserlichen Fiskal, findet sich wie in den vorhergehenden Kontrakten . Neu dagegen ist eine Bestimmung, die die Monopolinhaber verpflichtet, den Gold¬ schmieden in Kärnten und Krain „das queckhsilber und zinober, sovil sie dessen (zu täglicher notturft und nit weiter) selbs brauchen und haben muessen, in ain zimblichen press und werd zuesteher zu lassen“. Der Wunsch der Firma, sich vor unliebsamen Überraschungen zu schützen, der sich in der Schutzbestimmung dem Reichsfiskal gegenüber zeigt, ist auch aus anderen Paragraphen ihres Monopol¬ kontraktes von 1566 ersichtlich. So aus der Abmachung, der die Augsburger Handelsgesellschaft auf ihren Wunsch hin für den Fall von ihrem Vertrag entband, daß Krieg in jenen Gegenden tobte oder daß aus anderen Gründen die Straßen gesperrt seien. Einen interessanten Beitrag zu der Tatsache der allmählichen Objekti¬ vierung, der allmählichen Versachlichung der Finanzgeschäftex, bietet die folgende Abmachung in dem Monopolkontrakte der Haug, Langenauer & Co. vom Jahre 1566. Für den Fall, daß die Augsburger Firma das Idrianer Quecksilber-Großhandelsmonopol aus irgendeinem Grunde nicht mehr ausüben will, soll es ihr erlaubt sein, den Kontrakt ganz oder teilweise an einen oder mehrere Kapitalisten zu übertragen. Gefordert wird von seiten der Gewerken und der Regierung nur, daß die betreffenden Kapi¬ talisten „ehrliche und vermögende Leute“ sind, und daß sie ganz und gar in die kontraktlichen Verpflichtungen der Haug, Langen¬ auer & Co. eintreten 2. 1 Vgl. den Abschnitt „Die Kommerzialisierung des Wirtschafts¬ lebens“ bei W. Sombart, Die Juden und das Wirtschaftsleben, S. 60 ff. Siehe auch schon oben. 2 Die wichtige Stelle des Kontraktes lautet wörtlich: „Es ist auch verner lauter abgered, bedingt und versprochen worden, wo gedachter Haug, Langenauer und mitverwaudten disen gegenwürtigen kauf und vertrag und obbemelte queckhsilber und zinober ainer oder mer partheien gar oder ains thails ubergeben oder zuestellen wollten oder würden, dass si solches (doch gegen ecrlichen, verinügliclien leuthen und disen Sachen gemäss) zu thucn gucten fueg und macht haben sollen und wir und die gewerckhen denselben, auch iren erben und nachkhomen allermass gleichwie inen den kaufern hiemit verpunden, verpflicht ze halten und zu laisten schuldig sein sollen und wellen. Dagegen sollen auch der oder diseiben, denen so solcher khauf gar oder zum thail uebergeben würde uns und gemainen ge- Fünftes Kapitel : Monopole u. Kartelle im Idrianer Qnccksilberhandcl usw . 349 Welche Mühe Erzherzog Karl hatte , um im Jahre 1566 das Idrianer Monopol unterzubringen , erkennt man auch — außer an den schon genannten Konzessionen , die er den Haug , Langenauer & Co. machen mußte — an der folgenden Verabredung . Erzherzog Karl sowohl, wie die genannte Augsburger Firma wußten , daß die Herwart noch einen großen Vorrat Quecksilber besaßen . Damit nun die IIerwart nicht sofort , wenn sie von der Übernahme des neuen Kontraktes durch die Haug , Langenauer & Co. hörten , ihre Restbestände auf den Markt würfen und die Preise verdürben, verabredeten der Erzherzog und die neuen Monopolkontrahenten, ihren Kontrakt ein halbes Jahr geheim zu halten . Solange solle das Monopol unter dem Namen des Erzherzogs gehen . Man ging dabei von der Erwägung aus , daß es dem Erzherzog als dem an¬ geblichen Inhaber des Idrianer Quecksilber -Großhandelsmonopols leichter fallen werde, die Herwart zur Hochhaltung des Quecksilberpreises zu bewegen. Sollte es wider Erwarten trotz alledem unmöglich sein und sollten die Herwart über 10 oder 15 fl. unter den Anfang 1566 üblichen Quecksilberpreis heruntergehen , so würde den Haug , Langenauer & Co. ein entsprechender Preis¬ nachlaß im Einkauf des Quecksilbers gewährt werden. Schließlich sagte Erzherzog Karl der Gesellschaft Haug , Lan¬ genauer & Co. das Vorkaufsrecht bei einem neuen Monopol für den Fall zu, daß sie. dasselbe Gebot machten wie ein anderer Reflektant. Auf keinen Fall war es dem Erzherzog und den Gewerken gestattet, etwa mit einem neuen Kontrahenten in Verbindung zu treten , ehe die Haug , Langenauer & Co . das Quecksilber , das ihnen vertrags¬ weise zukam , abgenommen hätten. Es ist mir nicht möglich zu sagen , ob die Handelsgesellschaft David Haug , Hans Langenauer und Mitverwandte an dem Groß¬ handelsmonopol mit idrianischem Quecksilber große Summen ver¬ dient hat . Jedenfalls zeigte sich die Firma nach Ablauf des ersten Monopolkontraktes bereit , einen zweiten einzugehen . An Stelle von Georg llsung , der in der kaiserlichen Finanzverwaltung tätig blieb , aber in den Idrianer Akten seit dem 'Übergange des Berg¬ werks an Erzherzog Karl uns nicht mehr begegnet , schließt jetzt werckhen alles das, so diser aufgericliter kauf und vertrag inen den kaufern auferlegt , auch ze halten verpunden sein, doch dass si, die kaufer und ire erben uns und gemainen gewerckhen umb bezallung diser khaufsumma zu bcstimblen fristen guet und bezallcr seien.“ 2345 350 Drittes Buch: Monopole, Kartelle und Aktiengesellschaften im Mittelalte] Hans Khisl den Vertrag im Namen des Landesherrn1 mit den Ge werken und übergibt dann den Vertrag — unter Zuschlag eine tüchtigen Profits natürlich — der Augsburger Firma. Diese vei pflichtete sich, 7500 Zentner Quecksilber resp. Zinnober im Lauf von fünf Jahren abzunehmen. Der Kontrakt ist, soviel ich sehe leider nicht auf uns gekommen. Seine Hauptbestimmungen dürfte mit denen des ersten idrianischen Quecksilbermonopolvertrages de Firma übereinstimmen. £ * * Bis zum 8. April des Jahres 1574 ging das Monopolwesen ii Idria den vorgeschriebenen und gewohnten Gang. Die Haug Langenauer & Co. hatten bereits 5333 Zentner Quecksilber ab genommen, als plötzlich die Zahlungen der Augsburger Groß handelsfirma stockten. Der Aufschwung, den das Augsburger Handelsleben nach de: Bankrotten zu Anfang der sechziger Jahre des 16. Jahrhundert genommen hatte , war nur von sehr kurzer Dauer gewesen. Vor 1571—1575 setzte eine neue heftige. Krisis ein, die sich in zahl reichen Bankrotten bedeutender Handelsfirmen entlud \ Di» Krafter, die. Manlich, Hans Georg Baumgartner * und ander» * Augsburger Handelshäuser fallierten in jenen Jahren . Die Firm: David Haug, Hans Langenauer & Co., seit längerer Zeit schor auf einem ungesunden System vieler hochverzinslicher Depositer basiert % mußte ebenfalls ihre Zahlungen einstelien. Es scheint, als habe zu dem Ruin der Haug, Langenauer &. Co , am meisten der Felilschlag ihrer englischen Bergwerksunternehmungen6 bei getragen. Schon 1570 wurde in Augsburg ein Gerücht kolportiert, „dass es mit der englischen sach nit wo! stiende“. Schon damals wurden Depositengläubiger der Firma gewarnt 6. 1574 brach dann 1 5. Mai 1571; gültig vom 8. Oktober 1571 an. 2 Ehrenberg, Zeitalter der Fugger, II, S. 242. 3 Ausführliche Akten über diesen Bankrott im Rsichskammergerichtsarchiv zu Wetzlar; cf. Repertorium Bd. I, Nr. 141—146 ff. 4 Ehrenberg, Zeitalter der Fugger, I, S. 232. 5 Eh r e n b e r g a. a. O., I, S. 234 und derselbe, Hamburg und England im Zeitalter der Königin Elisabeth, S. 5 Anm. fi Vgl . den Brief Hans Langenauers an Hans Loner, den Haupt¬ vertreter der Firma in England, den ich als Beitrag zur Unternehmer¬ psyche jener Zeit im Anhang abdrucke. Fünftes Kapitel : Monopoleu. Kartelle im Idriancr Quecksilberhandel usw. 355 das Unglück unaufhaltsam über die Haug , Langenauer & Co. herein . Vergeblich hatten die Fugger die alte , angesehene Firma durch ein größeres Darlehn 1 zu halten versucht . Alle Bemühungen scheiterten an dem egoistischen Bestreben der Katzbeckischen Handelsgesellschaft , ihr Guthaben aus dem beginnenden Ruin der Haug , Langenauer & Co. zu retten . So wenig waren „ die Katzbeckischen “ der Tatsache eingedenk , daß sie erst vor kurzem durch eine finanzielle Hilfsaktion der Haug , Langenauer & Co . vor dem geschäftlichen Ruin bewahrt worden waren , daß sie nicht eher ruhten , bis David Haug , ihr eigener Schwager , von der Stadt in Haft genommen wurde 2. Vielleicht hat nichts mehr als gerade dieses rigorose Vorgehen der Katzbeckischen Handelsgesellschaft dazu beigetragen , die Bemühungen der Haug und Langenauer befreundeten Kreise aufrecht zu erhalten , die in dem Versuch einer Sanierung der zahlungsunfähigen Firma bestanden . Wir werden sehen , daß auch der Erzherzog sich diesen Bemühungen anschloß. Die Idrianer Gewerken gerieten über der Zahlungseinstellung der Haug , Langenauer & Co. in große Verlegenheit und starke Aufregung . Sie erwarteten , der Erzherzog werde in die Bresche springen , da ia er und nicht die Augsburger Firma mit ihnen den „ Quecksilberkauf “ abgeschlossen habe . Demgegenüber erklärte Erzherzog Karl , der alte Kontrakt habe durch die Zahlungsein¬ stellung der Augsburger Kontrahenten seine Endschafi erreicht. Er wolle aber sofort , um der Not der Gewerken ein Ende zu machen, einen neuen .,Quecksilberkauf “ auf sechs Jahre mit ihnen schließen und sich verbindlich machen , im Laufe dieser Zeit 6000 Zentner 1 Wofür sie sich freilich Quecksilber, das in der venctianisclien Faktorei der Firma lagerte, zum Pfand setzen ließen. Vgl. Hans und Marx Fugger an Erzherzog Karl. 21. Mai 1575. Original mit eigenhändiger Unterschrift und Siegel der Fugger in F. A. Wien. Innerösterr. Quecksilber 23. „Hierueber könnden Ew. f. dt. wir gehorsamlich nit bergen, dass genanntem Hans Langenauer und seinen mitverwandten zu erhaltung ires trauens und glaubens mit darstreckung einer großen summa, sovil uns erschwinglichen und möglichen gewest, treuherziger mainung die hand gereicht worden.“ 2 Die Firma Haug, Langenauer & Co. hatte 15fi5 mit den Katzbeck und den Fugger zusammen die „Gesellschaft des Jenbacher Bergund Schmelzwerkhandels“ inne. Vgl. A. Zycha in Vierteljahrs¬ schrift für Sozial- und Wirtschaftsgeschichte5 (1907), S. 281 Anin. 4. 352 Drittes Buch : Monopole, Kartelle und Aktiengesellschaften im Mittelalter. Quecksilber zum Preise von 24 fl. pro Zentner abzunehmen . Auch die Schuld der Hang , Langenauer & Co. an die Gewerken (in Summa 36 492 fl. 32 kr .) wollte der Erzherzog übernehmen . Er verpflichtete sich, die Summe im Verlauf der sechsjährigen Kontraktszeit ab¬ zubezahlen . So sehr den Gewerken dieser letztere Vorschlag gefiel, so sehr stießen sie sich an dem niedrigen Preis von 24 fl. pro Zentner Quecksilber . Sie forderten 28 fl. (resp . 32 fl. für Zinnober ), gingen aber später doch auf 24 fl. (resp . 28 für Zinnober ) herunter 1. Die Verstaatlichung des Idrianer Quecksilberbergwerks machte dem Feilschen ein Ende . Khisl , auf dessen Rat diese Neuregelung durchgeführt wurde , hatte schon länger daran gedacht , die Idrianer Quecksilbergruben in die absolute Verfügungsgewalt der Regierung zu bringen . Er hatte schon 1572 begonnen , im geheimen idrianische Kuxe aufzukaufen , da er aber nicht mehr als neun in seinen Besitz hatte bringen können , war er vorläufig von dem Plane ab¬ gegangen und hatte die Kuxe wieder verkauft . Jetzt trat Khisl an die „ Gewaltträger “, die Vertreter der Idrianer Gewerken mit seinem Vorschläge einer Verstaatlichung heran . Der Zeitpunkt war gut gewählt . Die augenblicklich ungünstige Konjunktur auf dem Quecksilbermarkte , die Schwierigkeit , an Stelle der Haug, Langenauer & Co. einen anderen Kapitalisten für das Monopol zu gewinnen und anderes nahm die Führer der Idrianer Gewerken durchaus für den Plan einer Verstaatlichung ein. Nur baten sie Khisl , bevor sie sich definitiv entschieden , mit ihren „Prinzipalen “, d. h . ihren Gewerkschaftsgenossen , Rücksprache halten zu dürfen. Die 144 Kuxe des Idrianer Quecksilberbergwerks waren damals wie folgt verteilt: Wolf Englprecht von Auerspergs Erben . . . . 9 3/4 Kuxe Jobst Joseph Freiherr vom Thum ...... 5 2/3 , , Hans Georg von Lambergs Erben . . . . . 6 1/2, J/42, 1/240 „ Hans Joseph Freiherr zu Eelch ........ 2, 1/252 , , Franz Joseph Freiherr zu Eelch Erben . . . . 2 , 1/252 „ Seyfridt Naspens sei. Erben ......... 2 3/i, 1/ 12 „ Leonhardt von Sigessdorff sei. Erben ..... 2 „ Franz Freiherr vom Thurn ......... 2/o> l/4, 1/6 „ Urban Ainlehurn . . Georg Haller . . . . Marco Stettners Erben 1 Alles nach dem Bericht Khisls vom 27 . Februar 1575. Fünftes Kapitel : Monopole u. Kartelle ira Idrianer Quecksilberhandel usw . Leonhardt Khreen........... Frau Katharina vom Thum....... Frau Anna Paradeisserin........ Michl Wolfperger und Frau Anna Saifnerin Frau Margaretha Neisserin sei. Erben . . Frau Cristina Wärlin sei. Erben..... N . Galln zu S. Georgenperg....... Georg von Obritschan sei. Erben..... Frau Katharina Gallin......... Frau Khirfelda Wittib ......... Mattheus Eelchen............ Cristoff Pfluegl............. Die Herwardten ............ Carl Villinger.............. Cristoff von Lamberg.......... Sigmund von Lamberg......... Ulrich von Lamberg.......... Marx Theen sei . Erben......... Abel Prasch.............. Hans Waiblinger............ Christoff Khronegger.......... Dr . Leonhard Jung........... Frau Katharina Hochstetterin Erben . . . Frau Barbara Neumanin sei . Erben . . Ein ersame Landtsehaft in Kärndten . . . Sigmundt von Dietrichstain....... Andre Eberhardt Stäuber........ Alexander Stäuber sei . Erben...... Andre Mägerls sei. Erben........ Bernhardt und Wilhalmb Leininger . . . . Jeronimus Khierchpuechers Erben . . . . Ludwig Alten und Bruders Söhn..... 353 19/2o Kuxe 1 99 1 99 V2> Vll2 99 V2> Vs 99 V., Va 99 1 Vs. V« 99 1/3 > V 48 99 1/a> V 48 99 Va, V« 99 / 22 4 99 16 V240 12 5 V2, V. 2 V4, Viü 2 Va, V15 99 99 99 99 99 V2> Vl5 99 1 Va, Vae 99 V, 99 V 22 4 99 1, V144 99 3 V2, V. 99 3 Va 99 /l68 , / 252 2 Va 5 V2, Ve 3 V2, Vis 3 V«, Vis V. 99 99 99 99 99 xu Vs 1 !. . . Summa 144 Kuxe Nach einer Schätzung der Führer der Gewerkschaft betrug der Gesamtwert der 144 Kuxe 200 000 fl. Khisl hoffte , „davon möchte ihnen leichtlich noch etwas abgebrochen werden “. Aber selbst wenn der Erzherzog den Kuxinhabern 200 000 fl. zahlen mußte, war er nicht übervorteilt . Khisl berechnete , daß aus dem augen¬ blicklich vorhandenen Erzvorrat mit einem Kostenaufwand von zirka 25 000 fl. leicht 14 000 Zentner Quecksilber gewonnen werden könnten . Setzte man den Zentner — nach Abzug von Fron und Wechsel — zu 19 fl. an , so ergab das schon 266 000 fl. Eine größere Schwierigkeit bestand nur in der Frage , woher sollte der Erzherzog Strieder , Studien z. Gesuh , kupitalist . OrganiaationBtormen . 23 354 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter, in Zukunft das Geld für den Verlag des Bergwerks nehmen. Auch hier wußte Khisl Rat . Nach seiner Meinung würde der Erzherzog ebensowenig eines eigenen Verlags zur Unterhaltung des Bergwerks bedürfen wie die bisherigen Gewerken. Die hätten seit 1524 keinen Pfennig aus ihrem eigenen Säckel darein verwendet, sondern allzeit die Idrianer Quecksilbergruben aus den getroffenen Monopol¬ kontrakten verlegt und unterhalten. Auf die Berechnungen und Vertröstungen Hans Khisls hin schloß der Erzherzog am 21. April 1575 den Handel mit den Idrianer Gewerken ab. Die letzteren überließen ihm für 170 000 fl. mit ihren Kuxen ihre sämtlichen Besitzrechte auf das Idrianer Bergwerk samt Vorräten usw. Die erste wichtige Aufgabe bestand nun für die erzherzogliche Regierung darin, einen Kapitalisten zu finden, der unter den veränderten Umständen das Monopol des Groß¬ handels mit dem Idrianer Quecksilber übernahm. Khisl schlug vor, das Monopol wieder der Firma Haug, Langenauer & Co. zu übertragen. Wahrscheinlich bestimmte ihn dabei — außer Nütz¬ lichkeitsgründen, auf die wir gleich zu sprechen kommen werden — auch ein Gefühl des Mitleids für die in Zahlungsschwierigkeiten geratene Augsburger Firma, besonders für Hans Langenauer. Der letztere muß — wenn man nach einigen uns von ihm erhaltenen Briefen urteilen darf — ein Ehrenmann gewesen sein, der unver¬ schuldet in seine mißlichen Geschäftsverhältnissegeriet. Auch die Habsburger waren nicht ohne Schuld dabei. So konnte Khisl sich nicht verhehlen, daß die Augsburger Firma den zweiten idrianischen Quecksilbermonopolkontrakt viel zu teuer übernommen hatte . Und auch das lag auf der Hand, daß der niederländische Krieg und die Eröffnung von Quecksilbergruben in Siebenbürgen1 einen schweren Schlag für Haug, Langenauer & Co. bedeutete. Dagegen muß es als eine Übertreibung bezeichnet werden, wenn die Firma behauptete, gerade die Verluste im Quecksilberhandel seien es gewesen, die ihr Falliment in erster Linie verursacht hätten. Ich glaube, hier liegt eine captatio benevolentiae vor, eine Über¬ treibung, um sich den Erzherzog, den Idrianer Regalherrn, günstig 1 15 . November 1572 (F . A . Wien . Innerösterr . Quecksilber 23). David Haug , Hans Langenauer & Co. beschweren sich , daß einige Nürnberger Bürger Quecksilber aus Siebenbürgen einführen und ihnen dadurch erhebliche Konkurrenz machen. Fünftes Kapitel : Monopoleu. Kartelle im Idrianer Quecksilberbandel usw. 355 zu stimmen und sich seiner Hilfe in dem Konkursverfahren zu versichern. Die erhoffte Folge trat denn auch wirklich- ein. Die Hilfs¬ bereitschaft der erzherzoglichen Beamten ging soweit, daß Kliisl, wie gesagt, vorschlug, der in Zahlungsschwierigkeitengeratenen. Firma auch dadurch die Möglichkeit einer Erholung zu geben, daß man ihr das Großhandelsmonopol für idrianisches Quecksilber zu einem sehr reduzierten Preise beließ. Freilich waren es nicht nur Gründe der Menschenfreundlichkeit, die Kliisl zu dem genannten Vorschlag bestimmten. Gründe der Berechnung kamen hinzu. So erklärte Khisl dem Erzherzog, daß es bei der augenblicklichen schlechten Konjunktur im Quecksilbergeschäfte und bei der Krisis im süddeutschen Handelsleben schwer halten würde, bald einen anderen Interessenten für das Idrianer Monopol zu bekommen. Besonders überzeugend aber mußten die folgenden Erwägungen wirken. Ließ man die Firma David Haug, Hans Langenauer & Co. fallen und übergab anderen Kapitalisten das Monopol, so hätte man das Geld verloren, das die Augsburger aus dem laufenden Quecksilberkontrakt noch den Gewerken und dem Erzherzog schul¬ deten. Das waren noch zirka 100 000 fl., von denen auf den Anteil der Gewerken 36 000 fl. entfielen. Letztere Summe hätte im Falle der Aufhebung des Haug-Langenauerschen Kontraktes von der Krone den Gewerken in bar ausgezahlt werden müssen1. Ein schrecklicher Gedanke bei der chronischen Finanznot jener Zeiten und allein schon zureichend, um den Erzherzog für den Plan Khisls zu gewinnen. Für einen Plan, der in einer Fortsetzung des HaugLangenauerschen Quecksilbermonopols , allerdings unter ' Neuord¬ nung und starker Reduzierung der bisherigen Preise bestand. Khisl hatte vorgeschlagen, den bisherigen Monopolinhabern in einem neuen Kontrakte auf fünf Jahre 3000 Zentner Quecksilber als sogenanntes „ Gnadensilber“ zu 29 fl. pro Zentner zu geben. Die. übrigen 3000 Zentner möge der Erzherzog den Haug, Langenauer & Co. „ wo nit um 40 oder 45 fl. so doch etwas weniger unter 50 fl. gnädigst überlassen“. Tatsächlich schloß Erzherzog Karl mit Llaug, Langenauer & Co. einen neuen „Kauf“ auf fünf Jahre (vom 1. Juli 1575 bis 1. Juli 1580) 1 Denkschrift Khisls vom 1. März 1575. F. A. Wien. Innerösterr. Quecksilber 23. 356 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. auf 6000 Zentner Quecksilber ab . Davon sollten 3000 für 29 fl. pro Zentner der Firma geliefert werden und 3000 Zentner zu 50 fl. pro Zentner , und zwar jeweils per Jahr 600 Zentner zu dem einen Kauf¬ preis und 600 Zentner zu dem andern . Freilich war das kein Kon¬ trakt wie der vorige oder wie die mit den früheren Kapitalisten eingegangenen . Der Vertrag war darauf zugeschnitten , der Augs¬ burger Firma die Möglichkeit zu geben , die Gelder, die sie dem Erzherzog und den Gewerken aus dem letzten „ Kauf “ schuldeten, ahzubezahlen 1. Zu diesem Zwecke war jeder Gewinn der Firma aus dem Quecksilberverkauf sogleich und unmittelbar zu ver¬ wenden. Zu diesem Zwecke war auch der Augsburger Firma eine auf möglichst hohe Gewinne zielende Preispolitik jetzt untersagt . Die Erfahrung lehre , hieß es in dem Kontrakt , daß es noch jedesmal zum Schaden des Idrianer Bergbaues ausgelaufen sei, „wann man die queckhsilber im press zu hoch spannen und den gewinn dabei gar zu übermäßig stöllen wollen“ . Darum sollten die Haug , Langenauer & Co. „in jetz wehrendem kauf sich desselben [d. h. einer scharfen Preispolitik ] enthalten und darundter ain soliche beschaidenhait gebrauchen , dabei si mit ehren und nach gebürlichen dingen besteen “ könnten . Das heißt , sie sollten sich „ an ainem zimlichen gwinn benuegen lassen “. Damit der Erzherzog die Befolgung der genannten Vorschriften genau kontrollieren könne, waren die Augsburger gehalten , bei jeder Quecksilberabnahme —- die übrigens nur gegen Barzahlung 2 stattfand — zu berichten , „wie hoch si allwegen ainen und den andern khauf , auch mit wem und umb wievil queckhsilbers beschlossen haben “ . An dem Monopolprinzip war auch jetzt festgehalten worden. Die erzherzogliche Regierung verpflichtete sich bis 1. Juli 1580 kein „idrianisches noch anderes fremdes quecksilber oder Zinnober dann allain das , darumb diser kauf getroffen an kainem ort noch end in ihren fürstentümern , herrschaften etc . verkaufen , hingeben, verschenken , noch durchführen zu lassen “ . Die Amtleute waren angewiesen , darauf acht zu haben , daß diesem Versprechen ge1 „Damit si unser bei inen noch ausstendige summa gelts desto gelegenlicher entrichten , sich etlichermassen widerumb erschwingen, wie auch bei andern iren trauen und glauben erholen und wider¬ bringen khönndten .“ Aus dem Vertrag. 2 Oder genügende Bürgschaft. Fünftes Kapitel : Monopoleu. Kartelle im Idrianer Quecksilbcriiandelusw. 35/ treulich nachgelebt wurde . Für eigenen Gebrauch , aber nur für diesen , sollte dem Erzherzog „etlich wenig phundt “ Quecksilber oder Zinnober von dem Bergwerk zu nehmen , freistehen . Außerdem mußten die Augsburger den Goldschmieden in Steiermark , Kärnten und Krain das Quecksilber für deren eigenen Gewerbebetrieb „ umb ain zimblich wert volgen “ . Eine Übertragung des Monopolkontraktes auf andere „ehrliche und vermögliche Leute “ sollte der Firma Haug , Langenauer & Co erlaubt sein. Doch sollte diese Übertragung mit Wissen und Willen des Erzherzogs geschehen und unter der Voraussetzung , daß die neuen Kontrahenten dieselben Bedingungen wie Haug , Lange¬ nauer & Co. auf sich zu nehmen gewillt seien. Auch das übliche Versprechen , die Monopolinhaber gegen Belästigungen durch den kaiserlichen Fiskal zu vertreten , findet sich in dem neuen Kontrakt unserer Firma . Hinzugefügt ist freilich die Bemerkung , „ doch weiter nit als was jetz gemelten queckhsilberkauf und desselben obangezogne conditionierte beschaffenhait betrifft “. Ich habe nicht näher untersucht , wie lange die Haug , Langenauer & Co. noch das Idrianer Quecksilbergroßhandelsmonopol besaßen . Es ist wohl an¬ zunehmen, daß der einmal begonnene Bankerott der Firma durch das Moratorium des Kaisers nicht aufzuhalten war l. Im Anfang der neunziger Jahre jedenfalls begegnet uns eine Firma Meinel als ihre Nachfolgerin in dem Monopol. Es ist ein Zeichen für den bekannten Rückgang der süddeut¬ schen kaufmännischen Unternehmungslust , wenn dann mit dem Jahre 1595 eine venetianische Firma an die Stelle trat , die fast ein Jahrhundert lang die Augsburger besetzt gehalten hatten . Der Venezianer Bartolomeo Bontempelo dei Calice, hatte dem Erz¬ herzog Ernst ein größeres Gelddarlehen bewilligt und sich dafür das folgende Versprechen geben lassen . Wenn der Kontrakt mit der Firma Meinel abgelaufen war und Bontempelo auf das Idrianer Quecksilbergroßhandelsmonopol reflektiere , so sollte ihm , falls er das , was „ andere etwo thuen auch laisten welle“ das „Vorkaufs1 Die Geschichte des Moratoriums wäre eine interessante Auf¬ gabe für einen geschichtlich gebildeten und für Archivarbeit fähigen Handelsrechtler. Auch hierbei würde sich ergeben, daß die Finanz¬ politik der Landesherren bei der Bewilligung von Moratorien— die ihnen zustand — eine große Rolle spielte. Also auch hier das Problem Fiskalismus und Kapitalismus! 12 358 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. recht “ zustehen . Würde etwa entgegen der Verabredung einem anderen das Idrianer Monopol übertragen , so solle Bontempelo „sein dariehen alsbald restituât “ werden . Tatsächlich machte Bontempelo auf das Monopol Anspruch und hat es auch vom 1. Juni 1595 bis zum 1. Juni 1599 (vielleicht auch länger ) von Erzherzog Ernsts Bruder Maximilian erhalten L Er sollte in den genannten fünf Jahren 7000 Wiener Zentner Quecksilber resp. Zinnober abnehmen , d. h. alljährlich 1400 Zentner zum Preise von 36 fl. (resp . 40 fl. für Zinnober ). Das Quecksilber wurde auf Kosten und Gefahr des Monopolinhabers , frei von landesherrlichen Zöllen und Mauten , bis nach Villach resp . Triest verführt . Die Zahlung hatte halbjährlich iu Venedig zu geschehen , gleichgültig, ob die Ware abgenommen war oder nicht . Um Bontempelo auch nach Ablauf des Monopolkontraktes noch eine zeitlang die Mög¬ lichkeit zu lassen , seine Ware zu Monopolpreisen abzusetzen 3, verpflichtete sich der Erzherzog Maximilian , bis zum 1. Juni des Jahres 1600 „ gantzlich stil zu stehen und mit khainem , wehr der imer seie, vor derselben zeit ainichen weitern khauf oder contract zu treffen noch zu schliessen“ . Bis zum Î . Juni des Jahres 1600 war es demnach den Habsburgern untersagt , idrianisches oder anderes Quecksilber und Zinnober zu verkaufen oder sonstwie ab¬ zugeben . In derselben Zeit sollte kein Quecksilber oder Zinnober die erzherzoglichen Lande passieren . Maximilian versprach auch, den Kaiser , seinen Bruder (Rudolf II .) und seinen Vetter , Erzherzog Ferdinand von Österreich , zur Grenzsperre gegen fremdes Queck¬ silber und Zinnober zu bewegen. Die noch von Erzherzog Ernsts Zeiten dem Venezianer ge¬ schuldete Summe von 55 000 fl. wurde auf 100 000 fl. von seiten des Darleihers erhöht . Die Schuld sollte durch Quecksilberliefe¬ rungen allmählich getilgt werden. Als charakteristisch mag bei unserer kurzen Besprechung des Bontempeloschen Kontraktes noch die Tatsache Erwähnung finden, daß darin ein Schutzversprechen gegen Angriffe des Reichsfiskals, wie wir es bei früheren Kontrakten fanden , nicht gegeben ist . Wir 1 Vgl. den Monopolkontrakt datiert Venedig 18. Mai 1594 . F . A. Wien . Innerösterr . Quecksilber 23. 2 In dem Kontrakt heißt es : „ Damit auch er , Bontempelo , solch queckhsilber und Zinnober desto bass verschlaissen und in würden erhalten müge “ . 1 Sechstes Kapitel : Einige sonstige Monopole besonders unter Ferdinand I. 359 müssen es dahingesteilt sein lassen, ob Bontempelo als Venezianer dessen nicht bedurfte , oder ob die Anti "Monopolistenbewegung einen gewissen Stillstand erreicht hatte. Mit Bontempelo beginnt die lange Reihe italienischer Firmen in dem Idrianer Quecksilbermonopol . Die wirtschaftliche Lage Süddeutschlands spiegelt sich in dieser Tatsache deutlich wieder. Obgleich z. B . Erzherzog Ferdinand von Österreich im Jahre 1604 alle Mühe aufwandte und auch seine Räte beauftragte , womöglich deutsche Kaufleute „ für den Kauf “ zu finden , so mußte doch auch jetzt wieder ein Italiener , Joh . Batt . Porro aus Mailand , einspringen . Dann folgen 1607 die Albertinelli , 1631 die Balbi usw. ’. Wir dürfen die Schilderung an dieser Stelle abbrechen . Von Srbiks mhaJtreiches Buch setzt hier ein. Sechstes Kapitel. Einige sonstige Monopole besonders unter Ferdinand I Schlußwort Wenn wir in der Finanzpolitik Österreichs und anderer Staaten des 17. Jahrhunderts eine große Neigung gewahren 2, das aus¬ schließliche Verkaufsrecht einer Ware an bestimmte Kapitalisten zu geben , wofür diese ihrerseits bestimmte jährliche Abgaben zahlten und sich auch durch Anleihegewährungen usw. dem Staate gefällig erzeigten , so ist das nichts neues. Bereits Ferdinand I. besonders hatte im 16. Jahrhundert oft auf die genannte Weise seine Finanzen aufzubessern gesucht . So erhielt um die Mitte des 16. Jahrhunderts . Anton Schmidt & Co. aus Danzig ein Privileg auf 15 Jahre , das die Firma befugte , allein Meersalz, das sogenannte Baiesalz , in Schlesien einzuführen und an Orten , wo es ihnen gut dünkte , umzusieden und dann zu verkaufen 3. Natürlich hatte 1 Von 1623 bis 1630 nahm noch einmal ein Deutscher , F. Over¬ holz das Monopol in die Hand . Vgl. von S r b i k , Der staatliche Exporthandel Österreichs, S. 4 ff. Das Werk Srbiks bietet in der Hauptsache eine Geschichte des österreichischen Quecksilber- und Kupfermonopols . Für clas 18. Jahrhundert vgl. auch Thorsch, a. a. O., S. 88 ff. und 97. 2 Vgi. von Srbik a. a. O, S. XXXI .f. 3 K. Wutke, Die Saizerscliiießungsversuche in Schlesien in * 3 6 360 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften irn Mittelalter. der Kapitalist dafür auch entsprechend zu zahlen . In Tirol be¬ schwerten sich wiederholt die Stände über die Monopole 1. Hatte doch Karl V. und Ferdinand I. selbst das allgemeine Hauen des Eibenholzes verboten und das Monopol dafür einem gewissen Balthasar Lurtsch übertragen 2. In derselben Weise war für das Land ob der Enns das Monopol, Eibenholz zu schlagen und daraus Bogen zu machen und nach England zu führen , dem Jobst Günther reserviert worden a. In charakteristischer Weise ist in dem Privileg des Jobst Günther von seiten der Regierung der allgemeine Nutzen, das Volkswohl zur Begründung des Monopols angeführt 4. Als Ehrenberg in den neunziger Jahren des 19. lalirhunderts sein „ Zeitalter der Fugger “ schrieb , da stand es ihm außer Frage , daß bei den Großhandelsgesellschaften des 16. Jahr¬ hunderts vielfach die Neigung vorhanden war , ihre Kapitalkraft zur Monopolbildung auszunutzen . Dagegen erschien es dem ge¬ nannten Forscher sehr zweifelhaft , in welchem Maße es den kapital¬ kräftigen Kaufleuten gelang , diese Neigung zu verwirklichen . Trotz vielfacher Bemühungen konnte Ehrenberg nur ein Beispiel wirk¬ licher Monopolbildung im 16. Jahrhundert ermitteln : Das schon öfters genannte Kupfersyndikat der Firmen Fugger , Herwart, Gossembrot und Baumgartner vom Jahre 1498 5. Mit Recht lehnte dabei Ehrenberg die Methode derjenigen Forscher ab, „welche sich bisher mit den Monopolen der großen Handelsgesellschaften des 16. Jahrhunderts beschäftigt haben “ . Diese sind bei der Be¬ hauptung , daß Monopole eine häufige Erscheinung des 16. Jahr¬ hunderts waren , „durchweg ausgegangen entweder von den gegen die Gesellschaften erhobenen Anklagen oder auch von den zu ihrer Verteidigung aufgesetzten Schriftstücken , jedenfalls also von Äußevorpreußischer Zeit. Zeitschrift des Vereins f. Geschichte u. Alter¬ tum Schlesiens, Bd. 28 (1894), S. 115. 1 F. B. von Buchholtz, Geschichte der Regierung Fer¬ dinand L, 8. Bd., S. 360. 2 Privileg vom 10. Februar 1521 (Worms). Wien, H. H. u. St.Archiv. God. Suppl. 414, p. 29 b—30 b. Ein Generalmandat des¬ selben Tages publizierte das Monopol. 1. c. p. 30 b—31. 3 Privileg und Generalmandat vom 10. Februar 1521, a. a. O., P- 27 b ff. 4 Vgl . den Eingang des im Anhang von uns abgedruckten Monopol¬ privilegs. 6 Ehrenberg, Zeitalter der Fugger, I, S. 395 ff., 399. 1 Sechstes Kapitel : Einige sonstige Monopole besonders unter Ferdinand I. 361 rungen, die durch Parteileidenschaft und Geldinteresse ihre Färbung erhielten“. Wenn aber demgegenüber Ehrenberg nur die „Tat¬ sachen sprechen lassen will, wie sie sich aus den Handlungs¬ papieren der Gesellschaften ergeben,“ so ist das methodisch nicht minder verfehlt. Die Bedrohung der Monopolisten durch die Reichs¬ und Landtage usw. brachte es mit sich, daß die Handelsgesell¬ schaften, die an Monopolbildungen beteiligt waren, sobald wie möglich, jedenfalls sofort nach Ablauf ihrer Monopolkontrakte, die betreffenden Papiere (Verträge usw.) vernichteten. Auch die Akten des Kupfersyndikats von 1498 waren nicht etwa aus dem Archiv der Fugger oder eines anderen der Kontrahenten des Kartells be¬ kannt geworden, sondern vielmehr aus einem Gutachten des Dr. K. Peutinger \ So wichtig also die kaufmännischen Archive und ihre Handels¬ akten für die Erforschung vieler Kapitel der Wirtschaftsgeschichte sind 2, so wenig direktes Material dürften sie im allgemeinen zur Geschichte der Monopole und Kartelle des 16. Jahrhunderts liefern. Hier müssen uns, wie wir bei der vorangehenden Darstellung ge¬ sehen haben, doch weder die staatlichen Archive weiterhelfen. Ich behalte mir vor, bei Forschungen, die ich besonders im k. k. Haus-, Hof- und Staatsarchiv zu Wien für andere Studien vor¬ nehme, auf weitere Monopole — wie auch weitere Kartelle und Aktiengesellschaften — des 16. Jahrhunderts zu fahnden. Hier nur noch ein ganz kurzes Schlußwort. In den vorangehenden Forschungen, die in keiner Richtung als abgeschlossen gelten sollen, vielmehr nach allen Seiten der Erweiterung aus archivalischem Quellenmaterial und der Ver¬ tiefung bedürftig sind, wurden gewisse kapitalistische Organisations¬ formen, insbesondere Kartelle und aktiengesellschaftliche Bildungen in einer früheren Zeit häufig vorkommend nachgewiesen, als ge1 Ehrenberg a. a. O., I, S. 396 Anm. 11 und Jansen, Jakob Fugger der Reiche, S. 52. 2 Vgl. meinen Plan einer großen Edition süddeutscher Handels¬ papiere des Mittelalters und des 16. Jahrhunderts, dem G. vonBe1o w und die historische Kommission bei der Kgl. bayrischen Akade¬ mie der Wissenschaften ihre wertvolle Unterstützung geliehen haben. Siehe den Aufruf etwa in der Vierteljahrsschriftfür Sozial- und Wirt¬ schaftsgeschichte, Bd. 11 (1913), S. 445 f. oder Schmollers Jahrbuch Bd. 37 (1913), S. 2151 f. 362 Drittes Buch: Monopole , Kartelle und Aktiengesellschaften im Mittelalter. wohnlich angenommen wird. Selbstverständlich legen wir auf diese rein „antiquarische“ Feststellung geringes Gewicht. Uns interessiert dabei lediglich die Folgerung aus dieser chronologischen Hinauf Setzung, die Frage, wie konnten sich so früh schon bei der starken Durchdringung der mittelalterlichen Gesellschaft mit der antikapitalistischen Moral der mittelalterlichen katholischen Kirche kapitalistische Organisationsformen von der Vollendung, wie es Monopole, Kartelle und Aktiengesellschaften sind, entwickeln. Wir fanden die Antwort in der Finanzpolitik der Päpste und der christlichen Staatsgewalten des Mittelalters. Die Geldnot, die finanzielle Abhängigkeit vom Großkapital zwang Kirche und Staat zur Konnivenz dern kapitalistischen Kauf manne gegenüber, der die strenge Wirtschaftsethik der Zeit durch eine mono¬ polistische Preispolitik übertrat . Mehr noch! Um sich möglichst hohe Einnahmen zu sichern, bat der Staat und die Kirche nicht selten selbst eine skrupellose monopolistische Preispolitik in „fiskalischen“ Werken oder in Produktionszweigen, in die der Landesherr hineinzureden hatte , getrieben1. Auf diese Weise sahen wir wirtschaftliche Organisationsformen von stark kapita¬ listischer Eigenart unter führender Mitwirkung der „Regierungen“ entstehen. Was hl. Levy für England unter Elisabeth, Jakob und Karl nachgewiesen hat 2, daß das Fürstentum direkt die Rolle eines spekulationslüsternen „Promotors“ kapitalistischer Monopolunter* nehmungen spielt, das gilt anderwärts schon viel früher. Im heiligen römischen Reiche deutscher Nation erreichte unter Karl V. und Ferdinand I. die entsprechende Tätigkeit des Königtums schon einen gefährlichen Umfang. Die Monopolhestrebungen gingen dort in. der ersten Hälfte des 16. Jahrhunderts nicht allein, nicht einmal vorzugsweise von den kapitalistischen Interessenten aus. Oft war der geldbedürftige Staatsleiter das treibende Element. Zumeist aber begegnen uns Fürst und Kaufmann hier in einem interessanten 1 Die Summen , die die Fürsten mit solchen Monopolisierungen verdienten , waren oft geradezu enorm . Siehe z. B. oben die Ge¬ winne der Habsburger aus der Vergebung des Idriancr Quccksilbermonopols. Was waren dagegen die Einnahmen aus Fron und Wechsel, den ordentlichen Regalgefällen! a. a. O., S. 68. Sechstes Kapitel : Einige sonstige Monopole besonders unter Ferdinand I. 363 Zusammenarbeiten am Aufbau der frühkapitalistischenWirtschafts¬ organisationi. 1 Das intime Verhältnis schon MaximiliansI. zu der Augsburger Großkaufmannscliaft ist ja bekannt (Ehrenberg, Zeitalter der Fugger, I, S. 190). Interessant ist der Versuch dieses Kaisers, die gesamte Tiroler Kupferproduktion durcit einen „ewigen Kupferkauf“, d. h. eine Zwangslieferung aller Gewerken in seine Hand zu bekommen und dann den Fuggern gegen Gewinnanteil weiterzugeben. Das Monopol (um 1498) ist an verschiedenen Schwierigkeiten, besonders auch daran gescheitert, daß die Fugger Max. I. nicht als Kompagnon wünschten. Jansen, Jakob Fugger der Reiche, S. 87. Anhang. Dokumente der frühkapitalistischen Wirtschaftsorganisation. I I. Allgemeine Akten zur Geschichte des Monopolwesens im 16. Jahrhundert 1. Bernsteinappaltvertrag zwischen Albrecht von Brandenburg und den HandelsgesellschafternNie. Pflaume und Georg Kramer zu Königsberg, Ebert Roge zu Danzig und Claus Lang zu Lübeck. — 1518(Jan . 9). Königsberg. — Entwurf : Kgl. Staats¬ archiv Königsberg. Von Gottes Gnaden Wir Albrecht etc. thun kunth u. bekennen mit disem brief, als wir mit den ersamen u. weyssen, unsern untlierdanen . . . Nyclas Pflaumen, Jorgen Cramer zu Königsberg unser stette , Ebert Rogenn zu Danzigk u. Claus Lang zu Lubeck bürgere in einer handlang unsers bernstains halben, den sie bishere ein zeit lang von uns genomen u. empfangen, gestanden, also haben wir uns in dato mit den oben angezaigten unsern underthanen . . . nach vleissig u. notdürftig besiehtigung aller vorige uffgerichte vertregen, schulden u. rechenschafften, weliche wir also volkomentlichen u. bestendigklichen durch die erbarn unsere rethe u. liben getreuen Ditherich von Schonberg, Cristoffeln Gattenhoffen, secretarien u. Cleophas Preuer, rentmaister berechnen u. übersehen lassen, also dass wir Uns mit den gemelten unsern underthanen liben, getreuen u. besonderen aufs neu vereinigt u. vertragen durch wellichen itzigen vertrag alle vorige vertrege u. verschreibunge sovil der von uns, unsern vorfaren u. unsern wege aufgericht u. verfast, aufgehoben, vernicht, crafllos u. unwirdig sein sollen. Als nemlich dan in disem vertrag 10,000 M. geringer Preusser montzs, die wir ihnen pflege u. schuldig sin, bits zu eines jeden tayls aufsagung oder abkundigung, weliche aufsagung wir als wol u. sic als wol als wir 1 jar lang zuvor ein tayl dem andern aufzusagen ordentlichen . . . macht haben soll unge . . . . u. unerfordert bits die zeit der abkundigung wie gemelt sich begibt u. eraigent ansteen pleiben sollen, die wir ihnen alsdan mit burnsteiu oder aber barem gelt 368 Dokumente der fr Uhknpi talistischen Wirtschaftsorganisation. unverzüglichen u. ungehindert im letzten jar hetzallen u. abkürtzen wollen lassen. Auf dass aber wir, desgleichen sie wissen mögen wie es mit der gewerung des burnstains auch mit der entpfahung des gelts da¬ gegen hinfurtter gehalten soll werden, wollen wir unsern burnstainmaister befehlen, wenn er des gemaynen staynes ein halben last u. des gutten ein thonen hat (u. ob etwas darüber were, dass es doch gantze volle u. nicht halbe thunen sein sollen), sollichen stain soll er aufs furderlichst auserhalb unserm schreiben herein schicken, der inen alsdan durch unsern rentmaister überreicht u. geliben soll werden. Darauf sie uns eins jeden jars soliehen stain auf 4 termin so vil sie des zu einer quattember zur andern entpfahen nach gutter rechenschaft mit barem gelt alsdan bezallen sollen. Wo auch die vilgemelten unsere underthanen u. lieben getreuen etc. vor dem termin des quartals was auslegen würden, soll inen auf dieselben quat.ember in alle wege nach redlicher rechnung abgezogen werden, darzue wellen wir inen den stain, wie sie denselben von uns u. unserm orden bishere gehabt, in gleichem kauf alle wege lassen. [Folgen einige durchstrichene Zeilen und das Datum : Königsberg, Sonnabend nach Dreikönigstag 1518.] 2. Kaiser Karl und Erzherzog Ferdinand verleihen dem Jobst Günther das Monopol des Eibenholzschlagsin dem Land ob der Enns. — Worms, 10. Februar 1521. -— K. u. k. Haus-, Hof- und Staats-Archiv in Wien. Codex suppl. 414 (weiß 719), fol. 27—28. lobsten Günthers Verschreibung umb das eybenholtz im land ob der Enns, daraus er bogen mag machen und die in Englland verfueren. Wir Karl etc. bekhenen fur uns, auch den durchleuchtigen fürsten, herrn Ferdinanden etc. Als wir vernomen und betracht, wie das eybinholtz in unser niderosterreichischen furstenthumben und landen an vil orten durch die underthanen, handwercher und pauerschaft, unordenlich on unser erlaubnus unnutzlich gefeldt, gehauen und zu unfueglichen Sachen gebraucht, dadurch unser vorst und weide verödt, auch uns und unserm camerguet damit nicht gedient wirdet. Das uns furter zu gestatten nit gemaint ist, sonder gedenckhen solher unser vorst und weide des eybinholz zu I. Zur Geschichte des Monopolwesens im 16. Jahrhundert . 369 verschonen , dieselben mit Ordnung zu gebrauchen , unser camerguet zu liieren und das iungst holtz daneben aufzuziehen . Dass wir uns demnach mit unserm getreuen lobsten Günther auf sein underthanig bit gnedigclich vertragen und ime des unser freihait, erlaubnus und gnad gegeben und gethon haben bis auf unser wolgefallen . Thun das auch wissentlich in crafft ditz briefs. Also dass er selbst oder durch seine handler , diener oder arbaiter , so er yetzuzeiten dartzu verordnet und prauchet und sonst niemandt anderer in unsern niderosterreichischen furstenthumben und landen alle vorst und wellde des eybinholtz in gebrauch haben , solch eybinholtz auf seinen cossten und schaden , auch vortail und gewin feilen, zu eybinpogen klieben , arbaiten und auf die Strassen nach Tantzig in Englland , doch in kamen weg, dass solhs den ungläubigen zuekeme , verfuren und vertreiben soll und mag , von uns und den unsern und sonst mcnigclich un verhindert und ungeirt . Dorumb soll er uns in unser camer von iedem tausent pogen holtz fünf gülden rheinisch in unser vitztumbambt in Österreich ob der Enns alzeit raichen und betzalen . Und sovil er zu yederzeit pogen ausberaiten und aus den vorsten und weiden wegfuren last , desselben glaubwirdig urkhundt von unsern vorstmaistern iedes orts nemen und mit dem dienstgelt in unser vitzthumbambt ob der Enns antwurten . Dartzu auch uns und sonst menigclich die gewonlichen zöll und meut auf wasser und land Vorbehalten . Damit soll er aber in ansehung berurts unsers camerzins zimlich und leidenlich wie von alter her gehalten und mit kainer neurung beswert werden. Er soll auch ausserhalb solher seiner arbait und handlung unser eybinholtz zusambt unsern vorstmaistern und vorstknechten uberal nach seinem vermugen hayen und bewaren , dasselb niemands anderm zu feilen, zu arbaiten , noch wegzufurn gestatten . Und ob er dar¬ über iemand betreten wurde , es sei in der arbait oder auf den Strassen, wasser und lande , der on unser erlaubnus und frevenlich solch holtz (zu was wergk das were) gehauen , gefeit , gcarbait oder weggefurt hette , denselben solch holz zu unsern handen nemmen , mit dem seinen behalten und vertreiben , doch uns davon den bestimbten camerzins , zoll und meut auch betzalen , daran er gegen niemand nicht verfrevelt , verfarn , noch verhandelt haben. Er soll auch selbst und durch die seinen unserer vorst und weide des eybinholtz schonen , dasselb nit wuestlich , noch zu ver¬ derben , veröden , sonder mit ordnung und beschaidenhait dadurch Strieder , Studien z. Gesell , kapilalist . Organisationsfornien . 24 370 Dokumente der frühkapitalisüselien'Wirtschaftsorganisation. das iung ertzogen 1 und solch holtz in steten gebrauch weien und behalten werden mug , handlen und gemainclieh in die und ander wege mit den weiden und vorsten des eybinholtz unsern nutz und pests betrachten und furderen , schaden und nachtail verhueten als ainem getreuen diener und handlet - gegen seinem herrn zu thun woll getzimbt und gebürt , wie er das gelobt und gesworn und sicli gegen uns verschriben hat . Und ob sich begeh, dass über kurtz oder lang ander personen furkomen , die solchen eybenholzhandl in höherem werd und zins annemen und besteen wolten , so uns dan lobst Günther , sovil als sich gründlich erfinden wurde , das wir von andern darumb haben mochten , auch thuen wolt , so sollen und wellen wir itne sollten handl in demselben werd und zins fur ander volgen und ine dabei bleiben lassn . Alles treulich und nngevetiich . Mit urkhundt ditz briet's etc . Datum Wormbs am. X . tag februarii , anno etc . im XXIten. 3. Kaiser Karl V. befiehlt dem Reichsfiskal , das Verfahren wegen Monopolaufrichtung einzustellen , das derselbe gegen Jakob Fugger , Andreas Grander , Christoff Herwart , Ambrosius Höch¬ stetter , Bartolome Welser, Andreas Rem und ihre Mitgesell¬ schafter eingeleitet hatte . Burgos , 15. Sept . 1523. — K . u. k. Haus -, Hof - und Staatsarchiv in Wien . Reichsregistratur Karl V. 3. Bd . fol. 234 f. Wir Karl etc. empieten dem ersamen unserm kaiserlichen pro¬ curator fiscalgeneral und des reiehs lieben getreuen Gasparn Marth, lerer der rechten , unser gnad und alles .gut . Ersamer gelerter, lieber getreuer ! Uns haben die ersamen unser und des reiehs lieben getreuen Jacob Fuckher , unser rath , Andres Grander , Chri¬ stoff Herwart , Ambrosius Hochstetter , Bartholomeus Welser und Andreas Rem für sich und irr mitverwannten clagsweis furbracht, wie Du si, all irr mitverwanten und geselschafter vor unserm kaiserlichen regiment oder camemchtern und beisitzern unsers camcrgerichts im hailigen reich mit unordenlicher iadung und vermainter clag furgenomen habest , als ob er Jacob Fuckher , auch Andreas Grander mit i r e r beder mit¬ verwanten und g e s c 11 s c h a f t e r n monopolien ge¬ übt und si , die andern all obgemelt , etlich un1 Das heißt , das junge Eibenliolz aufgezogen. I. Zur Geschichte des Monopolwesens im iG. Jahrhundert. 371 z i m1ic hund in rechtverpotten g e d i n g mit kau¬ fen und verkaufen gebraucht haben sollen , und uns darauf ferrer angezaigt , wie si solchen unordenliclien gerichtszwangk in kraft gemaiaer rechten und unser , aucli unser vorfarn reichsordnung , sunderlich auf dem reichstag zu Collen des 1512. iar aufgericht , aus meher , dan ainer gegrünten ursach (dem wir auch gnugsam unterricht und wissen haben ) zu waigern vermainten. Und uns darauf undertheniglich angeruefen und gebeten , so itzt durch die gemelte mass wider si furgenomen wurde , dass wir solchs alles ahthuen , nichtigen und abschaffen wolten , damit si bei ordenlichen und geburlichen rechten bleiben mochten und darvon nit gedrongen wurden . Und wiewol wir des willens und endtlicher meinung sein, dass im heiligen reich in zeit unser regiemng kein monopolien g e t r i beii , auch alle verpottne unz i m 1i c li kauf und verkauf abgethan werden, so khunnen wir doch diser zeit aus etlichen trefflichen und wolgegrunten Ursachen (uns darzu bewegend) kainswegs Zusehen noch ge¬ statten , dass dermassen wider obgemelt kaufleut gehandelt und procediert soll werden. Und demnach emphelhen wir Dir mit ernst und weilen, dass Du bei Vermeidung unser schweren ungnad gegen obgeschriebne kaufleut in khainerlai weg in diser Sachen weiter handlest , noch furtfarest und genzlieh stilstehest und si mit clag oder iadung obgnanter Sachen halben bis auf weitern misera bevelh nit an¬ sprechest noch betrübest , sonder die information und unterricht des handles , so Du wider obgemelt khaufleut hast , uns unter Deinem petschafft zuschickest und das nit lassest . Daran thustu unser ernstliche meinung . Geben Burgos in Castilien , den 15. tag des monats septembris , anno etc . im XXIIIsten, 4. Kaiser Karl V. nimmt die Erben Jakob Fuggers gegen jede Belästigung seitens des Reichsfiskals in Monopolan gelegenheiten in Schutz . Granada , 19. Oktober 1526. —■ K . u. k. Haus -, Ilof - und Staatsarchiv Wien , Reichsregistratur Karl V. 5. Bd . fol. 285- 288. Wir Karl V. etc . hekhennen offenlich mit disem brief und thun kundt allmeniglich : Als verschinen iar der ersam , gelert , unser kaiserlicher camerproeurator -fiscal-general und des reiclis lieber getreuer Caspar Marth vor unserm kaiserlichen stathalter und 24* 372 Dokumente der frühkapitalistischen Wirtschaftsorganisation. regiment im reich weilendt Jacoben Fugger , unsern rath , sambt seinen mitverwandten und zugehörigen neben und sambt etlichen andern citiert , furgenomen und beclagt hat , als ob si etlicli u nz imblich geding , kcuf und verkauf gethon, geübt und getriben betten , dardurch unser und des reichs underthonen und gemainer nutz grösslich beschwerdt , beschedigt und verhindert und derhalben im rechten monopolia genent und verpotten sein sollten , und wir aber den gedachten weilendt Jacoben Fugger , seine mitverwanten und zugehörigen eines erlichen , dapfern Wesens und gemuets alzeit erkendt und darzu ir gewerb und handtierung nit allain aufrichtig und unverweisslich , sunder auch uns, dem reiche und gemainer teutschen nation in vil weg frucht - und nutzbar befunden , so haben wir bemeltem unserm camerprocuratorfiscal vorlangst ernstlich geschriben und bevolhen , solcher seiner vermainten ansprach und clag gegen vorgedachten weilent Jacoben Fugger , seine verwandten und zugehörigen , auch alle andern abzustcen und si derhalben unangelangt und unbekhumert zu lassen. Und daneben gedachtem unserm stathalter und regiment im reich insunderhait auch angezeigt und bevolhen , solchs bei ime zu ver¬ fliegen und dise Sachen und process des fiscals abzustellen , bis wir inen derhalben ferrern bevelh geben und unser gemuet und willen entdeclchen wurden . Und darauf den egemelten weilent Jacoben Fuckher , auch seine bruedersöne , mitverwandten und zugehörigen , auch ir aller leib, kaufmanswar , hab und gueter (die si itzo haben oder noch khunftiglich uberkhomen ), in unsern kaiser¬ lichen schuz und schirm emphangen , die dermassen noch 1 also halten . In betrachtung , wo solch vermainte desselben unsers camerprocurator -fiscals citation clag und process dermassen gestat und zugelassen , dass si am vordristen weilent unsers lieben herren und anhern kayser Maximilians löblicher gedechtnus und des hailigen reichs Ordnung und abschid des gehalten reichstag zu Cöln im 1512. iar ausgangen (darin ein yeder in bürgerlichen und andern Sachen in der ersten instantz vor seinem ordenlichen richter , dar¬ unter er wonhaft und gesessen ist , beclagt , gerechtfertigt und ge¬ straft werden soll) und daneben auch unser selbs Statuten und Ordnung (die wir mit beisein, wissen und rath unserer und des reichs churfursten , fürsten und gemainer stendt derhalben fur1 Wohl Schreibfehler für auch. I. Zur Geschichte des Monopolwesens im 16. Jahrhundert . 373 genomen , aufgericht und ausgeen haben lassen) etlichermassen ent¬ gegen und wider sein möeht und darzu allen kaufleuten , gesellschai'ter und liandtierern ingemain gantz beschwerlich , und lestlich ein ursach sein wurde , ir gewerb und handtierung vil lieber zu underlassen , dan sich mit irer und der iren hab und gueter in ainich dergleichen beschwerlich anspruch und rechtiertigung unsers fiscals zustellen , molestiern und betrueben zu lassen . Das uns dan und dem hailigen römischen reich, auch dem armen gemainen man durchaus (der sich durch die kaufleut und handtierung vilfeltig underhelt und erneret ) zu grossem abbruch , nachtail und schaden kome und deshalben billicb und zeitlich ein- und fuersehung zu thun geburn will. Und demnach aus oberzelten und andern Ursachen uns darzu bewegend mit wolbedachtem muet , guetem , zeitigen rath und rechter wissen, auch aus kaiserlicher macht und volkomenhait syder und nach absterben des vorernanten weilent Jacoben Fuckhers unsere und des reichs lieben getreuen Reymunden , Anthonien und Jheronymussen , gebrueder und vetter , unser rethe , als sein nechstverlassen und instituirt . natürlich recht erben , auch all ir ver¬ wandten und zugehörigen , desgleichen ir aller und der iren leib, kaufmanswar , hab und gueter gegenwertig und khunftig von allen und yeglichen egerurten unsers stathalters und régiments , auch unsers camerrichters und beisitzer oder unser und des reichs fiscals und sunst meniglichs erkhantnus , ladungen , clagen, processen, con¬ fiscation , both und verboth , so wider si und di iren , ir leib, kauf¬ manswar , hab und gueter , wie vorbestimbt , obberurter Sachen halben erkent , furgenomen , gehandelt und ausgangen sein oder kunftiglich wider si ausgeen würden , wie und welcher gestalt das beschechen möcht , nichts ausgenomen , yetz als dan und dan als yetzo , gentzlich absolviert und entledigt und das alles und yedes aufgehebt , getodt , abgethon , vernicht und cassiert. Absolviern und erledigen si sambt iren leiben , kaufmanswaren, hab und gueter . Aufheben , thuen ab , vernichten und cassiern solchs alles hiemit wissentlich in kraft ditz briefs. Mainen , er¬ kennen , setzen , erclern und wollen, dass die obberurten Fugger gevetter und brueder , ir verwandten und zugehörigen , auch ir aller leib, kaufmanswar , hab und gueter , gegenwurtig und künftig, wie obstet , von der egemelten unsers und des reichs régiments, unsers camergerichts , fiscals und sunst von meniglich erkantnus, 374 Dokumente der frühkapitalistischen Wirtschaftsorganisation. ladung, clag, handlungen und processen, peenfal, strafen, buessen und sunst von allem andern darvon herrurend, so sich bisher diser Sachen halben zutragen und begeben haben oder hinfuro inneroder ausserhalb rechtens sich zutragen und begeben möchten, für und fur zu ewigen Zeiten und in alweg absolviert, entledigt, auch alles crafftlos, abgethon und aufgehebt, getodt und cassiert sein und darauf ferrer von bemeltem regiment, auch unserm camergericht, unserm und des reichs fiscal oder yemandts anderm weder inner- noch ausserhalb rechtens wider die egenanten Fugger, ir verwandten und zugehörigen, auch ir leib, kaufmanswar, hab und gueter, inmassen, wie oben erzelt, weder in gemain, noch in sunder weiter nichts furgenomen, erkanth, ausgeen, procediert, noch ge¬ handelt werden soll in kliain weise noch gestalt. Sunder si sich der berurten unser absolution, entledigung, restitution, aufhebung und cassierung gebrauchen und geniessen sollen und mögen von aliermeniglich unverhindert. Und ob auch hierüber wider die mergedachten personen ir leib, kaufmanswar, hab und gueter von wegen egedachter ladung, clag, handlung, process berurter Sachen halben oder dem, so daran hangt und darzu gezogen mocht werden, gar nichts darvon ausgenomen, an bemeltem unserm regiment, camergericht oder ye¬ mandts andern, ichts ferrer gehandelt ausgieng, furgenomen und procediert wurde, in was gestalt und form das geschehe, so wollen, erkhennen und ercleren wir itz alsdan und dann als ytzo aus obgemelter kaiserlicher machtvolkomenhait, dass solchs alles craftlos, untuglich, unwirdig und von unwirden gehaisen und sein und den obgemelten personen, iren leiben, kaufmanswarn, hab und gueter gegenwertig und khunftig ganz khainen nachtail, schaden, Ver¬ letzung noch Verhinderung bringen noch geberen soll. Und gebieten darauf allen und yeglichen churfursten, fürsten, gaistlichen und weltlichen, prelaten, graven, freien, herren, rittern, knechten, haubtleuten, landtvogten, vitzthumben, vogten, phlegern, Verwesern, ambtleuten, schulthaisen, burgermaistern, richtern, rethen, bürgern, gemainden und sunst allen andern unsern und des reichs underthonen und getreuen, in was wirden, standts oder Wesens die sein, und sunderlich unserm stathalter und regiment, camerrichter und beysizern unsers kayserlichen camergerichts im hailigen reich, auch unserm und des reichs fiscal, gegenwertig und khunfftigen, ernstlich mit disem brief und wellen, dass si die ge- I; Zur Geschichte des Monopohvesens im 16. Jahrhundert . 375 dachten Reymunden , Anthonien und Jheronymussen die Fugger gebrueder und vetter , ir verwandten , zugehörigen all ir und der¬ selben erben und nachkomen , auch ir aller und ir yeder leib, kaufmanswar , hab und gueter , gegenwertig und khunftig , bei diser unser Satzung, erclerung , absolution , aufhebung , cassierung und gnaden bleiben , die ruelich gebrauchen und geniessen lassen und daran nicht irren , noch verhindern , noch des yemandts anderm zu thun gestatten in khain weise, als lieb ainem yeden sei, unser und des reiclis schwer ungnad und straf und darzu eine peen , nemblich 20 marckh lottigs golds zu vermeiden , die ein yeder , so oft er frevenlich hiewider thete , uns halb in unser und des reichs camer und den andern halben tail obgemelten Fuggern , iren mitverwandten und zugehörigen , auch derselben erben und nachkhomen unablesslich zu bezalen verfallen sein soll. Mit urkhundt ditz briefs besigelt mit unserm kaiserlichen anhangenden insigel. Geben in unser stat Granaten am 19. tag des monats octobris nach Christi unsers lieben herren gebürt im 1526., unser reiche , des römischen im 8ten und der andern aller im 11. iarn. Carolus. Vidit Waltkirch . Ad mandatum Caesaris etc . W. Brantner. Taxata flor. rhen . 100 et in duplicato taxata nihil , quia dupli¬ catum. 5. Kaiser Karl V. bestimmt , daß die Kontrakte , die den Erz¬ großhandel in die Hände weniger reicher Kaufleute ausliefern, nicht als monopolistisch im Sinne der Reichstagsverhandlungen über Monopole angesehen werden sollten und dürften . Toledo (Kastilien ), 13. Mai 1525. -— K . u. k. Haus -, Hof - und Staats¬ archiv in Wien . Allgemeine Urkundenreihe . Repertorium 1. Original Pergament mit an schwarzgoldener Seidenschnur an¬ hangendem Siegel Kaiser Karls V. Das Duplikat , von dem unten die Rede ist , dürfte das im Fuggerarchiv befindliche sein (vgl. die dortige Dorsualnotiz ), das Jansen, Jakob Fugger der Reiche , S. 400 ff. gekürzt abgedruckt hat . Der ältere Abdruck bei F . A. Schmidt, Chronologisch -syste¬ matische Sammlung der Berggesetze der Österreich. Monarchie, 3. Abteil . Wien 1839, Nr . 52 ist voller Fehler und mit einer sinnlosen Inhaltsangabe versehen. 376 Dokumente der frühkapitalistischenWirtschaftsorganisation. Wir, Karl der funft, von Gots gnaden erwelter römischer kaiser etc. bekennen offenlich mit disem brief und thun khundt allermenigklich. Nachdem under andern notturftigen fursehungen, die uns als ainem römischen kaiser zu des hailigen römischen reichs und desselben underthonen und getreuen ere, nuz, wolfart und aufnemen ze thun und zu betrachten geburn, zu dem wir auch durch vleisz teglich nachgedenckhen aus kayserlicher miltigkeit begirig sein, unser kayserlich gemut nit wenig dahin steet, dass nit allein der gemein nutz, auch unser und des reichs underthonen zimlich aufnemen gefurdert, sonder auch niemands wider die billichait beswert, auch dasihen, was etwa aus ungnugsamer erfarnus oder undemcht geordnet oder bedacht were, nit zu nachtail noch schaden gebraucht werde und also aus dem, so in gestalt eins guten oder zu der gerechtigkait furgenomen wurd, nichts ungerechts, auch yemandts ainicher unbillicher nachtail entstee: Darauf so haben wir uns zu villen abschiden auf etlichen reichstagen, sonder¬ lich bei Zeiten weilant kaiser Maximilians, unsers lieben herrn und anherrn löblicher und seliger gedechtnus gehalten, ersehen und onder anderm darinnen etliche furnemen und einselien befunden, wie die furkeuf und monopolischen hendel, die undter anderm wider gemeinen nutz dermassen beschehen sollen, dass die waaren all oder der merer thail oft in ein handt verkauft und darnach dester in hoherm werdt oder gelt widerumb gegeben und verhandtiert wurden, abgestelt und gestraft mochten werden; auch daneben bericht empfangen, wie in solche furnemen kauf und handticrungen die bergkwerck, ertz und métal, so daraus erbaut werden, auch getzogen und die keuf, auch contract, so bemelter metall und ertz halben etwa in ein oder wenig handt ge¬ schehen sein oder noch geschehen mochten, für monopolisch geacht werden , auch wider recht und gemeinen nuz sein solten. Dieweyl nu (wie dan solchs wisentlich und offenbar ist, auch die erfarnus gnugsam zu erkennen gibt) die bergkwerck (der dan im hailigen römischen reich und teutschen landen mer dan sunst an keinem ort in der gantzen cristenhait erbaut werden) für ein stuck die groszt gab und nuzbarkait ist, so der almechtig teutschen landen mitgetailt hat , nit allain des grossen schätz halben, so daraus durch vil gold, silber, kupfer, zin, quecksilber, blei, eisen und ander mer métal (welches sich dan ungeferlich und ehe mer dan minder I. Zur Geschichte des Monopolwescns im 16. Jahrhundert . 377 icrlich auf zwaintzig mal hunderttausent guldin erstreckt) erbaut und gearbeit wirdet, sonder dass sich auch in teutschen landen etlich hundert tausent menschen, alt und jung, auch weib und kinder und sunst vil ir notturftige narung nit haben mochten, allein aus dem bauen, arbaiten und schmeltzen und darzu auch sunst durch verfuerung derselben métal in vil land in einer gemein vil ander leut als der fuerman, wirt, burger, baur und gemein handtwergksman allenthalben auf den strassen, dem landt und in steten, daraus menigfaltigklichen erneren. Deszgleichen die fürsten und herren ir einkomen an fronen und wexeln, zollen und meuten groszlich meren, auch erhöhen mer dan aus kainem ainigen liandel oder gewerb in gantzer teutscher nation und dem hailigen römi¬ schen reich. Also dass die bergkwergk auch derselben erbauungen und wesenliche bestendige, auch guete underhaltungen zum höchsten durch alle weg und mittel, die zu solhem imer dienstlich zu be¬ finden sein, gefurdert werden sollen. Dan ye mer solchs geschieht, ye grosser sich die oben gemelten gemain nuzbarkaiten dem hailigen reich, auch fürsten und herrn, darzu den gemainen man und dardurch gemainer nuz zu guttem erholiern und auszbraiten. Und herwiderurnb, wo solche furdrung nit geschieht, dass daraus nichts anders dan gewisser der bergkwerck abgang und abfal, auch verhindrung und mindrung der oben gemelten gemeinen und sondern nuzbarkeiten volgen mögen. Wie dan solhs in wenig Zeiten bei villen berckwerckhen durch die offen¬ bar erfarnus mit mercklichem nachtail befunden worden ist. Und aber under allen fursehungen und mitlen, durch die die bergkwerck erbaut und statlich, fruchtbarlich, auch bleiblich underhalten, gehandthabt , gefurdert, auch gehayet mögen werden, kein dienstlichers und bestendigers erfunden werden mag, dan das gold, Silber, kupfer, quecksilber und ander métal durch gut Ordnungen vil ehe in einem guten und gültigen gleichen, auch bestendigen, dan einem schlechten, geringen, nidern und ungleichem werdt zu verkaufen und zu kaufen, auch darnach die widerumb derselben gestalt zu verkaufen und zu verhandtiern und also die nit in vil, sonder in ein oder wenig hendt zu verkaufen oder körnen und ver¬ handtiern zu lassen. Wie dan solchs, als wir gruntlich bericht werden, lenger dan vor 40 iarn her an etlichen orten in unser grafschaft Tirol, da silber- und kupferbergkwerck erbaut werden, der gebrauch solcher massen mit verkaufung der silber und kupfer, 378 Dokumente der frühkapitalistischen Wirtschaftsorganisation. auch zum thail ein zeit lang des quecksilbers halben gewesen und noch also ist und ungezweifelt an andern orten etwa bei andern pergkwercken der und anderer mettal halben auch also geschehen ist und noch teglich beschehen mag. Dan wo gold, silber, kupfer und quecksilber, auch ander métal in einem guten gültigen werdt verfurt und verhandtiert werden (welches dan durch das verkaufen in ein oder wenig hendt am besten beschehen kan), so mögen auch dieselben métal des ersten kaufs dester in mererm hohem und gutem werdt durch die, so solch erz erbauen und zu métal schmeltzen, oder die sunst haben und empfahen, verkauft, auch aus solchem diejhenen, so bergkwerck bauen, dester mer zu bauen verursacht, auch lustig gemacht und also im grundt dardurch die bergkwerck, auch alle andere nutzbarkaiten, die, wie oben angetzeigt worden ist, daraus dem hailigen reiche und teutscher nation, auch gemainem nuz zu gutem volgen, desto grosser gefurdert und gemert werden. So ist auch das bergkwerckbauendarzu dasjhen, so von metallen daraus gefeit, solehs alles, wie ander gots gaben, die einem wachsen, nach notturft zu kaufen und verkaufen, zu verfurn, zu vertreiben und allenthalben zu verhandtiern von ime selbs ein gütliche, erbare und zuelessige handlung. Auch daneben billich vil erbarn leuten, so sich oft hoch und hart in bergkwerckhen verbauen und nach langer darstreckhung des iren allain der guten hofnung warten, alle notturftige furdrung zu erzaigen, auch dieselben bei gutem willen und iust und dardurch die bergkwerck zu bestendigem, auch beleiblichem wesen zu behalten. Sampt dem, dass auch solche métal, auch ertz und insonderhait kupfer und quecksilber gar eins clainen thails in teutschen landen verschlissen, aber wol in ander frernbde landt und nationen verfuert undt verhandtiert werden. Und das darzu solehs nit waaren oder gueter sein, der sieh der gemein man, als anderer gueter zu ainicher seiner notturft brauchen muesz. Dan obwol in teutschen landen doch ein clainer thail kupfer durch etliche handtwerckh verarbait, so wirdet doch denselben handtwerckhern in ainen weg als den andern hoch oder nider belont, darnach das kupfer gullt. So ist auch leichtlich und wol zu bedenckhen, wo die bergkwerck sonderlich durch die obgemelten und andere weg nit gefurdert werden solten, dass dar¬ durch der ertz und métal vil minder gemacht, auch ein grosser mangel daran sein und also ausz solchem noch in vil höheren werdt I. Zur Geschichte des Monopolwesens im 16. Jahrhundert. 379 gemeiner teutscher nation und derselben underthonen zu nit clainem nachtail wachsen wurden. Darumb und dem allem nach die gemelten pergkwerck als gaben des almechtigen , auch derselben nutzbarkaiten , so daraus, wie oben gemelt , entspringen , darzu gemeinen nutz noch mer zu furdern und herwiderumben die gedachten bergkwerkh vor abfal, auch sunst menigklich vor imbillichem naclitail zu verhueten und zu beschirmen : So setzen , ordnen , ercleren und wollen wir aus unser kaiserlichen machtvolkomenliait und rechter wissen, dass an allen den orten im haiiigen römischen reich , auch teutscher nation , wo bergkwerck gebaut werden , durch die gewerckhen , auch diejhenen , so bergkwerck bauen , die métal und erz ausz ainichen gerechtigkailen , vertragen oder handlungen zu empfahen haben, die métal und erlz , sonderlich die silber , kupfer und quecksilber in einig oder mer wenig hendt verkauft , auch sunst mit den kaufern auch herwiderumb durch die kaufet- mit den Verkäufern geding und pact , die, wie angezeigt , zu handthabung und furderung der berglcwergkh dienstlich seien oder erfunden werden mochten , allent¬ halben gemacht , deszgleichen darnach durch diejhenen , so dieselben erz und métal also von den gewerckhen , pergldeuten oder andern erkaufen , dieselben ertz und métal ferrer zum höchsten und nach den besten wirden , wie si des statfinden künden , nach irem gefallen verkauft und verhandtiert , auch in frembde landt und nationen verfurt werden mugeu ; und dass auch nit allain solchs, was hinfuro oben angetzeigter massen geschieht , auch gehandelt und ge¬ dingt wirt , oder hievor und bisher ytzgemelter gestalt mit erkaufung und verkaufung der métal in ein oder wenig liandt undt ferrer verkaufung , verbandtierung und verfuerüng derselben in ander frembd nationen geschehen , gehandelt oder deshalben fur geding gemacht were, kein unzimlich handlungen , kauf - und verkaufcontract , noch ainicli monopolia genent , gehaissen oder darfur ge¬ halten und vil minder dieselben , so merbemelter massen contrahiert geding und kauf oder verkauf gemacht , gehandelt oder die métal und ertz in ander nationen verfurt und verhandtiert betten oder solchs hinfuro thun wurden durch gantz niemands darumb gerechtfertiget , gestraft , gebuest , noch beswert werden sollen in gantz kein weysz noch weg unangesehen , obgleich das alles in etlicli weg etwa sondern personen fur nachteilig geacht oder verstanden werden soit . Dieweyl doch solchs sunst 380 Dokumente der frühkapitalistischen Wirtschaftsorganisation. in vil melier weg und dein gemeinen nutz dienstlich , fruchtbar und gut ist. Ob auch wider das alles einich ander fursehung , ordnung oder beschaiclt als aus ainem ungenügsamen bericht oder verstandt durch yemandt .s gemacht worden were oder furhin aufgericht, gemacht oder erfunden wurd , von wem solchs beschehe , solch alles wellen wir ytz alsdan und dan als ytz abermaln ausz unser kaiser¬ licher machtvolkomenhait und rechtem wyssen hiemit gentzlich und gar aufgehebt , abgethon , cassi ert , auch demselben allem dero¬ giert und erclert , auch geordent haben , dass nit allain dasselb wider dise unser ordnung , Satzung und erclerung an kainen orten oder gerichten gantz kein stat , wurckhung noch craft haben , darauf nichts gehandelt noch erkent , sonder gantz vernicht und von unwirden sein und haissen . Sonder ob wir ytz ainicherley Ordnungen zu abstellung der monopolien aufgericht und gemacht betten oder hinfuro machen oder aufrichten wurden , das dieselben Ordnungen und fursehungen in kaufen , verkaufen , handlungen , contracten, gedingen , handtierungen und verfuerungen der gemelten métal und ertz , sunderlich der kupfer , silber und quecksilber halben nit stat haben noch verstanden werden , sonder in denselben andern unsern Ordnungen und fursehungen gentzlich und gar auszgeslossen sein sollen. Darauf so gebieten wir allen und yegklichen churfursten, fürsten , gaistlichen und weltlichen , prela ten , graven , freien, heim, rittern , knechten , haubtleuten , burgraven , landtvogten , vitzthumben , vogten , pflegern , Verwesern, ambtleuten , schulteisen , burgermaistern , richtern , rethen , bürgern und gemeinden und sunst allen andern unsern und des reichs , auch unserer erblichen furstenthumben und landen underthonen und getreuen , in was wirden , stats oder wesens die sein, bei Vermeidung unser swern ungnad und straf und darzu einer peen, nemlich 50 marck lottigs goldts in unser kaiserliche camer unableszlich zu bezalen , ernstlich mit disem brief und wellen, dasz si solch obgemelt unser ordnung , Satzung, dé¬ claration und mainung halten , der geleben und nachkomen , da¬ wider auch weder inner - noch ausserhalb rechtens an gantz kainen orten gegen yemandt gantz nichts furnemen , handlen , sprechen, urthailen , exequiern , noch iclits auszgeen lassen . Auch solchs gantz niemantz zu thun gestatten , zueselien noch verhengen , als lieb einem yegklichen sei, obgemelte unser swere ungnad und peen zu vermeiden . Wo auch ichts wider das alles, wie obengemelt , in I. Zur Geschichte des Monopohvescns im 16. Jahrhundert . 381 einem oder mer von yemandts , wer die wem , furgenomen , ge¬ bandelt , getiion , procedicrt , geurtailt oder exequiert wurd , das alles wellen wir ytz als dan und dan als ytz hiemit abermalen ausz unser kayserlichen macht und volkomenha .it craftlosz , unpundig, nichtig und von kainen wirden zu sein erkent und erclert haben. Also dasz niemand ! demselben kain volg zu ühun oder gehorsam beweisen schuldig sein soi. Das ist unser ernstliche mainung . Mit urkhundt diz briefs, besigelt mit unserm kaiserlichen anhangenden insigel. Geben in unser stat Tolleten in Castilien am 13. tag des monats may nach Cristi unsers lieben herrn gebürt 1524, unserer reiche des römischen im 6. und der ander aller im 10. iarn. Carolus. Vidit Waltkirch. Auf der Plica : Ad mandatum Caesaree ac Catholice Mtis. pro¬ prium . W. Brantner m. p. Unter der Plica : Duplicata pro serenissimo domino archiduce racione minariarum et metallorum distractione. Rückwärts : R ta Obernburger. 6. Der kaiserliche Fiskal beklagt Bartol . Welser & Co. vor dem Kammergericht , Monopole errichtet und versucht zu haben, besonders durch eine Verabredung mit dem König von Por¬ tugal , laut welcher der König sich kontraktlich verpflichtete, niemandem Spezereien zu demselben niedrigen Preise zu ver¬ kaufen , wie er sie den Welser verkaufte . S. d. Aus dem Reichs¬ kammergerichtsarchiv in Wetzlar nach München abgegeben. K . allg. Reichsarchiv . Reichskammergerichtsakten Fase . 201 a. Wolgeborner , gnediger her ! Keis . Mt. camerrichter in Sachen sich haltend zwischen mir als keis. mt . und des heil. röm . reichs fiscal-general -clägern eins und Bartholome Welser und sein gesellschafter , beclagten anders teils uff ausgangen ladung erschein ich und bring nachvolgend dis meinung clagsweis für , doch nit in ge¬ stalt einer zierlichen clag sonder einer siechten sumarien geschiehterzelung wider gemelten Bartholome und sein gescllschafter oder ein yede ander person von irai wegen in gericht wie recht erschei¬ nend sag und clag dass, wiewol durch gemeine geschribne recht bei Verlust aller hab und guter auch Verweisung des lands höchlich verpotten , dass niemand monopolia schedlich handtierung und Wer¬ bung prauclien oder üben solle. Dagegen auch kein privilegia, begnadungen , freiheiten , statuta , verjarungen und gebrauch , so 382 Dokumente der frühkapitalistischcnWirtschaftsorganisation. dawider erlangt , gesetzt , eingezogen und angenomen weren , gantz von unwirden , nichtig , unerheblich , craftlos und in gantz keinen weg furtreglich sein sollen, sonder den schedlichen monopoliern und handtierern , so sich damit beschirmen wollen vil mer nach¬ teilig, streflich und derselbigen straf graviern und häufen ; alles inhalt gemeinen , geschi'ibnen recht , weihe ich als keis. fiscal hiemit gemeldet und angezogen haben will. Solhs alles doch unangesehen und ungeachtet , so hat Bartolome Welser mit seinen geselschaftern ein mercklich gross hauptgut und gelt allenthalben und mit grossem vleis aufgenomen und zuhandt gebracht von etwievil jaren her , jedes jar besonder und bis uff diss gegenwärtig XXIX . jar und monat decembris liegst verschinen im konigreich Portugal in der stat Lisibona mit dem konig von Portugal allerhandt kauf umb pfeffer und andere specerei angenomen und beslossen , solh gross aufgenomen und zusamenbracht hauptgut und gelt angelegt den eentner umb ein genante suma kauft , mit den geding und Vorworten, dass der konig zu Portugal in einem oder zweien ungeverliehen negstkomenden jaren nach beschehnem kauf, keinem andern kaufman pfeffer und andre specerei neher sonder vil teurer zu kaufen geben soll und obgleich darnach ander gesellschafter umb den konig auch kauft , haben si doch vil theurer kaufen müssen dann Welser und sein gesellschafter und darnach im reich teutscher nation vil ander ansetz und gevarlich auch gantz schedlich pact und monopolia geübt und gepraueht . Das hat so¬ lang gewert bis alle specerei in disen hohen werdt körnen ist wie e. g. vor äugen sehen und der pfeffer, imber , negell, muscatnuss, zimet , rörlin , muscatplut und anders durch solh ferlich vergeding Welsers und seiner gesellschafter in zwifachen werdt ungeverlich ersteigt und vertheirt . worden seind ime und seiner gesellschaft zu grossem vortheil , nutz und reichtumb und der gantzen gemein hochs und niders stands teutscher nation zu mercklichem grossem untregliclien nachteil und schaden reichent . Darumb die peen und strafen des gemaineu rechten wie obgemelt höchlich verwürckt haben etc. Demnach und dweil nun obgemelte ding war und beweislich seind , so ist an e. g. mein underthenig bit , gedachten Bartholome Weisem und sein mitgeselschafter in die peen der rechten zu ver¬ dammen , aller und yeder ier hab und güter , ligender , farender, nutzungen und Werbungen durch e. g. rechtlichen sprach gentzlich I. Zur Geschichte des Monopolwescns im 16. Jahrhundert . 383 zu entsetzen und abzusliessen , auch dieselbigen guter , nutzUngen und Werbungen keis. mt . und des heiligen reiclis fisco zuzuerteilen und in besess derselbigeu on alle verhin .de.rnus komen zu lassen, auch gemelten Welser und sein mitgesellschafter , Verächter derselbigen keis. rechten , umb solh sein eigennützige , schedliche handlung und Übung ewiglichen zu verweisen , deportiern , abzusundern und versetzen , alles inhalt geschribner rechten , darauf ich als keis. fiscal dise mein pétition erhalt , gegrünt und gesetzt haben will alles in der besten form , e. g. höchst richterlich ampt undertheniglich anruffendt vorbeheltlich aller notturft. Caspar Mart doctor , keis. mt . fiscal. Rückseite : Petitio sumaria des keis. fiseals gegen Bartholme Welser und sein mitgesellschafter . Speir 9. Marci 1530. 384 II. Materialien zur Geschichte der Spekulation mit Wertpapieren im 16. Jahrhundert 7. Einige Bestimmungen über den Kuxhandel in Sachsen während des 16. Jahrhunderts. Die Kuxhändler („Kuxkränzler“), deren immer nur eine be¬ stimmte Anzahl von der Obrigkeit verordnet wurden \ hatten folgenden Schwur zu leisten: „ Ich N. schwere, dass ich will meinem gn. Herrn, dem Churfursten zu Sachsen etc. getreu und gewärtig sein, seiner chfr. Gnaden und gemeines Bergkwergs Bestes treulich fördern, Schaden warnen und abwenden und meinem Dienst, so mir befohlen ist, nach meinem höchsten Vermögen und Verstand, treulich und fleissig vorstehen, iederman rechten, warhäftigen Bericht thun, was jedes Orts der gemeine Kauf ist, keinen Vortheil noch Betrug suchen noch gebrauchen, sondern mich gegen Armen und Reichen gleiclimässig und unverweisslich halten. Welcher mir am ersten Kuclcus zu verkaufen anbeut, oder zu kaufen begehrt, den will ich am ersten fördern, kein Geniess oder Nutz, dann so mir zugelassen ist, in dem allen gewarten, auch weder Gunst, Ge¬ schenk, Freundschafft oder Feindschafft ansehen, sondern mich an deme, was mir ein itzlicher aus gutem, freyem Willen zu Tranckgeld giebet, gänntzlichen genügen lassen und alles andere handeln und thun, das einem auffrichtigen Krentzler gebühret und zu¬ stehet, treulich und ungefährlich als mir Gott helff und sein heiliges Wort“. —- Aus der Bergordnung Kurfürst Christian I. zu Sachsen den 12. Juni 1589. Vgl. Codex Augusteus II. S. 198. 1Es„ soll in jeder unserer Bergstadt nicht mehr als ein KuxKrentzler sein und darzu absonderlich vereidet werden, selbiger aber jedesmahl bei Verkaufung der Kuxe den wahren Aufstand u. Be¬ schaffenheit der Zechen, von den Geschwornen unterschrieben , mit¬ bringen , damit derjenige, so dergleichen Kuxe an sich handelt u. annimmt , nicht betrogen werde.“ Diese Verordnung stammt aller¬ dings aus dem 17. Jahrhundert . Bergwerks-Dekret des Kurf . Joli. Georg II . von Sachsen, 6. Aug. 1659. Cfr. Codex Augusteus , II, S. 322. Vgl. auch S. 370, 383. II. Zur Geschichte der Spekulatiou mit Wertpapieren im 16. Jalirh . 385 Die folgenden Bestimmungen richten sich gegen irreguläre Kux¬ händler: „Ob auch wol ausserhalb der verordenten Kuckus -Krentzler, die dann durch unsere Ambtlcuthe darzu sollen vereydet werden, des Krentzelns und Kuckus -Vorkeuffens sich niemandts soll unter¬ stehen , so langt uns doch mannigfcltig an , dass etzliche , nicht alleine in unsern Landen und Furstenthumben , beföderst in unser Stadt Leipzig und andern Handels -Stedten , sondern auch ausser¬ halb derselben Kuckus und Berckteil zu vorkauffen sich anmassen, sonderlich an denen Örtern und Zechen, da vorlassene Gebeude seindt , oder auch , do sie ihre Keuffere solcher Theile derer wirderung , wie sie die ihnen angeben und verkaufft , nicht geweliren können , dadurch also die Leute betrogen , von dem Berckwerge abgescheuet und unsern Berckwergen grosser Nachtheil eingefurt wirdt . Solchs fortmehr zuvorkommen , ordnen und wollen wir, dass unsere Amptleute mit sonderlichem Fleis hierumb sollen Er¬ forschung und Nachtrachtung haben und do sie nun hinter jemandt kommen , der hierinnen verbrochen , oder aber würde durch andere , die er betrogen vor ihnen beklaget und uberweiset , sollen sie den oder dieselben zu Gefencknus einziehen und mit gantzem Ernst am Leibe straffen oder uns jederzeit davon Bericht thun, dass wir uns alsdann nach Gelegenheit gegen denselben mögen mit gebiirlicher Straff erzeigen. Sollten aber auch die vorordenten und geschwornen KuckusKrentzler gleich so wol die Leute , es weren Frembde oder Einlendische in ichte wider ire Pflicht und was die mit sich bringen, bevortheilen , wie solchs geschehen möchte , so soi es mit der Straff gegen sie gleicher gestalt , wie oben gemeldet , auch gehalten werden. LXXVIII . Art . („Krentzler und Kuckus -Partirer “ über¬ schrieben ) der Bergordnung Kurfürst Augusts von Sachsen („aus derer Hertzoge Georgens und Heinrichs , ingleichen Churfürst Moritzens zu Sachsen alten Articuln verbessert und gemehret “) vom 3. Okt . 1554. Abgedruckt im Codex Augusteus II . S. 140. Vgl. auch S. 174. Noch deutlicher erkennt man die betrügerischen Machen¬ schaften mancher Kuxhändler aus der Bergordnung Kurf . Chri¬ stian I. von Sachsen vom 12. Juni 1589. Dort heißt es im 74. Art. (Codex Augusteus II . S. 212) : Ob auch wohl ausserhalb der verordenten Kuckus -Krentzler, Strieder , Studien z. Gesell , kapitalist . Organisationsformen . 25 386 Dokumente der frühkapitalistischeil Wirtschaftsorganisation. die dann durch unsere Amtleute darzu sollen vereidet werden, des KrentzeJns und Kuckus -Yerkauffens sich niemand soll unter¬ stehen , so gelangt uns doch mannigfaltig an , dass etzliche , nicht allein in unseren Landen und Fürstenthumen , befoderst in unser Stadt Leipzig und andern Handels -Städten , sondern auch ausser¬ halb derselben Kuckus und Bergtheil verkaufen sich amnassen, sonderlich an denen Orten oder Zechen, do verlassene Gebäude sein oder auch do sie ihre Käufer solcher Theil , deren Wirderung, wie sie die ihnen angeben und verkauft , nicht gewären können, dadurch also die Leute betrogen , von dem Bergkwerge abgescheuet und unsern Bergkwergen grosser Nachtheil eingeführet wird , solches fortmehr zuvorkommen , befehlen und wollen wir, dass unsere Amt¬ leute mit sondern , ernsten Fleisse Erforschung und Naclitrachtung haben also, do sich iemand in oder ausserhalb unserer Lande unter¬ stehen und den Leuten ungebührlichen Partierens , betrieglicher hinterlistiger Weiss Theil aufhängen und höher , dann sie auf unsern Bergkwergen iedes Orts wirdig , verkaufen wurden , dass sie nach eingenommener glaubwürdiger Klage , solchen Betrieger gefäng¬ lichen einziehen und ihme auflegen sollen, dass er dem Käuffer alsbald sein Geld, darum er ihn betrogen , paar wiederum erlege und ihm den Partierer zum wenigsten 4 Wochen lang , auf sein eigen Unkosten gefänglich enthalten . Do er aber solch Geld, darum er den Käuffer betrogen , nicht zu erlegen vermöchte , soll er nach Endung der 4 Wochen auf gebührlichen Unfrieden unserer Bergk¬ werge auf etzliche Jahr verwiesen werden. Würde aber der Betrug grösser und höher bei ihme befunden und erweist , dass er Kuckus verkauft in den Zechen , so er nicht gemuthet , den Leuten Ertz weisen, do sie keines am Anbruch, kein Kuckus im Gegenbuch hätten , falsche Gewehr und Zupuszettel machen , Zupus einnehmen , do keine angeleget oder wol niemand wüste , wo solche Zechen gelegen und sollen unsere Bergkamtleulc mit Fleiss nachtrachten , dass sie einbracht und im Ge¬ fängnis härter dann andere enthalten lassen und verschaffen , dass sie denjenigen , so sie also aufgesetzt ihr Geld und aufgewandlen Kosten alsbald wiederum erstatten und zum wenigsten sie 8 Wochen gefänglichen enthalten und hernach , do das Geld nicht von ihnen erleget , des Landes verweiset werden. Wäre aber der Betrug dermassen beschaffen , dass die Strafe des Gefängnis nicht gnugsam , oder hievor damit gestraft und IÏ . Zur Geschichte der Spekulation mit Wertpapieren im 16. Jahrh . 3g',7- anderwert verbrochen , soll man dieselben mit Ruthen aushauen lassen und unserer Lande und Bergkwerge auf ihr Leben lang ver¬ weisen, damit männiglieh , dass wir ob ihrer Betriegerei Missfallen tragen. Sollten aber auch die verordenten und geschworenen KuckusKrentzler gleich so wohl die Leute , es wären Frembde oder Ein - . ländische in ichte wider ihre Pflicht und was die mit sich bringen bevortheilen , wie solches geschehen möchte , so soll es mit der Straff gegen sie gleicher Gestalt wie oben gemelt , auch gehalten werden “ . 25 * 388 III. Quellen zur Geschichte der Aktien¬ gesellschaft im 16. Jahrhundert. 8. Statut der Allgemeinen Eisenhandelskompagnie zu Steyr (Aktiengesellschaft ), 14. Sept . 1581, Nach einer Abschrift, die sich im Archiv der Stadt Steyr (Lade 3) befindet . Bl. 3 ff. mit Auslassung von Unwichtigem. Compagnia -Ordtnung 1 auf den Eisenhandl bey der Statt Steyr , so unter dem Namen und Tidl der Stadt Steyr solle ausgehen: Nemblich zum 1. So solle nun liinfürder aller und jeder Inner pergerischer geschlagener Zeug von Stahel und Eisen wie der Namen haben mag (darein auch die Schin-, Pflueg -, Plech - und dergleichen Eisensorter ., so in den khlainen Hamern nach der Enns heraus, die auf den klainen Pächen ligen und sowoll bey etlichen Hamerschmiden zu Holnstain aufgebracht und bissher hierher gegeben worden , zu verstehen ) allermassen wie hievor gegen den PrivatEisenhandlern alhie beschehen ist (in Crafft hieliger ersessenen Freyheiten und Rechts auch vermüg der alten und neuen Eisenordnungen ) allein hierher auf Steyr in und zu berürter Gsellschafft hernach bemelter Verwalter Händen (dartzue sie, die Gsellschafft bey der Enns ire sondere gelegenen Gewelb und Legstedt haben solle) in dem ordenlichen gesetzten Hamermaister -Khauff gelifert und sonsten an khain anders aussers Ortt noch hieligen PrivatBürgerspersohnen wenig oder vill gegeben und also weder ainem noch dem andern under hieligen Bürgersleuthen , haimlich oder öffentlich , ainiche Handthierung mit geschlagnen Eisentzeug , so¬ lang dise Gemainschaft und Compagnie werth , ze treiben gestattet werden. Und nachdem von villen langen Jaren der gestreckhte Stahel alhie. under etlichen Privat -Bürgerspersohnen in ainer Handt und 1 Ein anderes Mal heißt es in dem Schriftstück „ GemeinschafftOrdnung “ . III . Zur Geschichte der Aktiengesellschaft im 16. Jahrhundert . 389 Gesellschafft gewest und derselbige gestreckhte Stahel Inhalt der alten auch der mittleren und neuen Eisenordnungen und sonndern Endtschiedt l, so von der hochlöblichen niederösterreichischen Re¬ gierung und Camer des verschinen 59ten Jares zwischen berürter Gsellschafft und den Innerpergerischen Hammermaistern ergangen, andern auftzubringen verbotten und allain inen , den Gesellschafftern desselben gestreckhten Stahels in iren sondern Streckhämern zue machen zuegelassen worden . Und aber derselbe dem Eisenhandl auch anhengig und zu disem Wösen gehörig ist : So solle derselbe gestreckhte Stahel nunmers hinfürters auch in dise EisenhandlungsCompagnia , mit aller Gerechtigkhait so sy die vorigen Gsellschaffter liürüber gehabt , zugleich gehörig sein. Auch denjenigen Persohnen, so bissher drinen gewest, ir daselbs gehabtes Leggeldt sambt dem, so sie sonsten hürüber merers in dise jetzige völlige Gsellschaft thuen , nach Ordnung zue geschriben und solcher gestreckhter Stahel durch die Eisenhandlsgsellschafft -Verwaltter gleich wie all ander hieobbemelter Eisentzeug empfangen und hinwiderrumben verkaufft und verhandelt werden. Zum 3. Obwol! zu Erstattung angerürts Leggelt oder Capital diser Gsellschaft unnder der Rürgerschafft ain Gwisshait , was ain jeder unnder den Reichen , Mittlsvermiigigen und Ermern hierein underschiedlich und nicht darüber oder dahinder legen, item ob es auch aus aignem Guet beschehen oder was ainer sonsten anderwerts aufbringen khan , zuclässig sein, gleich alsbaldt jetzo anfangs gesetzt und bonent werden solle. Damit der Reich den Unvermügigern hierinen , wo dise Gsellschafft nutz - und gwinlich sein würde , nicht engen oder mit zu villen Leggelt an seiner Narung hindern thue . Weilln aber dises Wösen und das Haubtcapital wie verstanden sehr hoch und gross ist und man daher an jetzo der¬ gleichen Fürordnung zur Gewisshait nicht thuen khan , zumall das der Verschleiss mer im Steckhem als der Wierde der Zeit ist , daher das Gemainwösen zu Erhaltung der Stadt Freyhaidten , Mann¬ schaft und Narungen aines sterckhern Geldtzuesatz bedarff und also villmer dises Gsellschafftwesen ins Werch zuerichten die ge¬ westen Privat -Eisenhandler mit dem iren , so sy hievor in der Eisenliandlung gehabt sowohl auch andere als aus unns der andern vermügigern Bürgerschafft aufs sterckhist zuezusetzen zu bewegen, 1 Das heißt besonderen Abmachungen. 3Ô0 Dokumente der frühknpitalistischen Wirtschaftsorganisation. inderne dannocht khain Auskhumen , sondern hierüber noch ain grosse Suma Gelts viller 1000 Gulden umb Interesse auftzunemen ist , so khan demnach Solchen Leggelts wegen anjetzo kain gewisses Zill angeordent werden . Wann aber dises Compagniewösen in seinen Ganng und Übung khumbt und man mit etlichen Jaren siecht , wie es sich aalest und zuenimbt , solle alsdann nach Gelegenhait der Zeit hierinen auf ainen oder den anderen Weg zu Beförderung ' des hieligen gemeinen Nutz solche Ein - und Fürsechung beschehen , dass solch Leggeldt generaliter sein benentes Zill haben , und kainer drüber noch darhinder legen, noch ainer für den andern aigennutziger Weiss nicht beschwert . Aber underdessen und anjetzo anfangs ain Jeder so vill in dise Gsellschafft zu legen, als er selbs vermag oder anderwerts auf Interesse oder sonst aufbringen khan zuegelasscn werden . Doch dass es zum wenigsten under 100 Gulden nicht seie. Zum 4. Damit dise Eisenhandlsgsellschaiït nicht gleich im Anfang zertreut oder confundiert werde, welches sonderlich mit dem beschehen möchte wann ain jeder im Mitl oder nach Aus¬ gang des ersten oder andern Jars gestrackhs heraustrachten und sein Leggelt abfordern wolte : so solle alles und jedes , gross oder daines Leggeldt auf 4 gantze Jar , von jetzo diser Gsellschaft Anfang und beut datto antzuraitten , darinen unabgefordert ligen verbleiben und vor Aussgang derselben 4 Jar khainen Gsellschaftern von seinem Haubtleggelt niclits herausgegeben werden . On allain es erforderte aines erweisliche hohe und unentradliche Nottdurfft. Inderne die Gsellschafft ainen solchen nach Gelegenhait und billiclien Dingen , soweit es ir der Gsellschafft erschwinckhlich und dem gemainen Wösen unschedlich , sein Leggeld thaills oder gar (doch gegen Abtzug der verlornen Schulden so dise Zeit über in der Companie gemacht werden möchten ) hinaus zallen mag . Und was aida von den Gsellschaffts -Persohnen gemeldt , das solle zu¬ gleich auch verstanden werden , da ain Gsellschaffter mit Todt abgieng , Wittib , Kinder und Gläubiger verliess, auf deren ainer oder den andern Taill solch Leggeld durch Erb -Succession geschefft oder Vorwaisung gefallen, dass solche in obsteenden , zuelelligen, unvermeidlichen Nötten und Obligen auch vor Aussgang der 4 Jar zu bedenkhen. Zum 5. Weiln die Gsellschafft in dem Eisenhandl als aines hieligen bürgerlichen Gewerbs angericht wirdet , so solle in dieselbe III . Zur Geschichte der Aktiengesellschaft im IC. Jahrhundert . 39! weder jetzo noch khünfftiglicli niemandts annder , als die alhie geschvvorne Bürger seien, mit irem Leggelt , des auch jedertzeit (über das , was anjetzo bemelte vorige Privat -Eisenliandler und Gestreckhten -Stahels -Gesellschaffter in derselben ircr Eisenhand¬ lung und gehaltenen Gsellschafft richtig gemacht haben ), allain par und anders gar nicht beschehen solle, zuegelassen werden. Zum 6. Auf dass es mit Einnemung neuer Gesellschaftler auf khünfftige Zeit sein Mass habe , solle in Mittl des Jars und vor geschlossner Jars -Raittung khain Annemung — zu Vermeidung Irrung in der Raittung — geschehen . Aber nach Ausgang jedes Jars und geschlossner Raittung müg alle Jar , che die 4 Jar fürüber , neue Gesellen diser Ordtmmg gemäss angenomen werden. Und solches darumber , damit durch solcher neuen Gesellschaftler Einlag das auf Interesse aufbrachlc Gelt desto ehe wieder ab¬ gelegt . Item so auf solche Fall wie oben verstanden den alten Gesellschafftern oder iren Erben und Gläubigern vor Aussgang der 4 Jar in Nottfällen was in Abschlag zu zallen , dieselben Suma desto leichter wieder zuersetzen . Wie dann auch bey hernach volgenter 4 fürgesetzten Companies -Persohnen auch des Puechhalter und der Cassier Beratschlagung stehn solle, wann der neuen Gesellen in den ersten 4 Jaren zu vill khemen , dass es den ersten Gesellschafftspersohnen , so zum Anfang eingelegt und die Gfar des Anfang auf sich geladen oder mit tragen , beschwerlich und nachthaillig , auch sonst dem gemainen Wösen uit fürstendig wer, dass sie allain sovill annemben sollen als es nützlich und dienstlich ist . Wann aber die ersten 4 Jar fürüber , alsdann mag die An¬ nemung allzeit nach Ausgang 2er Jar beschehen. Zu Gesellschaffter sollen auch nicht allain die angesessenen Rüi’ger so Handlsleutb , sondern auch die andern , so unangesessen, darunder die Handwercher , angesessen und unangesessene und in genere alle diejenigen , so geschworne und gelibte Bürger seien bemaint zugelassen werden und liierinneii khainem der Zuetritt abgestrieckt sein. Was nun jeder Bürger für ein Leggeldt , obstehenden Inhalt gemess, zue der Cassa richtig machen wirdt — es scyc durch Pargelt oder Überschaffung solcher Posten , die vorher alberait in der Compagnia gewiss und richtig ligen und an solchen neuen Gsellschaffts -Persohnen durch ordenliche Mittl khumen —- darumben solle jedem von der Gsellschafft ain gefertigter Schein vermüg 392 Dokumente der frühkapitalistischen Wirtschaftsorganisation. der sonnderu verfassten und verglichnen Formbl, so hernach in¬ serieret, mit der Gsellschafft Petschafft verfertigt und von den nacbsteenden Puchhalter undertzaichnet, angehentigk werden. Inserirtcr verglichner Form des Scheins. Wir N. und N. die Gesellschaffter der Eisenhandlung zu Steyr bekhennen für uns, unsere Erben und Nachkhumen , als wir an jetzo dem N. dise Compagnia allermassen und gestalt wie es die desthalben aufgerichte Compagnia-Ordtnung mit iren Puncten und Articlen ver¬ mag, zu ainem Mitglidt und Gesellschaffter angenumen haben . Dass er hierauf zu seinen Leggelt N. Pfundt Pfenning auf Gwin undVerlust, wie in solchen Gesellschafftshandlungen gebreuchig ist , zu unserer Cassa erlegt hat , welche auch von datto antzuraitten , Inhalt an¬ geregter Ordtnung , er seie bey Leben oder schaide hietzwischen mit Todt ab, 4 gantze Jar unabgefordert oder unbetzalt stilligent bleiben sollen. Demnach zuesagen und versprechen wir für uns und unsere Erben und Naehkhomen , dass gedachtem; N. auf bemelte 4 Jar und so lang er hierüber in solcher unserer Gesellschafft sein, der ordenliche Gwin, was dessen auf angeregtes sein Leggelt jerlichen , vermüg der Raittung , gebüren oder fallen wirdet , eben wie all andere Gesell¬ schafts -Verwandten under uns mit Ausgang aines jeden Jars und aufs lengist innerhalb 14 Tagen hernach zu seinen sichern Hannden angehendigt werden soll. Und wann merbemelter 4 .Jar fürüber und er aus diser Compagnia begehen, auch mit halber Jars Zeit aufkhünden würde, so gereden wir verrer Inhalt der Ordtnung im und seinen Erben oder treuen Brieffs-Inhabern obberürt sein Haubtleggelt der N Gulden innerhalb 4 Jaren und alle Jar mit Ausgang desselben % daraus zu hetzallen . Oder wo auf bemelts N. Absterben seinen Erben oder andern , so er auf dises Leggelt weisen möchte , dise 4 jerige Betzallung zu lang sein wolte, alsdann inen dieselbe nach angerürter V2 Jars Aufsag in den darauf negst volgenden 2 Jaren zu thun und hietzwischen von dem in jedem Jar bleibenden Rest den pro rato gebürenden Gesellschaffts-Gwin obsteender Gestalt zu ralchen . Doch sollen in Abtzallung solchen Hauptleggelts die gar ungewissen und für verlobrn befundene Schulden, so nach Handelsbrauch und diser Gesellschafft und der Zeit Bescliaffenliait ir über angewenndten Vleiss nit gar vermitten bleiben kliünen , auch pro rato , was es auf dise Suma in Abraittung zuetrifft , liievon aufgeliebt werden. Truege sich dann zue, dass obgedachter N. vor Verstreichung der 4 Jar und seiner Aufkhündung mit Todt abgieng und diejenigen, an welche solches Leggeldt erblich oder rechtlich khäme , Bürgersleuth oder den bürger¬ lichen Wösen hie zuegethan weren, solle denselben nichts weniger der völlig Gwin (als ob der N. selbs im Leben und Bürger alhie hüben were) vom Leggeldt geraicht werden. So aber dieselben nit Bürgers¬ leuth oder sich von dannen setzten , solle inen nicht der Gwin, sonnder ein Interesse 5 % in Zeit das völlig Leggeit im Nutz ligt und auch hernach auf Endtrichtung abgehörter Jarsfristen von dem übrigen III . Zur Geschichte der Aktiengesellschaft im 16. Jahrhundert . 393 pro ratto biss es abtzallt , alles in parem gueten Gelt , landtleuffiger Wehrung , geraicht und hierinen der Compagnia -Ordtnung nach¬ gegangen werden . Des zu wahren Urklmndt haben wir vorernentem N . zu seiner Notturft disen Schein und Bekhandtnuss angehendigt. Mit unserin Compagnie -Handels -Petschafft verfertigt. So weit die Inserirung. Zum 7. So solle die Gsellschafft zu irer Cassa und Pueclihalterey ain wolbewarte Schreibstuben in hieligen Ratthaus , wo es mit newer Zuerichtung sein khan , oder aber sonnsten in der Stadt und dartzue ain aignes Handt -Pedtschafft in berürter Puechhalterey haben . Welches in der Visier gemainer Stadt Wappen, das Pandl -Thier haben und die Umbsehrifft also lautten sollet Stadt Steyr und die gemain Gesellschafft der Eisenhandlung aida. Damit alle Missife, Schein und was ier der Gsellschafft Notturfft ist — doch ausserhalb der Schuldtbrieff — gefertigt werden. Zum 8. So sollen jetzo im Anfang und dann alle Jar durch uns, ainen Rath 4 fürneme und taugliche Burgers -Persohnen , nemblich die zwen aus unsern Ratts und die andern zween von der anderen Bürgerschaffts Mittls , alle vier der Compania verwandt, erwöldt und aus denselben zur Umwexlung jerlichen die zwen eitern, so 2 Jar nach einander die Bemiiung getragen , verändert und andere auch durch unns ain Rath an ir stadt verordnet , dise Verände¬ rung aber die obvermelten ersten 4 oder doch zum wenigsten die 2 Jar sovil müglich underlassen . Auch solche 4 Persohnen von Jar zu Jarn der Gesellschafft Heubter sein und die Obhandt über dises gantze Gesellschaftswösen und deren hernach bemelte Diener und Verwalter haben , alle Sachen zum pestten bey den Undergebnen anordnen und zu gewissen Tagen und Stunden , deren sie sich vergleichen , wo nit jedertzeit alle vier , doch zwen auffs wenigist mit einander sich in der Schreibstuben finden und zuesehen , was allerseits fürgefallen und zue handeln von nötten , dasselbe auch also oft vleissig und fürdersam verrichten und wo sie samentlich die Sachen so wichtig funden , nach irem Gefallen und Bedenckhen noch etliche sondere Persohnen aus der Gsellschafft zu sich er¬ fordern , mit inen Beratschlagung halten und also in ainetn und dem andern an inen nicht erwinden lassen . Damit alles zu Befürdrung der gemainen Gsellschafft Nutz treulich gehanndlet und ir jedem für dise Bemüung zur Gleichhait und Gelegenhait durch die Gsellschafft alle Jar ain gebürliche Verehrung , aber sonnsten khain andere Besoldung gegeben werden. 394 Dokumente der frühkapitalistischen Wirtschaftsorganisation. Zürn 9. Wo auch die Notturfft erfordern wolte, dass man zu ainer oder der andern Zeit in die Gsellschafft Gcldt umb Interesse aufnemen müsste, so sollen bemelte 4 Ob-Persohnen daselb jedertzeit. mit Vorwisseu und Guethaissen unser, aines ersamen Raths thuen und sich darumben bewerben, auch die Schuldtverschreibungen umb aines oder das andere Anlehen von uns, ainem ersamen Rath und der Companie under gemainer Stadt Fertigung — Inhalt der abgehandleten Nottl so auch hienach eingeleibt oder wie man es von dem Darleiher haben khan — ausgehn. Inserirtc Schuldtbrieffs Formbl. Wir N. Biirgermaister , Richter und Rath der Stadt Steyr , auch wir die Bürgerschafft und Gesellschaffter des Eisenhanndls dasclben bekhennen für uns , unsere Nachldiomben und Erben hiemit diseni offen Brief wo der fürkhombt , dass wir dem N. und seinen Erben recht und redlich schultig worden sein benendliche N Gulden reiniscli gueter Landtswerung (jeden zu 15 Patzen oder GO Kreutzern zu rechnen). So er uns auf unser Bitten zu notturftiger Beförderung dises unsers Companie-Handls im parem Gelt dargeliehen u . in unser Gwaltsam behendigt , auch gewilligt hat , dise Suma Gelts von Jar zu Jarn gegen N Gulden per Cento järlichen Interesse stiliigen zu lassen. Wo aber er N. oder sein Erben uns solche Suma Gelts verrer nicht darleihen oder wirs lenger nicht gebrauchen wolten, dass ain Tail dem andern dasselbe y2 Jar lang zuvor glaubwürdigen u. auf¬ sagen solle. Darauf gereden, zuesagen u. versprechen wir für uns, unsere Nachkhumen u. Erben liiemit wissentlich u. in Crafft dits Brieffs nemblich dass wir gedachten N, seinen Erben oder getreuen Inhabern diss Brieffs, wann uns die beruert j4jerig Aufsag beschiecht, oder wir die selbs thuen wuerden , nach Vcrscheinung angeregtes y. Jars olme lengern Aufzug oder Waigerung , auch gantzlich ohn allen iren Uncosten u. Schaden zu iren sicheren Händen danckhbarlich betzallen u . hievon järlich u. aines jeden Jars besonders, so lang wir solche Suma Gelts inliendig haben , N Gulden obberürter Wcrung zu rechter Zeit die Vertzinsung alweg zuraichen u. ain JarsVertzinsung auf die ander nicht anstelin lassen wollen noch sollen. Und damit dann gedachter N. u. seine Erben angerüerter irer in Treuen unns dargelicbnen N. Gulden reiniscli sowoll auch dem hie¬ von gebüerenden jerlichen Interesse genugsamblicli versichert werden, so verweisen u. verschreiben wir inen durch diesen Brief unser der Statt Steyr u. unser der Bürger u. der gemainen Gsellschafft zu¬ sammen gelegte Guetter , darinnen nichts ausgenomen wo u. welcher Ennden die allenthalben zu finden sein, zu rechtem Für - u. UuderPfandt . Allso u. dergestalt da wir unsere Nachkhumen u. Erben vermelte N Gulden reiniscli über vorgehende Aufsag zu Endung des obbest halben Jars hietzwischen auch das angedeut jerlich Interesse obverschrihner Massen nit zallhaft machen , sonnder darmit wider III . Zur Geschichte der Aktiengesellschaft im 16. Jahrhundert . 395 iren gueten Willen säumig erscheinen u . er N oder seine Erben dessen »iniclien Schaden — Idain oder gross wie der Namen haben möchte — nemen Hielten , denselben Schaden allen , khainen redlichen aus¬ geschlossen , sollen sie sambt der völlig Haubtsuma u . dem aussstendigen Interesse haben , suechen u . bekhumen an u . auf denselben unnsern u. der Stadt Steyr u . unnser der Compania gesambten Gsellschafftsguettern ; wie auch die landlfürstliche Obrigkhait sy on alle weitere Clag oder Berechtung auf erstes Ersuchen bey diser Verschreibung zu richtigen ßetzallung mit steiffer , unverlengter Exe¬ cution handtliaben u . Mcwider in aincm nach dem andern nit be¬ schweren lassen sollen , bis sie obbestimbter Haubtsuma ü . Interesse sambt denen hiedurch erlidcnen Schaden zue guetem , völligen Benügen entricht u . zufridan gestclt worden sein . Alles u . jedes bey Verpindung des gemahlen lanudtleuffigen Schadenpundts , als ob der von Wort zu Wort nach lengs hierinen geschriben stiiende . Darwider u , dises alles unser Naehkhomen u. Erben khainerlei Eintrag noch Verwiderung wie die durch Menschen Sin im er erdacht werden möchten u . wir uns gebrauchen wollen oder würden nicht schützen, fürtragen , noch hievon entheben solle in khainerlai Weis , noch Wege, treulich one Geverde Soweit die Inserirung. Und nachdem diss Companie",yerch fürnemblich zur Erhaltung der Stadt Freyhaidten , Gewerb- und Manschafften hat fürgenumen werden müssen und villeicht die Frembten bey denen Geldt in Nottfällen auftzubringen — weil dieselben frembten Herleihern der Companie Beschaffenhait unbekhandt und dise Companie-Ordtnung zu eröffnen bedenckhlich — gemainer Stadt allain lieber, dann dass die Companie mit bekhennen und sich obligieren sollen, leihen möchten , so sollen in solchen Fällen die Scbuldtbrieff von uns N. Biirgermaister , Richter und Rath allain ausgehen , doch allzeit von der Compagnia für ir Gebüer , so weit sie darbey mit interessiert , ainern ersamen Rath ain Gegen-Recognition von der Puechhalterey angehendiggt werde. Zum 10. So solle ain Puechhalter aus der Bürgerschafft , so anders ain solche trügliche , verschwigne , vleissige und khundige Persohn zu bekhumen , behandlet . Da aber aus der Bürgerschafft khainen zu erlangen , alsdann ain Frembder (doch ain woll erkhandter Man, dem zu vertrauen ) auf billiche und zimliche Jarsbesoldung der Companie bestelt , uriÖ derselb in disem seinen Dienst vleissig und getreu sein. We.ill auch sonderlich die Veränderung eines Puchhalters zum Anfang ungelegen , geferlich und bedenckhlich, solle solch Persohn solang man khan continuirt und so sie hie nicht 396 Dokumente der Irühkapitalistischcn Wirtschaftsorganisation. Bürger were, sie doch dasselbe alsbaldt annemen. Welchem Puecli- lialter auch auf sein Verrichtung aus diser Haubt -Ordtnung ain sondere Instruction gefertigt, angehendigt werde. Aber sein Amts¬ pflicht dis nach inserirtem Inhalt sein solle. Inserirte Aidtpflicht: Ir werdet ainen Aidt zu Gott dem Allmechtigen schweren und X. Bürgemiaister , Richter u. Rath der Stadt alhie, desgleichen der Companie, von denen Ir zu ainem Puechhalter über solche EisenUandls-Gsellschafft aufgenumen , bey Euren Eliren und Treuen an¬ geloben, gedachten N. Bürgemiaister , Richter u. Rath und den Fürgesetzten der Companie getreu, gehorsamb und gewertig zu sein, Euren Puechhalter Dienst — nach Vermüg Eurer Instruktion , die Eucii angehendiggt — treulich , erbar und aufrecht zu verrichten, gemainer Stadt und der Compagnie Nutz u. Frumen , sovill an Euch u. Eures Dienst ist , zu fürdem , Schaden zu warnen und zu wenden, die Geliaimb dis Handls , wie die von ainer Zeit zur andern an Euch gelangt , biss in Eur Grueben zu verschweigen und nicht zu offen¬ baren , Euch auch mit niemandts Frembden in Handlung , Contract, Correspontentz oder Gemainschafft einzulassen, welches gemainer Statt und diser Geselb- u. Gemainschafft jetzo oder künfftig zu Nachteil u. Schaden geraiclien möchte u. sonst alles das zu thun u. zu lassen, das ainem erbarn , aufrechten Puechhalter und treuen Diener zu thuen aigent und gebürt . Darinen weder Muet, Gab, Freundschafft , Feindschafft , noch ichtes anders dann Eur Pflicht und die Erbarkhait antzusehen. Soweit die Inserirung. Ferer so will auch der Companie Nottdurfft sein, zu disem ansehlichen Werch zwen Cassier zu halten und sollen dieselben alletzeit nach Gelegenhait aus den Fürnemisten und Tauglichisten der Gsellschafft, die woll angesessen, behandlet und erwöllt. Der jeder solchem Ambt 1 oder 2 Jar fürstehn, und also offt ainer aus den zwaien nach Aussgang aines Jars gewexlet und ain anderer gesetzt, damit also ain alter und ain neuer Cassier Berichts halber im Ambt sein und der Elter im andern Jar durch den Jüngern enthebt khüne werden. Es solle auch ir jeder zu solcher Cassa ainen aignen Schlüssl haben und ainer ohne den andern nicht drain khumen und sie sonderlich, wan sie dise ire Ämbter antreten, uns ainen ersamen Rath für uns und anstat der Gsellschafft Handtglüb und Pflicht dahin thuen, dass sy bey disem Companiawösenim Einnemen und Ausgeben und sonsten allerseits getreu und vleissig sein, gemainer Stadt und der Companie Nutz und Frumen betrach¬ ten, Schaden und Nachteil warnen und wenden, die Cassa in gueter III . Zur Geschichte, der Aktiengesellschaft im 16. Jahrhundert . 397 Sorg und Venvarung halten , anders nlt als was dise Ordtnung zuelesst handlen , mit solcher vertrautten Cassa khain Gefahr oder Aigennutz — wie das Menschen sie erdenekhen möcht — gebrauchen oder yben , sonder dise Cassa in irer Verantwortung und Versperrung treulich haben und handlen wollen und sollen. Und auf dass sie aber solcher Müe und tragenden Veranworttung auch Ergötzlichkhait haben , solle inen järlich — nach Gelegenhait der Nutz bey der Gesellschafft gross und mittlmessig sein würde — ain solche Ver¬ ehrung , dass es der Gsellschafft erträglich , geraicht .und geben werden. Nachdem auch weiter von Nötten , dass taugliche Persohnen vorhanden , welche allen Zeug von den Hamermaistern und Hamerschmiden empfahen zu Steyr und von dannen verrer und inner, ausser Landts verhandlen , item , die die Gonträct schliessen und das Gelt umb solchen verhandleten Zeug einnemen und zur Cassa und Puechhalterey bringen , sollen deren hierzue 4 in der Antzall bestellt und aus den Gesellen oder aus der Bürgerschaft fürgenumen und sie nit baldt gewexlet , sondern bey iren Diensten und Ämbtern, so lang es immer ir und der Gesellschaft Gelegenhait geben mag, erhalten , auch ir jedem nach Beschaffenhait diser irer Amts¬ oblag, die dann zimblich starckh sein und neben Haltung aines aignen Dieners und Ross vill Bemüung auf sich tragen wirdet, ain billiche Jarsbesoldung , so der Gsellschaft erschwinckhlich ist, geraicht werden . Sy sollen auch zugleich ire sondere Pflicht haben, wie dann die dis Orts inserirt ist. Inserirte Pflicht. Ich gelob’ und zuesagc hiemit an Aidtsstal und bey meinet bürgerlichen Pflicht, dass ich gemaincr Stadt und der Companie als ein fürgenumener, besteiter u. besolter Diener, getreu, gehorsamb und gewärtig sein, der gemainen Compania Nutz u. Frumen, sovil an mir, befürdern, Schaden und Nachteil zeitlich warnen u. wenden, meiner Instruktion und Dienst treulich nachkhumcn, khain Gefahr hierinen brauchen , die Gehaim der Gsellschafft , so an mich gelangt und khombt, bis in mein Grueben verschweigen u. nicmandts oder denen es zu wissen nit gebüret , oder das der gemainen Companie zu Schaden geraichen möcht , hievon nichts offenbaren , sondern mich auf höchst , dass der gemainen Companie treulich u . nützlich ge¬ haust und geliandlet werde, befleissen u. sonst alles das thuen will, das ain treuer, besolter Diener seiner Pflicht u. der Erbarkhait nach zu thun schuldig ist und hicrincn weder Muet, Gab, Freundtschafft , Feindtschafft noch ichtes anders dann meine Pflicht u . die Erbarkhait antzusehen. Soweit die Inserirung. 398 Dokumente der frühkapitalistischen Wirtschaftsorganisation. Es solle auch zuegelassen sein, dass der vorbemelte Puechhalter , Cassier und dise 4 Händler sowoll als andere Bürger ire Legegeldt in der Gsellschafft mügen haben , auf dass sie umb sovil raer Ursach sich dahin zu bemüen , damit der Nutz und Aufnemen diser Gesellschafft befürdert werde. Damit dann auch solche Persohnen als Puechhalter , Cassier und Händler disen iren Ämbtern umb sovill mer abwarten khünen, sollen sy, so lang sie disen Officien beiwonnen , aller Gerhabschafften, Curratorien und ander bürgerlichen Ämbter entlassen sein und irer damit verschont werden. Dise Officien, Puechhalter , Cassier und Händler sollen denen hievor im 8. Artickhl verleibten 4 Persohnen underwürffig sein, auf dieselben iren Respect und Aufsehen haben und allerseits son¬ derlich in wichtigen Dingen mit irem Vorwissen handlen , inen auch der Puechhalter alle 2 oder zum lengisten 3 Monat oder so oft es begert und von Nötten , ainen Pelantz 1ziehen und fürbringen, und wie also alles Wösen mit Einnemen und Ausgeben , auch den Schulden so sie Gsellschafft zu thuen , item des Handls Gegen¬ schulden und was für Zeug unverkaufft , gestaltsam ist , gründ¬ lichen Bericht geben und sonderlich sie ire hiertzue bedürftige Leuth und Diener selbs aufnemen , underhalten und besolden . Doch, dass es solche Diener sein, denen in einem dergleichen Werch zu vertrauen . Wie dann inen den Cassiern, Puechhaltern und den anderen 4 Verwalttern derselben irer Diener halben die Verant¬ wort - und Erstattung obligt. In Sonderhait aber soll es mit verinelten 4 Verwaltern oder Handls -Persohnen in irer Verrichtung dise Abthailung und Underschaidt haben ; Dass durch die 2 das gantze Wösen , was die Radund Hamermaister mit Empfahung des geschlagenen Zeugs, auch Hineingebung des Geldts und sonsten in all ander Weg antrifft, und dann die andern 2 dasjenige so zum Verschleiss desselben Zeuges gehörig gehandlet und veri'ichtet , wie auch jedem Thaill hierauf ain sonder Instruction zugestellt solle werden . [Folgen genaue Bestimmungen über die Arbeitsteilung unter den 4 Fak¬ toren .] Und was nun also negstbenrelte 4 Verwalter oder Handelspersohnen in disem allen und vermüg angeregter irer abgesonderten 1Bilanz. III . Zur Geschichte der Aktiengesellschaft im 16. Jahrhundert . 399 Instructionen und sonnsten zu ainer und der anderen Zeit hanndien werden , und wie das gantze Wösen bey Rad - und Hamermaistern und irer Arbait so woll auch mit Raichung des verschribnen Eisentzeugs von danen her , item mit Wiederverhandlung desselben ge¬ schlagnen Zeugs und sonsten allenthalben geschaffen , den berürtcrx 4 Ob-Persohnen und dann sie die Ob-Persohnen dasselbe — und sowoll wie es bey der Puechhalterey und der Cassa steth — alle Jarsquart .al uns ainem Ratt ain schriftliche Relation übergeben. Zum 11. Was von der jetzigen hieligen Puppillen 1Geldt zur Befürderung der Gsellschafftshandlung aufgenumen wirdet , das solle inen mit 5 % verinteressiert und solche Verzinsung iren verordneten Gerhaben 2 jerlichen auszalt werden und dieselben Pup¬ pillen sonsten zu ainichem Gewin nicht zuelessig sein. Zum 12. solle diser Gsellschafft Handlung wegen mit Einnemen und Ausgeben aus Empfach und Wiederverhandlung des Eisentzeugs alle Jar — underschiedlich mit jedes Aussgang — gewisslichen und on ainichen Auftzug durch den Puechhalter richtig und ordenlicher Raittung geschlossen und dieselbe, alsbaldt sie fertig , ohne weiten Aufschub durch die vermelten 4 verordneten Gesellschaffts -Obpersohnen , auch noch dartzue aus den andern Gsellschafftern , 2 des Raths und 2 der andern Burgerschafft (so hiertzue tauglich und das maiste Leggeldt in der Gsellschafft haben) bestes Vleis aufgenumen und in diser Raittung auf alle Umbstendt woll Achtung gegeben und zu solcher Haubt -Raittungs Aufnemuiul Abhandlung durch dise 8, auch die andern 4 Persohnen , so den geschlagnen Eisentzeug empfahen und verhandlen , zugleich getzogen werden . Und was nun sie die ersten 8 oder sie alle 12 Per¬ sohnen solcher Raittung halber handlen und schliessen (doch dass sie unns ainen Ratt desthalben jedes Jars ir Relation thuen ), darbei soll es mit unnserm Wissen allerdings verbleiben und hierüber der gantzen Gesellschafft oder etlichen aus inen Ichain andere Relation beschehen , sondern sich ain jeder hieran benügen lassen und verrer die wenigsten Ein - oder Widerret zn thun befugt sein. Zum 13. Was den Gwin oder Überschuss so über der Gsell¬ schafft auf lauffenden Uncosten und Aussgaben auf angeregte beschlossne und aufgenummene Haubt -Jarsraittung verbleibt , be1 =Waisen. 2 =Vormund. 400 Dokumente der l'rülvkapltaHstlschen Wirtschaftsorganisation. trifft , derselbe Gwin solle jerlich , gestrackhs und so baldt solche Raittung beschehen und aufs allerlengist in 14 Tagen nach Verstreichung des Jars ordentlich und treulich ainem jeden Gesell¬ schafter sein Gebürnuss —was aufs Hundert jedesLeggeldt khombt — gegen Quittung ausgethaillt , zuegestellt und dasselbe (ausser sonderer Nott , so der Gsellschafft in ainem oder dem andern Fall zustehn möchte , welches doch auch mit aller Gesellschafter Wissen beschehn solle) weiter nicht vertzogen noch auch vor Verstreichung der 4 Jar oder in Mittl des Jars hievon khainem nichts hinaus ge¬ geben werden. Allso auch wann ain Gesellschaffter ableibt nnd sein Leggeldt auf sein Wittib oder Kinder khumbt und feldt , dieselben auch hie in bürgerlichem Wösen sein und bleiben , so solle solchen der völlige Gwin wie ainem andern Gsellschaffter , der ein geschworner Bürger ist , in Zeit vorberürter ersten 4 Jar oder so lang hernach dasselbe Leggeldt völlig oder taills in der Cömpania bleibt , passiert werden. So aber Wittib oder Kinder nit hie hauseten , oder den bürgerlichen Wösen nit eincorporiert weren , sollen sie des völligen Gwins nit, sonder des Interesse (5 %) fähig sein. Verrer so ain Gsellschaffter Gläubiger verliess, oder noch in seinem Leben kein Leggeldt auf dieselbigen Gläubiger khäme , solle auch allein das Interesse (5 %) und nit der Gsellschaffts -Gwin den Gläubigern von der Zeit sie des Gescllschaffters Gerechtigkhait am Leggeldt wirkhlich antretten , geraicht und geraitt werden . Doch wo der , so also das Leggeldt an seiner Schuldt von dem Gesell¬ schaffter anniinbt , auch ein Bürger alhie were, solle er zum Gsellschafftgewin zugleich seinen Zuetritt haben. Zum 14. Wann ain oder mer Gsellschaffter in seinem oder irem Leben oder auf Absterben deren verlassnen Wittiben und Kinder oder ir der Kinder verordnete Gerhaben so woll auch die Gläubiger (so sie allerseits wie gemeldt in bürgerlichen Wösen sein) auf Verstreichung ob eingefürter gewissen 4 Jar sein oder ir Leg¬ gelt hinaus nemen und verrer in diser Gesellschafft nit bleiben wolten , so solle der oder dieselben solches % Jar zuvor ir, der Geselschafft aufsagen und es alsdann auf denselben Fall für und für , solanng diese Ordtnung crefftig bleibt , also gehalten werden und sein billiche Mass und Zallfristen haben . Nemblichen dass man ainem jeden Theill dasjenige Leggeldt , so sich hinder oder auf 500 Gulden verlaufft , alsbaldt in Jarsfrist , aber das , so sich III . Zur Geschichte der Aktiengesellschaft im 16. Jahrhundert . 401 über solche 500 Gulden erstreckht (wie hoch es auch sein mag) hievon alle Jar den 4. Thaill — von berürter Aufsag antzuraitten — hinaus bezallcn solle. Zum Fall aber inen , den Wittiben , Kindern oder Gläubigern dise Zallfristen zu lapng sein, so sollen inen solche Betzallungen von derselben Aüfsag an in 2 Jaren , die negsten volgendt , beschehen . Wo dann auch die Sachen mit diser Gesell¬ schafft dermassen beschaffen wer, dass die Companie mit ainer oder der andern Ausstzallung auf weniger Jar oder grössere JarsZallfristen gehn, oder ainen und den andern Taill nach Gelegenhait gar und ainsten abfertigen möchte , solle es auch beschehen. Wann aber sie, die Wittiben und Waisen , also lenger in solcher Gesellschafft bleiben wolten , soll inen dasselbe statt getan und sie hievon wider iren Willen (alleweill sie in hieig bürgerlichem Wösen sein) nicht ausgeschlossen, noch hindan gefertigt werden . Wie dann auch hiegegen die Compagnia auf ir, der Wittib und Kinder Begern solche Betzallung gehörtermassen gar under ainsten zu laisten ebenmessig nicht verbunden solle sein. Doch sollen in den¬ selben Betzallungen des Haubt -Leggeldts die gar ungewissen und für verlohrn gerechneten Schulden (so nach Handelsgebrauch auch diser Gesellschafft und der Zeit Geschaffenhait ir über angewendten Vleiss nit vermitten bleiben khünen ) pro rato , was es auf dise Suma in Abraittung zuetrifft , hievon aufgehebt werden. Zum 15. Dieweill aus villen beweglichen Ursachen diss gross durchgeent Compagniewösen nit mag noch ldian so urpletzlich zu werch gericht und gestelt werden , in Bedenckhen , dass fürnemblicli bey jetziger Unwirde und andern Obligen die Geldts -Behandlungen zumall auf so grosse Suma so zu . . . . 1 schwer und weder für Ir kays . Majestät , die fürstl . Durchlaucht , das gemain Wösen, noch uns sein würde den Termin so khurtz antzusetzen , so man den mit der Vollstreckhung nicht erfüllen möcht und daher rattsamer , ain solch Zill und Frist zu erwölln drauf sich alle Taill zu verlassen haben . Demnach ist dasselb auf Martini des eingeenten 82. Jars verglichen , bestimbt und ernent . Also dass eben auf dieselbe Zeit dis völlig Compagnia -wösen sein Wirckhung und Anfang nemen und gewinen, das ist die Privat -Hanndlung des Zeugs auf Leistung der Verlag fortgehn solle. Wie dann auch wir der Magistrat under1 Unleserlich. Stricker , Studien z. Gosch , kapitulist . Organisationsforme ». 2(> 402 Dokumente der frühkapitalistischen Wirtschaftsorganisation. dess alles so dartzue notwendig propcrieren und in Beraitschafft richten sollen. Zum 16. Nachdem auch an dem gelegen, dass zu solcher obbestiinbten Zeit der Privat -Eisenhandler Abtrett - und hergegen der gemainen Compania Antrettung mit Ordtnung bey Radt - und Ilamermaistern geschehe , so solle ain jeder under inen (den PrivatEisenhandlern ) mit seinem Hamermaister die Abraittung also treffen , dass sie auf solchen Tag Martini gestelt sey und ain jeder den negsten zur Puechhalterey deshalber ain gefertigter Schein übergebe , was jeder by dein Wösen ligen habe , damit die Compagnia volgendts sich mit dem monadliclien Zuesatz , Empfahung des Zeugs fürnemblich in der Ordinari -Verlag auf die Ordtnungen zu hal¬ ten wiss. Und so vill den geschlagnen Zeug von Stahel und Eisen berüert , welchen biss auf den Abraithtag unverhanndlet jeder PrivatEisenhändler by den Hamern in Ladtstetten und auch zu Steyr liette , soll jeder zur Puechhaltery destwegen sowoll auch was ir der Eisenhandler verwonde Hamermaister für rauchen Zeug, so erst in den clainen Hamern für Khauffmans -Guct aufgebracht werden mues, haben ain Extract oder Austzug iiberraichen , damit man wiss, was unverhandlt in Vorrat und was für Sorten es seien und solle auch bey der Compagnia und ir der Privat -Eisenhänndler Gefallen, Bedenckhen und Vergleichung stehn , solchen Zeug taills oder gar oder aber nichts hierinen antzunernen oder dartzuegeben. Und so derselbe geschlagne Eisentzeug , so auf solchen Abraittag vorhanden sein, dan auch der so erst hernach aus angeregten rauchen Zeug aufgebracht wirdet , inen den Eisenhandlern bleiben würde, so mügen sie diesen gantzen Vorrat des Zeugs, nach Inhalt der Ordtnung wie zuvor , selbs woll verhandlen. Was dan die Formbel an ir selbs — das des Werch publiée gemacht — antrifft , darinen haben die röm . Khays . Majestät als Herr und Landtfürst . die Notturfft allergnedigist dero Gefallen nach zu verordnen. Zum 17. Und wiewoll in angerüerter 79järigen Eisencommis¬ sion durcli die Herren Commissarien neben berüerter Eisenhandlung auch den hieigen Messergewerb in ain Handt und Companie zu nemen tractiert worden , dartzue wier dann unsersthaills auch nicht ungewildt gewest ; wie dann etliche und vill aus unsers Rats Mitte und der andern Privat -Bürgerspersohnen in der fürgelofnen Be- III . Zur Geschichte der Aktiengesellschaft iin 16. Jahrhundert . 403 handlung gleich sowohl anfenckhlich vor unser als hernach .vor woligedachten Herren khayserlichen Commissarien ir Leg- und Hilfgeldt anderer Gestalt nicht bewilligt , als wan angedcute Messersowoll auch die hieige Segens 1- Sicliel - und Naglhandlungen neben angerüerten Eisengewerb zugleich in die Gemainschafft uiid Compania genumen werde . So hat sich doch darsiders in ir der Herren Commissarien und dann unseren weitern gehaltenen vleissigen Berathschlagungen allerhandt Umbstendt sovill befunden , dass man anjetzo mit vcrmeltem Eisenhandl denselben völlig in solche Com¬ panie zu ziehen umb der starckhen Summa Gelts willen, so man zue Verlag und stätter Herhaltung hierzue unendtrattlich bedürfftig davon dan hieoben in dem andern Articl rliser Ordtnung Auffürung beschehen , schwerlich und nicht woll aufkhumen khüne und solle man noch datzue angeregten Messer- Segen- Sicht - und Nagl -Handl auch in dieselbe Gesellschafft ziehen würde man umb so vill weniger mit der Nottdurfft Geldt . . . . gelangen mügen . Zumall weillen dieselben und sonderlich der Messergewerb eben sowoll ausser ainer grossen Suma Geldts viller 1000 Gulden nicht zu fiiren oder herzuhalten ist und also hiedurch aines das andere hindere , derowegen so haben anjetzo aus Nott obsteender und viller anderer Ursachen und Bedenckhen halber mer , so in gehaltenen Berat¬ schlagungen weitleuffig fürkhumen dieselben Messer- Segen- Sichlund Nagl-Handlungen von angeregter Compania abgesundert , aus¬ geschlossen und allein der Eisengewerb darein gezogen werden müssen . Inmassen dann hierauf und obgehörte Meinung dise Ordtnung gerichtet ist . Wann aber solche Eisen -Compania in iren Gang khombt und man hernach sieht wie sich die schickhen wolle und es für tuelich und erschwingelich geachtet wirdet den MesserSegens- Sichel- und Nagl-Gewerb mit einander oder thaills auch daher zu nemen , so khan und solle hiemit unbegeben lautter Vor¬ behalten sein. Zum 18. und letzten . So behalten wir uns hiemit lautter bevor dise Eisenhandls -Gesellschafft -Ordtnung nach Gelegenhait khünfftiger Zeit, Leuff und Fäll und wie es dises Gesellschaffts -Wösen aines und des andern Orts Notturfft und der allgemaine hieige Stadt Nutz erfordern wirdet in ainem oder mer Puncten 1 Sägen. 26 * 404 Dokumente der frühkapitalistischen Wirtschaftsorganisation. und Artikeln doch zugleich auch mit allergnedigisten Vor* wissen höchst ernenter röm. Khays . Mt. und Irer Khays . Mt. Eisengesatzordtnung one Schmellerung zu verändern , mündern und zu meren. Zu Urkhundt haben wir vorgemelte Bürgermaister , Richter und Rath unser und gemainer Stadt Steyr grösser Insigl hiefür ge¬ druckt . Geben und beschehen etc. 405 IV. Akten zur Kartellgeschichte 17. Jahrhunderts. des 14. bis 9. König Karl II . von Neapel befiehlt dem Seneschall der Pro¬ vence , in des Königs Namen das Salzvertriebssyndikat ab¬ zuschließen , das der Pächter der neapolitanischen Salinen in der Provence (das Florentiner Bankhaus der Bardi ) und der Pächter der kgl. französ . Salinen in Aiguesmortes und der area maritima von Aiguesmortes (das Florentiner Bankhaus der Franzesi ) mit einander verabredet hatten . 6. Dezember 1301. — Cfr. R . Davidsohn, Forschungen zur Geschichte von Florenz , III . Teil , Nr . 382. — Der folgende Abdruck nach R . Archivio di Stato in Napoli , Cancelleria Angioina, Registro 116, foglio 998 oder 298 tergo: Scriptum est Senescallo Provincie , fideli suo etc . Innotuit nobis quod inter Arbisum 1 Francesem militem aliosque officiales illustris regis Francorum pro parte ipsius et Bonaecursum de Tecco, receptorem fiscalis peçunie in Provincia pro parte nostra verba fuerunt de certo trattatu societatis ineundo inter Curias predicti regis Francorum et nostram super vendendo sale proveniente de salinis dicti regis Francorum quas habebat in Aquis mortuis et circa maritimam aream 2 ac de salinis nostris quas habemus subtus a Reate , in qua societate communis venditionis eiusdem magna utilitas utriusque Curie sicut nobis est expositum procuratur . Sperantes igitur immo pio firmo tenentes quod per industriam tuam et dicti Bonaccursi negocium ipsum laudabiliter ad utilitatem nostre Curie compleatur . Volumus et presentium tenore committimus una cum eodem Bonaccurso tractatum ipsum efficaciter exequaris et compleas prout tibi et ei pro meliori utilitate nostre Curie visum erit . In aliis autem casibus in quibus etiam utilitatem nostre Curie tu et idem Bonaccursus videritis posse tractari eam ambo prosequamini sicut videritis expedire . Nos 1 Statt Albizum. 2Aream „ aus Neapel hat dafür „illam “. “ liest Davidsohn , meine Kopie 406 Dokumente der frühkapitalistischen Wirtschaftsorganisation. enim ex nunc ut ex tunc ratificamus et approbamus quiequid per vos ambos attum et gestum fuerit in premissis , dicto autem Bonaccurso super hoc alias speciales nostras litteras destinamus . Datum Neapoli sub parvo sigillo nostro die sexto Decembris XVe Inditionis (1301). 10. Die bayrische und Tiroler (österreichische ) Regierung als Monopolinhaber der Reichenhaller bzw. Haller (Inntal ) Salinen und des dortigen Salzhandeis schließen ein Kartell , in welchem sie die Preise und Conditionen festlegen , unter welchen sie an die Großhändler verkaufen dürfen . Geschlossen zu Rosenheim am 5. Aug. 1649. — Original im Münchener Allgemeinen Reichs¬ archiv sub Tirol (fürstl . Grafschaft ) 19. fase. Bl. 7 ff. Das Kartell , war zunächst nur gültig vom 1. Dezember 1649 bis 1. Dezember 1651, ist in den nächsten Jahrzehnten aber immer wieder erneuert worden . Die Erneuerungen 1. c. Bl . 14ff. Zu wissen, demnach die durchleichtigisten Fürsten . . . Maximillian , Pfalzgraf bei Rhein , Hertzog in Ober- und Nidern -Bayrn etc. dann Herr Ferdinand Carl, Ertzherzog zu Österreich etc . bei etlich Jahren hero wargenommen , dass dieienige schweizerische und andere Saltzhandlsleith , so mit dem Reichenhallischen und Tyrollischen Saltz in vassen nacher dem Podensee und selbiger enden , auch gegen dem Schweitzerlandt trafficiert , in Einkhauffung desselbigen bei ainem und anderm Orth allerhand beschwerliche Ringerung in Tax, auch Zuegaben , lange Porg und Vörtl gesucht . Wardurch beederseiths chur - und ertzfirstliche Cammergeföhl , Nutzungen und Inter¬ esse nit befirdert werden khönnden , sonder den Khauffleithen der mehriste Gewün, Nutz und Yorthail zuegangen ist , dargegen die Saltzerzeug - und Verfertigungs -Uncösten merkhlich . hinaufgestigen. Und nun solchem Schaden verners zu verbieten für guet angesechen worden , dass beede ebur - und ertzfürstliche Heusser in Verhandlung dero Saltzes ainer melieren und bessern Verstenndtnus auch freundtvötterliche Correspondenz sich verglichen . . . .In [ dieser Er¬ kenntnis sind durch die fürstlichen Räte folgende Vergleichspunkte abgeredet worden ] : „Nemblichen als die churpayrischen Abgeordnete vorderist für ein Notturft gehalten und ihrer Instruction nach . . . begert haben, dass man sich der gewisen Saltzläger halber mit einander verstehen und ex parte Tyroll ain solchen Orth wie Reitta , welcher gegen IV. - Zur Kartellgeschichte des 14, bis 17. Jahrhunderts . 407 Landtsperg nacher Lindau ain proportion habe , benennen und den Khauffleithen von Herrschaft wegen die Liferung des Saltzs nit mehr an den Podensee und nach Lindau bescheclien solle ; die Tyrollische Abgeordnete aber solches, sintemahlen es derselben Instruc¬ tion zuwider , nicht einwilligen wollen, als hat man sich derent¬ willen mit ein ander dahin verstanden , dass, obgleich wollen die fürstl . Durchl . Ertzhertzog Ferdinand Carl zu Österreich etc . und ain yeder regierender Herr und Landtsfirst der fürstl . Grafschaft Tyroll wie anno 1615 und zuvor gewest , befuegt sein (wie es auch der sub dato 4. Mai anno 1617 zu Fiessen aufgerichte Vertrag zu erkhennen gibt und noch darbei das bestendige Verbleiben hat) dero Intallisch -Hallisch Saltz durch ihre Factores und Officianten nit nur nacher Reitta , sonder auch die Ober- und Understrassen auf Khempten , Lindau und Pemble verfertigen und daselbst verkhaufen ze lassen . So ist doch die negste 2 Jahr hinumb , d. i. vom 1. Decembris diss 1649. Jars angerechnet unnzt auf den 1. De¬ cembris dess 1651. Jahrs (gleich woll angedeitter ihrer fürstl . Durchl. habenden Gerechtsam und Herkhommen ins khonnftig unpraejudicierlich) dero Saltzhanndl in aigner Verlag nit weiter als bis auf gedachtes Reitta zu fiehren , allda die Haubtniderlag anzestellen und den Khaufleithen abzegeben beschlossen und verwilliget wor¬ den . In allweg aber solle an Orthen , wo baider Thail Saltz bishero seinen Gang und Vertrib gehabt , die geringste Verhinderung oder Eintrag wider die vorige Vertrag nit erzaigt werden. Und auf dies hin ist zum andern verglichen , dass denen Schweitzund andern Hanndlsleithen oder Particular -Persohnen firder von dem 1. Decembris diss Jahrs angeraith biss zu dem 1. Decembris anno 1650 als ain ganntzes Jahr zu Landtsperg ain chur -payrisch oder Reichenhailisch Vässl Saltz von 3 Scheiben oder 7 % Fiederl , wie es der Zeit gefierth wirdt , gefilter per 9 fl. 30 kr . Reichswehrung und zu Reitta ain Tyrollisch halbs Vässl Saltz von 1%. Hallischen Fuettern per 10 fl. angedeitter Reichswehrung mit hernach stehenten mehrern Conditionen verkhauft und abgeben werden . Yedoch sollen zu beeden Thailien die Vass an Mässerey des Saltzes d. i. ain Reichenhallisfh Vass von 7 % Fiederl , des Hall -Intallisch aber von iy 2 Fuedern gannz gleich und in khain mehr Saltz dann in dem andern gestossen und gefilt sein. 3. Was in solch ernentem Tax gewisen Haubthandlsleithen oder Stätten in grosso verhandlet wirdet , sollen baide Thaill selbige 408 Dokumente der frühkapitalistischen Wirtschaftsorganisation. Khauffer dahin obligiern, dass sye zu iedem Contract ainen 4. Thaill der belaufenten Khaufsumma baar in obbemelter gueter ReichsWehrung erlegen und die ybrigen % in den negst darauf volgenten 3 Potzner Märkhten iedes mahls % Thaill daselbst in Potzen guetmachen und bezallen . Da sich aber zuetriege , dass ain Potzner Markht etwa gleich 14 Tag oder 3 Wochen nach dem geschlossenen Contract oder Saltzpartida einfielle, khann dieselbige Marcktsfrist erst auf den negst darauf volgenten andern Markht anfangen , im widrigen aber es bei erstbedeiten 3 ersten Fristen sein Verbleiben haben. Fürs 4. sollen alle forthailhaftige Glegenhaiten , so den Khaufleithen mit Zuegaben , langen Porg und in ander weg gemacht worden , auch was sonsten ain Unglaichhait in obgemeltem ver¬ glichenen Pretio zu des andern Nachthail und Verhinderung seines Verschleiss causiern mechte , hiemit genntzlicli beederseiths aufgehebt und nit zuegelassen sein, dass ohne haar Gelt oder obgemelte Porg ainicher Contract , Saltzpartida oder ungewondlicher Verschleiss vorgehe , es seye auf was weis es welle zu practicieren ; vill weniger ist in Vorgesetztem verglichenen Pretio ain Ringerung oder Abschlag ze machen. Es solle aber zum 5. die Verstenndtnus und Vergleichung ainsmahls nit lenger als auf obangeregte 2 Jahr ihr Craft und Wirkhung haben . Dergestalt zwar , dass under wehrender diser Zeit von beeden chur - und ertzfirstlichen Heussern und deroselben Saltzbeambten guet Correspondenz gehalten was fir Hindernussen und Ungelegenheiten zu beeder , chur - und ertzfirstlichen Herrschaft Schaden und Steckhung des Saltzverschleiss firgehen und ob das Purgundisch , Lottring - und Frantzöschische Möhrsaltz an die Orth und Endt wo sonsten das Reichenhall - und Tyrolisch Saltz ver¬ schlissen worden , zuvil eingehen mechte , einander vertreilichen be¬ richtet und in Sonderheit nach Verschliessung des ersten Jahrs durch Schreiben oder da es auch die hoche Notturft und des Wercklis Wichtigkheit erforderte , persönliche Abordnung und Zusammenkhonften benennen , aida berathschlagen und beschlossen werden solle, welcher gestalt das ander darauf volgende Jahr das Pretium ze setzen und was sonsten vorzunemen thuenlichen sein mechte . Gleichwoll wirdt iedem Thail bevorgestellt wanns die Glegenheit zuelasst den Tax nach seinem Belieben zuerhöchen, solle aber solches dem andern Thail verthreilichen communicieren. IV. Zur Kartellgeschichte des 14. bis 17. Jahrhunderts . 409 6. Weillen Ire churf . Durchl . sich underm dato 27. Nov . anno 1648 gegen ihren Saltzhandlsleithen genedigist resolviert dero Yässl Saltz zu Landtsperg ains per 8 fl, 30 kr . ain gantzes Jahr — es schlagen die Uncösten auf oder ab — abvolgen zelassen , als muss es bei demselben allerdings verbleiben und dahero solcher Vergleich obgeherter Gestalt erst auf den 1. Decembris diss 1649. Jahrs seinen anfang nemen . Yedoch, sintemalen ordinarie Herbst¬ zeiten die mehriste Saltzconträct gemacht werden und zu befirchten, die Handlsleith , da sy von dieser Verstandtnus und vorhabenden Aufschlag Wissenschaft bekhämen , mechten zu ihrem Vortl und beeder Herrschaften Schaden die Zeit hierumb solche starkhe Conträct zu machen begehren , wardurch beeden chur - und ertzfürstl. Herrschaften der verhoffente Nutz woll auf ain gantzes Jahr lang entzogen werden khöndte . Dem aber zu begegnen ist für rhatsamb gehalten , dass diejenigen Conträct , so ain und andern thails beraith beschlossen und darum !) die Fristen benent und verglichen worden, bis auf den lessten ' negstkhommenten Monats Septembris gleichwoll ihr Richtigkheit und Verbleiben haben sollen. Was aber von dem darauf volgentem 1. Octobris biss zu End des lessten Novembris diss gemelten 1649. Jahrs in obgedachtem churpayrischen oder Tyrollischen bishero observiertem Pretio an gemeltem Vässl-Saltz erhandlet werden wolte, solle selbiges zu Landtsperg und Reitta anderer Gestalt nit , als gegen baar Gelt abgeben , damit die Khaufleith umb sovil mehr mit den Haubtcontracten auf gedachte Zeit dises Vergleichs Anfang gelaitet werden. Und nachdeme zum 7. die gemaine Uncösten in Lindau sowoll von dannen auf das Schifflohn yber den Podensee sehr gestaigert worden , welches neben dem ietzigen Aufschlag den Saltzhandls¬ leithen beschwerlich fahlen mechte , hat man fir guet angesechen, dass bei Anfang dises Vergleichs von baiden chur - und ertzfirstlichen Herrschaften an die Stadt Lindau Erinderungs -Schreiben abgehen solle, berierte neuerlich erhöchte Zoll- und Uncösten wie auch das Schifflohn in denienigen standt zu stellen , wie es vor Anfang des langgewehrten teutschen Khriegs gewest. Wann sich zum 8. zuetragen würde , dass ein Saltz -Khaufmann bei ainem Thail sich in ainen Conträct eingelassen hette oder noch ainen alten Rest , welchen er vor Aufrichtung dises Vergleichs schuldig verbliben , nit bezallen , auch khonftig mit den accordierten Fristen oder gebihrendem Laggio nit zuehalten wurde und daher 410 Dokumente der frühkapitaüstischen Wirtschaftsorganisation. sich von solchem Orth abschweifig machen und bei dem andern Thail anmelden wolte , als dann soll auf dergleichen zuetragenten Fahl derjenig von jedem Thail benant und ain solches zu wissen gemacht , deme aber so dergleichen abschwaif zu suchen begert, so lang und vill khain Saltz abgevolgt werden , bis er beglaubte Schein fürweisen , dass er hierumb gebihrundte Satisfaction er¬ stattet und alles richtig gemacht habe. Damit man auch jederweilen Wissenschaft haben khönnde was gestalten disem Vergleich nachgelebt und ob solcher observiert werde , oder was darwider in Einführung frembten Saltz und an¬ derem Vorgehen mechte , als stehet jedem Thail bevor ain aigene Persohn auf dero Costen zu Lindau oder anderwertig aufzustellen und ir derentwegen die Notturft zu bevelchen. Lesstlieh weil diser Vergleich allain auf beeder chur - und ertzfürstl . Durchl . genedigiste Ratificationen gestellt , als werden dero Resolutionen hieryber mit negstem ervolgen und haben sich vor verfliessung der 2 Jahr beede chur - und ertzfürstl . Durchl . zeitlich genedigist zu welern , ob, wasgestalt und wie lang forthann diser Vergleich weitters observiert und beederseiths bestendig gehalten oder was des Saltzpress \ Porghalber und in anderen Puncten dem Verschleiss zum besten und zu Vermehrung beeder Chur- und Ertzfirstl . Häuser Cammergeföhl und Nutzen verändert und weiters angestellt werden mechte. Dessen zu Uhrkhundt haben beede chur - und ertzfürstl . abgeordnete Commissarii sich mit aignen Händen hier underschriben und dero Pedtschaft fürgetruckht . So geschehen zu Rosenhaimb den 5. Augusti im Jahr 1649. [Folgen die Unterschriften der Kommissare und deren Siegel.] Zu dem erneuten Kartell -Vertrag von 1649 (erneuert am 5. October 1651 zu Kufstein ), der vom 1. Dez. 1651 bis 1. Dez. 1653 gelten sollte, wurde folgende bedeutsame Zufügung gemacht , die eine der wichtigsten Schattenseiten des Kartellwesens auch von heute noch zeigt : den billigeren Verkauf ins Ausland (1. c. Bl. 25 ff.) : „Obwohln nun vorangedeuter massen . . . widerumben beschlossen worden , dass . . . Rosenhaimbscher Vergleich abermahlen auf 2 Jar lang gehalten werden solle, weilen aber sonnderlich auf Chur-Bay¬ rischer Seiten vorgebracht worden , dass man daselbsten in dem i = Salzpreis. IV. Zur Kartellgescliichte des 14. bis 17. Jahrhunderts . 411 Saltzverschleiss gegen den Burgund - und Lotringischen Landen dise negst verstriehne 2 Jahr in den Verschleiss ain zimblichen Abgang befunden und selbiger Orthen zu befürchten , es noch merer ins khonftig beschehen mechte , als haben beede Thail innen Vor¬ behalten , was von ainem oder andern Salz über Bern , Basl und Solothurn weiter hinein gegen Burgundt und Lothringen verhandlet und durch angedeuter Stät glaubwirdige Urkhundten ordenlichen bescheindt wirdet , solches Salz nirgendts annderst wohin als in dieselbige verne Landt vertriben worden , dass ain und anderer¬ seits daselbsthin und auf ernennte Bescheinung jedes Vässl Salz zu Landtsperg und Reita umb 30 Kreizer ringer oder wolfailer als obgedachter verglichne Tax mit sich bringt , abgeben und verkhaufen mag . Jedoch dass in disem Fahl fleissiges Aufsehen be¬ stölt und gehalten werde , damit hierdurch von den Saltzhandelsleithen und Stäten die besorgende Vortailigkhaiten nicht veryebt, und selbiges Salz nit in die andere heerwerts gedachter 3 Stät ge¬ legne Lanndtschaften und Orth eingefiert werde und da solches in Wissenschaft khomen , alsdann dise 30 Kreizer Nachlass widerumben aufgehebt und beratschlagt werden solle, wie dem Werckh in ander Weg zu helfen. 11. Kartelivertrag der bayrischen und Tiroler (österreichischen) Regierungen als Monopolinhabern der Reichenhaller bzw. Haller (Inntal ) Salinen und des dortigen Salzhandels mit den Pächtern der burgundischen Salinen. 3. Nov. 1659. — Original im Münchener Allgemeinen Reichsarchiv sub Tirol (fürstl . Graf¬ schaft ), 19. fase. Bl. 67. Zu wissen. Nachdem die baide hochlöblichiste Chur- und Ertzfürstliche Heuser auf beschehens Ersuechen der jetztmahligen Fermiers des Burgundischen Salzwesens zu Salins sich gnedigist entschlossen und eingwilligt , einer mündlichen Conferenz des Salzverschleiss halb zu Khempten in Allgeu durch dero deputierte Rhäte mitzusein , also ist am heut hernachstehenden Dato zwischen hochgedachter Chur- und Ertzfürstl . Herrschaften Abgeordneten an einem und dann der beeden anwesenden Burgundischen Fer¬ miers ........volgendermassen abgeredt und beschlossen worden. Nemblich und für das erste haben beede Burgundische als Mitprincipale der Salinischen Salz-Admodration eröffnet , dass sy mit dem Orth und Stand Bern uf virthalb Jahr und dann mit 412 Dokumente der frühkapitalistischen Wirtschaftsorganisation. Solothurn , Freiburg in Jechtlande und Grafschaft Neuburg auf 6 Jahr lang in gewisen Salztractaten begriffen , welche sich auf khein Weise rescindiern oder zurugg nehmen lassen . Darbei man es disfals jedoch mit der Bedingung bewenden lassen , dass durch obgedachte 4 Orth alles das Salz, so sy von denen Burgundischen Fermiers jerlich nemen weithers nit als in jedes Orths oder Standts aignen District und Gebieth verbraucht und in khein ander Gebieth weder in Yessln noch dem Ausmass nach verkhauft werde . Welches sy, Fermiers , also zu effectuicren obligiert sein sollen. Und obwoln die Fermiers praetentiert und begert haben , dass ingleichen an Seithen der chur - und ertzfürstl . Heyser weder in Yass noch dem Ausmess nach in obbemelten Orth und Cantonen khain Reichen¬ hall - noch Hall -Intalisches Salz solle verkhauft werden , so haben doch die churbayr . und ertzfürstl . Abgeordnete die clausulam nit eingehen wollen, sonder allein ad referendum genohmen. Fürs ander ; die übrige Orth in der Schweiz betr . haben vor¬ gedachte Fermiers sich obligiert das Precium [desjenigen Saltzes, so sy nit in yetztgemelte 4 Orth sonder weithers in andere Cantones und Gebieth verführen und sie selbsten oder andere verschleissen mechten , dahin einzurichten , dass ein jedes Vessel ringer nit als per 19 guet Schweizer Gulden in Solothurn khomen und daselbsten solchergestalten verkhauft werden solle, weihlen das Reichenhall und Hall -Intalisch Salz bis nacher dem Baslichen und Solothur¬ nischen auch selbiger Enden wenigist bis auf 22 Gulden gedachter Wehrung steigen thut . Es soll zumahlen auch khain Tail ohne des andern Vorwissen und Consens umb ain geringem Tax ainiches Fessel nit verwenden. Ferners und zum driten ist verglichen , dass die Burgundischen Fermiers von iren Contrahenten vigore diser Verstendnus allain das baare Gelt und khain andere Werth oder Zallungsmitl , wie das Nahmen haben mag, für das Salz annehmen und ihnen die 1. Zallungsfrist : nemlich den 4. Tail der völligen Khaufsumma gleich bei Beschluss des machenden Contracts erlegen lassen , die übrige 3 Fristen aber in negst nacheinander darauf volgender 3 Jahrs - Quartale yedesmahl einen Teil zu empfangen haben . Auch baide chur - und ertzfürstliche Heuser wie nit weniger die Fermiers denen Saltzhandlsleuthen bei Machung der Conträct einichen For¬ theil , es sey mit Eingab , Lengerung der Porg oder wie es sich in ander Weeg directe vel indirecte begeben mag, nit ervolgen oder IV. Zur Kartellgeschichtc des 14 . bis 17. Jahrhunderts . 413 zu guetem khomen lassen sollen. Und soll diser Vergleich von Zeit der allerseiths eingeloffne Ratificationen in Crefften verbleiben ein ganzes Jar lang . Darbei bedingt worden , dass die Burgundische Fermiers deren Genembhaltung oder Erclerung zum ersten und zwar von Dato geraith innerhalb 6 Wochen zu Händen der ertzfürstl . Deputierten nacher Insprugg einsenden sollen, welche als¬ dann von dar nach Bairn zu schückhen und ihnen , Fermiers von baiden chur - und ertzfurstl . Herrschaften hinach die Ratification oder Resolution gleichsfahls unverlanget nacher Salin zu remittieren widrigen Falls soll obbedeihter Tractat nichtig sein ..... Zu Urkhundt sein dises Vergleichs 3 Originalia in theutscher und 3 in Französischer Sprach 1gleichen Lauts . . . geschriben von denen churbayrischen und ertzfürstl . Abgeordneten wie nit weniger beden Burgundischen Fermiers mit Handschrift und Pettschaft gefertigt und jedem Tail 2, nemblich ein teutsch und französisches Exemplar zu Händen gestellt worden. Actum Khempten im Allgew den 3. November Anno 1659. [Folgen die Unterschriften der Abgeordneten und Fermiers .] 1 Ein solches Original in französischer Sprache befindet sich im Münchener Allgemeinen Reichsarchiv. Loc. Tirol (fürstl. Grafschaft) 19. fase. Bl. 1 ff. 414 V. Dokumente der kapitalistischen Wirt¬ schaftsorganisation im sächsischen und böhmischen Zinnhandel des 16.Jahrhunderts. 12. Herzog Georg von Sachsen bestätigt die Gesellschaft des Zinn¬ handels . 6. März 1498. — Konzept des H . St . A. Dresden. Loc. 7414. Den Zinnhandel betr . Bl. 9. Wir George etc. bekennen : Nachdem durch unser liben ge¬ treuen ein geselschaft eins zcenhandels , keufens und verkeufins aufgericht , im besteu fürgenomen und uns vorgetragen ist mit de¬ mütiger bete , solchin aufgerichten und fürgenomen handel und geselschaft gnediglich zuzulassen und zu bestetigen . Dieweil wir dann mit zceitigem rate bedacht , dass solch fürnemen gemelter geselschaft erbar sei, aus gutem gründe zur fürderunge gemeins nutz der lande und zu aufrichtunge und merunge unser zcenden und bergkwergk fürgenomen wirdit , haben wir aus fürstlicher oberkeit und macht solchin handel und geselschaft gnediglich zcugelassen und bestetigt . Wullen auch solch geselschaft gnediglich hanthaben , schützen und vorteidungen gleich andirn unsern lantsassen und undirtanen , auch nachfolgende stügk und artikel der geselschaft zcu gute und nutz also gehalden haben : Nemlich dass die diner nichts vorpurgen als wi es inen von den verordneten der geselschaft befohlen wirt . Item die diner sollen auch denselbigen verordneten alle virteljars oder so oft solchs an inen gesonnen wirdt , wie der handel stet , underrichtung thun . Wir wullen auch, dass die diner der geselschaft einen slüssel neben dem wagkmeister zcu der wage uffm Aldinberge haben sollen, uff dass kein zcen hinder irem wissen in andir weise hinwegk gewogen werde , bis dass inen die bezcalunge getan wirdet von den, die inen schuldig sein und dass solch zcen, so daselbst gesmelzt , nicht an andirn orten gewegen noch ungewegen hinwegk gebracht werde. Item wir wollen auch , dass die diner angezceigter geselschaft allein des handeis warten und sich nicht sunderlicher eigener handel V . Im sächsischen und böhmischen Zinnhandel des 16. Jahrhunderts . 415 undirstehen . Darumb man inen destemehr Ions geben solle und ob sie geld in die geselschaft legen wullen, sal in irem gefallin stehen. Item die diner sollen auch die rechnunge alle jar uffs naw jar beslissen und den virzcenden tagk nach dem nawen jarstage obirantwurten . Und dieselbige rechnunge sal alle jar zu Dressen durch die hern der geselschaft , die darzcu körnen, ader ire vollcmacht schicken , verhört und denselbigen abschrift gegeben werden als vil inen not ist. Item wer der geselschaft beweissliche oder bekenntliche schuld schuldig ist , dcrselbige sal bezcalen . Wo das nicht geschieht , so wullen wir liiemit allen unseren amptleuten , die deshalb ersucht werden , befohlen haben , zeu irem gute , wu des nicht ginnge zeu irem leibe ane gerichts forderunge zeu verhelfen nach der gesel¬ schaft adir irer diner aneweisunge , uff dass sie irs hauptguts und Schadens daran bekomen . Wenn auch den dinern hendel und gescheft vorkomen , darin sie sich nicht wol zu vorwaren wüssten, sollen sie sich an denen so darzu verordent ader an den herren der geselschaft ader bei uns, ader bei unsern reten rates erholen, den wir inen auch gnediglich, so oft solches die nottorft erheischt , wollen mit teilen , auch diese obberiirte geselschaft allenthalbin fördern..... Zu urkund haben wir des gemelten unseres lieben herrn und vaters insigil, das wir hieczu gebrauchen , wissentlich anhängen lassen. Der geben ist zu Dresden am Dinstag nach Invocavit anno etc. XCVIII. 13. Gutachten des Dr. Tileman Brander über die Gesellschaft des Zinnhandels . Um 1500 abgefaßt . — Kopie im Kgl. Staats¬ archiv Königsberg . Msc. A. 34 fol. Bl. 204 ff. De societate stanni. Cristi nomine invocato casus facti proponitur . In territorio Missnensi est quedam societas negotiationis stanni et ex qualitate personarum et ex magnitudine pecuniarum plurimum insignis. Quae eidem negotiationi duos institores seu factores deputavit et ipsi negotiationi prefecit et praeposuit ad emendum , vendendum aliosque contractus ineundum . Ac institores nomine eiusdem societatis per plures annos emerunt , vendiderunt aliosque contractus per¬ fecerunt . Pecunias in usus societatis receperunt et dantes illas in societatem admiserunt , sociis eiusdem societatis scientibus , patien¬ tibus et non contra dicentibus . Praeterea dicti institores matri- 416 Dokumente der frühkapitalistischcn Wirtschaftsorganisation. culam in qua socii descripti 'sunt apud se habuerunt et. unumquem¬ que pro tempore ad societatem recipiendum cum pecuniarum summa inibi describere consueverunt . Et omnes de societate volentes scire qui essent socii ad matriculam illam recursum habue¬ runt . Deinde spectabilis et eximius utriusque juris doctor dominus Cristoferus Cuppener , sindicus senatus et communitatis Brunsvicensis ad eandem societatem recipi cupiens , ut esset tam lucri quam damni eiusdem pro rata summe pecuniarum suarum particeps eisdem institoribus duo milia fl. rh . in usum societatis huius modi •tradidit et assignavit et se in dicta matricula societatis describi obtinuit et in eadem societate per annum et sex septimanas abs¬ que cuiusvis reclamationibus seu contradictionibus quiete fuit et lapso anno distributionem lucri societatis pro rata summae 2000 fl. habuit et recepit . Ipsi etiam institores eandem pecuniarum sum¬ mam in usus societatis expenderunt et cum illa , negotiati sunt. Postremo nonnulli domini societatis praedictae socii accipientes institores praenominatos clam fugisse ipsum dominum Cristoferum a societate ipsa repellere conantur allegantes quod non sit ad eandem de voluntate sive consensu sociorun admissus . Ad quod respondet. Id factum esse de consensu sociorum attento quod pecunia sua in usus societatis conversa sit . Ipse etiam ut prefertur matricula lus distributionem lucri pro rata pecuniarum suarum pariter cuin ceteris de societate habuerit prout ex libro rationum ipsius societatis per¬ spicue liquere commemorat . Ex premissis dubitari contingit an prefatus dominus Cristoferus a societate prcdicta sit repellendus. [Folgt die juristische Begründung , daß Chr. Kuppener zu Recht Mitglied der Gesellschaft sei.] 14. Gutachten des Dr . Christoff Kuppener über die Gesellschaft des Zinnhandels . Um 1500 abgefaßt . — Kopie im Kgl . Staats¬ archiv Königsberg . Msc. A. 34 fol. Bl. 199 ff. Casus in consultationem oblatus talis est : Est quedam nota¬ bilis et specialis societas in terra Missnensi vulgariter nuncupata „de Gesellschafft des Zcynnhandels “, id est societas super negotia¬ tione stanni . Cuius quidem societatis domini eiusdem duos insti¬ tores seu factores sc. Titium et Sempronium ad contrahendum emendum et vendendum eidem societati praeposuerunt . Prout idem institores seu factores ita per plures annos contractus fece¬ runt , emerunt et vendiderunt . Necque unquam constabat , prout V. Im sächsischen und böhmischen Zinnhande ! des 16. Jahrhunderts . 417 nec hodie constare creditur, de limitato seu specivocato eorumdem institorum mandato, licet hodie domini societatis verbo hoc alle¬ gant sc. institores limitatum habuisse mandatum quod tamen pro¬ bare non possunt. Item matriculam illius societatis in quam domini eiusdem societatis matriculari consueverant iidem institores apud se habue¬ runt ad socios matriculandos; dantes nichilominus unicuique ex sociis citra matriculationem specialem recognitionem sue imposite summe etc. de manu et sigillis institorum. Accidit nunc quod Ticius 2000 fl. summam pro lucro et damno in eandem societatem stanni per organum et manus dictorum in¬ stitorum imposuerat ut clare litera per institores ipsi data hoc probaverit. Que pecunia sua longe ultra annum in eadem societate fuit et cum eadem ut creditur negociatum sit. Item matriculatus est in matriculam eiusdem societatis de eadem manu unius institoris de qua ceteri domini societatis matriculati sunt prout ipse se ita matriculatum personaliter vidit et legit. Item pecunia sua ultra annum et ebdomadas sex in eadem so¬ cietate fuit sine aliqua reclamatione dominorum de societate. Cum tamen iuxta principis terre Missnensis confirmationem super eadem societate traditam de quartali unius anni in aliud quartale semper domini eiusdem societatis rationis computum facere teneantur et defectus videre. Item post annum primum qui erat de anno domini etc. nona¬ gesimo octavo decursum sc. in diem circumcisionis anni currentis sc. nonagesimi noni quo tempore distributio lucri dominis de eadem societate iuxta uniuscuiusque summe estimationem fieri consue¬ verat, ipsi pro estimatione summe sue sine aliqua reclamatione dominorum societatis prout uni alteri ex dominis distributio facta et data est. Item in libro rationis et compute pro eodem anno decurso sc. nonagesimo octavo in quo libro de acceptis et expositis dominis singulis annis ratio consueta est fieri ita bene matriculatus invenitur et inscriptus cum sua summa prout alter ex dominis eiusdem so¬ cietatis. Item licet illustris princeps dominus Georgius dux Saxoniae eandem societatem stanni confirmavit et notabiliter privilegavit tamen pecunia sua longe ante principis confirmationem in eandem societatem fuerat imposta prout datum litterarum clare hoc ostenStrieder , Studien z. Gesoh . kapitaliat . Organ isationsformen . 27 418 Dokumente der friihkapitalistischen Wirtschaftsorganisation. derint etc . Queritur nunc quantum ad primum punctum scilicet cum Ticius 2000 fl. summa pro lucro et damno in eandem socie¬ tatem stanni per receptionem , organum et manus dictorum insti¬ torum imposuerat , utrum domini illius societatis qui tales institores huic negotiationi praeposuerant eundem Titium repellere possunt ab eorum societate quasi non consortium eiusdem societatis stanni et ita eidem in sua pecunie summa proiudicare . Et videtur michi dicendum quod non . Ex quo domini societatis passi sunt, per plures annos tales praepositos et institores a volentibus coire socie¬ tatem pecuniam recipere et eos societati immatriculari prout , litere et recognitiones eorundem institorum desuper sociis tradite et matri¬ cula societatis hoc clarissime probare nituntur . Cum scientia et pacientia eius qui prohibere potest pro consensu habetur . [Folgt juristische . Begründung .] 15. Das Monopolprivileg Herzog Georgs von Sachsen für die Ge¬ sellschaft des Zinnhandels . Datiert vom 14. Sept . 1500. — H . St . A. Dresden , Loc . 4491. Allerhand Privilegia und Befreyungen in Bergwereks -Sachen de ao. 1500—1681. Blatt 1 u. 2. Von gots gnaden , wir Georg, hertzog zu Sachsen etc . anstadt und in voller macht des hochgcbornen fürsten , unsers lieben hern und vaters , herren Albrechts , auch hertzogen zu Sachsen etc . be¬ kennen offintlich vor allirmenigklich an diesem unsern brive , nochdem durch unser retlie , underthan und lieben getrauen ein geselschaft eins zcinhandils , kauffens und vorkauffens im besten aufgericht und furgenomen , die uns furget.ragen ist , mit demuttiger bethe , denselbigen aufgerichten furgenomen handel und geselschaft genedigklich zuzulassen , zu vorgonnen und zu bestetigen . Dieweil wir dan mit zeeittigen rathe bedacht , dass sollich furnemen gemelter geselschaft erbar sei, auch aus guten gründe zu furderunge gemeins nutzes der lande , auch zu aufriclitunge und meninge unsers zeehenden und bergwergks furgenomen wird et : haben wir aus fürstlicher oberkeit und macht sollichen handel und geselschaft genedigklich zugelassen und bestetiget , lassen denselbigen handel und geselschaft zeu und bestetigen den aus angezeeigter unser fürstlichen oberkeit , macht und gewalt hirmit in craft dieses brives. Wollen auch solliche geselschaft genedigklich hanthaben , schützen und vorleidigen , gleich anderen unseren lantsessen und undirthan, aucli nochvolgende stucke und artickcl der geselschaft zu gute und V. Im sächsischen und böhmischen Zinnhandel des 16. Jahrhunderts . 419 nutze also gehalden haben : Nemlich allis zcen, das in unseren landen gewonnen ader uffbereit wirt , sali von dato drei jar langk den herren der geselschaft überreicht werden . Die sollen vor bereit zcen auch bereit gelt geben , vor itzlichen centner XI rh . gülden, halb meisenisch und den anderen teil behemisch gelt. Wellicher aber vorlegunge bedarffe , was derselbigen vorlegunge uff genügsamen vorstandt in jarßfrist zu bezcalen angenommen wirt , das sal nicht, andirs dan 1 centner an ein ort vor x fl. 1 bezcalt werden . Welliche aber auf ein halb jar Vorlegung annemen und uff gewilleten temin bezcalen , die sollen vor 1 centner x fl. haben , wu aber auf den ernanten termin des halben jars nicht bezcalung geschiet , alsdan sal es gleich den die vorlegunge uff ein jar genomen , gehalden werden . Alle die uffs gantze ader halbe jar vorlegunge nemen , dieweil sie nicht bezcalen , so sal allis zcen, was mitlerzce.it gemacht ader aufbereit wirt , der gcselschafft überreicht und an der schult abgerechindt werden . Und ab ymandt . bei anderen vorlegunge nemen worde , dennoch sal dasselbige zcen, wie obin angezeeigt , in die geselschaft gereicht werden . Und dass die diner nichts vorborgen noch vorleyhen , denne wie es ehn von dem meistenteil von der geselschafft befohlen wirt. Wir wollen auch , dass die diner der obberurtten geselschaft einen schlussel nebin dem wogkmeister zu der woge uffin Aldinberge haben sollen, uff dass kein zcinn liinder irem wissem in ander weise hinwegk gewegen, bis dass ehn die bezealunge von den die ehn schuldig sein, gethan wirdet , dass auch sollich zcin, so doselbst geschmeltzt , nicht an anderen örteren , gewegen noch ungewegen, hinwegkgebracht werde und dass die sehmeltzcr uff den Geusinge und andirswo , dohin gehaldin , dass sie alle wochin wöchentlich dem bergkmeister und zeendener angeben , wie vill pallen zcen und wem sollich zcen gemacht und geschmeltzst wirdet , domit unseren zeenden kein abebruch gescliee. Wir wollen auch , dass die diner angezeeigter geselschaft alleine des handeis warten und sich nicht sunderlicher eigener hendel undirstehen , dorumh man ehn distermehr lohens geben solle und ab sie gelt in die geselschaft legen wollen, sal in irem gefallen stehen. Die diner sollen auch die rechnunge alle jar auf den sontag exaudi beslissen und rechnung thucn und diesclbige rcchnung sali alle jar 1 Das heißt 10 11 — t ort - 9 % ll. 21 * 420 Dokumente der frühkapitalistischeil Wirtschaftsorganisation. zu Leiptzigk durch die herren der geselschaft , die darzu kommen ader ire volmaeht schigken, vorliort und denselbigen abschrift , als vil ehn not ist , gegeben werden. Wer auch der geselschaft beweisliche ader bekentliche schult schuldigk , derselbige sal bezcallen . Wo das nicht geschieht , so wollen wir hinnit allen unseren amptleutten , die derhalben ersucht werden , befolen haben , zu yren gutteren ader wo die nicht genug¬ sam weren, zu iren leiben , nach der geselschafft addir irer diener anweisunge , ane gerichtsforderunge zu vorhelfen , uff dass sie ires habtguts und Schadens daran bekommen mögen . Wo auch den dineren hendel und gescheffte Vorkommen, dorinne sie sich nicht wol zu vorwaren wüsten , sollen sie sich an dem, so darzu vorordint, ader an den herren der geselschaft , addir bei uns, addir unseren rethen rathts erhoben , den wir ehen auch genedigklich , so oft das die notturfft erfordert , wollen mitteilen. Ob auch ymandt sein gelt gar ader eins teils widerumb aus der geselschaft nemen wolt, der sal das ein gantze jar zuvor den vorordenten addir dineren angezceigter geselschaft abeschreiben. Alsdenn, nach abschreybunge des jars , sal ym sein eingeleit gelt an barem gelde, an zcinn und an schult mit gewinne und vorlust, wie den der handel angezceigter geselschafft auf die zceit stehin wirdet , oberantwort und gereicht werden . Und domitte sollichs allinthalben diste fleissiger gehaldin werde , wollen wir die angezceigte geselschaft allinthalbin genedigklich furderen , schützen und handthaben , als treulich und ungeverlich . Zu urkunde haben wir des gemelten unsers lieben herren vaters insigel, des wir hirzu ge¬ brauchen , wissentlich an diesen briff hengen lassen , der geben ist zum Schellenberg am montag exaltacionis sancte crucis anno do¬ mini XV cten. 16. Projekt einer großen sächsischen Zinnkaufsgesellschaft und eines Kartells mit den Beherrschern der Schlackenwalder Zinn¬ produktion . 1518. Kleines Papierheft . Auf dem Umschlag: „Ein bedencken wie ein zinnkauf auf dem Altenberg wiederum angerichtet werden könnte 1518“ . — H . St . A. Dresden , Loc. 7414. Den Zinn -Handel betr . 1497—1544. Bl. 23 ff. Auf fürstlicher durchlauchtickeit begeren volgt das bedencken wie und durch wen widerumb ein zinnkauf auf dem Aldenbergk angericht und getrieben ; darmit seiner fürstl . gnaden zehenden V. Im sächsischen und böhmischen Zinnhandel des 16. Jahrhunderts . 421 und daselbst das volck erhalten und aus der not, darinnen die armen leut yetz sein, erlöst werden möchten. Darzu gehören vor¬ nehmlich vier ding: Erstlich, dass nicht allein dasselbig, sonder alles zin, so im fursthenthumb gemacht in disen kauf und also in ein hand gebracht und dass von dem landesfürsten ein zimlich mittel getroffen, darmit in stendem kauf kein ander ader frembde zin in dem fürstenthumb verkauft oder auch dardurch in die Schlesien, die Marek, in Sachsen1oder an die sehe gefürt. Und dass sollicher handel auf ein christenliche ursach gebauet, also dass darinnen vornemlich ein gemeiner und nicht a i g n e r nutz gesucht und niemandes wider die billickeit zu rechten gedrungen werd. Zum andern: so gehören darzu leut, die ein summa gelts zusamenlegen zu gwin und Verlust, und dieweil der kauf stehen würde, bei einander liegen lassen. Dieselbige summa müsste auf die summa des zinn gestalt, so viel man sich vorseche, dass ein jahr tausent Zentner möcht geliebert werden. Wirt ge¬ achtet, dass man auf jedes tausend Zentner ain 12 000 fl. legen. Dan man müsste das zinn bar bezallen und meisttheils verborgen, auch auf furlon und ander unkost gelt im vorrat haben. Und müsst das zinn allenthalben über 2—3000 Zentner das jar nicht ge¬ macht, sollte es anderst bei disen schweren läufften und der großen unwirde widerumb zu wirden gebracht und mit nutz Vortrieben werden. Das würde an disem ort auf 36 000 fl. laufen. Zum dritten : So müsste man zu Leiptzig ader anderswo zween hendler erwelen und vermögen, dass sie denselbigen handel, faktor und diner, allenthalben regirtten. Denen müssten zween von des fürsten rethen zugegeben werden, die sunst in den merckten von s. f. gn. wegen zu Leiptzig sein, ob Sachen vorfielen, darzu ihnen ihr radt und hilf von nötten, dass ihnen der mitgeteilt würde. Und dass sie nicht allein in den merckten, sonder auch darzwischen, wann es ihnen von nötten, ihre Zuflucht zu ihnen haben möchten. Und zum vierten : dass eine guete Ordnung und ein kreftig Verschreibung darüber aufgericht würde, wer rechnung halten, wer dieselbigen vorantwurten, wan und vor weme man die thun, wie man es mit austeilung des handeis, wann der kauf ein ende lief, halten sollte sambt anderen notdürftigen punkten und artikeln. 1Das heißt niedersächsischer Kreis. 422 Dokumente der frülikapitalistischenWirtschaftsorganisation. Den ersten artickel muss man gar wol bewegen. Wan man von der rechtvorstendigen1schon ein gueten grundt hat, dass sollicher einhendiger handel sambt Sperrung und Stei ¬ gerung der war utnb des ennthalts der armen und anderer ursach willen one alle beschwerung der gewissen getrieben werden, dass man auch sollichs erhalten möchte. Also wan man schoen die stras an einem orte sperte oder mit aufsalz eines Zolles die war draussen behielte ader in also steigerte, dass man des landes wahr darmit keinen fahl machen mochte; dass sie nicht etwan durch das Churfürstenthumb ader auch durch die krön zu Behem darmit in die Schlesien, Marek ader Sachsen und also ire war an die sehe und fort ins Nyderlandt bringen möchtt (auf Nuremberg und von dannen aufm Maien oder Rein bis gen Köln bette ich nicht gross sorg, dan es were dan nocht weit auf dem landt, so hat es auch am Rhein grosse zoll). Wa man aber die oberkeit im churfürstenthumb durch zimliche mittel (als dass sich etzliche dem land und ihnen, auch zur fürderung des zehenden in diesen handel begeben) dahin bewegt werden möchte, daselbst auch darüber tzu halten, so wurde es meins achtenns keinen fall haben. Dan auf Nürmberg künde das frembde diesem zinn wenig schaden thun, so wurde es meins achtens durch die krön zu Behemen auch weitleftig an die see und in Sachsen 2zu bringen. Aber in der Schlesien möchte es diesem handel dannocht schaden, da sein dan viel Vortrieben wirtt. Wan aber die zu Sehlackenwaldt, dahin zu bewegen weren, dass sie des jars nicht mer dan an 2—3000 Zentner machen und auch glauben halten und sich die auf dem Aldenberg an 1(4—2000 Zentner des jars zu machen, genügen lassen und zu Eberdorf 1000 zentner, so weite ich dannoch achten man solte des jars zu Nürembergk 2000 zentner Schlackenwalder zinn mit guettem nutz vorschleissen und 2000 zentner im Nyderlandt, das übrig hie im landt ader auch 1000 zentner in als. Und es were fürwar meines achtenns denen von Schlackenwalde nützer, 3000 zentner mit zimlichen nutz zu vortreiben dan 6000 zentner mit nachteil. Können ihnen doch die zwitter 3 nicht entlaufn, so kriegt die herrschaft 1 Das heißt von den Kennern des kanonischen Rechts. 2 Soll heißen niedersächsischer Kreis, Magdeburg usw. 3 Zinnzwitter = die Zinnstein enthaltende Gesteinsmasse. 3* V. Im sächsischen und böhmischen Zinnhandel des 16. Jahrhunderts . 423 mit der zeit ihr gebur auch darvon , so können sie auch ain Zentner wohl eins florin neher 1 erzeugen , weder 2 die ziner im fürstenthumb . Wan es dan durchaus ein kauf, so were es inen ja wol treglich und darnach nicht von nöten das landt zu sperren, damit die geleit zu schmelern und den Leiptziger marckten abbruch zu thun. Auf den andern artickel müsste man auch gedacht sein, wie leutte und gelt aufzubringen weren . Und demnach Bartlome Welsers geselschaft gar nicht mehr gewilligt ist , sich voriger gestalt zu diser zeit in einen zinnkauf einzulassen und villicht Publer mit seinem anhang das seine auch an andere ort gewant hat , so müsste man meins achtenns aus dem fürstenthumb hin und wider leut auslesen , da jeder ein zimlich summa (die ihme an anderen seinen handeln unverhinderlich were) legte . Und zu forderst als ein beschützer des handeis: Mein gnediger herr , hertzog Georg zu Sachsen mit 4000 fl., herr Ernst von Schönberg 3000 fl., herr Rudolph von Bünau 2000 fl., der von Karlewitz 1000 fl., der Kantzier 8 1000 fl., der von Thaubenheim 2000 fl., herr Hans von Werter 2000 fl., und sunst noch ein Tunf vom adel 5000 fl. Summa : 20 000 fl. Zu Leiptzigk. Andres Pflug , ambtmann 2000 fl., M. Publer und geselschaft 4000 fl., Heinz Scherle 2000 fl., Straub und gesellschaft 2000 fl., Barttlame Welsers hüttengesellsehaft 2000 fl., die Breutgamen 1000 fl., der Pucher gesellscliaft 2000 fl., Kilian Reit.twiser 2000 fl., Dr . Breitenbach 2000 fl., etzliche doktores und bürger 4000 fl., die Preusser 2000 fl. Summa : 25 000 fl. F r e i b e r g. Mertenn Manwilz 2000 fl., G. vom Steyg 1000 fl., die Albecken 2000 fl. Summa : 5000 Gulden. Kemnitz. Der Gleitzman 2000 fl. und sunst zween bürger 1000 fl. Summa : 3000 fl. S a 11 z a. Etwan ein vier bürger 2000 fl. Summa : von allen stedten sambt dem hauptman zu Leiptzig: 38 000 fl. Thut zusamen 55 000 fl. Darmit künde man meins achtens 4—5000 Zentner zin vorlegen und vorhandeln . Wa aber 1 =als. 2 =billiger. 3 Aus disen mussten zween erwelt werden , den regirer des handeis rettlich u. hilflich zu sein. 424 Dokumente der frlihkapitalistischen Wirtschaftsorganisation. mer zinn angenommen, so würde man auch mehr gelts haben müssen. Und aus den angezaigten bürgern allen müsste man zweene erwelen, die dem handel vornemlich vorstunden. Die andern müssten ein jeder in der stadt , da er gesessen ader die ihme am gelegensten were, bei den kannegiessern und anderen, den man zinn verkaufte, die schulden helfen einmanen, den Vor¬ stehern überreichen und berechnen. Wa man aber under diesen ader andern so vil gelts nicht auf¬ bringen, so möchte man wa kirchen ader hospital feiernd gelt bei ihnen ligen hetten, das man sunst auf zins ausleicht, da es villeicht kaum so sicher und göttlich als es aida sein möcht, auch darzu gebrauchen und denen, so die arbeit theten von dem nutz, deren so gar feierte zimlicher mas ergetzung tun. Auf dem dritten und vierdten artickel were gut mas zu finden, wie man den handel mit leutten und andern bestellen und wie vorbriefen. Gelaub auch, dass Barttlame Welsers hauptgesellschaft auf ansuchen auch 1000 oder 2000 fl. darzu legen, so er anderst guet darbei sein soit, dem faktor im niderlandt durch sein leutt furderlich zu sein. 17. Privileg des Herzogs Georg von Sachsen für die Gesellschaft des Zinnkaufs, datiert Dresden, 20. Dezember 1520. — Kgl. Haupt¬ staatsarchiv zu Dreden, Loc. 9826. Altenberg: Statuten oder Ordnung der Stadt 1515. it : Bergk-Sachen, Ordnung und anders ufm Altenberge, Contract mit Raupennest wegen eines Stollen 1534, Mandat wegen des Zinn-Kauffs 1520. Bl. 34 ff. Wir Georg von gots gnaden hertzog zu Sachsen etc., bekennen hiemit an diesem unserm offen brive, fur allen desselben ansichtigen und thun kundt : Nachdem und als sich die bergkgebeude auf dem Aldenberge, auch an etlichen andern orten in unsern landen und furstenthumben faste tief und schwehr machn und die zcin etlich jaer in einen mergklichen abfal komen, dass auch diejhenigen, so die zcin erbauet, schwerde halben der gebeude schwerlich dobei haben pleiben können und sich wol zu besorgen (wo dem nit notturftiglich vorgedacht) dass die bergkwergk in einen schweren falh komen ader gantz mochten liegen pleiben und alsdan die leute sich an den örtern nicht mehr erhalten können und gemeinen unsern landen ein mergklicher schade und nachteil hiraus erwachsen wurde; solchs alles zuvorkomen und gemeinen unsern landen zu guthe, V. Im sächsischen und böhmischen Zinnhandel des 10. Jahrhunderts . 425 haben wir mit reifer, tiefer betrachtung mit denjenigen , so auf dem Aldenberge , auch allen andern örtern in unsern landen und furstenthumben zcinwergk bauen ader fluetwergk haben und geprauchen , die auch uff unsern vorbeschiet den mehern teil durch sich ader ire volmacht erschinen , als vorkeufern an einem und etlichen kaufleuten als keufern am andern teile handlunge furgewendt und einen rechten , redlichen , uffrichtigen und bestendigen kauf und vortrag mit irer allerseits volwortte und vorwilligung drei jar langk zwuschen inen besprochen , aufgericht und beslossen, nachfolgender gestalt und meinung : Nemlich dass gedachten keufern alle zcin, so in unsern furstentumben und landen auf bergkwergken, fluetwergken ader seiffemvergken ytzo ader kunftigklich in zeeit der dreier jaer erbauet ader gemacht , auf den Aldenbergk in die wage sollen überreicht und überantwort werden . Ausgeslossen die zcin, so zu Erberstorff und doselbst umb gefallen ; die sollen die geselschafter aldoselbst auf der flösse in gleichm kaufe und gewichte annehmen . Aldo die keufer den vorkeufern baer kegen baer vor einen itzlichen centner bergkgewichte , der zuvor sal geeicht und gerechtfertiget werden , 11 gülden in muntze , halb sechssisch, ye 21 zcinsgroschen fur 1 gülden und halb behmisch , 24 behmische groschen fur 1 gülden , alles landtswehrung geben sollen. Docli an enden , do man nicht behmisch geldt nimrnet und die gesel¬ schafter doselbst die betzalunge mit eitel sechssischem gclde thun musten , sollen sie auch darkegen von ydem Aldenberger bergkcentener ein orth eins gülden weniger geben und also den centener umb 11 gülden minus ein orth betzalen. Es haben auch die bergkleuthe ader zciner , so under uns ge¬ sessen und uff dem Muckenbergke zcin erbauen , bewilliget , die zcin, so sie des orths bestimpte zeeit erbauen werden , in angezeeigtem kaufe auch aldohyn auf den Aldenbergk in die wage zu leveren und zu überreichen ; welche aber under uns nit gesessen, dieselbigen mögen die Muckenbergischen , Graupenischen und ander zcin nach irem gefallen ausserhalb unser lande und furstenthumb anwerden . Wo sie aber die im furstenthumb vorkeufen ader vor¬ treiben wollen, sollen sie die, wie angetzeiget in bestimptem kaufe der geselschaft in die wage uff den Aldenbergk überantworten , das wir hirmit wollen vorordent haben. Und sal solcher zcinkauf auf den sontag oculi in der heiligen fasten schirsten angefangen und drei jar nach einander weren. 426 Dokumente der frübkapitalistischen Wirtschaftsorganisation. Und ein itzlicher angetzeigter parteien , der den fürder zu halten nit gemaint , soi es dem andern teile ein halb jar fur aussgange der dreier jar schriftlich uff- und abkundigen . Und ab von den zcinern vil ader wenig zcin albereit auf liverunge vorkaufet were ader zwuschen hie und oculi vorkaufet wurde , was desselbigen in der zceit nit gelivert wurd , das soll alles bei unden ausgedrugkter pene von zcinern der geselschaft in die wage auf den Aldenbergk , wie obstehet , überantwort werden. Die schmeltzer und huttenmeister solln auch alle den geselschaftern voreidt werden , dass sie nicht dornicht ader ander zcin, das nit kaufmansware ader gut ist , ingiessen 1 und sunst allent¬ halben getreulich handeln und alle zcin in die wage bringen wollen und sal ein itzlicher auf seine kupperschicht sein zceichen schlahen. Es sollen auch die zcinherren schuldig sein, ungeferlich ein dritten teil alles zcines, so sie machen , nach ordenung und notturft der geselschaft in stucke ader gatter zu giessen lassen , wie es der gesel¬ schaft gelegen sein wirdt. Die wage auf dem Aldenberge sal mit zweien schlossen bewart und vorslossen werden ; zu dem einen schloss sal der zcehender und zu dem andern der geselschaft factor einen schlussel haben. Wo auch ymant etwas von zcin nach angehender geselschaft , es were zuvor zwuschen hir und oculi ader dornach uff liverung vorsprochen und ehr oculi nit gelivert ader sunst in andere wege heim¬ lich ader offenberlich durch sich ader andere vorkeufte ader sunst hinweg brechte und in die wage auf dem Aldenberge ader kegen Erberstorff wie vormeldet in die geselschaft nit bringen wurd , der soi, so oft er des uberkomen , desselbigen zcins vorlustig und dasselbig zcin uns die helfte in unsere cammer , die ander helfte der kirchen auf dem Aldenberge verfallen sein. Solchs alles haben wir, domit sich ein yder , der sich zcinwergks ader dasselbigk in unsern landen und furstenthumen zu vorhandeln gebraucht , auch sunst mennigklich dornach zu richten , seine hendel , gewerbe und Sachen anzustellen , auch dieser unser Ordnung und aufgerichten vortrage zu geleben und sich vor schaden zu Vorhuten habs öffent¬ lich anschlahen lassen . Zu urkunde mit unserm unden aufgedrugk1In„ die Stücke oder Gatter giessen“, heißt es in dem Vor¬ anschlag des Privilegs. Siehe Emest .Gesamtarchiv Weimar , Reg. T. Bl. 273/4, Nr . 3—6. V. Im sächsischen und böhmischen Zinnhandel des 16. Jahrhunderts . 427 tem secret wissentlich besiegelt und geben zu Dresden dornstags nach Lucie anno domini 1520. 18. Konvention zwischen den sächsischen Fürsten , den Schlick (Joachimstal ) und den Pflug (Schlackenwald ) zur Regulierung des Angebotes und der Nachfrage von Arbeitern auf den Berg¬ werken der Kontrahenten usw. 1521. •— Kgl . Hauptstaatsarchiv zu Dresden , Loc. 4486. Bergwercks -Sachen de ao. 1487—1599. Bl. 72—74. Von gots gnaden wir Friderich etc . churfurst etc ., Johanns und Georg, gebrudern und vettern , alle hertzogen zu Sachsen etc ., be¬ kennen fur uns, unser erben und nachkomen und thun kundt allermenniglich mit diesem unserm brive . Nachdem die wolgeborne, edeln, unser lieben besondern Steffan Slick, graf zu Passaun , her zu Weiysskirchen und Slagkenwerd etc ., sampt andern seinen ge¬ brudern und Hans Pflugk , her von Rabenstein auf Petzschaw , mit iren herschaften und bergkwergken an unser furstenthumber und bergwergk ruren und stossen , haben wir in bedacht desselben und umb sonderlicher gnad willen, die wir zu inen tragen , auch des¬ halben , damit von unsern und iren bergkgenossen und underthanen entporung , todtslege und ander unthat mocht verhuet und also gut fried , recht und ainigkeyt allenthalben gehalden und die berg¬ wergk treulich und vleissig gefurdert werden , uns mit gedachten Slicken und Pflugk nachfolgender artickel gnediglich vorainigt und vortragen , vorainigen und vortragen uns derselbigen mit inen hirmit wissentlich in craft ditzs briefs. Nemlich zum ersten , dass wir , obbemelten churfursten und fürsten zu Sachsen, desgleichen die Slicken und Pfluegk auf unser allerseits bergwergken vleissig achtung haben und uns in keinen weg dahin bewegen und dringen lassen sollen, dass auf denselben der lohn erhöhet , sonder auf allen bergwergken ein gleich lohn nach wert der muntz , so in eins yeden land genge ist , gegeben werden . / Zum andern sal keinem hauer gestattet werden zwu schiebt zu faren , dan nit wol möglich ist , wo einer zwu schicht faren und volkomen lohn darumb nhemen wil, dass er seiner arbeit umb seinen lohn genug thun möge. Zum dritten , nachdem sich auf bergwergken vil mutwilliger, boeser todtslege begeben , ob nue hinfurder beschehe , dass einer 428 Dokumente der frühkapitalistischen Wirtschaftsorganisation. ader mehr mutwillige todtslege thetten und also von einem bergkwerg auf das ander weichen und sich villeicht gegebener freiheit trösten wurden , dass derselben keiner , der nach gethanem todtslag auf eyn ander bergwergk weichet , anders dan zu recht aldo ge¬ sichert und mag gleichwol derselb auf ansuchen des nachfolgers 1 gefengklich angenhomen , wider den auch gestrackts rechtens soi gestat werden. Zum vierden , ob ein Schichtmeister , steiger ader ein ander den gewergken , im zehenden ader den erbeittern schuldig bliebe und also von einem bergwergk auf das ander weichen wurde , dass die¬ selben keiner freiheit noch Sicherung gemessen , sonder der amptman , wo sie ankomen , von des ampts sie enttrunnen , auf sein geburlich ansuchen angenhomen werden , yedoch das derselb entweicher , so er zu betzallung Versicherung thuet , viertzehen tage geglait werde. Zum fünften , ob sich auf berurter bergwerg einem begebe, dass sich etlich understundcn bey gemeinen bergleuten Unwillen, aufsteen und aufrur zu entporen und dieselben also mit ürsach darinne vormerckt und auf solchem bergwergk abgelegt und vor¬ weist wurden und sich auf die ander bergwerg begeben wolten, sollen sie auf der andern bergwergk keinem , wie oben gemelt , mit arbeit gefurdert werden. Zum sechsten soll auf eins jeden regirenden hern bergwergks allen bergkmeistern , amptleuten und geschwornen bei vormeidung schwerer straf ernstlich eingebunden und befolhen werden , auf die bergwergkarbeiter und hauer gutte achtung ze haben , dass sie geburliche Schicht halten , bei irer arbeit vleis thun und zu rechter zeeit aus - und einfahren und dass die steiger zu yeder schicht gegenwertig uff der zcechen sein und den arbeitern selbs unslet und eisen geben sollen, domit sie wissen, welcher arbeiter zu geburlicher zeeit an - und aussfehrt . Und soi den arbeitern bier¬ schichte zu halden , in kein weglc gestattet werden. Zum siebenden , ab sich nue darüber begebe, das , die knapschafft und ander bergkarbeiter ein gemein aufsteen machen , auf welchem bergwergk einem das geschehe und sich also aus einem lande in das andere zu beschwerung der underthanen mit einem häufen niderlassen und legen wolten , dass alsdan wir obgedachten 1 Wohl nach bars? V. Im sächsischen und böhmischen Zinnhandel des 1(5. Jahrhunderts . 429 churfursten und fürsten , desgleichen die Slicken und Pfluegk solchs nit gestatten , mit macht dawider trachten , die entwichene zu der gerechtigkeit halten und irs mutwillens strafen sollen. So auch einer auf unser , der churfursten und fürsten zu Sachsen, bergwergk verfest und geechtigt , der sal auf bemelter Slicken und Pfluegk bergwergk auch verfest und geechtigt sein und wider denselben soll sunder anregen mit straf verfaren werden und widerumb sollen genante Slicken und Pfluegk auf unsern bergwergken desselben auch gewertig sein. Wan auch unser , der churfursten und fürsten zu Sachsen, abtrünnige mishendler und beschediger sich in der Slicken und Pfluegk herschafften u'nderslaiffen enthalden, ader darinne betretten wurden , desgleichen widerumb , ob der ge¬ dachten Slicken und Pfluegk abtrünnige mishendler und beschediger in unser , der churfursten und fürsten zu Sachssen, furstenthumben und landen underslaiffen enthalden , ader darinnen betretten wurden, darinnen soll sich ein teil gegen dem andern halten und ertzaigen nach vormoge der erbeinunge zwuschen der chron zu Behaim und dem haus zu Sachsen etc . aufgericht. Und nachdem die Slicken uns angetzaigt , wie sie dieser zeeit muntzen , mit undertheniger pitt , dass wir dieselb muntze unser allerseits landen und furstenthumb wolten nehmen und gangkhaftig sein lassen, welchs wir inen zu sondern gnaden dermassen bewilligt , also dass wir ir gemuntzte silbern groschen , der einer auf ein gülden , der ander ein halben und der dritt auf ein ort geslagen wirdet , in berurten unsern landen und furstenthumben wollen gangkhaftig sein und nhemen lassen . Doch dass bemelte Slicken mit solcher irer muntz unser körn und schrot halten und damit nit fallen sollen. Und welcher thail über kurtz ader lange zeeit in diesem vortrag weiter nit steen wil, der soll es dem andern ein halb jar zuvor aufzeschreiben macht haben . Und des zu urkundt , steter und vhester haldung , haben wir obgedachter Friderich , churfurst fur uns und unsern brudern , hertzog Johansen und wir Georg, hertzog zu Sachsen , unser insiegel an diesen brief wissentlich hengen lassen, der gegeben ist am dinstag nach unser lieben trauen tag irer besuchung nach Christi unsers lieben hern geburdt , funftzehen hundert und im zwainzcigisten jharen. 430 Dokumente der frühkapitalistischen 'Wirtschaftsorganisation. 19. Herzogliche Bestätigung des Dienstvertrages , durch den die Gesellschafter des Zinnkaufs den Hans Alnpeck jr . zu ihrem Faktor annehmen . Datiert 4. März 1521. — Kgl . Hauptstaats¬ archiv zu Dresden , Loc. 4500. Bergwercks -Sachen zu Frey¬ berg bel. Vol. I. 1453—1543. Bl. 25/26. Wir von gots gnaden Fridrich der jüngere , hertzogk zcu Sachsen etc ., bekennen hirmitte und thun kundt , dass wir durch unsern liben he.rn und vatern heimverordente rethe , nemlich her Ceser Pflugk , ritter , doctor Johanns Kochel , cantzier und Segemundt von Maltitz zcu Dipesswalde zwischen der geselleschaft des zcinhandels , welche wir itzo von nauern haben aufrichten lassen und Hansen Allenpegken zcu Freibergk , den man den jungem nennet, nachvolgenden vertragk haben bereden , beslisen und aufrichten lassen . Nemlich dass gedachte geselleschaft Hansen Allenpegken zcu einem factor und diner volgende drei jar langk angenommen hath . Alzo dass sic irne dieselbigen drei jar nach einander itzlich jar zweihundert gülden rh . muntze zcu jarsolde geben sollen. Darkegen hath Hans Alienpegk der geselleschaft alle seine ligende und farende gutter zcu Freibergk , auch alle ander gutter , zo vil und an welchem ende er die hath , ader kunfticlich gewinnen wirdt, vor alle seine handelunge , zo er von wegen gedachter geselleschaft und in irem dinste handeln wirdet , zcu einem verstände und willigen pfände vor allen andern seinen gleubigern ingesatzt . Dass sie sich irer scheden , die sie seiner handelunge ader sunst seinethalben entphaen wurden , vor andern seinen gleubigern doran zcu erholen haben sollen. Es hath auch Hans Allenbegk bewilligt , dass er der geselleschaft vor seine person alleine dinen und keinen andern handel treiben , sundern alleine der geselleschaft handel getraulich handeln , warten und ausrichten , auch der geselleschaft schaden , ab er den erfure , offenbarn , denselhigen vorhutten und nach allen seinem vormugen iren frommen und besten schaffen und fordern wolle und solle. Und worzcu ime die geselleschafft gebrauchen wil, dorzcu sali er sich bei tagk und nacht gebrauchen lassen. Hans Allenbegk sali auch der geselleschaft geldt ane irem wissen und willen nimandes verleien , auch in sein eigen nutz nicht gebrauchen . Er sali auch keine nacht ane wissen und willen der geselleschaft von Aldenberge ligen . Item das zcin, zo in die wage kommet und uberantworth wirdt , dorauf er auch vleisigk achtunge geben soll, sali er auf seine eigene unkost zeusampne zcu tragen, V . Im sächsischen und böhmischen Zinnhandel des 16. Jahrhunderts . 431 zcu heben , iuzcufaren , von einem hause ins ander zcu tragen , zcu laden etc . verschaffen und alle ander geringe unitost , zo dorauf gehen wirdt , von seinem gelde entrichten und sali dieselbige über ine gehen . Er sali auch auf seine eigene unkost , zo offte das die nottorfft erfordert , kegen den Grauppen reiten und aldo der gesclleschaft zcin keufen und bestellen . Dessgleichen sali er in 10 ader 14 tagen ader zo offte es die not erfordert , einen vor die hutten reiten , vleisiclich sehen und forschen , was vor zcin gemacht und wo es hinkommen , welchs auch auf seine eigene unkost besehen sali. Hans Allenpegke sali sich auch auf seine unkost mit einem eigen hause auf dem Aldenberge versehen , dorinne der geselleschaft gut verwardt sein möge, er keuffe es ader mutte es, wi er das haben kan , doch alzo dass ime die geseileschafter , obbemelt , zo vil muglich, dorzcu furderlich sein sollen. Und sali die geselleschaft in solchem hause mit herbrige und lager vorsehen , doch alzo dass sie sich mit futter , haffer , haw , stroe etc . selbst versorgen und ime die kost beigeben sollen. Item er sali auch auf sein eigen unkost ein pferdt halden und alle jar einmal ader so offte es die geselle¬ schaft begern wirdt , rechnunge zcu thun , auch der geselleschafft heymliclikeyt nicht zcu offenbarn , sundern , wie oben vormeldet, iren schaden zcu warnen und besten zcu fordern schuldigk und pflichtigk seyn . Dissen vertragk haben die geselleschaffter durch ire geschigkten . . . . 1 und Hans Allenbegk , vor sich und seine erben traulich zcu halden geredt , gelobt und mit handtgebenden trauen zeugesaget , alles traulich und ungeferlich . Zcu urkunde mit unsern liern vatern zeurugk aufgedrugktem secret , das wir hirzcu gebrauchen , besigeldt und gegeben zcu Dresden montagk nach oculi anno 21. 20. Die Leipziger Kaufleute Michael Puffler und Ulrich Mordeisen verabreden mit den Gewerken von Geyer, Thum und Ehren¬ friedersdorf einen Zinnkauf auf 3 Jahr und eine Verlagsgewäh¬ rung (zinslos) von 600 fl. pro Jahr . 24. April 1525. — Kgl. Hauptstaatsarchiv zu Dresden , Loc . 4500. Das Bergwerck zu Ehren -Friedersdorff bei. 1377—1538. Bl. 13/14. Anno domini 1525. Uff Montag nach quasimodogeniti ist mith den geschickten vom Thume , Erinfridessdorff und Geyer diese 1 Einige Namen , von denen ich nur Michael Puffler , Bürger zu Leipzig mit Bestimmtheit entziffern kann. 432 Dokumente der frühkapitalistischen Wirtschaftsorganisation. abrede und untherhandelung zu aufrichtunge eins bestendigen zinkaufs uff drei jhar lang gescheen, also das anzufahen sontags cantate. Michell Puffeller und Ulrich Mordeisen, bürgere zu Leipczigk iczlichem der drei stetlein mith zweihundert Reinische gülden und also in summa mith sechshundert gülden anfänglichen vorlegen, doch also, dass es inen zusampt fürstlicher gunst genugsam vor¬ sichert werde . Davon sal man ihn keinen geniess geben 1 und zu ausgange der dreier jhare mith gelde widderumb ahne seumnus bezcalen . Ap auch ein stetlein under den dreien die Vorlegung nicht dürfen wurde und die andern beide die uffnehmen , sollen dieselbigen, so das gelt enpfahen , solche Vorsicherung bestellen. Die zcinkeufer sollen vor 1 centner blangk zcin 12 % gülden geben . Vor ein centner weiss zcin 11 gülden und ein ort und baer umb bar bezcalen . Die bezcalung sali halb mith furstenmuncze und halb mith Behemischer muncze geschehen. Ap einer ader mehr von den zcinhern ader gewercken ein centner, ein halben , ein virttell adir eczliche pfundt zcin vor sein haus zu gesesse bedorffen wurde , mag er von dem seinen darzu woll ge¬ brauchen , doch also, dass es der zcinkeuffer factorn angezcaigt werde und dasselbige zu andrem handel zu seinem nucze nicht ge¬ braucht. Diese artickell seindt von den zcinkeuffern und zcinern also bewilligt , aus vorhengniszs unsers g. h. lierczogen Hainrichs etc. bis uff wolgefallen unsers g. h. herezogen Georgen zu Sachssen etc. 21. Vorschläge eines Leipziger Bürgers ( ?), wie den Leipziger Kauf¬ leuten die Herrschaft über den mitteleuropäischen Metallhandel verschafft werden könne . Um Michaelis 1527. — Kgl . Haupt¬ staatsarchiv Dresden , Loc. 10 532. Leipziger Händel etc. 1525 bis 1551. Bl. 131 ff. Es ist gewiss, dass durch den fhall der von P r a g a w 2 und des landes zu Behemen die niderlage und merckte zu Leiptzig gross haben zugenummen und merglich seint gepessert worden. Dan wo Praga in seinem wesen bliben , were der handel zu Leiptzig 1 Das heißt unverzinslich. 2 „Man sehe Praga, Regenspurg u. andere stete, da vor handel gewest, wie sie in armerung kommen". Aus Münzstreitschrift von 1530 bei W. L o t z , Die drei Flugschriften über den Münzstreit der sächs. Albertiner und Ernestiner um 1530, S. 20 f. V. im sächsischen und böhmischen Zinnhandel des 16. Jahrhunderts . 433 schwerlich also gemeret und gepessert worden . So ist es auch kunt und offenbar , dass die niderlage und merckte zu Leiptzig durch den handel zu Eislauben eine lange zeit und bisher seint erhalden worden ; aus deme, dass der kaufmann seinen Wechsel durch die Eislaubischen hendeler hat bestellen können gen Nürmberg , Aussburg , Franckffurt , Antorff und andere ortter mehr. Weil aber die grafen zu Mansfelt einen grossen seiger-handel selbst aufgerichtet und das silber, so sie aldo zu wegen brengen, vormünzen , also dass dasselbige gemeine hendeler entzogen , derwegen der gemeine hendeler seinen Wechsel nicht vormag zu be¬ stellen . Darum zu besurgen , wie es dan auch gewiss, dass der handel zu Leiptzig in ein abnemen must gefürt werden. Wie nun zu disem der grafen von Mansfelt vornemen die von Pragaw sich auch understunden , den handeil pei inen wider auff zu richten , wie inen dan von kuniglicher durchlauchtikeit zu Behem dartzu hülff . . . geschehen und sich auch albereit etliche hendeler niderlage doselbest zu halden vorgenummen , so musste von not wegen volgen, dass niderlage und handel zu Leiptzig . . . schaden leiden musste. Hirauss wolt erstlich volgen, dass u. g. h. an seinen zollen und geleiten merglichen abgehen muste . Item es must auch volgen, dass alle stete im fürstenthumb durch welche die War gefurt wirt, darinnen die furleute und andere geczert , an irer narung musten abnemen . Item die stadt Leiptzig und alle ire inwoner mussten mercklichen nachteil an irer narung erleiden , dieweil die stadt und inwoner sunst keine narung hat , dan von Merckten. Disem nach dem willen Gotes vorczukommen , ist vor gut und notdürftig angesehen , dass vleis darauf zu legen sein soit , damit man den Bleikauf zu Gosslar, die behemische kupfer und silber, das Schlackenwaldische zinn und das zinn im fürstenthumb Sachssen alle in eine liant und an die inwoner zu Leiptzig bringen mocht, Hiraus worde erstlich volgen, dass der graffen von Mansfelt vornemen dem handel und niderlage zu Leiptzig kein schaden oder ye nicht so gross thuen konde . Angesehen, dass der hendeler seinen Wechsel durch die obenberurte metall zu bestellen hette und obgleich die grafen v. Mansfelt dis thun nicht vorgenummen heften, so were es doch gut , dass die hendel pei einander wereu ; dan , wan die hendel also zusamen bracht , wurden die grafen v. Mansfelt mit der münz nicht dermass trotzen dürfen , wie vilmals bisher gescheen. Strieder , Studien z. Geaoh. knpitalist . Organ isationsforinen . 28 431 Dokumente der frühkapitalistischen Wirtschaftsorganisation. Item es konde auch die niderlage zu Pragaw nicht aufgericht werden, dergestalt , dass sie diser zu Leiptzig sunderlichen schaden thun mocht. [Es würden Zölle und Zehnten hochbleiben . Der Leipziger u. a. Landeseinwohner Verdienst nicht sinken etc .] Weil dann nun dise h endel alle sollen zusammen bracht werden, ist vor gut geacht , dass man erstlich und vor allen dingen den bleikauff vom herczoge zu Braunschweig, ader den von Goslar haben musste . Dan durch den bleikauff weren die Behemen zu dringen , dass sie ire metall czum bleikauf musten volgen lassen . Derwegen hat burgermeister Wolff Wideman sampt seiner geselschafft sich bevlissen, den bleikauf pei den von Gosslar zu erhalden , wie ime dan der vom rathe daselbest zugeschriben und von doctor Krausen müntlich zugesaget . Allein ist des Untrils 1 halben ein aufschub gemacht bis uff irer aller Zusammenkommen, wie dan solches unserm g. h. in einer supplication noch der lenge angeczeiget wirt . Weil aber die von Gosslar itzo den kauf Wolffen Wideman fast abeschreiben und die grafen v. Mansfelt als graf Albrecht und graf Gebehart disen itzigen marckt zu Leiptzig mit den hüttenhendelern haben gehandelt , dass sie den Saclissen 2 hinfür kein kupfer vorkaufen sollten , darein dan Heinrich Scheille 3 Kuntz Keller , Moritz Bücher und alle andere bis uff Wolffen Wide¬ man und Lucas Straube gewilliget und die Sachssen 2 itzt die merckt am statlichsten besuchen , dan sie bringen pfeffer, kommel 4, gewallt, reiss, allaune , czugker , allerlei fische, rauche wäre 5, czobel und marder , vil wolle und speckis, das sie alles aldo vorkaufen und ir gelt wider anlegen an silber , kupfer , zinn u. a. des sie des jhars von 8—9000 ctr . kupffer gekauft , auch wol an 3000 ctr . zinn , das sie alles zu I .eiptzig mit gutem gelde bezalt . Wo sie nun ir gelt nicht wieder anlegen kontten , umb wäre, die ine dienet , würden sie mit gewalt von Leiptzig getriben . So haben auch die gemein hendeler disen marckt an u. g. h. und den rath supplicirn wollen und sich 1 Vitriols. 2 Den Einwohnern der alten sächsischen Kreises (Hansen ). 3 Wohl Scherl. 1Wohl Kanel = Zimt. 4 Rauchwaren. Sachsengaue , d . h . des nieder¬ V. Im sächsischen und böhmischen Zinnhandel des 16. Jahrhunderts . 435 beclagen , dass sie czum Wechsel nicht kommen konden ; darvon sie czu disem mall an gut sein gewisen worden. Derhalben wil nunmher von nöten sein dise hendel zu¬ sammen zu brengen und sunderlichen mit dem bleykaufe nicht zu seumen, sundern u. g. h. zu ersuchen und zu bitten in aller unterthenikeit, burgermeister Wideman sampt seine mitgesellschafter gnediglichen und ernstlichen zu vorschreiben, dass die von Gosslar ime und seine mitgesellschaftern den kauf halten mussten. Dan wo dis nicht geschiet und die von Gosslar den bleikauf behilden, so weren die andern metall dester schwerer ader gar nicht zuerlangen. Item über dis ..... worde auch volgen, dass eine naw und grosser steigern mit dem blei werden muste, dan vor ye gewest were; wie dan solchs aus dem briefe von der gesellschaft geschriben weiter zu vornemen. Dem zinnkauf umb das Schlaekenwaldische czin nach zu trachten vor dem tag, der den vom Altenperg und den hendelern angesatzt. Dan das zinn uff dem Aldenperg were sunst nicht anczunemen. Hieronimus Walther ist itzt pei dem von der Weigmüle des kupfferkaufs halben und im fal ob er dene nicht crhilde, das u. g. furst und herr Hansen von Schonburg czu königl. durchlauchtikeit zu Behem schicken wolt, sunst wossen die hendeler czum kauf nicht zu kommen zu disem mall. 22. Herzog Georg nimmt auf 3 Jahre den Zinnkauf in Altenberg, Lauenstein und Bernstein in seine Verfügungsgewalt. 27. Mai 1538. — Hauptstaatsarchiv Dresden, Loc. 7414, den Zinn¬ handel betr. 1497—1544. Bl. 33 resp. 35 u. 38. Georg von Gots Gnaden etc. Fügen allen und jedem gewercken des zcinbergkwerges aufm Aldcnberge, Bernstein und Lauens.tein, so dosclbst oder anderswo wonhaftig sein und sunsL meniglichen hiemit zu vornehmen: Nachdem und als ir, die gewercken des zcinbergkwerges aufm Aldenberge wonnhaftig uns vilfaltig angelangt, einen bestendigen zcinkauf aufzurichten, der¬ halben wir dan Euch sampt den andern so zu Freibergk und annderswo wonnhaftig alher vor uns beschaiden. Und aber Euch desselben undereinander nicht hapt vergleichen, noch vor biliiehen ermessen können, dass Ir daselbst aufn Aldenberge, Bernstein und LauensLein 436 Dokumente der frühkapitalistischen Wirtschaftsorganisation. wohnhaftige gewercken der andern und frembden gewergken halben Schadens solten gewarten, dardurch auch die zcinbergkwerge mochten vorwüst werden: So seindt w'ir vorursacht worden aus fürstlicher macht und oberkeit ein ordenunge und satzunge des zcinkaufs auf¬ zurichten . . . und gebieten, dass alle diejenigen, so das zcinbergkwergk uffm Aldenbergk Bernstein und Lauenstein bauen, daselbst oder anderswo wonhaftig und gewergken sein, sollen auf nechst künftig Johannis sonnewende anzufahen schuldig sein, alle ir zcin, das sie daselbst erbauen und zubereiten, auch das flut-zcin in die wage auffrn Aldenberge zu antworten, und einen zcentner umb 11 gülden unserer müncz, wie dieselbige in unserer kämm er genomeu und ausgegeben wirdet, zu geben und zu vorkeufen. Die wir auch einem jeden bhar kegen bhar, doch als fern es kaufmanns¬ gut befunden, also entrichten und beczalen lassen. So wollen wir auch uns hiemit Vorbehalten haben, diese unsere ordenunge und ausgesatzten zcinkauf zcu unserer gelegenheit widerumb aufczukündigen, zu vorendern und zu vorbessern. Welcher sich aber derselbigen zu enntkegen wirdet unterstehen das zcin sunst zu vor¬ tauschen, teurer zu vorkeufen, ader in andere wege anzuwenden, den wollen wir darumb nicht ungestraft!, lassen . . . Dresden, mon¬ tags in der creutzwoehen anno domini 1538. 23. Herzog Georg übergibt Michael Büffler den Zinnkauf auf dem Altenberg, Bernstein und Lauenstein auf 3 Jahre (nur die Alnpeck ausgenommen). 26. Mai 1538. — Hauptstaats¬ archiv Dresden. Kop. 103 Bl. 30, auch Loc. 7414. Den Zinn¬ handel betr. 1497—1544. Bl. 36. Wir von Goths gnaden Georg, hertzog zu Sachssen etc., thuen kundt und bekennen hinnit vor uns, unsere erben, nachkomen und allermennigklich, dass wir aus bewegenden Ursachen und vorbetrachtung auf unserm bergkwerg, dem Aldenberg, auch zum Bernstein und Lauenstein einen bestendigen zcinnkauf vorordent und ausgesatzt, auch an ader zu demselben zcinkauf unsern burger zu Leiptzigk und lieben getreuen Micheln Bufflern drei jar langk, nebst Johannis anzufahen, haben, doch mit dem bescheide kommen lassen, dass er einen yeden zcendtner und alsso aller gewergken zcin, so doselbst gewonnen und gemacht, alleine der Alnpecken zcu Freibergk ausgeschlossen, alspalt der in die wage geantwort und als kaufmansguth befunden, umb 11 gülden unserer muntz, wie die V. Im sächsischen und böhmischen Zinnhandel des 16. Jahrhunderts . 437 in unserer kammer genohmen und ausgegeben wirdet , annehmen und also bar umb bar geben und betzalen sali. Aber dennoch diesergestalt das ausgangs zweier jar bemelter Büffler , sein erben, ader wer sonnst solchen zcinkauf die zceit haben wurde , uns antzeigen sollen, ab sie denselbigen lenger behalten wollen, ader nicht, dormit wir uns in gemelter unser ordenung wissen zu halten . Des¬ gleichen uns auch freistehen und hiemit Vorbehalten wollen haben, ihnen vilgemelten zcinkauf aufzukundigen ader lenger zu lassen und im falh , dass er nach endung der dreier jar zugehen und ein ander aufgericht werden soit, alsdenn gedachter Büffler , so es ihme anders gelibet , vor einem andern dorann den vortrit haben . Das wir ihme aus besondern gnaden hiemit Zusagen, treulichen und ane geferde . Zu urkunde mit unserm aufgedrugktem secret besigelt und geben zu Dresden sontags vocem jocunditatis nach Cristi unsers lieben herren gebürt tausent fünfhundert und im acbtunddreissigsten jare. 24. Herzog Georg macht seinen Bergbeamten von der Verleihung des Zinnkaufs an Michael Puffler Mitteilung , befiehlt den öffent¬ lichen Anschlag der Verordnung und den Schutz und die Unter¬ stützung Pufflers bei der Ausübung seines Privilegs . 26. Mai 1538. — Hauptstaatsarchiv Dresden , Loc. 7414. Den Zinn¬ handel betr . 1497—1544. Bl. 34. Georg von Gots gnaden etc . Lieber getreuer , als und nachdem wir aus Ursachen auf unserem bergwergke dem Aldenbergke , Bernnstein und Lauenstein einen zcinkauf ausgesatzt und verordent , wie wir Dir dann die ordenung hibei übersenden und aber an solchen zcinkauf unsern bürgern zu Leipzigk und lieben getreuen Michel Bufflern 3 jahr lang haben von Johannis schirst anne zu heben kommen zu lassen , so begeren wir , Du wollest solche ordenungen öffentlichen anschlagen und verkünden lassen , auch dem Buffeier dabei schützen und handthaben , desgleichen die gewercken alle bescheiden und ihnen von unseretwegen gebieten , dass ein jeder auf ein jeden gatter . seines Zeins sein eigen zceichen schlagen lasse, damit ob er falsch befunden , man darumb hätte zu reden . Auch den smeltzern bei ihren eiden, die Du von ihnen neh men wirdest , ernstlichen einbinden , lauter und nicht dornicht zcin zu giessen, oder zu machen, bei vormeidunge unserer straf ; daran beschiet unser mainung. Datum Dresden , Sonntags vocem jocunditatis anno domini 1538. 438 Dokumente der frûhkapitalistischen Wirtschaftsorganisation. 25. Herzog Georg vermittelt zwischen Michael Puffler und den Ge¬ werken von Ehrenfriedersdorf , Thum und Geyer einen Zinnkauf auf 3 Jahr . 29. Mai 1538.— Hauptstaatsarchiv Dresden , Kop . 103, Bl. 33. Auch Loc . 7414, den Zinnhandel betr . 1497 bis 1544. Bl.37. Von Gots gnaden , wir Georg, licrtzog zeu Sachsen etc ., thuen kundt , dass wir aus bewegenden Ursachen und Vorbetrachtung durch unsere amptleuthe zeu Radebergk , Meissen und Sanct Annaperg , rethe und lieben getreuen , Jeorg von Karlewitz , Ernsten von Miltitz , Hainrichen von Gerssdorf und hern Ludovicum Fachsen, der recht doctorn , auf unsern zcinbergwercken Ernfriedersdorf, Geier und Thum , zcwuschen unsern lieben getreuen , den gewercken des zcinns , so doselbst erbauet , an einem und Michaeln Bufflern, bürgern zeu Leiptzigk , anders theils , mit irer baiderseits vorwilligung einen bestendigen zcinkauf auf drei jar lang nehst sonnabent nach Johannis antzul 'ahen , aufgericht und darauf — doch lenger nicht — sie volgender weis vortragen : Dass bemelter Bufler einen jeden centner schons umb 11 gülden und einen orth und ein zeentner weis zcin umb 11 gülden unserer muntz , wie die in unserer camhmer genohmen und ausgeben wirdet , sobalt es in die flos geantwurt und als kaufmansgut befunden , annehmen und also par umb par geben und betzalen soi. Aber danne diesergestalt , dass ausganges zewaier jar bemelthem Bufler , seinen erben ader wehr sunst solchen zcinkauff die zeeit haben wurde , desgleichen auch den gewercken obgedacht freistehen solchen zcinkauf lenger zeu halthen ader aufzcuschreiben . Dass dann auch jeder theil dem andern auf die benante zeeit also antzeigen soi, darnach hab zeu richten . Es wollen aber die gewercken des orts eins gülden halber , den sie itzo nachgelassen , nach endung dieses kaufs freistehen und hiermit nichts begeben haben und im fal, dass nach endung der dreier jar der zcinkauf dermassen nicht bestehen und ein ander aufgericht werden soit , alsdann soi Bufler , so es ihme geliebt , vor einem andern daran den vortrit haben . Das wir ihme zeu gnaden hiemit zeusagen, treulich und ane geferde . Zeu urkundt mit unserm aufgedrucktem secret besiegelt und gescheen auf Sanct Annapergk , mittwochs nach vocem jocunditatis anno domini funfzeehenhundert und im achtunddreissigsten. 26. Verlagskontrakt Michael Pufflers mit einigen Zinngewerken zu Altenberg , 1539. — Kgl . Hauptstaatsarchiv Dresden , Loc. V. Im sächsischen und böhmischen Zfnnhandei des 16. Jahrhunderts . 439 4493. Bergsachen den Altenberg und Gießhübel betr . 1509— 1697. Bl. 73/74. Uff heut suntag am tage Mathei des 1539. jhar , sindt vor mir Werner von Nossa, die zeit amptman uffm Aldenberge und Glassheutten erschinen , auch in beisein Matlies Morgenstern , die zeit rieh ter : mit namen Hanns Gemperlein , Paul Puckschka , Wolf Meuerer erben , Mathes Morgenstern , Hans Taubenheim , Paul Peyer, Pihlep Engelhart , Asmus Heidenreich , Gorge Heit , Hans Beier , ' Nickell Gemperlein , Michell Heidenreich , Gorge Kunell , Hans Frauenstein , Stheffen Herclozs , Cristoff Rullingk , Michell Tauben¬ heim, alle diejenigen , so izo ader hinfore und so lange der zinn¬ kauf sein bleibens haben mocht , so von dem erbern , namhaftigen Michell Pueffler , burger zu Leipzigk ader seinen befehlhabern vorlcg bekumen mochten und haben sich aida eintrechtiglich vorwilliget (ein yder vor sich selbst mit vorzeihung alles behelfs, ausrede ader berckrechtens ), dass sie alles dasjhenige , so inen gedachter Michel' Pueffler ader die seinen leihen ader vorstrecken wurden (welches doch zu seinem selbst gefallen gestelt sein soll) zu gutem danck mit zine entrichten wollen, auch niemandts anders zine zustellen wollen noch sollen, dan ime ader den seinen und auch von iin selbst nit mehr geldes entpfahen , noch unser keiner sich mit imandts anders in vorlege einlassen wollen, dass im an seiner ersten bezallung zu nacliteil ader vorzuck reichen mocht . Es sei denn , dass ehr zuvor des vorgestrackten und gelihen geldes ganzs und gar mit zine eines iden beredung und vorwilliguug nach vorgenuget und bezalt sei. So aber einer ader mehr in dem pruchigk befunden , also dass Pueffeler ader die seinen befinden mochten , dass wir ader einer unther uns sein zine anders den im zuwenden wolthen , das doch nicht sein soll, Michell Puffler ader die seinen in ader ausserhalb der wage macht haben , alsbalde das zine zu seinen handen zu nehmen , an alles weithers ersuchung und erlaubnus und einen iden betreffent an seiner vorlegten summen abkurzen. Doch so bergk - ader mull -erbetter 1 iren lohn , so sie scheinbarlich zu erweissen, an vorlegtem zine ausenstehen haben wurden •— also dass es nicht aide ader ander schult , so nicht uff dem berekwergk ader mollen auch uff die zeith ein is iden vorleges gegannen ader gewant sei — so soll es alsdan nac .i berckordenunge den er1 Arbeiter. 440 Dokumente der frühkapitalistischen Wirtschaftsorganisation. bettnern zuvor an dem zine bezalt und vorgenuget werden . Und dan die Übermasse gedachten Michel Puffler und sunst niemandts anders zugestelt werden , bis so lange ehr von einem iden bezalt wurde . Welches alles ein ider , wie oben stehet , mit hande und munde zugesaget , deme allen getreulich nachzukumen und stete und vehste zu halden . Das wir den auch amptshalben zugelassen, gelube angenuhmen und bestetiget haben . Bestettigen auch sulches hirmite und in kraft unsers ampts mit zusagunge darüber auch eines iden seiner vorpflichtung nach vehstiglich zu halden und stattzugeben . Des zu sicherheidt ist sullches mit uffgetrugtein mein, Wirners von Nossa, angeboren pezschafft ins gerichtsbuch vorleibet , gesehen im jhar und tagk wie oben etc. 27. Michael Puffler beklagt sich bei Herzog Heinrich von Sachsen, dass einige Altenberger Zinngewerken , die er verlegt hat , ihr Zinnprodukt ihm nicht übergeben wollen. 1541. — Kgl . Haupt¬ staatsarchiv Dresden , Loc. 4493. Berg -Sachen den Altenberg und Giesshübel bei. 1509—1697. Bl. 72. Durchlauchter , hochgeporner furst und herre . E . f. g. werden aus inligender copeie zeu vornemen haben , wie ich zeum zcinkauf ufm Altenberge kommen ; dass ich par umb par das czinn doselbst beczalen soll, wie denn bieshero geschehen unde noch geschiet. Dennochs habe ich uf e. f. g. vorschrieft etlichen gewercken , so hirbei vorczeichent , das zcin zeuvor und eher sie das gemacht, beczalet . Doch dergestalt , dass sie mir solch zcinn mit erst 1 sie es machen , zcustellen sollen ; wie ich dan das mit inen lauts ingelegtes Vortrags, so durch den ambtman doselbest aufgericht , einigk wur¬ den byn. So wollen sie nuhn solch zcinn inen zeu nucz den erbeiter vor ir lohn zeukommen lassen unde mich mit iren gutem beczalen. Dieweil es aber ein kauf unde ich zcinn unde nit guter beczalt , so vorhoffe ich, dass gedachte gewercken mich nit unpillig mit meiner beczalten wahr unde mit sunsten nichten anders zeufriede stellen. Derwegen an e. f. g. unterthenige bi et, e. f. g. wolten mit ernst vorschaffen , dass sie mir solche beczalte wahr , sovil mein ausgegeben kaufgeldt erstreckt , zuestellen und antworthen und sunsten nimandes anders zcuczukommen gestaten , hiemite aufgerichte vortrage 1 sobald = als. V. Im sächsischen und böhmischen Zinnhandel des 16. Jahrhunderts . 441 nachgegangen werde . In ansehen , dass ich solches uf e. f. g. begeren untertheniges gefallens gethan habe , so bin ich auch gesetiget, dass ich geduldt habe , bies sie zcinn machen können . <Doch dass sie das erste , so sie machen , mir behendiget werde . Ob sie aber auch einige einrede ader behelf vorwenden wurden , so kann ich vorbeschiedt und vorhor vor e. f. g. ader dêrselbigen rethen erleiden, tröstlicher zcuvorsicht e. f. g. werden mich hirauf genediglichen bedencken , dass ich mein erkauft gut dencklichen magk bekennen. Das umb e. f. g. byn ich untertheniges gehorsames fleises zcu vor¬ dienen gefliessen. Datum Leipczigk , sonnabents nach letare anno etc . 41. E . f. g. untertheniger und gehorsamer Michell Bufeler, burger zcu Leipczigk. Dem durchlauchten und hochgepornen fürsten und herren , hern Heinrichen , herezogen zuw Sachssen etc. 28. König Ferdinand überträgt dem Augsburger Bürger Conrad Mayr das böhmische Zinnhandelsmonopol auf 3 Jahre . Prag, 6. Dezember 1549. — K , und k . gemeinsames Finanzarchiv in Wien . Gedenkbuch Böhmen 1549—50 Nr . 305, fol. 112— 115. Entwurf dortselbst Fasz . Böhmen M. u. B. 1540—1590 Nr . 16 407. Wir , Ferdinand etc . bekennen für uns und unser erben offendlich mit disem brief und tuen kund allermenigelichen , das wir uns mit wolbedachtem muet auch gueten zeitigen rat mit unserm und des reiclis lieben getreuen Conraden Mair, burger zu Augspurg , unsern rate , fur sich, seine erben und nachkumen aines zinnkaufs halben nachvolgunder massen und gestalt verglichen und beschlossen. Und nemblichen und erstlich gedachten Conrad Mair, seinen erben und nachkumen alle und jede zinn , geschmeidige und ungeschmeidige, so diser zeit und hinfuran auf unsern perkwerchen in der chron Behaim allenthalben gemacht und uns zuesteen werden auf vier jar lang , die nechsten nachainander volgunt , so sich zu eingang nechst kumends funfzehen hundert fünfzigsten jars anfachen und zu ausgeenden dreiundfunfzigisten jar enden werden und sollen, 1 Darnach könnte es scheinen , als ob nur das Zinn aus den könig¬ lichen Gruben in Frage käme , aber in dem Revers des C. Mayr, in welchem er sich an den Kontrakt bindet , heißt es klar und deutlich: „alle zinn , so jetzo u. kunftiglich allenthalben in der chron Behaim gemacht werden .“ 442 Dokumente der frühkapitalistischen Wirtschaftsorganisation. keuflich zuesteen und volgen zulassen , versprochen und zuegesagt haben. Benenntlichen ainen jeden cennten geschmeidigs zinn Schlackenwalder gewandlichs perkgewichts , so wir ime zu jeden nachvolgunden fristen aida zu Schlackenwald durch jeczigen oder künftige unsere amtleut daselbst uberlifern lassen sollen umb drei und zwainzig gülden , und den cennten hërdt , ungeschmeidig zinn umb 22 gülden, alwegen vierzechen paczen oder sechsundfunfzig kreuzer für ain gülden gerechendt . Auch die liferungen berurter zinn , so allent¬ halben in ermelten vier jaren auf angeregten jeczigen und künftigen unsern berkwerchen in der chron Behaim gemacht werden , alwegen zu zwaicn monaten , sovil deren gemacht worden sein, zu Schlacken¬ wald durch gegenwärtig oder künftig unsere ambtleut zu sein Conraden Mairs, seiner diener oder bevelchhabern handen richtiglich tuen und antwurten lassen sollen und wellen. Dagegen soll er, Conrad Mair das bedingtlich kaufgelt , sovil dan inen , den gwercken , zu irem tail umb die anzal zinn , welche ime zu jeder liberung geantwurt werden , geburen wirdet , daselbst zu Schlackenwald par in allerhand gold und münzen , wie die in der chron Behaim gib- und gangpar sein werden , erlegen und bezalen , doch die gwercken mit grosser anzal klainer münz über ir gelegenhait nit beschwären . Was aber über das losunggelt an denselben zinn uns vorstendig sein wirdet , soll er Mair alwegen zu halben jaren mit einander under ainest zu Schlackenwald uns auch mit parem gelt in allerhand gold und grober münz , so im kunigrcich Behaim jeder zeit gib - und gangpar sein wirdet , one abgang erlegen und bezallen. Wir sollen und wellen auch auf jeczt nechstkumend weinachten und furan zu jeder zinliberung durch unsere Verwalter oder ambt¬ leut daselbst zu Schlackenwald ime, Conraden Mair oder seinen leuten anzaigen lassen, was zu der nechsten liferung ungeverlich fur zinn gefallen werden , sich mit der bezallung und in ander weeg seiner notturft nach zu versehen haben , dergleichen auch verfuegen darob und daran sein, dass gemelte zin baider gattung , geschmeidige und ungeschmeidige , wie zuvor und bisheer gebreuchig gewesen, rechte , guete werung kaufmansguet und wolgearbaidt seien. Es soll uns auch ermelter Conrad Mair im anfang und eingang des jiczt künftigen funfzehen hundert und funfzigisten jars zu Ver¬ lag der zinn dreissig tausend gülden vorgemelter gestalt und werung V. Im sächsischen und böhmischen Zinnliandei des 16. Jahrhunderts . 443 daselbst zu Schlackenwald zu handen unser ambtleut oder Ver¬ walter gegen derselben quittungen erlegen und bezalen , und die¬ selben bis zu ausgang ermelts zinkaufs der vier jar lang on ainich intéresse oder ergoczlichait ligen lassen . Und damit dan gedachter Conrad Mair, seine erben und nachkumen des ermelten furleichens der dreissig tausend gülden , wie pillich gnuegsamblichen verwisen, auch derselben widerumb habhaft und bezalt werden , so haben wir mit zeitigem rat und gueter Vorbetrachtung ine, den merermelten Conrad Mair, seine erben und nachkumen auf die berurten unsere zinn , geschmeidige und ungeschmeidige , so furohin auf unsern perkwerchen in der chron Behairn allenthalben gemacht , uns zuesteen und gefallen werden , verwisen und versichert . Tuen auch solches hiemit wissentlich und in craft dicz briefs , also und der¬ gestalt , das wir die ernennten dreissig tausend gülden ime, Conraden Mair, seinen erben und nachkumen , in den leezten zwaien zinliberungen des ausgeenden drei und funfzigisten jars , jeden der zwaien letsten liferungen funfzehen tausend gülden in hiefor ermelter gangparer gold- und grober münz ausrichten , bezallen und ine dieselben auf ernennte zeit der zwaien liferungen an dem kaufgelt , so er uns umb die zinn zu bezalen schuldig sein wirdet, selbs aufheben und innenhalten lassen sollen und wellen on ainichen auszug , Verhinderung und einreden. Ferer soll ernennter Conrad Mair unserm gnedigisten begern nach denen von Amberg jarlichen und jedes der vier jar besonder fünfhundert centner zinn Schlackenwalder gewicht in dem kaufund allergestalt , wie er Conrad Mair dieselben von uns an sich ge¬ bracht und auch gegen parer richtigen bezalung zu Schlackenwald folgen lassen , die si allain zu irem plechhandl verarbaiten und ge¬ brauchen sollen. Wo aber die von Amberg genannte zin gar oder tails anderstwohin verkaufen , verfueren oder verhandlen und nit alle zu irem selbstaigen plechhandl gebrauchen und verarbeiten wurden , so soll er Conrad Mair macht , gwalt und guet fuegen haben , welches wir ime auch hiemit gnedigist bewilligt und zuegelassen haben wellen, wo er oder die seinigen solche zinn also erfarn oder betreten wurden , dieselben aufhalten arrestiern , zu iren handen einziechen und damit ires gefallens als mit irem angefahnem aigen guet handlen sollen und mugen. Gleichergestalt soll er auch unsern undertanen , den zinngiessern, in perkstetten zu Schlackenwald und Grauppen gesessen, auch ain 444 Dokumente der frühkapitalistischen Wirtschaftsorganisation. anzal von zwai in drei hundert ccntner und nicht darüber jarlichen, die si auch sonst nindert hin vergeben noch verkaufen , sonder allain zu irer selbst handarbait , dem zinngiessen , prauchen und verarbaiten sollen bei der peen , wie obsteet , wo si darwider taten und handleten , gegen parer bezalung in obangeregtem kauf und gewicht, auch allergcstalt , wie er Conrad Mair dieselben von uns an sich gebracht , folgen und zuesteen lassen. Weiter sollen uns aus obangeregten verkauften zin zu unser selbst notturft des geschucz und andern gebrauch jarlich in die fünf hundert centner und nicht darüber Vorbehalten sein. Also und dergestalt , dass wir von solchem vorbehaltnem zinn allain der gwercken gebuer und ferer nichts zu bezalen schuldig sein sollen. Gleichfals soll auch er Conrad Mair umb dieselben zinn , so wir zu unserm gebrauch , wie obsteet , nemen wurden , über der gwercken geburnus oder losunggelt auch nichts zu bezalen schuldig sein. Wover wir aber dasselbig zinn gar oder tails nit bedürftig wurden, soll er Mair dasselbig im wert und kauf , wie das ander anzunemen schuldig sein. Er Conrad Mair soll auch die ernennten , von uns erkauften zin in allen unsern auch andern kunigreichen , landen und orten seinem pesten nucz und gelegenhait nach verfueren , verkaufen , vertreiben und verhandlen mögen . Wir sollen und wellen auch ine und die seinigen in unsern kunigreichen und landen in gnedigistem schucz und schierm halten und haben ; also das er und die seinen in den¬ selben sicher und on alle gefarliche beschwarung handlen und wandien mögen und sollen. Ob ime aber über das bei andern und frembden etwas irrung , eintrag oder ansprach zu nachtail oder Ver¬ hinderung des handls begegnen wurde , in solchem sollen und wellen wir ime und den seinen auch gnedigiste furderung erzaigen und beweisen. Vilbemelter Conrad Mair soll auch mit bemeltem zin an allen zollen, meuten und aufschlegen , wie die namen haben mochten, allenthalben in der chron Behaim allerding frei sein und dan in den andern allen unsern kunigreichen und landen in zeit des werenden kaufs und solang bis er solche von uns erkaufte zin gar verfuert und vertriben haben wirdet , über die gewondtlich zoll, maut, dreissigist und aufschleg , wie die jeezo sein und abgefordert , nicht gestaigert oder beschwert werden. Und sover wir nach ausgang obvermelts kaufs der vier jar V. Im sächsischen und böhmischen Zinnhandel des 16. Jahrhunderts . 445 den zinkauf weiter verlassen wolten , sollen und wellen wir den¬ selben gedachtem Conraden Mair, wo ime gelegen sein wird , utnb dieselben sovil als sich ander zu geben und zu bezallen anbieten wurden , zu geben, vor allen andern zuesteen und erfolgen lassen. Wo wir auch ainich gelt auf unsern bevorstand , so wir von disem zinnhandl haben werden , anticipiern und aufpringen wolten, sollen und wellen wir solches an gedachten Conraden Mair gelangen lassen und uns darauf fur ander mit ime in zimblit'he, leidenlichc handlung einlassen. Und ob gedachter Conrad Mair diser zinhandlung halber , als ob er ain verbotne oder monopolische handlung damit getriben und geuebt het , vor der Röm . kais . Mt., derselben camergericht oder kaiserlichem viscall oder andern orten furgenomen , beclagt und umbgetriben werden solte oder wurde , so sollen und wellen wir ime, Mairn in demselbigen allen geburliche hilf und furdrung erzaigen und beweisen. Wir sollen und wellen auch mit dem churfursten von Sachsen seiner, des churfursten zin halben die sach dahin handlen , damit er, churfurst dieselnen zin, so in sein furstentumben und landen ge¬ macht werden , auch auf den wert und preis pringe , und nit nachrer verkaufe oder den gwercken zu verkaufen gestatte , als wir unsere zin gegen ime Conraden Mair hinbracht und verkauft haben. So wir aber solches bei dem churfursten über allen angewendten vleiss nicht erlangen oder dahin bringen mochten , so sollen und wellen wir alsdann bei ime, churfursten dahin handlen lassen, das dieselben zin an ende und ort verfuert und verschlissen werden, aida si unsern behambischen zinen und ime, Conraden Mair daran im verschleiss und verkaufen nicht nachtailich noch verhinderlicli sein mugen. Und wover über furgewendten vleiss solchs bei gedachtem churfursten auch nicht erlangt noch erhalten oder fueglich weg darzue gefunden werden mochten , so sollen und wellen wir doch alsdann in allen unsern kunigreichen , furstentumben und landen mit ernst gebieten und verschaffen , auch darob halten lassen , das man berurte Sächsische noch andere frembde , gemachte noch un¬ gemachte zin die obvermelten vier jar lang und solang bis er Conrad Mair seine von uns erkaufte zin gar verkauft und vertriben haben wirdet , in ermelte unsere kunigrcich und lande nicht fucren , dar¬ innen weder verkaufen , kaufen , verarbaiten noch durchfuren lassen 446 Dokumente der frühkapitalistischen Wirtschaftsorganisation. sollen noch wellen, bei Verlust derselben auslendischen zinn . Es soll auch er, Conrad Mair oder die seinigen, wo si dieselbigen frembden und auslendischen zinn in ermelten unsern kunigreichen und landen betreten oder erfarn wurden , ansprechen und als ain ver¬ fallen guet zu unsern handen einziechen und arrestiern mögen. Des wir inen zutuen hiemit macht und gwalt gegeben haben wellen. Davon ime, Mairn alsdann der drittail folgen und die übrigen zwen tail uns beieiben sollen. Wir haben auch dem gedachten Mair in dise handlung in ansechung des furlcichen der dreissig tausend gülden on interesse und dann auch aus sondern gnaden in zeit der ersten drei jar dises zinkaufs , jedes derselben besonder , ain hundert centen geschmeidig zin Schlackenwalder gewichts frei on ainich bezalung in den ganzen zinkauf ze geben und volgen zu lassen bewilliget , die wir ime auch als jecztgemelt alwegen zu halben jaren fünfzig centner bis zu erfüllung der dreihundert centner zuezustellen verordnen und ver¬ fliegen wellen und sollen. Und beschliesslich , wo sich zuetruege , das Got lang gnediglich verhueten welle, das in obangeregten vier jaren in der chron Behaim dermassen kriegsleuf entstuenden , dardurch gedachter Mair die gemachten zin mit sicherhait nicht erheben noch vertreiben wurdt künden und das gelt zu bezalung der zin gen Schlackenwald unsicherhait halben auch nicht bringen möcht , so sollen und wellen wir uns nach gelegenhait der leuf gnedigist mit ime vergleichen, damit er Mair derselben zeit mit annembung und verfurung der zin nit beschwart werden sollen. Alles getreulich und ungeverlich mit urkund diczs briefs, besiglt mit unserm kuniglichen anhangen¬ den insigl. Geben Prag , den sechsten tag decembris anno etc . im XLVII Ilten. 29. Errichtung eines Zinnkaufs zwischen den Gewerken des Alten¬ berger Radeschacht und den Leipziger Zinnhändlern Georg Huttherr , Michel Jerniger , Hans Eibe , Dominicus Breun , Adrian von Hilsen und Christoff Walburger unter Vermittlung Wolf von Schönbergs , des Hauptmanns des Erzgebirges und Mathias Arnolts , des Amtsverwesers . 6. Mai 1562. Original des Ver¬ trags mit den eigenhändigen Unterschriften und Siegeln der Kontrahenten im Hauptstaatsarchiv Dresden , Loc.36080 Nr.666. Bl. 75 ff. V. Im sächsischen und böhmischen Zinnhandel des 16. Jahrhunderts . 447 Demnach und als bishero uf dem Altenberge viel grosser be¬ schwerlicher unnöttiger unchosten ergangen , das auch die gewercken derwegen in grosse scheden und beschwerung kommen , als haben ich Wolff von Schönberg zu der Neuensorga uf den ertzgebirgen hauptman etc . und Mathias Arnollt , ambtsvorweser doselbsten, heut dato die zihenhendtlcr von Leipzigk als Jorge Hütern x, Michel Jernigern , Hans Eibe , Dominicus Breun , Adrian von Hilsen und Christoff Walburger semptlichen und sonderlichen vor eins, auch die gewercken des Altenbergers zum radeschacht gehörig anderstheils vor uns erfordert , diese unerdregliche und unmugliche beschwerungen mit ihnen beiderseits auch zu beforderungen cf. zehents gehandellt und geredet und entliehen auf nachvolgendc , mittel und Wege sie von beiden theiln vorgliehen und vortragen. Demnach kein ander besserung und Ordnung kan oder mag — dass das Altenbergische bergkwerg wider in aufnehmung kommen möchte — gefunden werden , als dass die zihenhendler den wöchent¬ lichen Vorlagen auff- und zu sich nehmen . Welchs dann die obbemelten zihenhendler wie volget , dessgleichen die gewercken bis ahn unsern gnedigsten churfursten und herrn confirmation auf beiden theiln angenohmen und bewilligt . Dergestallt : Es sollen und wollen die zihenhendler von Exaudi des 62. jars bis wider Exaudi des 65. also 3 jar alle wochen mit gutter seclisischer muntz unsers g. h. schrots und korns auf dem Altenberge die berggebeuden , butten, muhlen , kohl, holtz , pocheisen etc . und in summa alles, was zu dem bergwerge dissorts gehöret , nichts ausgeschlossen , das sich dann in gewieser summa wöchentlichen nicht über 700 gülden erstrecken soll, darlegen und vorsetzen etc . Und sollen die obbemelten zihenliendtler zu fernerem wöchentlichen Vorlagen nicht gedrungen wer¬ den . Und do auch die ernantten 700 fl. wöchentlichen zu solcher Vorlage nicht von nöten , so sollen dieselben aucli nicht von den zihenhendelern abgefordert werden . Do es sich aber zutruge — wie wier dann zu dem lieben Gott hoffen — dass die bergwerge des orts in besserung und aufnehmung kommen möchten , so sollen und wollen die obbenanten zihenhenndtlcr mit geburlicher wöchentlicher vorlag über die 700 fl. was den radeschacht belangt auch zu vor¬ legen nit in wegerung stehen . Dagegen sollen sie die zihen sovil dero ein jede woche disorts und innerhalb eXaudi des 62. bis exaudi 1 Schreibt sich in der Unterschrift Georg Huttherr. 448 Dokumente der frühkapitalistischen Wirtschaftsorganisation. des 63. jars den ctr. vor und umb 12 gülden obbeschriebner muntz zu sich aus der wage entphahen und nhemen. Vollgende 2 jar als von exaudi des 63. und 64. bis auf das 65. ermeltes sontags exaudi sollen und wollen die ziehnhendler ein jeden ctr . zihen, soviel dero gclieffert umb und mit 12 gülden ein ort den gewercken zhalen, vorgnugen und zu sich losen. Weil auch sontags exaudi anno im 61. die obgemelten ziehn¬ hendler den Altenbergischen gewercken des radeschachts 4600 gülden uf vorlag dargethan und vorgestrackt und dis jar über ahm datto von jederm centner zihen ein halben gülden abgekurtzet und innen¬ behalten worden, so haben oft gedachte zihenhendler bewilligt und angenhomen, dass sie dieselbe abgekurtzte jaressumma des halben guldens, sovil dero centner zihen gelieffert worden und diss die rechnung geben wirdet, alsbalt und uf einmhal widerumb mit bharem gelde den gewercken erlegen und hinausgeben wollen. Dass also den gewercken 4600 gülden in muntz widerumb ausserhalb des wöchentlichen vorlagens vorgesetzt werden möge. Dagegen und herwiderumb haben die oft gemellten gewercken des Altenbergs zum radschacht gehörig, dass sie solche 4600 gülden in muntz, inen von den zihenhendlern vorgesatzt, exaudi des 62. anzufahen bis exaudi des 65. also lange diese summa der 4600 gülden erreichen thut , von jedem centner zihen ein halben gülden durch ihren vorordenten factorn abzukurtzen und innenzubehalten, inen geliehen und gefallen lassen, auch entliehen bewilliget. Und demnach auch die ernanten zihenhendler diese beisorge getragen und haben, dass, do etliche gewercken so wöchentlichen vorlag uf ire bergtheile (wie oben vormelt) entpfangen, was ahn zihen ihnen den zihenhendlern von quartain zu quartain also in jaresfrist mit ersetzungen des wöchentlichen vorlags der zihen, was hinterstellig vorplieben wurden (sie des also nicht in wenigen schaden und nachtheil gesetzt möchten werden), also haben des bergwergs ufm Altenberge zum radeschacht gehörenden bauenden gewercken hiemit und in kraft dieses schiedes semptlichen und sonderlichen bewilligt und ahngenhomen, dass sie vor dieselben alle und jede aussenstendige vorpliebende zihene und schulden diese 3 jar über als selbscbulldige purgen und zhaler des wöchent¬ lichen vorlages kegen den obbemelten zihenhendtlern zu hafften eingelassen und sich hiemit wie es am krefftigsten vorpflichtet haben. V. Im sächsischen und böhmischen Zinnhandel des 16. Jahrhunderts . 449 Diweil aber Mathes Rode und die Buehfurrischen 1 kein wöchentîiclien vorlag begehren , und sich desselben eussern, so sollen sie auch mit der gesampten burgschaft verschonet und nicht gemeinet sein. Doch sollen und wollen sie dem churfursten zu Sachsen etc . zu underthenigstcm gehorsam und volge ire zihen gleich andern gewercken des radeschachts in diesen kauften liefern und geben und sich all anderer artickel gemess verhalten. Zudehme , wollen auch die zihenhendler und gewercken alle quarta ! oder viertheil jar , der eingenhomenen zihen und wöchent¬ lichen vorlag richtige rechnungen miteinander thun und halten. Es wollen auch die zihenhendler zu erhaltungen und besserungen des radeschachts besonders zu beforderungen u. g. chur¬ fursten und herrn etc . zehenten uf unser underhandlung und der gewercken vleissiges ansuchen 200 gülden muntz künftig exaudi nach datto den gewercken des radeschachts zu steur der gebeuden doselbsten ohne einige widerentgeldt gutwilligklichen darlegen und ordtnen. Mher sollen und wollen die gewercken ufm Altenberge alle zihen zum radeschacht gehörende , so sie von sonntags exaudi ahn, der do ist der zehende monats Mai dieses lauffenden 62. jars bis wieder uf exaudi des 65. und also 3 jar lang in ein geordnet giess¬ haus aufm Altenberge stuckweis ungegattert fhuren , darinnen die zihen durch eines jedem geschwomen schmeltzers gartern und rein ausgiessen lassen, also dass kein dornicht , feistes, tattelhaftig oder unkauffmans guth soll darein gegossen werden , auch durch solche vereidente schmeltzer und giesser auf einem jederm und alle gattern das Altenbergische Wappen, den leuen wie bishero breuchlichen ge¬ wesen und auch darneben ein geordnetes Zeichen, dass es in die radeschachtgebhure gehöret , neben eines jedem gewercken Zeichen gemercket werden . Was dan fur feistes dornichts oder andere böse zihen , so die sau pfleget geheissen zu werden , unausgegossen bleibet, dass sollen solche ausgiesser einem jedem gewercken neben seinem gegaterten zihen und kretz zustellen und von den gewercken den zihenkauffern oder irem factor antworten . Dann sollen die zihen durch einen vereideten butner oder einem anderen tüchtigen in die vass eingeschlagen und das rechte Altenbergische Zeichen, den leuen , darauf brennen lassen ; auch kein ander zihen, dann solche 1 Valtin Buchführers Gesellschaft. Strieder , Studien z. Gesch . kapitalist . Organisationsformen . 29 450 Dokumente der frühkapitalistischen Wirtschaftsorganisation. ausgegossene und gegaterte zihen in die vass schlagen und sollen auch dieser Verordnungen des ausgiessens nach vorfliessungen der obgemellten 3 jar die gewercken ires gefallen freistehen. Es solen auch die gewercken je und allewege recht Altenbergisch gewicht , wie es durch den churfursten zu Sachsen in der wage ge¬ ordnet und bishero gehalten worden , zu reichen und zu liefern schuldig sein, hiemit kein klag derhalben furfalle. Yohr ausgehung der dreier jar sollen und wollen die gewercken ein halb jar zuvor , den hendtlern den zihenkauff weiter ahnbieten und ihnen vor allen anderen gönnen , sofern die hendler sich mit ihnen ferner vergleichen können . Auf den fhall aber , dass solchs nit geschehe, dass sie sich vorgleichen würden , alsdann soll es den gewercken mit andern sich einzulassen freistehen. Und so einig gewerck vor exaudi des 65. jars ahn seinem antheil der 4600 gülden in diesen dreien jharen mit etwas seumig gefunden, derselbige gewerck soll das hinterstellige rest ahn seinem zihen, so er zum nechsten darnach einbringen und liefern wirdet , abrechnen lassen . Und so ein gewerck ahn lieferung der zihen solche seine zhalungen nicht erreichen wirdet , desselbigen guter bergtheile, hutten und muhlen sollen zu einem willigen underpfand umb mherer vorsicherung darauf hafften und damit gentzlichen vorgnuget und bezhalet werden und sollen keinen anderen keuffern ire zihen fur diese zeit der bezhalung liefern oder zukommen lassen. Es sollen auch die gewercken den vorrath , so sie itzt anfengklichen dies kauffs halben oder biss uff endung der dreier jar gewinnen werden , nicht mutwilligklichen unauffbereitet und ungeschmeltzet den hendtlern zu nachtheil ligen lassen , vorsturzten 1 oder sonsten Vorhalten , sondern wollen denselben mit allem vleiss sovil muglichen uffarbeiten , damit den zihenliendlern der obgewilligte zihen¬ kauff gehalten wirde . Do dann dem amptsvorwalter befehlch ge¬ schehen und so einer des befunden , dass er darumb soll gestraft werden. So sollen und wollen auch die hendler semptlichen und sonder¬ lichen diese 3 jar über zu solchem zihenkauff sowol also die ge¬ wercken vorbunden sein und sich weder mit krieg , raub , nahm oder sterbenden leuffen zu behelffen haben . Welches also beiderseits gewercken und hendler solches alles zu halten mihr , dem haupt= fortscliaffen, verderben. V. Im sächsischen und böhmischen Zinnhandel des 16. Jahrhunderts . 451 mann der Ertzgebirge und dem amptsvorwalter mit hand und mund angelobt und zugesagt haben. Entliehen, die vielgemelten gewercken semptlichen, dass sie m. g. churfursten und herrn confirmation hierüber aus und zuwegen pringen, uff sich genhommen haben. Seind also diesmal derer verschriebenen artickel zu grund aus entschieden und vortragen, haben auch solch die benannten ziehnhendler allsampt, dessgleichen die gewercken zu steter, vhester haltungen beneben mir, dem hauptmann und amptsvorwaltern als hendlern, mit unsern angebornen und gewonlichen petschaften hier unden ufzudrucken, wissentlichen diesen gezwiefachten schied bemercken lassen. Geschehen und geben Freiberg, Mittwochs nach Philippi Jacobi, den 6. Mai des LXII. [Es folgen die Unterschriften und Siegel des Berghauptmanns, des Amtsverwalters, der bedeutendsten Gewerken und der Zinn¬ händler.] 30. Kurfürst August verspricht dem Hans Jenitz, Hans Harrer, Wolf Prager, Hans Biener und Hieronymus Krahwider nach Ablauf des Kontraktes mit den Leipziger Zinnhändlern den Zinnkauf auf dem Altenberger Radschacht auf 8 Jahr unter der Bedingung, dass sie den Dr. Blasius Grunwald und Domini¬ cus Breun aus Leipzig in die Zinnkaufsgesellschaft aufnehmen. 24. Aug. 1564. Konzept im Hauptstaatsarchiv Dresden, Loc. 36 080, Nr. 666, Bl. 86 f. Von Gottes gnaden wir Augustus, herzog zu Sachsen, churfürst etc. thun kundt hirmit öffentlich und bekennen fur uns, unser nachkommende erben und sonst menniglich, dass wir unsern lieben ge¬ treuen Hansen Jenitzen, secretarien, Hansen Harren, cammer¬ meister, Wolff Pragern, zehendnern zu Freibergk, Hansen Bienern, muntzmeistern und Hieronimussen Kralnvidern, cammerschreibern unser gnedigsten Vertröstung nach den zcinkauf uffn Aldenberg nach ausgang unserer gunst, so wir elzlichen hendelern zu Leiptzigk zuvorn hiruber geben und sich auff exaudi des kunfftigen 65. jars enden würdett, 8 jhar lang, die nechst volgenden nacheinander, gnedigest gegönnet, versprochen und verwilliget haben. Gönnen, vorsprechen, vorwilligen und vorschreiben auch obbenannten unsern dienern und iren erben solchen zinkauff auff 8 jhar freiwillig und wohlbedechtig in und mit crafft dis brieffs. Doch dass sie unsern 29* 452 Dokumente der frühkapitallstischen 'Wirtschaftsorganisation. leibartzt Dr . Blasium Grunewaldt sampt seinem aiden Dominicum Breun zu Leiptzigk in diese ire gesellschaft die zeit lang , so wir ihnen beiden gewilliget , zu sich ziehen , damit dieselbige unsere erlangte bewilligung ungeschwecht in iren krefften bleibe . Dagegen sollen obgemellte geselschaffter die ziengewercken uffn Aldenberge zum radeschacht gehörig wie bishéro wöchentlich zur notturfft vor¬ legen, sich auch allezeit von zweien jharen zu zweien jharen eines gewissen zinkauffs nach gelegenheit der leuffe mit den gewercken vergleichen , welche Vergleichung wir dann auch jedesmals gnedig confirmiren und sie ires vorlags aus dem berckwerg und andern der gewercken semptlich unvorschiedenlich haab und guttern ge¬ nugsam vorsichern und dermassen vorgewissern wollen, dass sie nach endung dieser bewilligung von dem zinkauff und Vor¬ sicherung durch niemandt abgedrungen werden sollen, sie seindt denn irs auffgewenten vorlags zuvorn gentzlich und volkomlich vergnügt. Und in diesen 8 jharen wollen wir nicht bewilligen noch ge¬ statten , dass sich einige gewercke im radeschacht — er bedörffe gleich verlag oder nicht — von den andern absondern , sondern es sollen obgemelten Vorlegern alle zin vom radeschacht unzerteilt in einem kauft volgen und gelassen werden . Wollen dann die geselschafter oder Vorleger daneben auch etzliche feldtgebeude 1vor¬ legen und befurdern , das soll bei inen auch freistehen . Und mögen sich mit den gewercken derselben feldtgebeude des zinkauffs halben wie sie können zum besten vorgleichen treulich und ohne alle gefehrde. Zu urkunde haben wir diese begnadung und befreihung des zinkauffs mit unserm auffgedrucktem secret besigelt und unsern eignen handen unterzeichnet . Geschehen und gegeben auf unserm schloss Kemnitz , den 27. August nach Christi , unsers erlosers gebürt im 1564. jliare. 31. Die Gesellschaft des Altenberger Zinnkaufs , bestehend aus den in der vorigen Urkunde genannten Mitgliedern , schließt mit den Gewerken des Altenberger Radeschachts wiederum einen Zinnkauf auf 2 Jahre ab . 27. Nov. 1566. Urkunde im Haupt¬ staatsarchiv Dresden , Loc . 36 080. Nr . 666, Bl. 89 ff. 1 Außer dem Radcschacht. V. Im sächsischen und böhmischen Zinnhandel des 16. Jahrhunderts . 453 Demnach die bauenden gewercken des Aldenbergs zum rad¬ schacht gehörig eines notturftigen zienkaufs und vorlags uf 2 jhar lang , nemblichen von exaudi des 65. bies uf exaudi künftiges 67. mit den achtbarn , ernvhesten und erbarn hern Blasius Grunewalden, der ertznei doctorn , Johan Jhenitzen , cammersecretarien , Johan Harrer , cammermeistern , Johan Biener , muntzmeistern , Hieronimus Krahnwider , cammerschreibern , Wolf Pragern , zehndnern , alle churfurstliche sechsische hofdienere und Dominicus Breun bei¬ stendig geschlossen und aber nunmehr solche zwei jhar künftig exaudi beruertes 67. ihr entschaft haben und abgehen , also seint auf entpfangenen churfurstlichen sechsischen , meines gnedigisten herrn , befehlich beides obbemelte heren zienhendeler und die ge¬ wercken des radschachts ufm Aldenberge volgendermas uf vor¬ gehende beschehende vorbescheidung und vleissiger underhandtlung durch mich Wolffen von Schönberg auf Knauthan und Nauensorga, liauptmahnn der ertzgebierge aller nachvolgenden notwendigen artickel mit irer sembtlichen , wolbedachten willen ufs naue wiederumb gentzlichen vorglichen und vorlragen werden . Also: Es haben die gewercken des radschachts den obbemelten hendelern alle ire zien, sovil dero ufm Aldenberge zum radschacht ge¬ hörig gemacht , den zehndtner zien um 12 gülden muentze des churfursten zu Sachsen, unsers gnedigisten herrn , schrots und korns, oder deme ahm werth gemes, darunter der dritte th eil grob gelt sein soty, bahr umb bahr , zugk um zugk uf zwei jhar langk , also von exaudi des 67. bies wider exaudi des 69. entliehen zugesaget und vorsprochen. Und weil die gewercken nach vormoge jetziger gehaltener abrechnung 7196 gülden muntz schuldig blieben und aber itzo wiederumb zur notturfftigen vorlag und erhaltung irer gebeude ein ahntzanl geldes behilfig, also haben die oftgedachten hem zienhendler ihnen , den gewercken sembtlichen 3804 gülden auf künf¬ tigen nauenjharsmarck ahngehendes 67. jhars zu den vorigen vorbliebenen 7196 gülden zu erlegen und also widerumb 11 000 gülden muntz vortzusetzen bewilligt . Was aber mittler zeit von datto ahn bis uf exaudi des 67. von zien gemacht wirdet , daran soll vermöge der vorigen vergleichunge den hendlern ahn jedem zehndtner 1 gülden abgeezogen und inebehalten werden und den gewercken ahn den Vorgesetzten 11 000 gülden wiederumb ablauffen und sollen also weitter auch die zwei jhar über zu keiner 454 Dokumente der frühkapitalistischen Wirtschaftsorganisation. fernem übrigen vorlag und vorsetzung vorpfliehtet noch vorpunden sein. Dagegen soll den herrn Vorlegern die zien des radschachts, soviel deren diese zwei jhar über gemacht , wie zuvorn geschehen, alle gevolget und sonsten von den gewercken oder jemandes anders dieselben zien nicht verkauftet , verhandelt oder undergeschlagen werden. Damit aber vorgemellte herrn vorlegere irer vorgesatzten und dahrgestrackten summa geldes widerumb behendiget und habhaftig gemacht wurden , haben die gewercken des radschachts bewilligt — wie sie dan hiemit gütlichen und einhellig versprochen und zugesaget — dass von jedem zehntner zien, so sie diese zwei jhar über gemacht und in die wage geliefert wirdet , zwene gülden ahn der bezahlung ihnen lassen und ablegen wollen, welche durch iren vorordenten factor abgekuertzt und innebehalten werden sollen. Doch soll ihrem factor ehe und zuvorn die alten zienhendler ires benanten hienderstandts , dero sie vom zehntner einen halben gülden ablegig gewarten , gentzlich vorgnueget , mehr nicht , dan anderthalben gülden volgcn . Nach abzahlung aber derselben sollen vom centner jederzeit diese zwei jhar über zwene gülden abgehen und innenbehalten werden. Weiln aber nach vorflissung derer zweier jhare ahn ablegung der 11 000 gülden was wichtiges hinderstellig vorbleiben wirdet, also haben die vilgemellten bauenden gewercken des Aldenbergs zum radschacht gehorigk auch ihnen geliehen lassen und in kraft dies schiedes sembtlich und sonderlich bewilliget und mit hand und mundt gelobet , dass sie vor dieselben alle und jede aussenstendige vorbleibende zien und schulden die zwei jhar über als selbschuldige bürgen und zahler der 11 000 gülden oder des restes gegen den obgcmellten herrn zienhendlern zu haften eingelassen und sich hiemit wie es am krefftigisten vorpflichtet haben. Damit sie aber die hendler des rests , so ihnen künftig vorbleiben mechte , umb soviel deste mehr vorsichert , so sollen ihnen alle der gewercken bercktheile , huetten , muhlen und vorrath zu einem willigen underpfande hafften und stehen. Es sollen und wollen auch alle gewercken des radschachts, welche eigene feltgebeude haben oder noch in obberurten zweien jharen bekommen mechten , alle ire zien, so sie diese zwei jhar von denselben machen , den hendlern in disem kauf zu lassen vorpflichtet V. Im sächsischen und böhmischen Zinnhandel des 16. Jahrhunderts. 455 sein. Dagegen wollen die hendler einen jeden nach gelegenhcit seines vorradts , welchen er jedcsmals .durch den berckmeister und geschwornen mit vleiss besichtigen wirdern und ihme hierüber ein Zettel ahn den factor geben lassen soll, vorlegen. Zudeme haben die vielgemellten gewercken vorsprochen und zugesagt , dass sie den zienkauff künftig exaudi des 69. den jetzt¬ gedachten zienhendlcrn vor allen andern ein halb jhar zuvor ahn¬ bieten , gönnen und zukommen lassen wollen. Do sie sich aber des von beiden theilcn nicht vorglcichen werden können , auf den fhal soi den gewercken mit andern sich einzulassen freislehen . Doch soi den itzigen hendlern der rest von den 11 000 gülden , soviel des nach gehaltener abrechnung verbleiben wirdet , ahn zien oder bahrem gelde exaudi des 69. fur abtretung des zienkauffs ahne allen vorzugk bahr und auf einmahl bezahlt und erleget werden. Es haben auch die gewercken damals sich vorpflichtet und zugesaget , dass sie forthin iren arbeitern mit bahrem gelde lohnen wollen, damit zwietter gewonnen , zien gemacht und das bergkwergk gefordert , sich auch künftig die arbeiter oder sonst jemants dcrhalben nicht habe zu beklagen und auch derwegen unser gnedigister herr , der churfurst zu Sachsen, die ambtleutte , auch die hendeler unuberlauffen und unbelanget bleiben . Sollte aber solcher von einem oder mehrern vorbleiben und von ihme nicht gelohnet, so soi uf den fahl nach vermöge m. g. herrn ordnunge gegen den¬ selben mit ernster straff Vorfahren werden. Mehr sollen und wollen die gewercken ufm Aldenberge alle ihre zien zum radschacht gehörende , so sie von sonntags exaudi des 67. bis widerumb exaudi des 69. und also zwei jhar lang machen werden, in das geordente giesshaus ufn Aldenberge stuckweis , ungegattert fueren, darinnen die zien durch einen jedem geschwornen schmeltzer gattern und rein ausgiessen lassen , also dass kein domichts , feistes thadelhaftiges und unkaufmannsguet soll darein gegossen werden. Auch durch solche schmeltzer und ausgiesser , welche damals von nauem in gegenwardt der hendler oder ires befehlichhabers sollen vorcidet werden , woferne solches nicht albereit geschehen , auf einem jedem und alle gattere das Aldenpergische wappen , den leuen , wie bieshero breuchlichen gewesen und auch daneben ein geordentes Zeichen, dass es den gewercken des radeschachts und in diese Ver¬ gleichung gehörig , neben eines jedem gewercken Zeichen mit vleiss unnachlessigk gemercket werden . Was dann fur feistes, dornichts 456 Dokumente der frühkapitalistischen Wirtschaftsorganisation. andere böse zien oder hardtwerck , so die sau pfleget geheissen werden , unausgegossen bleibet , das sollen solche ausgiesser einen jedem gewercken neben seinen gegatterten zien und kretz zustellen und von den gewercken den zienkauffern oder irem factor treu¬ lichen geantwortet werden . Dan sollen die zien durch einen dazu voreideten buttner oder einen anderen tüchtigen in die fass ein¬ geschlagen und das rechte Aldenbergische Zeichen, den leuen , darauf brehnnen lassen , auch kein ander zien, dan solche ausgegossene , gegatterte zien und welche zuvom mit den geordenten drei Zeichen vormerclcet , in die fass schlagen . Und sollen auch dieser verordenung des ausgiessen nach verflissung der zweier jhare den gewercken ires gefallens freistehen. Es sollen auch die gewercken je und allewege recht Aldenbergisch gewicht, welche jherlich sollen ufgezogen und justificirt werden, wie es durch den churfursten zu Sachsen in der wage geordenet und bisher gehalten worden, zu reichen und zu liefern schuldig sein, hiemit kein klage derhalben furfalle. So haben auch ferner die hendeler bewilliget , dass sie von jederm Zentner zien, so inen die zwei jhar über von den gebeuden des radeschachts geliefert , 3 pfennige , wie dan die gewercken gleichergestalt auch gewilliget, solche 3 pfennige vom Zentner zien irestheils zum Gotshaus und erhaltung der kirchen geben wollen. Welche Steuer durch der hendtler factor alle halbe jhar abgekurtzt und den kirchvettern mit der gewercken vorwissen zugestelt soll werden. Die gewercken wollen auch den vorradt , so sie vor der zeitt und inkunftig der zweier jhar gewinnen werden , nicht vorsetzlich unaufbereittet und ungeschmeltzt , den hendlern zum nachtheil liegen lassen , vorsturtzen oder sunst Vorhalten , sondern wollen den¬ selben mit allem vleiss sovil muglich aufarbeiten , damit den herrn zienhendlern das zien volkomlichen geliefert und der abgewilligte kauft gehalten werde . Do dan dem ambtsvorwalter befehlich ge¬ schehen soll, so einer des befunden , dass ehr darumb sal gestraffet werden. Desgleichen sollen und wollen auch die herrn hendeler sembtlich und sunderlich diese zwei jhar über zu solchem zienkauff sowol als die gewercken beiderseits verpunden sein und sich weder mit krieg, raub, nahm oder sterbenden laufften, wie das einen nahmen haben magk, zu behelffen haben. Im fahl aber durch vorhengknus Gottes des almechtigen etwan durch kriegs- oder sterbenslauffe das V. Im sächsischen und böhmischen Zinnhandel des 16. Jahrhunderts . 457 zien in solch stecken kommen wurde , dass die hendeler zu schieiniger vorpflichter zahlunge nicht kommen konden , so wollen die gewercken desfalls mit ihnen auch freuntlichc , gutwillige gedult haben . Hier¬ gegen aber haben sich die herrn hendler gegen den gewercken freuntlich und doch unvorbuntlich vornehmen lassen , da es ihnen aus mangel des Wassers hinderung und nott vorfallen wurde , dass sie sich nach gelegenheit und befindung des vorradts mit einer gut¬ willigen- vorlag willferig erzeigen wollen. Welches also beiderseits die herrn hendler und gewercken solches alles zu halten mir , dem hauptmahn der ertzgebierge etc . mit hand und mundt angelobet und zugesagt haben. Entliehen haben die oftbenumbten gewercken sembtliche hier¬ über meines gnedigsten churfursten und herrn confirmation aus und zuwege bringen uff sich genohmen. Und seint also diesmahls derer vorbeschriebenen artickel zu grundt aus entschieden und vortragen , haben auch solches die benanten zienhendler ahlsambt , desgleichen die gewercken zu stetter, vhester , ahngenehmer haltung beneben mir, dem hauptmahn als underhendler mit unsern ahngebornen und gewonlichen petzschafften herunden ufzutrucken , wissentlichen diesen zwiefachten schiedt bemercken lassen. Geschehen zu Pirnau , Mittwoch nach Katharine -, den 27. tag Novembris nach Christi gebürt im 1566. jhar. 458 VI. Dokumente der kapitalistischen Wirt¬ schaftsorganisation im Idrianer Quecksilber¬ handel des 16. Jahrhunderts. 32. Ferdinand I. und die Gewerken von Idria schließen mit den Höchstettern (Augsburg ) einen Quecksilbermonopolkontrakt. 1. Januar 1525. —- F . A. Wien . Gedenkbuch 21, Bl. 248 ff. Vertrag des queckhsilber - und zinoberskauf in Ydria zwi¬ schen fürstl . durchlaucht , den gwerckhen daselbs und Am¬ brosien und Hannsen den Höchstettern gebrueder zu Augs■purg. Kundt vnd zu wissen sei gethan menigklich , dass ..... herr Ferdinand etc ., auch die wolgebornn , edl, vesten , fürsichtigen , ersamen und weisen herr Hans von Auersperg , herr zu Schönberg für sich selbs und als volmechtiger gewalthaber des erwirdigen fürsten und herrn , herrn Cristoff, bischof zu Laybach etc . herr Sigmunden von Dietrichstain , freiherrn zu Hollenburg und Vinckhenstain etc. Wilhalmen Newmqns und ander etlicher mer gewerckhen , der gwalt er hat , Blasii Hölczl , fürstlicher durchlaucht rat vnd plileger zu Vellenberg , Cristoff Herbst , phleger zu Toblach , Steffan Käserer, Valtein Kutler von Salzburg vnd Liennhardt Ruepp , die all für sich selbs vnd als gewalthaber annderer irer mitverwandten vnd gwerckhen des perckhwerchs in Idria mit zeitigem rat vnd wolbedachtem muet für sich, ihr erben vnd nachkomen gnedig vnd vnnderthenigclich miteinannder veraint auch entslossen und den vesten , furnemen Ambrosien vnd Hansen den Höchstettern gebruedern zu Augspurg vnd iren mituerwandten ainen queckhsilbervnd zinoberkauf von und aus dem perckhwerch Ydria gegeben vnd kaufweis zugestelt haben , wissentlich in craft diz briefs . Also dass den gemelten Höchstettern vnd iren mituerwanten von der fürstlichen durchlaucht vnd den gwerckhen zuesteen vnd eruolgen soll : nemlich alles queckhsilber vnd zinober , sovil des auf heutigen tag gemachts guets vorhannden ist und zusambt demselben von VI . Im Idrianer Quecksilberhandel des 16. Jahrhunderts . 459 denen queckhsilbern vnd zinobem in negstvolgenden vier jarn ge¬ macht werden, benentlich in ainer suma vmb 300 000 guldin werdt guets an queckhsilber vnd zinober. Also vnd dergestalt, dass solh queckhsilber vnd zinober den Höchstettern von allen gwerckhen gemachts guets vnd auch von obberürter fürstlichen durchlaucht fron vnd Wechsel vnd andern der gwerckhen quekhsilber vnd zinober, so si in Ydria haben werden bei gedachtem perckhwerch in Ydria sollen uberantwurten und zustellen nach rechten geschwornen zimmenten wienischen gewicht ain yeden zenten queckhsilber vmb 30 gülden rh. und ainen yeden zenten zinober umb 35 gülden rh. Alles mit gueter gemainer landsgebreuchlichermünz nachvolgender gestalt zubezalen. Aber mit abziehung der fei vnnd fassl solls gehalten werden, wie bisher vnd vormals mit dem Phliegl, Newman oder andern kaufleuten gehalten worden ist. Und nachdem gwer¬ ckhen und verwandten zu sand Achazien, Katherein und denselben verwandten grueben mass und gerechtigkaiten ainen treffenlichen vorradt gemachts queckhsilbers vnd zinober in Idria zu Cronburg, Laybach, Venedig oder andern orten ligen haben und ob ir fürst¬ lichen durchlaucht und die obgemelten gewerckhen zu iren geburenden tail auch etlich queckhsilber oder zinober in Idria hetten, so soll den gemelten Höchstettern durch obgemelte und dieselben gwercken und verwandten der genant vorradt von stund an in abslag diser kaufsuma der 300 000 gülden rh. zugestellt und gegen iren quittungen uberantwurt werden, doch was furlon vnd uncossten darauf ganngen ist aus Ydria bis in die leger, da yezundt das guet ligt, des sollen die genanten Höchstetter den gewerckhen auch bezallen vnd si darumb zufriden stellen. Was aber die fürst¬ liche durchlaucht und gemain gewerckhen den Höchstettern und iren mitverwandten über den angezaigten vorradt zu völliger erstattung der 300 000 guldin werdt queckhsilber und zinober noch zuuberantwurten schuldig werden, das sollen gemelte fürstliche durchlaucht und gemain gwerckhen den Höchstettern vberantwurten und zustellen. Dergestalt nemlich in-dem nachvolgenden 1526, 27 und 28 jar zu quattember Zeiten nach gelegenlicher, geburlicher eintailung was sich dann nach anzall der suma auf yedes jar zu quattember Zeiten gepurt. Und von wegen der bezalung ist es also beredt vnd durch die Höchstetter bewilligt, welhs auch von der fürstlichen durchlaucht vnd den gemainen gwerckhen angenomen ist : dass die Höchstetter auf solhen kauf yezunder be- 460 Dokumente der frühkapitalistischen Wirtschaftsorganisation. zallen sollen 40 000 gülden rh. und die gen Innsprugg erlegen und den vorgemelten gwerckhen von Sannd Achazien, Katherein und derselben gemessen pürg 1 und gerechtigkait in abslag der bezalung irs vorradts gegen iren quittung vberantwurten. Und auf nechstkünftigen Sannd Jacobstag dits 25. jars sollen gemelte Höchstetter und ir mituerwanten anfahen und bezallen, nemlich mit der zeit wie si stat haben mugn bis auf weinnechten ausgang des 25sten jars benentlich 35 000 gülden rh. und dieselben zu hannden der oftgemelten gwerckhen zu Sannd Achazien und derselben gerechtig¬ kait gegen iren oder irer diener und gewalthaber quittung auch in abslag des vorradts, den si inhalt dits Vertrags empfangen haben mit parem gelt uberantwurten und bezallen, damit si die vorge¬ melten gwerckhen von den gemelten Höchstettern auf dits 25. jars in abslag irs emphanngen queckhsilbers und zinobers bezalt werden, wie vorgemelt ist. Nemlich 75 000 gülden rh. Dieweil aber noch, wie vorsteet, mer queckhsilber vnnd zinober zu erstattung ganzer kaufsuma soll geantwurt werden, ist also beredt und entlieh be¬ sessen worden, dass im 26. jar soll angefangen und zu quattemberzeiten die geburend anzal queckhsilber und zinober geantwurt werden inmassen, wie hieuor gemelt ist. Aber die bezalung der¬ selben queckhsilber vnd zinober soll albeg ain jar nach der emphahung vnd uberantwurtung der gueter angeen dergestalt zuuersteen, dass soll angefanngen werden die erst quattember des 27. jars vnd soll bezalt werden das quecksilber und zinober, so die erst quatember des 26. jars empfangen und uberantwurt worden ist, und also furtan für und für gehalten, uberantwurt und auch bezalt werden albeg zu quattember Zeiten, nemlich des 27., 28. vnd 29. jar, yedes jar 75 000 gülden rh. zu gannzer bezalung der 300 000 gülden rh. werdts guets, so inhalt des vorgemelten artigcls in disen bestimbten 4 jarn an queckhsilber und zinober uberant¬ wurt worden sein. Doch ist das dar und lauter geredt worden, wo das perckwerch in abfal käme und sich mit kriegsleuffen oder in ander weg, — das Got lanng verhueten well — vrsachen zu¬ trugen, dass nit souil queckhsilber und zinober gemacht wurde, als yez hievorgemelt verkauft ist, so sollen die fürstliche durchlaucht, auch herrn und gwerckhen den Höchstettern nit verpunden sein, die erstattung mit queckhsilber und zinober auf die angezaigt 1Das heißt abgemessenes Gebirge. VI . Im Idrianer Quecksilberhandel des 16. Jahrhunderts . 461 suma der 300 000 gülden rh . zuthun . Auch die Höchstetter den gemelten gwerckhen nit mer zubezallen verpunden sein, dann die Höchstetter an queckhsilber und zinober von den gwerckhen emphangen haben . Verer nachdem fürstliche durchlaucht zu erhaltung irer fürstlichen durchlaucht camerguet , auch den gemelten gwerckhen und verwandten des perckwcrchs nit albeg an ainer stat die bezalung gelegen sein will zuemphahen , ist lauter beredt worden , das die Höchstetter und ir mituerwandten der fürstlichen durchlaucht und den genanten gewercken die bezalung sollen thun und raichen zu yeder zeit , wie die fristen benent sein in der stat ainer als zu Wien in Österreich , zu Salzburg , zu Hall im Intal oder zu Villach in Kerndten , wo das irer fürstlichen durchlaucht etc . und den herrn vnd gwerckhen gefellig vnd gelegen sein will zuemphahen. Darauf bekennen wir Ferdinand, ..... auch wir vorgemelt gwercken in Ydria für uns selbs und als volmechtig gwalthaber der andern unsern mituerwandten und gwerckhen bemelts perckhwerchs und wir Ambrosy und Hanns gebruder die Höchstetter als für uns selbst und unser mituerwandt auch all ander unser erben und nachkommen wissentlich hiemit und in craft dicz briefs also dass wir solhem verkauf und kauf für die vorgeschriben 300 000 gülden rin queckhsilber und zinober , wie der in allen und ypden vorgeschriben puncten vnd artigkln gestelt und begriffen ist , vesst, stet und ungewaigert halten , auch demselben mit uberantwurtung gemelts queckhsilbers und zinobers und dargegen mit der bezalung zu yeder vorbenenten zeit getreulich und erbarlichen nachkomen und geleben sollen vnd wellen. Wir obgenandt Erzherzog Ferdi¬ nand und obgeschrieben gwerckhen sollen vnd wellen auch in obbestimbter zeit der vier jar ob mer als fur die 300 000 gülden queckh¬ silber und zinober gemacht wurde bei und vnder verpfundung 300 zennten queckhsilber und 200 zennten zinober niemand anderm durch uns oder von unsern wegen kain queckhsilber oder zinober zustellen , antwurten noch verfuern , sonder also stilligen und den kauf inirderHöchstetterainigehanndt erstgedach¬ ter 4 jar beleihen lassen . Und ob sich aber das aus vergessenhait oder sonst in annder wege wie sich sölhs zutragen möchte , dass ainicher verkauf ausserhalb der Höchstetter bescheche , welches doch in dhainen weg sein soll, begebe , so soll doch solcher verkauf, Zustellung, oder wie das genent werden möcht , kain wirckung , craft noch macht haben , sonder die vorgeschriben verpindung der 300 462 Dokumente der frühkapitalistischen Wirtschaftsorganisation. zenten queckhsilber und 200 zenten zinober die fürstliche durchlaucht und die gwerckhen den Höchstettern on underlass zu bey allen vnd nichtdestminder den verkauft für die 300 000 gülden vesst und stet zuhalten schuldig sein. Doch Hansen Paumgartner zu Augspurg , Wilhelmen Newman und weiland Hansen Phluegls erben ir erkauft queckhsilber und zinober , so in disen vorgeschriben gemainen kauf oder den gemainen gewerckhen nit zugehört , Vor¬ behalten und hierinn aussgeslossen , also dass si irem gefallen nach damit handlen mugen . Souer si aber ainich queckhsilber und zinober erkauft , oder in ander wege das billich und inhalt des Ver¬ trags , so zwischen vns, erzherzog Ferdinand und uns, gemainen gewerckhen jungst zu Wien am 28. tag Augusti anno etc . im 24. aufgericht ist , in disen kauf gehöret , bei hannden hetten oder noch zu iren hannden pringen möchten , dasselb soll auch in disen kauf feruolgt vnd uberantwurt werden laut obgemelts Vertrags . Wir sollen und wellen auch gedachten Höchstettern , iren erben vnd mituerwandten nach verscheinung obgemelter vier jar souer wir, erzherzog Ferdinand , auch gcmain gewerckhen solhe vorgeschribne war des queckhsilbers und zinobers wider in ain hanndt zu verkaufen willens sein und sich die Höchstetter in disem yezigen vierjerigen kauf uns und dem perckwerch erschiesslich halten und das bcmelt queckhsilber und zinober in ainem mererm oder hohem kauf weder diser yezgemelter kauf der gestalt ist , nemlich der zendten queckhsilber auf 30 und zenten zinober auf 35 gülden pringen , denselben Höchstettern ainen neuen kauf vor menigclichen vergannen und albeg ain yeden zendten ob¬ gemelter war umb 30 kreuzer nehner dann yernands andern ver¬ folgen lassen . Weiter so wellen wir, oftgenanter erzherzog Fer¬ dinand für uns, unser erben und nachkomen gemelten Höchstettern, iren mituerwandten und erben gegen der römisch kayserlichen maiestät , unserm gnedigisten lieben herrn vnd bruder , oder seiner kayserlichen mayestät und des reichs regiment oder desselben fiscal auch sonst gegen menigclichen von welichem si solhs kaufs halben angesprochen und dardurch in schaden zufuern vnnderstanden wurden , ir gnediger herr , schirmer vnd vorsprech er sein, auch deshalb ganz schadloss halten . Weihe war fur die gemelten 300 000 gülden rh . queckhsilber und zinober sollen und mugen auch obbemelte Höchstetter und ir mitverwandt zu yeder , in krigs oder fridlichen Zeiten, wann inen das fuegt , irem freien willen, ge- VI . Im Idrianer Quecksilberhandel des 16. Jahrhunderts . 463 fallen, nuz und notdurft nach verfuern , verkumern und verkaufen, on unser , unserer erben , nachkomen und mituerwandt auch menigclichs von vnsern wegen irrung , hindernus und widersprechen . Doch uns die gewondlichen zoll und meut , wie dann die von alter und bisher von solher war gegeben und abzalt worden sein, darüber wir si auch in dhainen weg zu staigern understeen noch gestatten wollen Vorbehalten ; alles treulich vnd vngefehrlich . Zu vrkundt sein diser kauf und Vertragsbrief 3 in gleichem laut gemacht und under vnser erzherzog Ferdinand gewondlichem handtzaihen vnd furgedruckhten secret verfertigt . So haben wir Blasy Holzl, Cristoff Herbst , Steffan Kasserer , Valtein Kutler vnd Liennhardt Ruepp fur uns selbs und anstat gedachts herr Hannsen von Aursperg und dero, so er, wie obgemelt ist , gwalt hat , und uns in seinem abwesen zuuerfertigen beuelh vnd gwalt geben hat und dann alle ander unser mitgewerckhen uns mit aigner hannd underschriben und unser aigen petschaft aufgedruckht . Dergleichen hab ich, Ambrosy Höchstetter für mich, mein brader Hannsen vnd alle annder meine mituerwandten mich auch mit aigner hannd underschriben und mein gewondlich petschafft furgetrackht . Geben zu Innsprugg , am ersten tag Januarii, anno etc . 25. 33. Verabredung eines idrianischen Quecksilberhandelsmonopols zwischen König Ferdinand , den Gewerken von Idria und Hans Baumgartner . 1. August 1539. -— Wien , Finanz -Archiv. Innerösterr . Quecksilber 22, Nr . 18 320. Khundt und ze wissen sei gethan menigclich , dass der allerdurchleuchtigist herr Ferdinand , römischer khunig etc . und die edlen etc . herr Joseph von Lamberg zu Schneeberg , ritter , röm. ku . mt . rat und derselben gemahel oberster hofmaister , herr Melchior von Lamberg , hochgedachter ku . mt . rat und hofmarschalch , bed für sich selbs, herr Niclaus Ribeisen zu Neu -Kiemnitz , frau Barbara Neumanin zu Wasserburg , Wilhelm Rasp , Marx Stettner und Niclaus Mossner für sich selbs und als volmechtig gewalthaber aller anderer gewerckhen , so im landt zu Kherndten und Crain gesessen sein, Ulrich Geltinger für sich selbs und als volmechtiger gewalthaber der gewerckhen , so in der grafschaft Tirol gesessen sein, und weiter obgemelter herr Niclaus Ribeisen , Wilhelm Rasp und Cristoph Cronneckher als volmechtig gewalthaber aller gewerckhen im stift Saltzburg sesshaft , in chraft irer habenden genuegsamen und vol- 464 Dokumente der frühkapitalistischen Wirtschaftsorganisation. khomener gewaldt für sich selbs und alle ire prinzipal, auch alle andere abwesendt herrn und gewerckhen des perkhwerchs in Ydria, Sandt Achatzien Rechtn, mit zeitigem rat und wohlbedachtem muet für sich, ire erben und nachkhumen mit einander veraint, auch beslossen und dem edln und vesstn Hansen Paumgartner von Paumgarten zu der hohen Schwangaw und Erbbach, röm. kais. und ku. mt. rat und seinen erben oder nachkhumen ainen queckhsilber- und zinnober-khauf von und auf dem berckhwerch Idria und desselben jetz gemachten und khunftigen Vorrats und guetern gegeben und zuegestellt haben von primo Januarii nechstkhunftig auf 5 jar lang, die nechsten nacheinander volgendt, wissentlich in chraft ditz briefs. Benenntlich umb 75 000 fl. rh. in muntz queckhsilbers und umb 25 000 fl. rh. in muntz zinobers. Nemblich je ain Centen queckhsilbers umb 26 fl. und je ain centen zinober (alles wienisch gewichts) umb 30 fl. in muntz, je 15 patzen 60 kr. oder wie jeder¬ zeit 1 fl. in muntz gelten und gerechnet wirdet, in gueter, landtleufiger, unverruefter muntz oder italianischen gueten cronen oder ducaten, wi di jederzeit zu den fristen der bezallung genng und gab sein werden. Bringt in summa queckhsilber 2889 centen und an zinnober 833 und % aines centners wienisch gewichts. Und nachdem aber die gegenburtig zeit und leuff so gef erlichen und schwär, sonderlichen diser obgemelten waar halben, so haben die hochgedacht ku. mt. und di obgenannten herrn gewalthaber für sich selbst und ire prinzipal aus den und andern gueten, beweg¬ lichen und ansehenlichen Ursachen, auch neben anderm der praticen halben, so der Paumgartner in vorigen kheufen diser handlung zu guetern geen Alcheuro und Constantinopl mit etwas grossen chosten, wie ehr glaubhaft dargethan, gefuert hat , sonderlichen auch damit si den Paumgartner zu aiüem solchen grossen khauf anzenemben bewegt, genedigistlichenund freundtlich bewilligt, be¬ slossen, zuegesagt und versprochen dem gedachten Paumgartner oder seinen erben oder nachkhumen in disem obgemelten khauf jetz nachgeschribne auf- oder eingab, nemblichen 687 centen queckh¬ silbers und 166 und % aines centner zinobers wienisch gewichts frei on aiuich khaufgelt oder bezallung auf- und einzegeben. Und soll auch mit abziehung der feil und fasl gehalten werden wie bisher und vormals mit andern gehalten worden ist. Und haben sich sonst zu beden tailen inmassen und gestalt wie hernach volgt verglichen und vertragen. Namblich so sollen VI . Im IUrianer QuecksiMerhandel des 16. Jahrhunderts . 465 die hocligedacht ku . mt . und Herren und gewerckhen in Idria dem obgenanndten Hansen Paumgartner und seinen erben oder nachkhumen , oder wem si an ihrer stat des verordnen , solche obbestimbte summa der queckhsilber und zinober wie obstect in queckhsilbern und Zinnober uberantwurten und übern von, auf, in und bei benieltcm pcrcklnverch Idria auf ir oder irer verordenten erstes er¬ fordern gar oder zum tail , wie inen jederzeit gefellig und gelegen und auch derselben zeit des gemachten Vorrats halben in der ge¬ werckhen vermugen ist on allen Verzug und saumbnus. Und ob Herrn und gewerckhen jetzo zu Triest , Villach oder ainichen andern orten queckhsilber oder zinober hetten , aida dem Paumgartner , seinen erben oder iren verordenten solche queckh¬ silber und zinober anzenemben gelegen und gefellig, das soll inen gestattet und von herrn und gewerckhen an solchen orten unverzogenlich auf ir begeren zucgestellt werden . Allain soüen der Paumgartner , sein erben oder gewalthaber den herrn und gewerckhen bezalln den ungeverüchen unchosten , so aus dem perckhwerch Idria bis zu denselben orten fucrlons und ordinari -unchostens halben auf solche queckhsilber und zinober an dieselben ort ze bringen auf¬ gelaufen wäre . Es sollen auch deshalben di ku . mt . und herren und gewerckhen in Idria gegenwärtigen und lchunftigen Verwesern sondern bevelch thuen , dem Paumgartner , seinen erben oder nachkhumben oder iren gewalthabern also jederzeit nach irem begern, wie obsteet , di obbestimbteu queckhsilber und zinober zu libern und zu geben. Und sollen insonderhait der Paumgartner , sein erben und nachkhumen macht und gewalt haben under solchen obbestimbten summa queckhsilbers und zinobers aines jeden ze nemben sovil inen gefellig und gelegen. Das ist zu versteen , dass si nit verpunden sein sollen (so es ir gelegenhait nit wäre) die drei viertl queckh¬ silber und den vierten tail Zinnober ze nemben . Sonder si mugen, als jetzt obsteet , des ainen wenig und des andern sovil dester mer nemen nach irem gefallen ; doch ain jedes in seinem khaufgelt alle obsteet . Und ob aber der Paumgartner , sein erben oder naclikhumen des zinobers mer dann ain viertail der khaufsumma mergemelter 100 000 fl. rh . haben wollten , so sollen si als dann den herrn und gewerckhen solches 4 monat zuvor verkhunden und anzaigen und als dann inen die herren und gewerckhen sovil inen in vorbestimbter zeit zu machen muglich ist , zu geben und täglich Strieder , Studien 7,. Gesch . kapitalist . Organisationsformen . 30 466 Dokumente der frühkapitalistischen Wirtschaftsorganisation. auf ir begern zu libern schuldig sein. Doch die obbestimbt einund aufgab , so inen on khaufgelt und bezallung frei ze geben be¬ willigt ist , di sollen si ir jedes in der obbeschriben anzall ernpfahen, damit disfalls die Sachen wie obsteet lauter und richtig beieiben; des sich der Paumgartner zu thuen bewilligt hat. Und dargegen soll der Paumgartner , seine erben oder nachkhumen di bezallung also thuen : Nemblichen soll er, Paumgartner oder sein erben jedes jars 20 000 fl. zu zwaien fristen und benanntlich am ersten tag Junii nechstkhumendt 10 000 und am ersten tag Decembris des nechstkhunftigen 40. jars aber 10 000 und also auch hinfuran jedes jars bis auf den ersten tag Decembris des 44. jars bezallen (an welchem die lesst frist angeregter bezallung beschehen) und damit soll di völlige khaufsumina der 100 000 fl. also entricht und bezalt werden. Und zu jeder obgemelter frist soll der Paumgartner ku . mt. gepurendt fron und wechsl, nemblich den zehenden phening für fron und den achten phening fur wechsl, trifft allmal von 10 000 fl. 2125 fl. von und aus disem khauf und allmall von den 10 000 fl. zu jeder frist , wem solches ir mt . Verweisung nach gepurt , entrichten und bezallen lassen. Nachdem auch die herrn und gewerckhen der röm . ku . mt. jetzt von wegen nachlass des halben wechsl 18 000 fl. untertheniglicli verert und diseiben 18 000 fl. irer mt . aus disem khauf in den nechsten dreien jarn zu den gemachten fristen von irer gepur zu bezallen unterthenigclichen bewilligt , demnach soll der herr Paumgartner zu den ersten 6 fristen , nemblich zu jeder frist 3000 fl. von der ge¬ werckhen angepur irer mt . gegen genuegsamen Quittungen bis zu völliger bezallung der obgemelten 18 000 fl. entrichten und bezallen. Solche jetz gemelte 18 000 fl. wellen die gewerckhen ime, Paum¬ gartner oder seinen erben fur richtige bezallung passieren und an der khaufsumma abraitten lassen. Und sollen die bezallungen , sovil den gewerckhen daran gepurt, durch den Paumgartner oder sein erben jede der obgemelten fristen in nachgestellten zwaien stetten beschehen . Nemblich zu Villach in Kherndten , sovil den gewerckhen , in den niderösterreichischen landen gesessen, daran gepurt , sambt 6000 fl., so jedes jars auf die sambchost und verleg des perckhwerchs , sonderlich damit das arzt zu laistung dises khaufs zu khaufmannsgut gemacht werde , verordent sein. Und zu Schwatz in Tiroll der ku . mt . gepurendt fron und VI. Im Idriancr Quecksilberhandel des 16. Jahrhunderts . 4ß7 wechsl und die angepur der gewerckhen , so in den Öberösterreichi¬ schen landen , auch dem stift Saltzburg , der stat Augspurg oder sonst oben im reich gesessen sein, zu handen irer verordenten gegen geburlichen quittungen erlegt werden , albegen in landtleufiger bezallung . Nämblich zu Villach in gueter weisser und zu Schwatz in daselbst gengiger und gäber unverruefter muntz oder italianischen cronen in dem werdt wie vorsteet und alsdann albegen 4 kr . ( fur ainen patzen und 15 patzen fur ainen guldin oder wie ain guldin in muntz derselben zeit gérait wirdet. Es soll auch Wilhelm Rasp , jetziger Verweser in Ydria ge¬ dachtem Paumgartner inner 6 Wochen, den nechsten , ainen lautern auszug , inhalt , undt was und wievil er, Paumgartner an jedem der gedachten fleckhen , Villach und zu Schwaz, bezallen und was ainem jeden gewerckhen daraus gepurt , uberschickhen und zuestellen lassen. Und ob aber Paumgartner , sein erben oder nachkhumen über khurtz oder lang der ku . mt . oder herrn und gewerckhen auf ir genedigist und freundtlich handlung und Vergleichung an diser obund vilgemelten khaufsumma der 100 000 fl. etwas vorein bezallen und erlegen wurde , so ist jetzo beredt , bedingt und angenumben, dass der Paumgartner , sein erben oder nachkhumen dasselbig zu jeder den obgemelten fristen und zilen der bezallungen inenbehalten und aufheben . Es soll und mag auch der Paumgartner , sein erben oder nachkhumen insonderhait an jeder solcher bezallung nit allain aufheben und inenbehalten dasjhenig , so inen auf irer guggus 1und tail , so si in disem perckhwerch haben oder khunftig uberkhumen, gebürt , sondern auch dasjhenig , so si villeicht ainichen gewerckhen in chraft des articels hernachgemelt dis khaufs halben vor den fristen auf Vergleichung voreingeben oder bezallt hetten . Doch soll er, Paumgartner oder sein erben in die 6000 fl., so ain jedes jars zu underhaltung des perckhwerchs wie obsteet zu haben von notten , khainem gewerckhen nichtzit voreinbezallen , sondern allain in der ubermass wie jetzt oben gemelt ist . Und seint auch sonst in disem queckhsilber - und zinnober -khauf und vertrag furnemblich dise hernach volgendt articl durch hochgedacht ku . mt . und herrn und gewerckhen an ainem und dem Paumgartner am andern tail lauter abgeredt , gencdiglich und vestigclich bewilligt , angenumben und zuegesagt worden: 1 Das heißt Kuxe. 30* 468 Dokumente der frühkapitalistischen Wirtschaftsorganisation. Erstlich nachdem zwischen and sider ausgangen des Paumgartners jungstgehalter zwaier queckhsilber- und zinnober-kheuf der 32 000 und der 10 000 gülden bisher von queckhsilber und Zinnober etwas zu aintzing 1verkhauft worden, des doch herrn und gewerckhen ires Wissens nit über 10 oder 12 säm ungefcrlich angeslagen, so ist demnach dem Paumgartner gewislich zuegesagt, dass zwischen und nach solchen zwaien vorigen kheufen über 20 säm nit verkhauft, sonder damit stillgestanden seie. Verrer dass die ku. mt . und herrn gewerckhen ire Verweser noch sonst jemandts von iren wegen innerhalb obbestimbter zeit der 5 jar khein idrianisch queckhsilber oder Zinnober an kheinem ort noch ende nit verkhaufen noch verfuern, dasselb auch niemants gestatten noch inen darin durch ainigerlei weg eingriff thuen oder zuefuegen lassen. Dass auch in sonderhait die hochgedacht ku. mt. sonst niemandt andern dann bemeltem Paumgartner oder wer des von im bevelch hat in werenden obermelten 5 jarn khain queckh¬ silber oder zinnober durch ire mt. erblandt und derselben erblandt gepiet nit passiern lassen und auch bei verlierung desselben ernstlich verpietten und deshalben allen ambtleuten ernstlichen bevelhen, guet aufmerckhen ze haben und also in disem faal herrn und ge¬ werckhen auch den Paumgartner, seine erben und nachkhumben gcnediglichen handthaben und ob inen halten wellen. Doch hier¬ innen ausgenummen di queckhsilber und zinober, so dem Paum¬ gartner und seinen erben und nachkhumen in chraft ditz khaufs zuegehörig oder von inen oder mit irem gueten wissen und willen von disen idrianischen queckhsilbern und zinnober erkhauft sein. Darzue sollen auch die ku. mt. der hispanischen und beharni¬ schen queckhsilber halben, damit diseiben dem idrianischen quechksilber khainen nachthail bringen, sovil ir mt. mit fueg erheblich ist gn. verholfen sein. Und wellen ir ku. mt. der röm. kais. mt . auch schreiben und pitten, dass ir kais. mt. in irem hispanischen queckhsilberperckhwerch verordnen wellen, dass dasselbig nit dermassen, wie ain zeitheer angefangen, uberpaut, sonder wie von alther ain mass gehalten und bedacht werde, damit dise bede perckhwerchen wie hievor neben einander unvernachtailt in langwirigkhait hinkhumen mugen. 1Soll vielleicht heissen: im einzeln. VI . Im Idrianer Quccksilberhaiuiel des 16. Jahrhunderts . 469 Es sollen auch furnemblich der hochgedacliten ku. mt. und lierrn und gewerckhen gegenwurtig und kliunftig Verweser in Idria gegen gedachtem Paumgartner, seinen erben und nachkhumben jetzo und khunftigclich jedesmals in chraft dises Vertragsbrief verschriben und obligiert sein, mit dem verkhaufen nit allain wie jetzt obsteet still ze steen, sonder auch die ambtleut und Verwalter bei Iren ambts- und aidspflichten gewisslichen des verhueten und fuerkhumen gleichermass als ob di jedesmals sondere Verschreibung gegeben und die ku. mt. und herrn und gewerckhen die sonderlich eonfirmiert betten, des si hiemit in chraft ditz briefs tliuen. Ob auch dem Paumgartner , seinen erben oder nachkhumen die queckhsilber und zinober von jemandts , wer der oder die weren, von wegen ainiclier sprach und vordrung , so diselbèn oder der zu hochgedachter ku . mt . oder den gewerckhen Sachen halb des perckhwerebs queckhsilber oder zinober aus Idria belangend t hetten, haben möchten oder zu haben vermainten , angesprochen würden, in was wege das beschähe , so sollen alsdann die ku . mt . und herren und gewerckhen ime, dem Paumgartner , seinen erben oder nach¬ khumen solch queckhsilber und zinober on iren (des Paumgartners, seiner erben oder nachkhumen oder wen er, Paumgartner oder seine erben die verkhaufen würden ) costen , schaden und nachthail wider ledig und frei machen und halten. Und ob sich auch zuetraeg , dass sich der Paumgartner , sein erben oder nachkhumen in zeit dis Vertrags mit ainem oder mer gewerckhen vertragen und vergleichen , also dass in demselben seinen gepurenden tail vor der zeit geben würden , des sollen si guet macht und gewalt haben , doch sovil den notwendigen samcosten und verleg des perckhwerchs (als nemblich jedes jar 6000 gülden gerechnet ) betrifft , wie ob angezaigt , ausgeslossen . Und soll dadurch derselb von den andern gewerckhen in ander weg noch herrn und gewerckhen von dem oder denselben nit abgesondert sein, auch in sonderhait der gedacht Paumgartner , sein erben oder nachkhumen dasselbig also wie obsteet zu jeder frist an der bezallung innen¬ behalten und aufheben. Es ist auch hierinnen furnemblichem genedigclichen abgeret, bewilligt , angenumen und zuegesagt worden : So sich begeben , dass der Paumgartner , sein erben oder nachkhumen mer queckhsilber und Zinnober dann umb die 100 000 gülden werdt in der obbestimbten zeit bis zu endt der 5 jar begeren und haben wellten (wie 470 Dokumente der frühkapitalistischen 'Wirtschaftsorganisation. er sich das auf das maist sovil muglich zu versleissen allen gueten vleiss furzekhern, doch unverpuntlich erpotten hat), so sollen die ku. mt. und herrn und gewerckhen inen sovil si des begern in dem precio und mit der ein- und aufgab nach antzall wie in disem khauf der 100 000 fl., auch mit abzug der feil nach gebrauch und gewonhait, alles wie obsteet, sovil sie des im Vorrat haben und inen ze machen muglich ist, zu geben und zu libern unverzogentlich zu jeder zeit schuldig sein. Doch dass Paumgartner, seine erben oder nachkhumen solch ubermass dreiviertl jars vor ausgang dises gegen¬ wärtigen khaufs empfahen und sollen das in vorbemeltem preis und albegen nach der antwurtung in jarsfrist nemblich halbs zu ausgang des 6. monats und den andern halben thail zu endt des jars in muntz oder cronen auf muntz wie obsteet, wie dann ain gülden in muntz gérait wirdet, auch an orten und enden wie vorbemelt, zu bezallen schuldig sei. Doch ist dises jetzobeingeschribens articls halben weiter obgeredt: So der Paumgartner oder seine erben in der zeit ditz khaufs über di vorbemelt khaufsumma ain merere antzall queckhsilber und Zinnober begern und emphahen und aber dasselb in zeit ditz ersten khaufs nit gar verkhaufen, sonder inen daran etwas uberbeleiben wurde, das sollen alsdann der Paumgartner oder sein erben nach ende des khaufs der ku. mt. und herrn und gewerckhen, inmassen solchs emphangen haben, wider zu antwurten und zu geben, auch sie, die ku. mt . und herrn und gewerckhen anzenemen schuldig sein, in abschlag der bezallung und in dem precio wie er, der Paumgartner, von inen erkhauft hat. Doch sollen die ku. mt. und herrn und gewerckhen alsdann dem Paumgartner oder sein erben insonderhait auch erstatten und er, Paumgartner, inenbehalten den ordinarie unchosten, so auf solch queckhsilber oder zinober, was das an di ort, da es ligt, und den gewerckhen solches widerumb emphahen sollen, geloffen. Wo aber der Paumgartner die ubermass des queckhsilber oder zinobers gar oder zum tail bezallt het und die ku. mt. sambt den herrn und gewerckhen das queckhsilber gegen widerbezallung der hinausgegeben summa gelts begeren wurden, alsdann und sonst nicht sollen dieselbe hinausbezallt summa gelts die ku. mt . und herren und gewerckhen den Paumgartner und seinen erben in 2 monaten, den nechsten nach ausgang der 5 jar mit parem gelt zu Augspurg wider in dem werdt wie vorstet zu bezallen schuldig sein. Wo aber die ku. mt. sambt den herrn und gewercken solches VI. Ira Idrianer Quecksilberhandel des 16. Jahrhunderts . 471 in bestimbter zeit nicht volzugen, alsdann soll Paumgartncr und sein erben und nachkhumen mit demselben queckhsilber solches seinem nutz nach zu vertreiben on ku. mt. und der gewerckhcn Verhinderung frei sein. Verrer als in beden vergangen kheufen und derselben vertragen dem Paumgartncr der vorkhauf verschriben gewesen und nun die gewerckhen sicli gentzlichen versehen , dass Paumgartner etwas mereres uber die 100 000 gülden in denen 5 jaren versleissen werdt, deshalben haben die ku . mt ., auch herrn und gewerckhen gegen ge¬ dachten Paumgartner und seinen erben jetzt nachvolgender meinung bewilligt : Nemblichen sover er, Paumgartner oder sein erben , sich in disem fünfjärigen khauf gemainen herrn und gewerckhen erschiesslich 1 halten und das queckhsilber und Zinnober in ainen hohem preis dan es jetzo ist , bringen werden und ' in 6 monaten vor ausgang des khaufs bei den gewerckhen oder irem Verwalter umb ein weitern khauf ansuechen , oder ob die gewerckhen zu solcher zeit ine Paumgartner oder seinen erben umb ein weitern khauf anlangen wurden , so mag der Paumgartner selbs oder durch seine gewalthaber mit inen sich aines weitern khaufs halben vergleichen und denselben besliessen, darin sich die ku . ml . und die gewerckhen gegen dem Paumgartner fur ander genedigclich und freundtlich halten und erzaigen wellen. Sover er aber vor ausgangs ditz gegenwurtigen khaufs khein weitern khauf mit inen besliessen wurdt, so wellen alsdann die röm . ku . mt . und herrn und gewerckhen weiter gegen ime, Paumgartner oder seinen erben deshalben nichts mer verpunden sein, sonder ires gefallens jemandts andern ain khauf geben oder selbs verkhaufen . Doch wellen si in disem werenden khauf niemandts ainigen khauf zuesagen noch mit je¬ mandts solchen khauf vor ausgang ditz gegenwärtigen khaufs be¬ sliessen. Und ist hierin auch furnemblichen gegenwurtigen schwären leufen nach bedacht , abgeret , bewilligt und obligiert worden : Ob sich zuetragen wurde , dass in dem perckhwerch durch ainigerlai krieg , einfall oder ursach eingriff geschehe , also dass gcdachts pergkwerchs queckhsilber oder Vorrat durch jemandts geschedigt, angriffen oder ausser des herrn Paumgartners , seiner erben und nachkhumen verfuert oder verkhauft wurden (das doch in khainem i = ersprießlich, nützlich. 472 Dokumente der Irühkapitalistischen Wirtschaftsorganisation. weg sein soll), alsdann in solchem fall, so es wissentlich gemacht wurde und sonst nicht in des khaufers, seiner erben und nachkhumen wilkliuer, macht und gewaldt steen, weiter queckhsilber und Zinnober in diesem vertrag und khauf zu empfahen oder des muessig zu steen. Und so si also in solchen fallen weiter in rue und muessig steen wollten, sollen sie alsdann weiter hernach der ku. mt. und herrn und gewerckhen noch deren erben oder nachkhumen ainich khaufgelt fur das, so si nit emphangen haben, zu bezallen nit schuldig, sondern in albeg allerding ledig und mues¬ sig sein. Und ob also durch die hochgedacht ku. mt. und herrn und gewerckhen oder dem Paumgartner diser zeit obgeschriben vertrag und verainigung aus vergessen oder sonst nit gehalten, sondern dem zuwider in ainichen weg geschehen oder gehandlt wurde, (des doch nit sein) so sollen alsdann die ku. mt. und herrn und ge¬ werckhen oder der Paumgartner, welches tails halben dann hier¬ innen mangl befunden wurde, dem andern tail allen nachtail und schaden gentzlich widerkhern und abthuen. Wo sich aber alsdann solcher widerkherung guetlich nit vergleichen möchten, so soll je ein tail dem andern derhalben vor hochgedachter ku. mt. ungewaigerts rechtens sein und alsdann dabei also unverwaigert beleihen. Es ist auch bei den lauter abgeret, bedingt und versprochen worden, ob gedachter Paumgartner, sein erben oder nachkhuinen ainichen andern partheien in disem vertrag und khauf thail mit¬ lassen, gar oder ainstails ubergeben und zuestellen wolten oder wurden, dass er, sein erben oder nachkhumen des zu thuen guet macht haben und herrn und gewerckhen, derselben erben und nach¬ khumen denselben allen gleich wie inen, den Paumgartner hiemit verpunden, verpflicht zu halten und zu laisten schuldig sein sollen und wellen. Dagegen sollen auch der oder diseiben, den solcher khauf gar oder zum thail ubergeben wurde, der ku. mt. und herrn und gewerckhen alles das, so diser vertrag dem Paumgartner oder seinen erben auflegt, auch ze halten verpunden sein. Es ist auch hierin insonderhait beredt und genedigist und unterthenig bewilligt und zuegesagt worden, dass die hochgedacht ku. mt. und herrn und gewerckhen etliche volmechtige gewaldthaber verordnen, darzue die ku. mt. derselben rat und tirolischen camermeister, Grégorien Maschwannder, iresthails jetzo benennen und verordnen und dann die obgedachten herrn und gewerckhen VI . Im Idrianer Quecksilberhandel des 16. Jahrhunderts . 473 zu jetzigem und khunftigen perckhwerchsverwesern noch ainen oder zwen zu Villach und dann zu Schwaz auch ainen oder zwen setzen und verordnen und aufs wenigist 3 monät vor der ersten frist angeregter bezallung dem Paumgartner oder seinen erben schriftlich verkhunden, anzaigen und benènnen sollen, der oder di die bezallung von dem Paumgartner, seinen erben oder nachkhumen zu jeder frist wie obsteet annemben, empfahen und dargegen notturftige quittung geben. Diselben quittungen auch also genuegsam und chreftig und hiemit ratificiert sein sollen, als ob die von der ku. mt. und allen herrn und gewerckhen verfertigt und aufgericht worden; alles und jedes wie jetzt obsteet gcnediglich, getreulich und ungeferlich, die geverde hierinnen in allweg vermitten sein sollen. Und darauf bekhenen wir, Ferdinand von Gottes giiaden römi¬ scher kunig etc. und wir, die gewerckhen und gewalthaber als ob¬ steet fur uns selbst und als volmechtig gewalthaber der andern unsern mitverwonten gemelts perckhwerchs in Idria etc. und ich, Hans Paumgarten von Paumgartner etc. für mich, mein erben und nachkhumen wissentlich hiemit in chraft dis briefs also dass wir solchen verkhauf und khauf der vorgeschriben 100 000 guldin werdt queekhsilber und zinober mit ein- und aufgab und allen an¬ hangenden conditionen und clausulen und was sunst diser vertrag in allen obgeschriben articln inhalt und begreift, vesst und ungewaigert halten, auch denselben und allen seinen inhaltungen genediglichen, getreulichen und redlich nachkhumen, vollziehen und geleben sollen und wellen. Und insonderhait wir kunig Ferdinandt, der genanten Hansen Paumgartners, seiner erben und nachkhumen gegen der röm. kais. mt. oder irer mt . und des reichs regimendt oder derselben viscall und sonst gegen menigclich, von welchem sie solches khaufs halben angesprochen und dadurch in schaden ze fuern unterstanden wurden, ir genedigister herr, schirmer und vorsprecher sein und deshalben gantz schadlos halten. Solch queekhsilber und zinober sollen und mugen auch der gemelt Paumgartner, seine erben und nachkhomen jederzeit, wann es inen gelegen, irem freien willen, gefallen, nutz und notturft nach verfuem, verkhumern und verkhaufen on unser und unser erben und nachkhumen, mitverwonten und sonst mennigclich von unsern wegen irung, hiridernus und widersprechen. Doch uns kunig Fer¬ dinanden die gewonndlichen zoll und meut wie dan von alter heer 474 Dokumente der frühkapitalistischen Wirtschaftsorganisation. von solchen waaren gegeben und bezallt worden sein, darüber wir sie auch in khainem weg zu staigern untersteen noch gestatten wellen, Vorbehalten. Genedigclich ungeferde zu warem urkhundt so haben hochernennte ku. mt. ir mt. secret und die merbemelten herrn und gewerckhen, auch der oft gedacht Hans Paumgartner ire pedschaft an disem Vertragsbrief, der drei in gleichem laut aufgericht sein, gedruckht und sich mit aigen handen underschriben. Geschehen zu Wienn, den ersten tag Augusti anno etc. im 39. Ferdinand. Joseph von Lamberg, Melchior von Lamberg. Niclaus Ribeisen. Barbara Neumanin. Wilhelm Rasp. Hans Paumgartner von Paumgarten. Marx Stettner. Niclas Moser. Ulrich Geltinger. Cristoff Cronegger. 34. Hans Langenauer, der Chef der Firma David Haug, Hans Langenauer & Co. zu Augsburg, ersucht seinen englischen Faktor , Hans Loner, von der Königin Elisabeth eine größere Geldsumme zu verschaffen, da sonst die Firma über ihren englischen Bergwerksunternehmungenfallieren müsse. Speier, 22. September 1570. Gleichzeitige Kopie nach dem Original. Im Augsburger Stadt¬ archiv bei den Akten der englischen Unternehmungen der Lange¬ nauer etc. Laus deo 1570 adi 22 September in Speier per post. Ersamer, lieber Hans Loner, Euch sei mein freundtlich grues und alles guts zuuorn. Dis mein schreiben an Euch geschieht umb Euch anzuzaigen, nachdem ich hie bei dem herrn landvolgt Ilsung umb bezalung angehalten, welche dann fürwahr nit fortwill. Dann gar kain gelt bei Ir Mt., noch vil weniger in Augspurg und jetzt vergangen in Frankfurt zu bekummen, dass ich gleich nit wais wie ich doch umb das aufkündt gelt zu zaln wil aufkummen. Dann under anderm zaigt mir herr landvogt an, dass im vertreulich von Augspurg aus wurde zugeschriben, dass unser sach in Engelland nit wol stiende. Derhalben wo er gelt bei uns hett wolt man in gewarnt haben. Hierauf hab er denselben zugeschriben, er werde übel bericht sein und er wiss wol, dass unser sach wol stande etc. Das zaig ich Euch derhalben an, dass Ihr sehen könnet, was uns dise englische handlung für mercklichen nachthail und schaden pringt. Und wo Ir mit der königin nit handlet, dass sie uns mit ainer summa gelts zu hilf kumpt, so müssen wir laider, Gott er- VI . Im Idrianer Quecksilberhandel des 16. Jahrhunderts . « 475 barms, zu spot und zu schänden werden. Dann kain gelt ist zu bekummen und müssen wir bis primo tutto december ob 40 000 fl. zaln. Ich hab mich gegen dem herrn landvogt vernemen lassen, dass er mir den 1 anzaig , wöll in nit vermeren, allein damit ich mich vor im hüten könne. Das hat er aber nit thuen wölln, aber sein vetter hat ims von Augspurg, geschriben. Ich sorg ich werd zu Antorf etwan auch durch solche gotlose meüler verklainert und also gar umb trauen und glauben kummen. Pitt derohalben, Ir wolt Euchs lassen angelegen sein und der künigin sampt herrn secretari und grafen von Lessester selbst unser not anzaigen, ir Mt. umb Jesu Christi willen ansprechen, dass sie uns bei unserm alten trauen und glauben erhalten wolle, dann wir je von irer Mt. willen umb wir uns mit unserm und ander leut gelt in ir land begeben in disen labyrinth kummen. Wir hoffen zu Gott, sie werd dises christ¬ lichen gemüts sein und uns nit also in disen unsern nöten verlassen. Ich wais meiner kain rath , das soit Ir mir gäntzlich glauben, darumb hab ich Euch das anzuzaigen nit underlassen wollen, damit Ir solches auch ain wissen hapt und desto statlicher dazu thuet . Lasst Kesswick Kesswick sein und gept diser sach vor allen dingen ain austrag. Wann Ir schon der künigin disen brief verdeutscht oder verdolmetscht, ist nichts daran gelegen, dann wann ich von haus köndt und nit den argkwon desto grösser machet, wolt ich selbst hinein, all soit es mich mein leben costen. Dann Ir hapt zu gedencken wie es mir geth. Ich darfs kainem freund klagen; ich müst sonst all mein thun und lassen zaigen und eröfnen und wer der sach nit damit geholffen. Ach Gott, Ir glaubt mir nit, wie mir so angst und bang ist, wolt lieber tod sein dann also zu spot, und zu schänden werden, das wa es geschieht es je nun aus woltrauen, das wir dem Höchstetter und Euch also wol vertraut haben. Gott der allmechtig geh gnad, dass es nit geschehe und wöll diser künigin ir herz eröfnen, dann sie je uns aus disem ruof on all iren nachthail und schaden helffen kan, wölt das gelt und die kupfer bei einander behalten, damit doch nichts mehr auf uns genummen werde. Datum ut supra. Hans Langenauer. 1Das heißt denjenigen, der das Gerücht verbreitet hat. Orts= und Personenregister. fl (ae), ö (oe) und ü (ue) sind im Anlaut und Inlaut wie a, o, u rubriziert, y im Inlaut wie i. Die Bezeichnungen Deutschland (Süddeutschland usw.), die fast auf jeder Seite Vor¬ kommen, sind nicht in das Register aufgenommen. Aachen 110. Aimaberg 37. 239. 247. 274. 284. 438. Acciajuoli 166. Antwerpen 12. 69. 103. 107. 109 f. 152. Achaia 78. 155. 182. 231. 253. 309. 322. 326. Adam 224. 328. 330 f. 334. 336. 340 « . 433. 475. Adler 79. Arabien 168. Adrianopel 203. Archangel 203. Armenien 168. Aiguesmortes 69. 164. 405. Ainlehum 352. Amolt 283. 446 f. Alberti 161. Amstadt 29. 48. 109. 159. Asien 32. Albertinelli 359. Albrecht II., deutscher Kaiser 92. Asphaltsee (Judaea ) 78. Auersberg , von 292 f. 352. 458. 463. Albrecht von Brandenburg 34. 367. Albrecht der Beherzte 213 ff. 224. 415. Augsburg 7. lOf. 1Z 14. 20f . 28. 32. 34f. 418. 36. 41. 47. 49. 62. 73 f. 77. 79 f. 85 « . 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Barcelona 203. 343 f. Ancona 203. Bardi 69. 108. 161. 164. 166. 405. Anjou 163. Orts- und Personenregister. 477 Breun 283. 446. 451 ff. Bärenstein 251 ff. 435 ff. Basel 88. 193. 197. 228. 411 ff. Brügge 167. 174. 177. 180. 301. Bauer, Merten 10. Brugkner 30. Baumgartner 25. 27. 32. 95. 296. 298 f. Brüssel 204. 307. 310. 313 ff. 360. 462 ff. Bücher s. Pucher. , Buchführer 228. 256. 262. 282 f. 449. Bauten 9. Buchholz 274. 284. Becherer 148. Bülder 148. Beier 439. Beirut 204. Bünau , von 247 f. 423. ßuonsignori 161. Belgien 204. Bellanti 181. Burgos 73. 370 f. Berberius 187. Bürgstein 205. 207. Berckha 280. Burgund 178 ff. 197 ff. 408. 411 ff. Burkhart 49. 106. Bern 197 f. 411. Bernhardin von Siena 90. Cadix 203. Bernstein , Dr. 255. 277. Calatrava 304. Bernstein , Hans 263. Calw 200 f. Beumel 148. Canäle r 148. Biener 283. 451. 453. Capistrano 189. Bilbao 203. Caste 11. Bimmel 25. 28. 49. 96. 106. Castilien 371. 375. Bindinello 181. Castro, Joh . de 169 f. Bircke von der Daube 219. Castro 293. Byzanz 56. Öechtitj 208. Bodensee 194. 409. Centurioni 180. Böhmen 8. 24. 36. 117. 143. 180. 185. Chemnitj 10. 224. 231. 247. 273. 423. 202 ff. 212. 221. 234 ff. 238. 244 ff. 452. 257 ff. 303 f. 311. 333. 335. 338. 347. Chigi 181. 414 ff. 422. 432 f. 441 ff. Chios 112. Böhmisch -Kamnig 205. 207. ' Christian I., Kurfürst von Sachsen 3S4 f. Bonacorsi 164. '166. 405. Cypern 112. Bontempelo del Calice 357 ff. Civitavecchia 169 ff. Bordeaux 122. 203. Clopfer 148. Bottner 37. ■ * Coldit5, von 217. Bourgneuf 195. Conserans 41. Bozen 195. 408. Cornwall 7. Brabant 179. Coruiia 203. Brandenburg 2 )1. 421 f. Cronnegger 463. 474. Brander 220. 415. Dänemark 99. 112. Brantner 375. 381. Brasilien 32. 340. Danzig 34. 99 f. 103. 359. 367. 369. Braunschweig , Stadt und Herzöge von Datini 162. 31. 33. 37. 245. 247. 416. 434. Dechant zu Freiberg 224. Dell ’Antella 161. Bräutigam 230. 233. 423. Devonshire 7. Breisgau 174. Breitenbach 231. 423. Dicke , von 99. Breitenloer 148. Dietrichstein , von 292 f. 342 f. 345 f. 353. 458. Bremen 203. Breslau 37. 100. 219. Dyet5 148. 478 Orts- und Personenregister. Dilln 26. Dordrecht 203. Dortmund 7. Drachstedt 29. Dresden 33. 186. 214 ff. 22Z 224. 227. 252. 257. 273 f. 415. 424. 427. 430 f. 436 f. Dubraius 92. Duns Scotus 90. Ferrante , König voh Neapel 70. 171 ff. Ferrol 203. Flandern 13. 15. 56. 60. 167. 179 ff. Flick 24. Florenz 14. 60. 161. 164 ff. 405. Fornari 305. Forsterin 148. Framura 169. Frankfurt a . M. 32. 37. 74. 77. 100. 109 f. 114. 146. 152. 154 f. 204. 433. Eck, Dr . 121 f. 474 f. Eckkeil 148. Frankreich 41. 56. 67. 69. 112. 146. 152. Eduard 111. von England 7. 160. 164. 166. 195. 204. 208. 210. Eduard IV . von England 178. 339 f. 405. 408. Egkh , von 293. Franzesi 69. 164. 405. Eelch , von 352. Frauenstein 439. Eelchen 353. Ehrenfriedersdorf 238 ff. 251 ff. 274. 284. Freiberg in Sachsen 33. 217 f. 227. 231. 239 f. 254. 423. 430. 435 f. 451. 286. 422. 425. 431. 438. Freiburg i. Uechtland 197 f. 412. Eibe 283. 446 f. Freihainer 333 f. Eibenstock 274. 286. Freißlich 148. Eisenerz 16. 119. 125 ff. Friaul 178. Eisfeld 21. 109 f. Friedrich III. , deutscher Kaiser 103 f. Eisleben 233. 433. 141. Elisabeth von England 362. 474 f. Friedrich der Große 123. Elsaß 197 f. Friedrich Wilhelm , der Große Kurfürst Elsterberg 210. 201 f. Engelhardt 256. 439 f. Friedrich , Kurfürst von Sachsen 427. England 7. 15. 41 f. 51. 56. 64. 67. 100. 111. 113. 152. 156. 158. 160. 166 f. Friedrich , Herzog von Sachsen 430. 178 ff. 182. 204. 212. 221. 250. 288. Friedrich August I. von Sachsen 114. Friedrich , Pfalzgraf 146 ff. 269 f. 350. 369. 474 f. Friedrich der Jüngere , Herzog von Enns , Ostreich ob der 360. 368 f. Tirol 128. Erasso 330. 345 f. Friesland 179. 240. Erbach 313. Fuchsin 148. Ergasilus 158. Füger (Fieger ) 18 f. Ernst , Erzherzog von Österreich 128. Fugger 5. 7. 9f . 11 f. 15 f. 25. 27. 32. 357 f. 40. 43. 46 ff. 56. 68. 73. 79. 80 f. 95. Fachs 254. 273. 438. 102 f. 105 f. 109 f. 111. 169. 215 . 236 f. Fano 178. 262 ff. 299 ff. 304 ff. 318 ff. 333 ff. 341 ff. 351. 360 ff. 370 ff. Ferdinand I., deutscher Kaiser 10. 71. 73 f. 78. 80. 182. 185. 246. 258 ff. Fünfkirchen 15. 276 ff. 293. 295 ff. 299. 303 . 305 ff. Furcapaß 197. 311 ff. 323 ff. 328 ff. 343 ff. 359 ff. 362. Fürer 34. 84. 110. 159. 233. 235. 368. 441 ff. 458. Füssen 407. Ferdinand von Aragonien 304. Ferdinand , Erzherzog 272. 278. 358 f. Gaetanis , de 169. Ferdinand Karl , Erzherzog von Öster¬ Galicien 41. Gallin 353. reich 406. Orts- und Personenregister. 479 ; Haller 224. 352. Galln 353. Gastein 40. 141. ! Hallsladt 141. Gattenhofen 367. Hamburg 100. 168. 203. Geyer 24. 213. 224. 238 ff. 251 ff. 274. Hansa 7. 8. 67. 96 ff. 167 ff. 177. 195. 243 f. 284. 431. 438. Geiler von Kaisersberg 62 f. 189 ff. 202. Harrer 99. 283. 451 . 453. Harstal , von 146. Geising 216. 221. 228. 419. Geldern 307. Hartmann , Dr . 146. Harz 6. Geltinger 463. 474. Gemperlein 439. Haug 7. 12. 28. 48 f. 96. 106. 262. 280 f. 346 ff. 354 ff. 474 f. Genua 60. lllf. 115 . 162. 168f . 307. Georg der Bärtige , Herzog von Sachsen Haußner 211. 6. 73. 214 ff. 220 ff. 225 ff. 236 ff. Haustein , von 264. 240 ff. 244 ff. 385. 414 ff. 423. 424 ff. Hecker 148. 432. 435 ff. Hegner 149. Heyden 148. Gerndorf , von 181 ff. Heidenreich 262. 270 ff. 439. Geringer 143. Heinrich VI ., deutscher Kaiser 30. Gersdorf , von 254. 438. Ginori Lisci 161. Heinrich , König von Portugal 108. Giorgio (Zorzi ) 177 f. Heinrich VIII . von Neapel 166. Glashütte 439. Heinrich , Herzog von Sachsen 240. 385. 432. 440 f. Glaß 255. Gleißmann 231. Heinrich von Braunschweig 33. 247. 250. Glenigk 256. Goslar 9. 27. 31. 33. 37. 100. 244 ff. Heit 439. 433 ff. Hengst 258 ff. Gossembrot 299. 360. Hennegau 179. Gossensaß 28. 32. Henckel von Donnersmark 11. Hennel 223. Gothenburg 209. Herbrot 110. Gotsgab 258 ff. Herbst 262. 293. 458. 463. Gotthardpaß 197. Gräfenthal 109. 159. 233. Herkloß 256. 439. Granada 81. 371. 375. Herwart 25. 28. 49. 73. 95. 106. 110. Grander 73. 370. 262. 281. 299. 317 ff. 322 ff. 346. 349. 353. 360. 370. Graupen 238. 261. 425. 431. 443. . Hessen -Kassel 117. Gregor XIII . 121. i Heßler 224. Grimaldi 305. Heuptel 148. Gropp 148. Grunwald 451 ff. Hilbrant 224. Guadalcanal 319. 324. Hilsen , von 283. 446. Himmelreich 224. Gunterode , von 224. Günther 360. 368 ff. Hippo 78. Guttierez 16. 304. Hirschfogel 25. Hödistetter 41. 73. 81. 102. 270. 295 ff. Guzmann 320 f. 328 f. 343 f. 353. 370. 458 ft. 475. Hof 224. Haag 203. Habsburger 6. 13. 67 f. 78 f. 105. 240. Hofer 293. 279. 301. 354. 362. Hohenschwangau 313. Hohenstaufen 163. Hall 19. 141. 193 ff. 297. 406 ff. 461. 480 Orts- und Personenregister. Holland 57. 112. 134. 159. 167. 203f . 206. ] Karl der Kühne 178 f. Karl II. von Neapel 69. 164f . 405. Hölfel 293. 458. 463. Karl , Erzherzog von Österreich 131. Hopfer 264. 346 ff. 407. Hörniann 345. Hubmair 148. Karlowitj , von 254. 287. 289. 423. 438. Kärnten 11. 49. 326. 348. 353. 357. Hufnagel 148. 461. 463. 466. Huinpiß 25. Hussiten 171. Karpathen 8. 26. Käserer 458. 465. Huttherr 283. 446 f. Katjbeck 49. 106. 351. Jakob I., König von England 362. Kawertschen 66. Janowo 15. Keller 243. 434. Idria 22 f. 36. 80. 141. 270. 281. 288. Kempten 198. 407. 411. 413. 292 ff. 362. 458 ff. Keßler 228. Jenbach 49 f. 106. 351. Keswick 7. 475. Jenits 262. 283. 288. 451. 453. Kettwig 37. Jemiger 283. 446 f. Ketjler 223. Iglau 142 ff. Khirfelda 353. Ilsung , Dr . 122. Khierpuecher 353. Ilsung , Georg 279 ff. 287 ff. 291. 326 ff. Khisl 345. 352 ff. 349. 474 f. Khreen 353. Imhof 25 Khronegger (cfr. auch Cronneger ) 353. Indien 69. 91. 309. Kleinasien 168. Ingolstadt 151. Knebbel 29. Innsbruck 19 f. 293. 413. 460. Kochel 430. Joachimstal 28. 42. 44. 100. 117. 236. Koler 9. 245. 247. 427. Köln 7. 60. 71. 73. 74 f. 85. 110, 235. Jobstei 214 ff. 219 f. 222. 371 f. 422. Johann , Herzog zu Sachsen 252. 427. König , Hans 149. Johann Georg II., Kurfürst von Sachsen Königsberg (in Ungarn ) 26. 384. Königsberg i. Pr . 34. 367 f. Irland 41. Konstantinopel 168 f. 203. 310. 464. Ischia 70. 171 ff. Konstanz 76. Italien 13. 15. 55 ff. 60. 65. 68. 89. 111 f. Kopenhagen 203. 115. 146. 160 f. 168. 182. 203. 303 ff. Kospott , von 247. 359. Kö &ing 146. Juden 66. 113 ff. Krafft 305. Julius II. 181. Krafter 350. Jung 353. Kräh wider 283. 451. 453. Krain 348. 357. 463. Kaff 258 ff. Krakau 8 ff. 26 f. Kairo 204. Kramer 34. 367. Kalabrien , Herzog von 166. Kratowo 15. Kamploff 224. Karl V . , deutscher Kaiser 3f . 6. 35. Kraus , Dr . 434. 64. 69. 71. 73. 74. 75. 77. 78. 81. Krell 224. 82. 83. 84. 85. 87. 89. 264. 298 ff. Kremnit ; 26. 208. 305. 312 f. 360. 362. 368. 370 ff. Kreuziger 21. Kronburg 297. 459. Karl I., König von England 362. Krott 236. Kar ! VII . von Frankreich 67. 181 Orts- und Personenregister Li vorno 203. Löbl 143. Löffler 19. Loi » 99 Ï. 101. Loeklein 264. Lombardei 178. Lombarden 66. 177. London 178. 203. Loner 350. 474 f. Laibach 297. 458 f. Lothringen 197. 408. 411. Lotter 262. Lamberg , von 292 f. 352 f. 463. 474. Lübeck 34. 98 f. 100. 168. 203. 367. Landsberg 194. 198. 407. 409. 411. Lucca 163. Lang 34. 367. Langenauer 7. 11 f. 28. 48. 96. 106. Luder 29. 281. 346 ff. 354 » . 474 f. Luderstadt 21. 48. 109. 159. Ludwig XV . von Frankreich 210. Langhans 214. 218. Languedoc 210. Ludwig der Reiche 42. Lauenstein 214. 228. 251 ff. 435. Ludwigsburg 200 f. Lausi tj 185. Lüneburg 100. Lurtscli 360. Lauterbach 258 » . 292. Leeuwarden 204. Luther 61. 62. 79. 82. 88. 225. Le Havre 32. Madrid 75. 78. 204. 328. 334. Leicester , Graf 475. Madtstedt 236 f. Leiden 167 f. 203. Leimbach 10. Mägerl 353. Magyaren 56. Leininger 353. Leipzig 10. 20 » . 29. 33 f. 47. 100. 108. Mähren 143. 114. 146. 154. 201. 211. 222 » . 228. Mailand 122. 204 . 359. 230 f. 233. 235. 237. 239 » . 243 ff. Mainz 60. 273. 276. 283. 290 f. 385 f. 420 ff. Mayr 258 » . 276 » . 291. 441 » . 432 » . 446 ». Malaga 203. Leo X. 181. Maltitj , von 430. Leoben 6. 24. 125 ff. 347. Manlicb 11. 28. 34. 95. 262 f. 281. 305. Lesbos 168. 314. 337 « . Leubel 21. 228. Mansfeld , Grafschaft und Grafen von 6. 8. 17. 24. 29 f. 34 f. 44. 46 ff. 109. Leutenberg 30. 42 f. 109. 159. Levante 15. 56. 168. 305. 309. 314. 322. 159. 233. 235. 243 ff. 433. Libau 203. ManwiÇ 231. 255. 423. Libethen 5. 26. Margarete von Parma 183. Lichtenstadt 258 » . Marienberg 37. Lienz 27. Marseille 203. 305. 309. Marth 73 . 87. 370 f. 383. Limburg 183. Lindau 193. 407 ». Maschwannder 472. Lindenau , von 34. Mathias , König von Ungarn 26. Link 11 f. 28. 48. 96. Maximilian I. , deutscher Kaiser 80. Lintacher 33. 182. 188. 292 f. 299 f. 301. 363. 372. Linz 146. 376. Lyon 204. 336. Maximilian II., deutscher Kaiser 276. Lissabon 108. 203. 382. 279 ff. 287 f. 289 ff. 327. 329 f. 342. Küchenmeister von Waldenburg 224. Kufstein 32. 196. 313. Kunell 439. Kunze 223. Kuppener 214 ff. 220. 224. 416. Kurz 323 ff. Kutler 458. 463. Kuttenberg 92. 182. 185. 243. 246 » . Strieder y Studien z. Gosch , kapitalist. Organ ituvlionsformen . 31 m Orts- und Personenregister. Maximilian , lir/ .herzog von Österreich Neapel 164 ff. 171 ff. 405. 358. Neidhart 35. 95. 262. Maximilian , Pfalzgraf hei Rhein 406. Neißer 353. Medici 161. 170Jh 179 ff. Neuburg (Grafschaft ) 198. 412. Neumann 293. 296. 353. 458 F. 462 F. 474. Medina , Johannes de 911'. Medina 318. Neuschloß 205. 207. Meinel 357. Neusohl 10 f. 15. 26. 43. 47. 300. Meinhart 148. New York 204. Meißen 221. 289. 415. 417. 43S. Niederlande 51. 108. HOF. 146. 160. Meistersdorf 207. 182 1 235. 307. 326. 354. 422. Nigschwig , von 224. Memmingen 193. 264. Mendlin 148. Noirmoutiers 195. Mertens 34. Normanen 163. Mesopotamien 168. Nossa , von 439 f. Nowobrdo 15. Messina 305. Nubien 168. Metscbberger 262. 270 lf. Meuerer 439. Nürnberg 81 21. 29. 32. 34. 47. 77. 84. 96. 103 f. 1091 146. 152. 154 fl Meuting 32. Mexiko 5. 204. 184. 201. 204. 219. 228. 233. 235. 243 fl 260. 262. 269. 2791 288. 3111. Middelburg 203. 326. 354. 422. 433. Miltig , von 219. 254. 43S. Minckwig , von 224. Oberliebich 205. 207. Mock 148. Obernburger 3S1. Modler 14S. Obritschan 353. Moldaugegenden 203. Ochselmayr 148. Moler 148. Ofen 301. Molina 62. 90 ff. Olsnig 210. Mordeisen 223 ff. 237. 240. 242. 248 ff. Oporto 203. 252. 431 f. Orient 56. Morgenstern 256. 439. Ortenburg , Graf , s. Salaniaiica. Morig von Sachsen 100. 257. 272 if. Österreich 131. 178. 20S. 303. 316. 328. 275 ff. 385. 340. 343. 359. 389. 406 ff. 461. Morne wech 98. Ostland 108. 160. Moßner (auch Moser ) 463. 474. Ostseelünder 108. 202. Moskau 203. Overholz 359. Mückenberg 238. 258. 425. Paderborn 204. Mühltroff 210. Paläologen , die 16S. Mulbergk 146. Palästina 78. Mulner 148. Palermo 203. München 186. 199. Münsterberg , Herzöge von 6. 31. 33. Paller 11. 262. Paradeißer 353. Münzthal 204. Paris 204. Passau 141. Nadler 146. 151. Paul II. 70. 181. Nancy 204. Paul IV. 114. Narwa 99. Pausa 210. Naspen 352. Pedek 9. Naumburg 201. Navarrus 122. Peyer 439. Orts- und Personenregister. 483 Preußor 231. 423. Peringer 258 ff. Probst 223 ff. Perotto , Aniello 172. Peru 5. Provence 164 f. 405. Przbram 36. Peruzzi 161. 166. Publer 423. Petjolt 224. Peutinger 62. 69- 70. 74. 75. 78. 80. Puch er 230. 233. 243. 423. 434. 82. 84 f. 86. 120. 157. 165. 264. 361. Puckschka 439. Puffler 228 ff. 233. 236 f. 240. 242 If. Pfalz -Zweibrücken 117. 335. 248 ff. 251 ff. 431 f. 436 ff. Pfalz -Zweibrücken , Wolfgang , Pfalz¬ Pukancz 26. graf von 333. Putsch 293. Pfinzing 29. Pfister 264. Pflaume 34. 367. Radeberg 43S. Ragusa 162. Pfluegl 296 . 353. 459. 462. Pflug , von 6. 44. 230. 236. 247 f. 250. Raiser 319. 269. 423. 427 ff. 430. Rammeisberg 9. 31. 33. 245. 247. Ramsteiner 103. Pforzheim 200 f. Randerott 253. Philipp II. von Spanien 328. 343 ff. Philipp der Schöne von Frankreich Rasp 293. 463. 467. 474. 69. 182. Ratiborzicz 36. Philipp der Schöne , Erzherzog von Rattenberg 27. Räuber 292 f. Österreich 182. Philipp , Herzog von Pommern 100. Raupennest 255. 424. Phokäa 169. Rauscher 33. Pirna 457. Ravensburg 96. Pisa 60. 163. 169. 187. Regensburg 60. Pius II. 169. 180. Reichenhall 42. 185. 193 ff. 405. Pius V . 122. Reichenstein 33. 37. Platten 258 ff. Reichstadt 207. Plauen 210. Reitwieser 231 . 423. Plautus 158. Rem 73. 370. Pockler 29. Remscheid 204. Polen 8. 10. 108. 160. 201 f. 250. Reutte 194. 198. 406 ff. Ponte 296. Reval 203. Porro 359. Rheinland 7. Portinari 179. Rholingk 273 fl. Portugal 35. 57. 68. 69 . 87. 91. 107 f. Riebeisen 463. 474. 160. 170. 195. 203 ff. 324. 339 f. 344. Riechen , von 148. 381 f. Riga 203. Potosi 5. Rincke 29. Pozzuoli 171. Ringsmaul 19. Prag 100. 143. 185. 245. 259. 432 f. 441. Roge 34. 367. 446. Rom 6. 42. 65 . 166. Prager 283. 451. 453. Romagna 178. Prasch 353. Romania 305. Prato 162. Römer , Martin 18. Prechter 79. Rosenberg , von 28. 36. Preuer 367. Rosenheim 194. 405. 410. Preußen 31. 34. 367. Roth 223. 255. 282 f. 449. 31’ 484 Orts- und Personenregister. Rolt , Konrad 107 F. 160. Schneeberg 18. 22. 24. 28. 33. 37. 431. 225. Schnöd 25. Rotterdam 203. Rueclai 180. Schondel 148. Rudoll 1)., deutscher Kaiser 36. 142ff. Schönbach 311. 358. Schonberg , von 367. Schönberg , von 2S3. 286. 423. 446 Ï. 453. Rudolph , König von Böhmen 92. Ruepp 458. 463. Schönburg , von 246. 435. Schönfeld 258 ff. 292. Rullingk (cfr. Rholingk ) 439. Schrenck 224. Ruland , Ott 103. Rurer 148. Schuster 184. Rußland 167. 202 f. 204. Schüren , von 308. Rußwurui 219. Schüfe 10. 247. Schwaiger 145. Saatfeld 117. Schwarz , Matheus 66. 266 Ï. Sachsen 6. 8. 16. IS. 24. 30 If. 36. 37. Schwarza 48. 109. 159. 44. 114. 117. 156. 185 f. 208. 2 )0 ff. Schwarzenberg 274. 286. 272 ff. 384 ff. 414 ff. 433. 445. 449. Schwarzwald 16. 30. 194. Schwaz 18 ff. 27 f. 32. 40. 42. 48Ï . 105. Saifner 353. 300. 466 f. Saylcr 148. Salamanca , Gabriel , Graf zu Ortcn- Schweden 7. 112. 208 f. Schweiz 194. 197 ff. 406 ff. burg 292 f. Schwerfecl 223. 256. Salhausen , von 214 Ff. 224. 228. Salicetus 187. Seydtwife , Weygsch 224. Seife 264. Salinas , de 305 ff. Salins 411. 413. Sender 102. 301 f. 307. Salza 231. 423. Serbien 15. Sevilla 4. 35. 203. 318 I. 323 . 344. Salzburg 30. 141. 297. 461. 463. 467. Sarazenen 56. Siam 32. San Jago 203. 304. Siebenbürgen 143. 203. 354. San Lucar 319. Sigesdorf , von 352. San Sebastian 203. Sigismund , deutscher Kaiser 76. St . Petersburg 203. Sigismund I., König von Polen 10. 100. Santander 203. Sigismund von Tirol 32. 299. Sauli 181. Sigwein 19. Scheille (?) 434. Simon (Jude zu Frankfurt ) 152. Syrien 305. Schellenberg 420. Sixtus IV . 180. Schelm , Ph . von Bergen 146. Sixtus V. 122. Schemnife 26. Sizilien 70. 78. 163. Scherl 21. 48. 230. 233. 243 ff. 423. Slamerstorff , von 146. Sehet ) 182 f. 253. Scheuer ! 24 f. Smith , Adam 125. Schlackenwald 25 f. 44. 235 ff. 246 ff. Smyrna 169. 204. 258 ff. 292. 420 ff. 427 ff. 433 ff. 442 ff. Soyter , Dr . 146. Solothurn 197 ff. 411 f. Schleinil ), von 217. Schlesien 19. 24 f. 31. 212. 219. 235. Sonnebrunn 43. Soranzo 161. 359. 421 f. Schlick 6. 44. 236. 311. 427 ff. Spanien 4. 6f . 15. 41 f. 57. 75. 160. 170. Schlüsselfelder 284. 195. 203 f. 304 ff. 312 ff. 319 ff. 324 ff. Schmidt 359. 333 ff. 339 ff. Orts- mul Personenregister. 485 Thurzo 8 ff. 11 ff. 15. 26 f. 40. 43. 46 ff. Sparsbroi 224. 106. 299. Speier 85. 383. 474 f. Spengler 214 ff. 219 f. 222. Tidemann van Limberg 7. Starschedel 224. Tyeffenbach 148. Stäuber 353. Tirol 5. 6. 8. 18 ff. 27. 31 f. 40 f. 46. Staufmehl 223. 49 f. 67. 80 f. 83. 106. 193 ff. 299 ff. 360. 363. 377. 406. 463. 466. Steher (Stäber ) 311 ff. Steiermark 6. 16 f. 24. 357. Toledo 81. 85. 89. 298 ff. 300. 375. 381. Steyg , G . von (am ) 231. 256. 423. Tolentis , de 178. Steinach 48. 109. 159. Tolfa 16. 70. 169 ff. Toscana 78. 166. Steinberger 129 ff. -Steinhäuser 152. To filer 148. Steyr 24. 42. 119. 125 ff. 142 f. Traunstein 185. 388 ff. Treviso 178. Stelle 29. Triest 203. 358. 465. Tücher 307. Stellwagen 29. Türkei 10. 146. 169. 171. 176. 180. 324. Stephan , Bischof von Lucca 178. Turkestan 32. Sterzing 28. Stettin 99 f. Ulm 77. 264. 279. 327. Stettner 352. 463. 474. Ulrich 224. 228. 236. Stockholm 203. Ungarn 5. 8 ff. 10 ff. 26 i. 40. 43. 46. Stralsund 203. 48 f. 106. 110. 143. 203. 300 f. 307. Strafiburg 79. 192. 204. Straub 33. 230. 233. 244 ff. 250. 423. Unterfranken 225. Urspringer 148. 434. Utrecht 204. Streubl 148. Strigl 293. Valencia 203. Stüber 224. Valladolid 204. Stuttgart 200. Varna 203. Sulzbach 16. 145 ff. Venedig 14 f. 32. 60. 80. 161 f. 174. Suntgau 197 f. 177. 178. ISO. 266. 292. 296 f. 303. Tabor 36. Tännfil 18 f. Taubenhaim 439. Taubenhain , von 224. 423. Täufers 83. Tetsehen 207. Theen 353. Thiele 224. Thilenius 185. Tholzanus , Petrus Gregorius 92. Thomas von Aquino 5S ff. Thorn S. Thrazien 168. Thum 238 ff. 251 ff. 274. 2S4. 431. 438. Thüringen 11. 46. 10S. 159 f. 204. 233. Thum , vom 352 f. 309 ff. 316. 322, 324. 327. 334. 336. 351. 357 ff. 459. Venier 310 f. Vigo 203. Villach 296 f. 312. 316. 337. 346. 35S. 461. 465 ff. Villinger 293. 353. Voglmair 19. Vogt 293. Vogtland 211. Vöhlin 25. Volkmayr 148. Volterra 17t. Vom Ende 224. Vordernberg 125 ff. 347. Waiblinger Walburger 353. 283. 446. 486 Orts- und Personenregister. Wallachei 203. Walliser Land 197. Wallwitj 224. Walter 224. 231. 236 f. 240. 242. 246. 248 ff. 252 f. 435. Waltkirch 375. 381. Wiiriin 353. Wasserburg 185. Wasserneuburg 296. Wechsler 14S. Weigmühle , von der 435. Weyhmann 223. Weiß 11. 148. Weißenfels 201. Welschland 197. 300. Welser 7. 25. 42. 73. 79. 81. 87 ff. 95. 159. 224. 229 f. 237. 243 f. 246. 301. 308. 370. 381 ff. 423 f. Wenzel II ., König von Böhmen 185. Werter , von 423. Witlebach , von 217. 223 f. Wiedemann 33. 236, 244 ff. 434 f. Wiederkehrer , gen . Probst 225. Wien 289. 296 f. 303. 318. 325 . 327. 329 f. 344. 347. 461. 474. Wigkell 303. Willanzheim 225. Wirsberg , W . von 150. Witrer 148. Wittenborg 98. Wixstetter 152. Wolfberger 353. Wolgast 100. Wolkenstein 240. Worms 368. 370. Wucherer 119. 141. Zeno 157. Zips 327. Zorzi (Giorgio ) 177 f. Zott 296. Nachträge. Strieder , Studien z. Gesch . kapitalist . Organisationsformen. 32 * S. 489 Erster Nachtrag. Das Kupfersyndikat der Firmen Fugger und Manlich (geschlossen im Jahre 1548). In der Geschichte der europäischen Montanindustrie stellt sich die Zeit der ersten Jahrzehnte des 16. Jahrhunderts als die Epoche einer Vorherrschaft der Fugger auf dem Kupfermarkt der Welt dar. Die Fugger hatten damals die ungarische Produktion vollständig in der Hand , die Tiroler beherrschten sie händlerisch und kapitalistisch wenigstens zum größeren Teil 1. Zweifellos hat die Augsburger Firma dabei ein glänzendes Geschäft gemacht . Wenn trotzdem Anton Fugger im Jahre 1545 daran ging, seine Vorrangstellung im ungarischen Kupferbergbau durch Kündigung der Pacht der Neusohler Kupfer¬ gruben aufzugeben , so waren es verschiedene , wichtige Gründe all¬ gemeinerer und besonderer Art , die ihn hierzu veranlaßten . Im allge¬ meinen neigte damals Anton Fugger dazu , den sachlichen und geo¬ graphischen Bereich der Geschäfte seiner Firma soviel wie möglich einzuschränken 2. Es versteht sich, daß er dabei die riskantesten Unter¬ nehmungen zuerst abzubauen begann . Hierher gehörte die Pacht der ungarischen Kupferbergwerke . Wie rücksichtslos und gewalttätig die nationale ungarische Eifersucht einer Überfremdung ihrer wichtigsten heimischen Industrie durch das deutsche Unternehmertum entgegenarbeitetc , das hatten die Fugger des öfteren , besonders aber im Jahre 1525 sehr zu ihrem Schaden erleben müssen s. Inzwischen waren in dem politisch zerrissenen Land die Verhältnisse noch weniger günstig für große Kapitalinvestierungen geworden. Neben innerpolitischen Kämpfen der verschiedenen Thronanwärter und ihrer Parteigänger, die natürlich die industriellen Anlagen der Fugger öfters in Mitleiden¬ schaft zogen , bildeten drohende Türkeneinfälle eine beständige Ge1 MaxJansen, Jakob Fugger der Reiche . Studien u. Quellen I. 3. Heft der Studien zur Fugger -Geschichte. Leipzig 1910. S. 98 und sonst. 2 Richard Ehrenberg, Das Zeitalter der Fugger . Geld¬ kapital und Kreditverkehr im 16. Jahrhundert . 2 Bde . Jena 1896 und seitdem in mehreren unveränderten Neudrucken . I. Bd . S. 144. 3 J a n s e n , a a. O. S. 179 ff. und vorher. 1 F . Dobel, Der Fugger Bergbau und Handel in Ungarn. Zeitschrift d. hist . Ver. für Schwaben u. Neuburg . 6. Jahrg . (1879) S. 48. 32* 490 Nachträge. fahr für die Bergwerksbetriebe der großen Augsburger Firma in Ungarn. Öfters heißt es in den Fuggerschen Korrespondenzen schon um das Jahr 1543 und früher : Es stünde zu befürchten , daß die Türken — da die Grenze bei den Bergstädten schlecht besetzt sei — einen Streifzug nach Neusohl machen und die dortigen Bergleute in Gefangenschaft führen würden 1. Unter solchen Umständen versteht man es, wenn Anton Fugger im Frühjahr 1545 König Ferdinand die Pacht der ungarischen Berg¬ werke kündigte 2. Ungern sah Ferdinand die Fugger aus dem unga¬ rischen Bergwerksbetriebe , in dem sie sich mehr als ein Menschenalter immer großzügiger betätigt hatten , scheiden . Zwar hätte er gern das Einkommen der Krone aus dieser ertragreichen Quelle noch mehr gesteigert , als es in der Zeit der Fuggerschen Pacht möglich gewesen war. Dem König, wenn auch nicht dem modernen Kenner der Schatten¬ seiten des fiskalischen Betriebs , mochte die Übernahme der Kupfer¬ gruben in staatliche Regie als taugliches Mittel hierfür erscheinen. Wenigstens dann , wenn die Fugger durch Vorschuß der nötigen Be¬ triebsmittel und durch feste Abschlüsse auf Abnahme des Produkts, also durch sogenannte Kupferkäufe oder Kupferkontrakte dem König ihre Kapitalkraft und ihre Welthandelsbeziehungen für den Absatz des Kupfers zur Verfügung stellten. Zunächst hat sich auch Anton Fugger zu einem solchen Betriebs¬ vorschuß und zur Verpflichtung der Abnahme der Neusohler Kupfer¬ produktion bereit gefunden 3. Dann aber zog sich die Firma auch von dieser indirekten Teilnahme am ungarischen Bergbau zurück . Der König mußte sich nach anderen Kontrahenten für den Neusohler „Kupferkauf “ umsehen . Neben sonstigen Bewerbern scheinen im Jahre 1545 die Nürnberger Kaufleute Bonaventura Furtenbach und Hans Ebner die Nachfolgerschaft der Fugger in Ungarn angestrebt zu haben. Ein Brief Anton Fuggers (Original im Fuggerarchiv 2, 3, 10) berichtet von diesbezüglichen Unterhandlungen , die die Nürnberger durch ihren Faktor Veit Holzschuher am Königshof führen ließen . Zu einem Ab¬ schluß haben sie nicht geführt . Anfang des Jahres 1548 begegnen wir vielmehr einer Augsburger Firma mit Namen Matthias Manlich und Mitverwandte als Kontrahentin des Neusohler Kupferkaufs , d. h. als alleinigem kontraktlichen Abnehmer (natürlich gegen Vorschüsse) der ungarischen Kupferproduktion 4. Die Manlich verpflichteten sich 1 Fuggerarchiv 2, 3, 10. 2 Do b el , a. a. O. S. 49. Der Entwurf des Kündigungsbriefes, datiert v. 23. März 1545 im Fuggerarchiv 2, 3, 10. 3 D o b e 1, a. a. O. S. 49 und H . J . Kirch, Die Fugger und der Schmalkaldische Krieg . 5. Heft der Stud . z. Fuggergeschichte . München und Leipzig 1915. S. 162 ff. 4 Vgl. in dem voranstehenden Werk S. 11. In den Quellen wird der Augsburger bald Matthias , bald Matheus genannt. Erster Nachtrag. 491 in dem Kontrakt während der nächsten drei Jahre (1548—1550) dem König 39 000 Neusohler Zentner ungarisches Kupfer abzukaufen, und zwar in folgender Verteilung : 1548 waren 12 000 Zentner , 1549 ebenfalls 12 000 Zentner und 1550 waren 15 000 Zentner abzunehmen. Das Kupfer wurde in Krakau und Teschen von unserer Firma in Emp¬ fang genommen. Mit dem Eindringen der Firma Matthias Manlich und Kompanie in den ungarischen Kupferhandel war eine starke Konkurrenzbewe¬ gung und eine damit zusammenhängende Preisunterbietung auf dem stark mit Ware übersättigten Kupfermarkt zu erwarten . Die Fugger hatten noch große Mengen Neusohler Kupfer unverkauft in ihren ver¬ schiedenen europäischen Faktoreien liegen. Dazu erhielten sie auch weiterhin einen beträchtlichen Teil der Tiroler Kupferausbeute. Bisher zwar hatte das größte Augsburger Handelshaus eine Katastrophe auf dem Kupfermarkt dadurch zu verhüten gewußt , daß es trotz mannigfacher Absatzstockungen 1 den Kupferpreis hochhielt und lieber weniger , als zu einem niedrigen Preis verkaufte . Auch in der Produktion selbst h-atten sich die Fugger in den Zeiten geringerer Nachfrage eine gewisse Zurückhaltung auferlegt . In einem Schreiben ihrer Faktoren Hans Dernschwam 2 und Sebastian Sauerzapf an König Ferdinand ist es direkt ausgesprochen , daß unsere Firma ihre Kupfervorräte in der letzten Zeit ihrer ungarischen Bergwerkstätigkeit darum „ so hauffend wachssen vnnd zusamen khumen lassen, damit sy in hohem preiss vnd werdth “ blieben . Durch eine solche Warenzurückhaltung sollten die „ pergwerckli nit allain in Hungern , sonnder in Tyroll vnd andern Ewer kunigl . majestet kunigreichen vnd erblanden , auch gannzen teutschen landen in wierden vnnd aufnehmen erhalten werden “ 3. Um den Preis des Kupfers auf der Höhe zu erhalten , rieten die genannten Fuggerschen Faktoren dem König nunmehr dieselbe Politik der Warenzurückhaltung auch für die Zukunft lebhaft an. F erdinand möge jetzt , wo die Fugger den ungarischen Kupferbergbau nicht mehr kontrollierten , wo der König die Neusohler Gruben in eigener Regie betrieb , die Kupferproduktion und den Kupferhandel nicht — anarchisch werden lassen . So wenigstens würden wir im Sprach¬ gebrauch der modernen Kartellbewegung uns ausdrücken . Das liege 1 Die sehr gesteigerte Nachfrage konnte schließlich doch oft nicht der noch schneller gesteigerten Produktion nachkommen. 2 Hans Dernschwams interessantes Tagebuch einer Reise nach Konstantinopel und Kleinasien (1553—1555) hat jetzt nach der Ur¬ schrift im Fugger -Archiv Franz Babinger als 7. Heft der von mir herausgegebenen Fugger -Studien veröffentlicht . München und Leipzig 1923. 3 Fugger-Archiv 2, 3, 10. Auf der Rückseite des Briefes ist die Bemerkung angebracht : „Abkhundigung des khupfer -khaufs adi 18. July des 1547 ten .“ 492 Nachträge. nicht nur im Interesse der königlichen Finanzen , sondern der Volks¬ wirtschaft des ganzen heiligen römischen Reiches deutscher Nation, dessen „ bedeutendste handtierung und gewerb“ der Bergbau sei. Denn, so argumentierten diese Kaufleute ganz richtig , „wo durch machung großer summa kupfer vnordenliche verkhauffung bcsehehe , vnd die kupfer vilen vnd vnuermuglichen leudten, die der zeit damit nit erwardten vnd nit auf hohen preiss halten khundten , verkaufft , würde es nichts gewisers als den abfall der kupfer geberen vnd bringen . Was alsdan Ewer kunigl . majestet desselben bey allen ihren pergwerkhen in Hungern , Behaim , Tyroll vnd andern an. irem chamerguet vnd der manschafft , auch lannd vnd leut für schaden vnd nachteil nemen wurde , das haben Ew . kuniglich majestet als der hochverständigist bey ir selbst genedigist zu erwegen“ . Die Produktions - und Preispolitik , wie sie hier durch die Fuggerschen Faktoren ihren Augsburger Instruktionen gemäß vertreten und empfohlen wurde, war vergleichsweise einfach durchzuführen , solange die Fugger den ungarischen Bergbau als Monopol in der Hand gehalten hatten . Auch dann noch mochte das gehen, solange der König zwar die Produktion im Regiebetrieb führte , die Fugger aber durch den unga¬ rischen Kupferkaufskontrakt die Höhe der Produktion und die Ver¬ kaufspreise sehr stark mitbestimmen konnten . Anders wurde erst die ganze Sachlage auf dem Kupfermarkt , seit die Manlich, wie oben be¬ richtet , den Neusohler „ Kupferkauf “ in die Hände bekamen . Wie, wenn jetzt diese Augsburger Firma mit ihrem Neusohler Kupfer die Fugger unterbot ? War dann Anton Fugger — wollte er im Kon¬ kurrenzkampf bestehen — nicht gezwungen, zu niedrigeren Preisen als der junge Wettbewerber zu verkaufen . Nicht nur für die beiden kon¬ kurrierenden Augsburger Firmen , sondern auch für den ganzen Berg¬ bau des heiligen römischen Reiches mußte das sehr schlimme Folgen haben. Aus diesem Dilemma konnte nur ein Kartell herausführen . König Ferdinand , der Herr von Tirol, Ungarn und Böhmen , hatte das leb¬ hafteste Interesse daran , daß es nicht zu einem Konkurrenzkämpfe der zwei genannten Firmen auf dem europäischen Kupfermarkte kam. Seine Finanzpolitik zum mindesten , wenn nicht seine Sorge um das Wohlergehen eines wichtigen Zweiges der Volkswirtschaft seines Reiches zwang ihn , den Kartellgedanken aufzugreifen und zu verwirklichen zu suchen . Wir haben an verschiedenen Stellen des vorausgehenden Werkes gezeigt, daß Ferdinand I. vor solchen privatkapitalistischen Monopolorganisationsformen der Wirtschaft nicht zurückschreckte, mochten sie noch so sehr der herrschenden Wirtschaftsethik seiner Zeit und den klaren Bestimmungen der Reichstagsbeschlüsse entgegen¬ stehen . Unbeirrt von wirtschaftsethischen Gedanken nahmen Ferdinand und seine Kammerräte als willige und gelehrige Schüler der großen Kaufleute des Zeitalters des deutschen Frühkapitalismus das Syndikats¬ projekt auf. Mehr noch, sie setzten ihrerseits viel Mühe und Arbeit 3 Erster Nachtrag. 493 daran , um cs einer Verwirklichung entgegenzuführen . Auf den Rat Anton Fuggers hin hatte Ferdinand I. bereits 1548 in den Kupfer¬ kaufskontrakt mit der Firma Matthias Manlich und Compagnie 1 die Bedingung gebracht , daß die Manlich den Kupferpreis auf derselben Höhe halten sollten, wie er zurzeit auf dem wichtigen Kupfermarkt Antwerpen gang und gäbe war . Nur dann durften die Manlich im Preise herabgehen , wenn die Fugger das ihrerseits tun würden . Damit aber letzteres nicht geschähe, wies der König in einem interessanten Schrei¬ ben (Augsburg , den 21. Januar 1548 datiert ) Anton Fugger an , den bisherigen Kupferpreis „ erhalten zu helfen“ . Man sieht aus dem Briefe absolut eindeutig , wie hier von Ferdinand ein Preiskartell der Fugger und Manlich vorbereitet , ja direkt anbefohlen wird 2. Es war also durchaus der Wahrheit entsprechend , wenn ein Augsburger Gutachten des 16. Jahrhunderts die Behauptung aufstellte : Monopole, Kartelle und andere privatkapitalistische Organisationsformen würden nicht nur von den Kaufleuten angestrebt , auch das Fürstentum betätige sich umfassend nach dieser Richtung hin 8. Das von Ferdin and I. gewünschte und eingeleitete Kupferkartcll zwischen den Fuggern und der Firma Matthias Manlich und Companie ist im Februar des Jahres 1548 in Schwaz, wo Anton Fugger in diesen unruhigen .Jahren (schmalkaldischer KriegI ) öfters weilte, zwischen Anton Fugger und Matthias Manlich persönlich abgeschlossen worden. Es stellt sich in seinen einzelnen Paragraphen teils als Preiskartell, teils als Gebietskartell dar . Der Wortlaut der Abmachungen ist fol¬ gender: 1 Der Kontrakt ist mir nicht zur Hand . Ich vermute , daß er im Finanzarchiv zu Wien liegt . Im Fugger -Archiv (2, 3, 10) findet sich ein Auszug, dem ich folgende, auf das Kartell bezügliche Stelle ent¬ nehme : „Vnnd damit der kauft vnnd precio des kuphers , inmassen durch die Fuggerischcn zu Anntorff gephlcgen würdet , in ainer gleichait erhalten werde, so solle vilbemelter Mathcs Mandlich vnd sein mituerwondten , wie er sich ze thuen bewilligt , erpoten vnd zugesagt hat, mit diesem erkhaufften kupher auch in kainen weg auf ain wenigers fallen, sonnder bey demselben vesst vnnd statt beieiben . Dagegen wellen die kunigl . majestät mit dem Fugger lianndlen von wegen der kupher , die er diser zeit noch bey hannden hat , damit er, oder dicjhenigen , so kupher von ime kauffen , im yctzigen precio nicht fallen. Wo aber solches durch ine, Fugger , oder die, so das kupher von ime kauffen , beschehe, vnnd das genuegsamlichen ausfundig gemacht würde , so solle er, Mandlich, vnnd sein mitverwondt , dises artickels halben auch vnuerpunden sein.“ 2 Der bedeutsame Brief ist weiter unten in extenso von mir ab¬ gedruckt. 3 Vgl . im voranstehenden Werke S. 79, S. 362 und sonst. 2 * 494 Nachträge. „Vergleichung , so herr Antoni Fugger mit dem Matheus Manlich gemacht , betreffent das verkauften des vngrischen kupher. Auf der römischen ku . mt. 1, vnsers allergenedigisten herrn beuelch, das sich Anthoni Fugger mit dem herren Matheus Manlich vnd mituerwandten soll vergleichen von wegen des kupfer verkauffens , haben sy sich pede a verglichen , wie nachuolgt: a) Erstlich zu Cracaw, Presslaw , Thorn vnd Tanizka 3 sollen die Fugger kain [Kupfer ] aida verkauften von disen sortten : der gmain gossen vnd in schrot , der Vierkant , mittl vnd rund scheiben. Was aber anndere sort , sollen die Fugger an gemelten ortten im verkauften vnuerpunden sein vnnd soll sich die zeit anfachen , wann die Manlichischen ire kupfer an dise ort pringen. b) Im Niderlandt , als zu Annttorff 4 vnd Ambsterdamb , sollen bede tail frey sein vnd mugen verkauften ires gefallens. c) Was aber für Hispania dienlich, haben die Fugger hiezuuor ain vertrag , der soll inen also pleiben ; vber vnd ausserhalb desselben Ver¬ trags soll yeder tail mit verkauften , auch selbs ze schickhen vnd contrahieren der kupfer per Hispania frey sein. d) Mit dem kunig von Portugal haben die Fugger ainen kauff, der solle inen also peleiben ; aber furohin soll jedem tail zu contrahieren vnd verkauffen disem kunig , auch kupher daselbsthin zeschickhen, frey sein nach yedes gefallen. • e) Mit dem verkauffen für Franckhreich oder an dieselben ort zeschickhen von Annttorff aus, soll den Manlichischen allain erfolgen, ausserhalb das die Fugger an die ort im Adriatischen mör, in Franckh¬ reich vnnd ander ort in Ytalia mugen frey schiffen, wo es inen gelegen ist. f) Es sollen auch pede tail im precio pleiben in allem verkhauffen im Niderlandt , wie die Fugger bisher verkaufft haben , vnd darinn gar nit fallen, sy kondten es dann ainer höcher verkauffen , das soll demselben zu guet körnen. g) Die Manlichischen sollen an andere ort , da die Fugger kupher haben , mit schickhung ircr kupher in rue steen , biß die Fugger ire kupher gar verkaufft haben. h) Die Fugger sollen auch hinfüro im Niderlandt kain kupfer mer auf finanz verkauffen , das dieselben kupfer aida in ringerm 6 precio, dann wie sys yetzo geben, verkhaufft werden . Wo solchs vnd so offt das beschehe , sollen sy den Manlichischen von yedem eenten 6 ain guldin reinisch münz bezalen on alle widerred. Dagegen sollen auch die 1 =Kgl . Majestät. 2 «= beide. 3 Danzig. 4 Antwerpen. 6 geringerem. = 6 Zentner. Erster Nachtrag. 495 Manlichischen im Niderlandt auch kain kupher auf finanz geben, das wider in ringerm 1preys möcht verkaufft werden , auch ire kupfer , so sy in Polln , Schlesy vnd Preissen werden verkhauffen , dermassen handlen , das die nit ins Niderlandt gefuert . Wo aber die Manlichischen im Niderlandt die kupfer auf finanntz , wie oblaut , geben, oder von gemelten ortten deren ainiche ins Niderlandt gefuert vnd betretten wurden , so sollen sy den Fuggern von jedem centen ain guldin reinisch in münz zutzalen verfallen sein, so offt das beschicht , on alle widerred. i) Ynnd soll dise beredung pleiben vnnd weren, solang der Man¬ lichischen vertrag werdt. k ) So bewilligen die Manlichischen , das die ku . mt. 2 vnangeschen irs yezigen Vertrags den Fuggern die 12871/a ctr . gossen kupfer erfolgen lassen. Dises alles war, vesst vnnd statt zehalten , haben obgemelte Antoni Fugger vnnd Matheus Manlich an einander zuegesagt . Zu vrkhundt sein diser Vergleichung zway in gleichem laut geschribcn , die jeder mit aigner hanndt vnderschriben hat . Beschehen zu Schwatz , am Werten tag Februari , im achtvndvierzigisten jarc “ 3. Ein Brief Anton Fuggers 4 an seinen hochbegabten und treuen Faktor Hans Dernschwam , der sich viele Jahre lang in den ungarischen Bergwerksunternehmungen der Fugger bewährte und der auch die Unterhandlungen über das Kartell mit König Ferdinand und seinen Kammerräten zu führen hatte , gibt uns noch etwas ausführlichere Einsicht in die einzelnen Paragraphen des Syndikatsvertrags , als es der Wortlaut der endgültigen Fassung zu tun vermag . Nach § a) des Syndikats sollte der Firma Matthias Manlich und Companie in Krakau, Breslau , Thorn und Danzig der Verkauf bestimmter gegossener und geschmiedeter Kupferprodukte Vorbehalten sein. Andere Sorten sollten dortselbst auch die Fugger verkaufen dürfen. Der § b), wie er in dem Kartellstatut vorliegt , war das Produkt längerer Verhandlungen gewesen. Matthias Manlich hätte es gern ge¬ sehen, wenn die beiden Firmen in Antwerpen und in Amsterdam ihr Kupfer gemeinsam verkauft hätten , so zwar, daß bei den jeweiligen Aufträgen jede der beiden Firmen die Hälfte der bestellten Ware zu liefern hatte . Aber Anton Fugger wollte hiervon nichts wissen. Er wies 1 =geringerem. 2 Königl. Majestät. 3 Fuggerarchiv 2, 3, 10. 4 Datiert Schwaz , 7. Februar 1548. F . A. 2, 3, 10. Über das Zustandekommen des Kartells berichtet Anton F’ugger an Dernschwam: „ . . . . Matheis Mandlich hatt mich erstlich angesprochen , das er hab sambt sein mituerwonten den kupherkauf mit der khunigl . majestät getan , vnnd weil wir auch vil kupher haben , wolt er sich mit mir ver¬ gleichen, damit khainer dem anderm im verkauften schaden thette. Darauf ich ime anzaigt , das ich dißhalb von der khunigl . majestät beuelch hab .“ 496 Nachträge. darauf hin, daß die eine Firma mit der anderen darunter zu leiden hätte , wenn ihre Faktoren an schlechte Zahler verkauften . Hieraus müßten Vorwürfe und Streitigkeiten entstehen . So wurde denn für die Niederlande nur verabredet (§f), daß man ein und denselben Verkaufs¬ preis wahren , im übrigen aber selbständig handeln sollte. Um den Antwerpener Kupfermarkt nicht mit Ware zu überschwemmen, wurden die Manlich verpflichtet , bei Verkaufsabschlüssen in Polen, Schlesien und Preußen dafür zu sorgen, daß die Ware nicht nach Ant¬ werpen ging. Geschah das doch, so hatten sie eine Konventionalstrafe^ in der Höhe von einem rheinischen Florin pro Zentner an die Fugger zu zahlen (§ h). Auch die folgenden Bestimmungen des Kartellstatuts hatten den Zweck, den Kupferpreis gerade in den Niederlanden zu stützen . Aufs strengste sollte beiden Kontrahenten verboten sein, in den Nieder¬ landen Kupfer „ auf Finanz “ zu verkaufen , d. h. sie sollten jene häufig geübte Verquickung von Finanz - und Handelsgeschäft unterlassen, wobei einem geldbedürftigen Fürsten statt Bargeld eine gangbare Ware in der Weise geliehen wurde , daß er nach bestimmter Zeit eine ent¬ sprechende Geldsumme dafür zurückzahlen mußte . Dem Kaufmann waren solche Geschäfte zunächst angenehm . Er verdiente doppelt dabei : einmal an den Zinsen des Darlehns und zweitens an dem Ab¬ satz der Ware . Auf den Markt freilich vermochten diese Manipulationen nicht gerade günstig zu wirken . Nur in der ärgsten Geldverlegenheit pflegten sich die Fürsten auf sie einzulassen . Es versteht sich von selbst, daß der fürstliche Warenempfänger oft in seiner Finanznot die ge¬ liehene Ware zu Schleuderpreisen abstoßen mußte , um seine leeren Kassen notdürftig aufzufüllen . Aus diesem Grunde verbot das Kartell das Verkaufen von Kupfer „ auf Finanz “ . Wenigstens im allgemeinen. Wünschte einer der Kartellkontrahenten dennoch solche Geschäfte zu machen , so mußte er seinem Komparenten einen rheinischen Florin Buße für jeden „ auf Finanz “ verkauften Zentner Kupfer geben. In den romanischen Ländern waren die Verkaufsbeschränkungen der beiden kartellierten Firmen verschieden geregelt . In Spanien hatten sowohl die Fugger wie die Manlich feste Verkaufsabschlüsse mit einzelnen Firmen getätigt . Die sollten ihre Gültigkeit behalten. Im übrigen engte keine Bestimmung dort die freie Betätigung der beiden Handelsgesellschaften ein. In Portugal war vonseiten der Fugger mit dem König ein fester Kupferlieferungsvertrag — das Kupfer ging zumeist in die portugiesischen Kolonien — abgeschlossen worden. Der Vertrag blieb voll in Kraft . In Zukunft sollte es jedoch auch den Manlich freistehen , mit der portugiesischen Krone Kupferlieferungs¬ verträge abzuschließen . Auch der übrige Kupferhandel der beiden Firmen in Portugal wurde durch das Kartell nicht berührt. In Frankreich , wo die Fugger vergleichsweise geringe geschäft¬ liche Beziehungen seit jeher unterhielten , die Manlich dagegen alte und weitverzweigte Verbindungen besaßen , in Frankreich,wohin die Fugger 3* Erster Nachtrag. 497 insbesondere bisher nie Kupfer gesandt hatten , blieb den Manlich das Feld im wesentlichen überlassen . Die Fugger verpflichteten sich in dem Kartellstatut , kein Kupfer von Antwerpen auf dem Landwege nach Frankreich zu bringen . Dagegen scheint Anton Fugger seiner Firma die Verschiffung von Kupfer nach Frankreich in dem KartellStatut offengehalten zu haben . Wenigstens wird an der Stelle des KarLellvcrtrags , wo davon die Rede ist, daß die Fugger ihr Kupfer in die Städte am adriatischen Meer 2 und sonst nach Italien frei verschicken dürfen , als erlaubter Bestimmungsort Fuggerscher Schiffsladungen mit Kupfer auch Frankreich genannt (vgl. § c). Ich vermute , daß cs sich um Häfen in Südfrankreich dabei handelt . Dort war Marseille wenigstens in den siebziger Jahren des 16. Jahrhunderts ein wichtiger Ausfuhrhafen für deutsche Metallartikel (sogenannter Quincailles d’Allemagne) in die Levante 8. Aber auch andere südfranzösische Häfen können die Fugger gemeint haben , in die sie ihrem Tiroler Kupfer etwa von Genua her den Seeweg offenhalten wollten. Das Kartell von 1548 bot den Fuggern die erwünschte Gelegenheit, ihre ungarische Handlung vollends zu liquidieren , d. h. ihre ungarischen Kupfervorräte in Ruhe abzusetzen . Einschließlich dei- 1287l/s Zentner gegossenes Neusohler Kupfer , die sie unter Zustimmung der Manlich noch vom König zu fordern hatten (§ k). Eine rücksichtslose Kon¬ kurrenz konnte ihnen dabei von der Firma Manlich nun nicht mehr gemacht werden , denn über die genannten Abmachungen hinaus er¬ hielten die Fugger in dem Syndikat von 1548 von ihrem wichtigsten Kupferkonkurrenten noch das Versprechen , daß die Firma Manlich an andere als die im Statut genannten Plätze kein Kupfer schicken dürfe, bis die Fugger das ihrige verkauft hätten (§ g). Als Dauer des Kartells sind in dem Statut vorläufig drei Jahre vor¬ gesehen. Auf dieselbe Zeitdauer war ja auch der Manlichsche Kupfer¬ kaufskontrakt mit Ferdinand I. abgeschlossen worden . Der Syndikats¬ kontrakt war in doppelter Ausfertigung ausgestellt und eigenhändig von den beiden Kontrahenten unterzeichnet. Das Kartell von 1548 bietet ein interessantes Gegenstück zu dem Kupfersyndikat vom Jahre 1498, an welchem die Fugger führend 1 „Dieweil er, Manlich, in Franckhreich vil khenung vnd khundt•chafft hatt , vnd wir nie kupher hinein gfüert . . 2 Anconaz. B. war eine Stadt an der Adria , die sehr oft den Fuggern als Einfuhrhafen für Kupfer diente , das an die Päpste ver¬ kauft worden war. Vgl. Alois Schulte, Die Fugger in Rom 1495 bis 1523. 2 Bdc. Leipzig 1904. I. Bd . S. 191 f. 2. Bd . S. 149. 3 Vgl . Paul Masson, Histoire du commerce française dans le Levante , au XVIIc siècle. Paris 1897. S. XI . Dazu Jakob Strie¬ der, Levantinische Ilandelsfahrtcn deutscher Kaufleute des 16. Jahr¬ hunderts (Meereskunde Heft 149). Berlin 1919. S. 17. 498 Nachträge. beteiligt waren, und auf das bereits Ehrenberg aufmerksam gemacht hat 1. Es bietet auch ein Gegenstück zu den kartellistischen Abma¬ chungen, die wiederum unter starker Anteilnahme, ja man darf sagen, unter dem Druck des deutschen Königs im Jahre 1515 die Fugger und die Augsburger Firma Gebr. Höchstetter u. Co. vereinbarten. Damals war den beiden großen Handelshäusern für die Zeit von 1520—1525 gemeinsam ein Handelsmonopol für die gesamte Schwazer Kupfer¬ ausbeute (die weitaus bedeutendste Tirols) von Maximilian I. über¬ tragen worden. Gegen eine bedeutende Anleihe selbstverständlich! Damit nun im Kupferhandel die Höchstetter gegenüber den Fuggern, die daneben die ungarische Kupferproduktion absolut beherrschten, nicht allzusehr ins Hintertreffen gerieten, vereinigten sich die zwei Firmen in einem Kartell zu folgenden Sicherungen der Absatzmöglich¬ keiten der Tiroler Produktion. In Hochdeutschland und Italien sollte nur Tiroler Kupfer verkauft werden. Ungarisches Kupfer durfte während der Vertragsdauer nicht nach Hochdeutschland oder Welsch¬ land gehen. Dagegen waren die Niederlande für die Zufuhr von ungarischem Kupfer freigegeben. Wenn die Fugger dorthin die Ausbeute ihrer ungarischen Bergwerke und der mit diesen zusammenhängenden Hüttenwerke versenden wollten, so sollte ihnen dabei auch der Trans¬ port durch Hochdeutschland erlaubt sein. Voraussetzung— unter An¬ drohung der Konfiskation der Ware durch den Kaiser— war, daß die Fuggerschen Beauftragten kein Kupfer unterwegs in Hochdeutsch¬ land verkauften. Selbst jenes ungarische Kupfer, das sich nach Ab¬ lauf des vierjährigen Kartellvertrags noch innerhalb Hochdeutsch¬ lands auf dem Wege ins Niederland befand, sollte nicht in Hoch¬ deutschland oder Italien verkauft werden dürfen, sondern ins Niedcrland weitertransportiert werden. Nur ein paar Ausnahmen zugunsten der Fugger wurden in den Syndikatskontrakt aufgenommen. Die eine bezog sich auf den Verkauf ungarischen Kupfers der Fugger in Thüringen. Der größte Teil der in den ungarischen Bergwerken geschürften Schwarzkupfererze wurde in den Hüttenwerken des ungarischen Handels der Fugger in Neüsohl (Oberungarn) und in 1E h r e n b e r g, a. a. O. I. Bd. S. 369 ff., S. 417 ff. Vgl. auch Jansen, a. a. O. S. 52 ff. Schon 1494 berichtete Hans Maltitz, oberster Bergmeister in Österreich an die Statthalterei in Innsbruck, die Fugger brächten ungarisches, in Kärnten von ihnen gesaigertes Kupfer nach Venedig zum Verkauf. Sic wollten damit einen Druck auf die Baumgartner ausüben, die den Schwätzer Kupferkauf inne¬ hatten . Die habsburgischen Behörden erkannten bereits damals, daß durch einen Konkurrenzkampf des Tiroler und ungarischen Kupfers in Italien die Habsburger als Regalherren den größten Schaden hätten. 1494. 6. August. Statthaltereiarchiv Innsbruck. Kopie. Pestarchiv XXXIX . 103. Erster Nachtrag. 499 Hochklrch (bei St . Georgentlial in der Nähe von Ohrdruff itn Thü¬ ringer Walde) gesaigert 1. Nun hatte Jakob Fugger vom Kaiser die Erlaubnis erhalten , von seiner Thüringer Hütte Hochklrch (oder St . Georgenthal wie sie auch genannt wurde ) aus die Thüringer Kesselschmiede mit Kupfer zu versorgen 2. An diesem Rechte sollte auch der Kartellvertrag nichts ändern , obwohl er den Fuggern im allgemeinen den Verkauf von ungarischem Kupfer nur in den Nieder¬ landen gestattete. Eine zweite Ausnahme erlaubte den Fuggern für einen mehr privaten als kaufmännischen Bedarf 1000 Zentner ungarisches Dach¬ kupfer auf der Donau nach Süddeulschland zu schicken . Das Kupfer war für die Bedachung Fug'gerscher Häuser und der Heilig-KreuzKirche in Augsburg bestimmt 3. Über die Wirksamkeit des Kupferkartells von 1515 (Abschluß¬ jahr ) ist ebensowenig wie über diejenige des Syndikats von 1548 etwas aus den mir zur Zeit zugänglichen Akten zu erkennen . Auch so erhellt schon aus der Tatsache ihres Abschlusses manches Wichtige für die Kartellgeschichte . Zum mindesten das Syndikat von 1548 war eines von denen, die der Zeit einer schweren wirtschaftlichen Depression auf einem bestimmten Marktgebiet ihr Dasein verdankten, also eines jener „Notstandskinder “, von der in der Streitfrage der¬ jenigen Nationalökonomen gesprochen wird , die sich mit den Ent1 Näheres auch über diese Technik bei J . Strieder, Die In¬ ventur der ^ Firma Fugger aus dem Jahre 1527. Tübingen 1905. S. 44 f. 2 Aus dem Wortlaut der Aussagen Jakob Fuggers („ Die weil mir Kays . Maiestät . . . verwilligt hat , das ich auff meiner hüten zu Jörigental daselbst im lannd den Kesselschmiden zu verarbeiten Kupher verkauften . . . mag) ist nicht klar ersichtlich , ob es sich um Verkauf oder um verlags-systematischc Versorgung der Thüringer Kesselschmiede mit Kupfer handelt . Die Ausdrucksweise von Jansen a. a. O. S. 115 ist irreführend . Jedenfalls haben die Fugger einige Zeit später Kupfer - und Messingkessel in großen Mengen herstellen lassen, um sie in die portugiesischen Kolonien an der Goldküste Afrikas zu verkaufen . Ich komme auf diese noch völlig unbekannten kolonialen Handelsbeziehungen der Fugger in der 1. Hälfte des 16. Jahrhunderts näher zu sprechen in meinem Buch : Aus Antwerpener Notariats¬ archiven . Regesten und Urkunden zur deutschen Wirtschaftsgeschichte des 16. Jahrhunderts . Das Werk , das mit Unterstützung der histo¬ rischen Kommission bei der bayrischen Akademie der Wissenschaften in den Jahren 1917 und 1918 in Belgien entstand und Ende Oktober 1918, kurz vor unserem Rückzug aus Antwerpen fertiggestellt wurde, konnte der Ungunst der deutschen Wirtschaftslage wegen bisher leider nicht gedruckt werden. 3 Jansen , a. a. O. S. 115 f. 500 Nachträge. stehungsgründen der Kartelle beschäftigt haben 1. Wichtiger er¬ scheint mir das Folgende : Gerade auch das Kartell von 1548 zeigt uns das, was ich als Leitmotiv großer Teile des voranstehenden Werkes immer wieder betonen mußte . Es zeigt den innigen Zusammen¬ hang auf, der zwischen der staatlichen Finanznot und der Genesis jener kapitalistischen Organisationsformen besteht , die wir Kartelle nennen . Der Fürst , der sein Bergwerksregal vor einer Wertminderung bewahren will, kommt , von Erwägungen der staatlichen Finanzpolitik ausgehend , zu derselben Forderung eines Syndikats , zu der die Unter¬ nehmer ihr privatwirtschaftlicher Gesichtspunkt drängte. Beilage 1. Ferdinand I. an Anton Fugger . Der König bereitet das Kupfersyndikat zwischen den Fuggern und der Firma Manlich vor . Augsburg , 21. Januar 1548. Fugger -Archiv 2, 3, 10. Ferdinand von gottes genaden römischer , auch zu Hungern vnnd Behaim etc . khunig etc. Lieber , getreuer ! Wir wellen dir genediger maynung nit ver¬ halten , das wir vns yetzo mit vnserm vnnd des reichs lieben getreuen Mathiasen Mandlich vnd seinen mituerwondten alhie in Augspurg aines kupferkauffs halben in handlung eingelassen vnd denselben also auf drey jarlang mit etlichen conditionen abgehanndelt vnd beslossen. In welcher kupherkauffsvergleichung wir aber mergemeltem Mathiasen Mandlich vnd mitverwondten mit ernst auferlegt vnd eingepunden, das sy bestimbte zeit in solchem kupfferkhauff , wirdigung vnd precio deßselben , in massen wie der yetzo zu Annttorff gangper ist , aller¬ ding peleiben vnd dauon khaines wegs fallen, noch in demselben ainiche minderung , merung oder enderung thuen oder machen sollen etc . Wie sy sich dann sollichs vnder annderm genuegsamlich gegen vnns verobligiert vnd verschriben haben. Vnnd dieweil dann sollich hievorgeschribner Vorbehalt der wir¬ digung vnnd precii angezaigtes kupherkhauffs gegen vermeltem Mand¬ lich vnd mituerwondten mit deinem guettem wissen vnd sonderlich auf dein selbst Tätlich guetbedunckhen beschehen vnd erfolgt ist , so gelanngt demnach vnser genedigs beger an dich, du wellest also gleichermassen sampt denjhenigen , so kupfer von dir erkauffen vnd annemen, in dem khauff nicht fallen oder abweichen, sunder die wirdigung vnnd precium des kupfferkhauffs hieuorgeschribner gestalt neben vnns , fürnemblichen in bedacht , dieweil solliches allen vnsern Tirolischen , Newsollerischen vnd anndern kupferperckhwerchen zu wolfart , nutz vnd guettem geraichen mag, erhalten helfen. Vnd sunderlichen haben wir mit ernenntem Mandlich auf vorbemelt . dein beschehen anzaigen vmb souil ernstlicher handlung gephlegen , damit er von yetziger wirdigung vnd precio des kupferkaufs nicht fallen solle, im fall aber , wo das durch dich oder diejhenigen , so 1 Vgl . J . Strieder, Ein Kartell deutscher Kaufleute aus dem Jahre 1743. Historisches Jahrbuch 1911. S. 50 Anm . 3. Erster Nachtrag. 501 das kupffer von dir kauften , beschehe (das doch nit solle), so hat ime gedachter Mandlich lautter vnnd austruckhenlich beuorbehalten, darinnen alsdann auch vnverpunden zu sein. Derwegen wird est du dich sampt denjhenigen 1hierinnen der notturfft nach zuuerhalten wissen, damit es also bey angezaigter wirdigung vnd precio erhalten werden muge . Wie wir vnns dann soliches gantz gnedigclich zu dir versehen , auch das hinwiderumb in gnaden vnnd guettem erkhennen vnnd bedennckhen wellen. Daran beweist du vnsern sundern angenemen willen vnd gefallen. Geben in vnser vnd des reichs stat Ausgpurg , den ainundzwaintzigisten tag Januari anno etc . im achtundviertzigisten , vnserer reiche des römischen im achtzehenden , vnd der anndern im 22ten . Ferdinand. Ad mandatum domini regis proprium : Philipp Breyner , Melchior von Hobereckh , M. Zeller. An Herren Anthoni Fugger außganngen. Auf der Rückseite : 1548. Copi der kon . mt. 2bevelch an herrn Antoni Fugger , sich mit dem Mathias Manlich des kupherkauffes halb zü vergleichen . 283. Beilage 2. Kupfersyndikat der Augsburger Firmen Fugger und Höchstetter zum Zwecke der Monopolisierung des hochdeutschen und des italienischen Marktes für das Schwazer Kupfer . Der Kartell¬ kontrakt ist abgeschlossen i. J . 1515. Das Kartell soll 1522 beginnen. Original-Papierhandschrift besiegelt mit dem aufgedrückten grünen Wachssiegel der Aussteller . Haus -, Hof- und Staatsarchiv , Wien . All¬ gemeine Urkundenreihe. Wir nachgeschriben mit namen Jacoben Fugger für mich unnd anstat meiner gebruedersson , der ich mich hierinn annymb unnd mächtig , auch all mein unnd ir erben , auch wir Ambrosy unnd Hanns die Höchsteter gebruèder fur unns , unnser mitverwanndten unnd erben bekennen : Als der allerdurchleuchtigist , grossmächtigist furst unnd herr , herr Maximilian römischer kayser , zu allen Zeiten merer des reichs in Germanien , zu Hungern , Dalmacien , Croacien etc . konig, erzherzog zu Österreich , herzog zu Burgundi , zu Brabannt unnd Phallnzgrave etc. unnser allergenedigister herr yezo abermals ainen Silber- unnd kupherkauff von newem auff vier jar lanng , die sich zu eingeendem funfzehenhundertisten unnd zwainzigisten anfahen unnd zu ausgeenden funffzehenhundertisten unnd dreyundzwainzigisten jarn enden werden , mit unns beslossen unnd auffgericht hat , darinn unnder anderm lauter begriffen ist , das wir unns genugsamlich verschreyben , damit wir in obberurter zeit kain annder frömbd kupfer in Hochtewtschlannd noch Welschlannd fueren noch vertreyben sollen 1 Hier hat der Kopist wohl ein paar Worte übersehen , die etwa lauteten : so das Kupfer von Dir (Anton Fugger ) kaufen. 2 =Kgl . Majestät. 502 Nachträge. noch wollen, das demnach ich offtgenanter Fugger fur mich unnd anstat obbestimbter meiner gebrueders son, all mein unnd ir erben, auch wir Ambrosy unnd Hanns die Iiöchsteter für unns und unnser mit verwandten unnd erben Zusagen, geloben unnd versprechen wissent¬ lich hiemit in crafft dits briefs also, das wir, noch kainer unnser mitverwanter , noch erben , noch yemands annderer von unnsernwegen noch in unnserm namen die berurten vier jar lanng dhain 1 annder frembd , dann allein die Swazer kupher in Hochtewtschlannd noch Welschlannd fuern noch vertreyben sollen noch wollen. Unnd nachdem unns aber hierinn das Niderlannd Vorbehalten ist, ob wir dann annicherlay frembd kupfer auff Strassen durch Tewtschlannd in das Niderlannd fuern wurden , so sollen und wollen wir dhains 1 derselben frembde kupher , so wir auff denselben angezaigten Strassen fuern werden, daselbst die berurten vier jar lang nicht verkauften, sonnder allain inn das Niderlannd fuern unnd vertreyben . Unnd ob aber derselben frembde kupher , so wir dieselben vier jar lanng gefuert heten , etwas uberbeleiben wurden auff den Strassen, wa die legen , so sollen wir dieselben uberbeliben frembde kupher in Hoehtewtschland nach ausgang der obbestimbten vier jar auch nicht ver¬ kauften , sonnder in das Niderlannd fuern . Wo aber , als doch kains wegs sein noch beschehen soll, ainnich annder frembd kupher durch unns , oder yemands annderer von unnsern wegen, oder in unnserm namen in berurter anzal jar unnd kupherkaufs in Hochtewtschlannd und Welschland annders dann wie obstet durch unns gefuert , ver¬ kauft und vertriben gefunden wurden , so soll alsdann sein kays . mt. gut macht , fueg unnd recht haben , unns darumb wie sich gebürt zu straffen unnd sonnderlich dieselben frembde kupfer , wa die betreten und gefunden wurden , als verfuert und verfallen gut , zu irer mt. hannden zu nemen unnd behalten. Doch mir Jacoben Fugger unnd mein gebrueders sonen hierinn Vorbehalten : Dieweil mir kays . mt . hierüber ain sonndern verwilbrie ff gegeben unnd darinn verwilligt hat , das ich auff meiner hüten zu Jörigental , daselbst im lannd den kesselschmiden zu verarbaiten, kupher verkauften , auch ain tausend centen tachkupher von Unngern auff der Thonaw — innhalt seiner kays . mt . passbrieff — herauff fuern mag, so soll mir dise mein verschreybung daran kain Ver¬ hinderung noch nachtail bringen . Alles trewlich unnd ungevarlich. Des zu urkundt haben wir obgenanten Jacob Fugger , auch Am¬ brosy und Hanns gebrueder die Hochsteter unnser yeder sein aigen innsigcl hiefurgetruckht . Beschehen am newnzehenden tag des monats Novembris nach Cristi gebürt funffzehenhundert unnd im funffzehennden jare. 1 d . h. kein. 2 503 Zweiter Nachtrag. Anton Fugger und das mitteleuropäische Zinn¬ monopolisierungsprojekt vom Jahre 1550 ff. Auf Seite 264 ff. des voranstehenden Werkes habe ich ausführ¬ lich über den kühnen Versuch des Augsburger Kaufmanns Konrad Mayr berichtet , um die Mitte des 16. Jahrhunderts durch Kartellierung der böhmischen und der sächsischen Zinnproduktion ein mittel - und osteuropäisches 1Zinngroßhandelsmonopol aufzurichten . Die Tat¬ sache, daß von zeitgenössischen Kennern der Verhältnisse behauptet wurde, die Fugger hätten bei dem großzügigen und ungemein riskanten Projekt hinter ihrem langjährigen Faktor Konrad Mayr gestanden, die Tatsache ferner, daß Matheus Schwarz, der Hauptbuchhalter der Fugger , dem Konrad Mayr ein Buchführungsformular für das Zinn¬ handelsunternehmen einrichtete und anderes mehr s, brachte mich auf den oben auch ausgedrückten Gedanken , daß wir es in dem wag¬ halsigen Beginnen mit einer versteckten Spekulation der Fugger zu tun hätten . Neue Funde im Fürstlich und Gräflich Fuggerschen Familien - und Stiftungs -Archiv zu Augsburg bestätigen mit einem hohen Grad Von Wahrscheinlichkeit diese Vermutung . Hierüber soll die folgende kurze Abhandlung Bericht erstatten. Das Monopol- und Kartellprojekt Konrad Mayrs, zu dem Ferdi¬ nand I ., deutscher Kaiser und König von Böhmen , durch Finanz1 In Westeuropa spielte auch damals noch das englische Zinn eine so große Rolle, daß nur durch die Einbeziehung auch dieser Produktion ein Zinn-Weltmonopol hätte geschaffen werden können. Im Mittelalter haben zwar zeitweise hansische Kaufleute eine bedeut¬ same Stellung im englischen Zinnbergbau und Zinnhandel einge¬ nommen — so waren im 14. Jahrhundert die fiskalischen Bergwerke von Cornwallis zeitweise an Kölner Kaufleute verpfändet — im 16.Jahr¬ hundert jedoch scheint der deutsche Einfluß hier gering gewesen zu sein , wenn auch noch im Zeitalter der Königin Elisabeth deutsche Technik und deutsche Kapitalkraft für die Hebung des englischen Bergbaus nutzbar gemacht worden ist . Vgl. oben S. 7 näheres darüber. 2 In einer Urkunde vom 31. Juli 1548 (Fugger -Archiv 5, 1, 1, Bl. 8) nennen die Gebrüder und Vettern Anton , Hans Jakob , Georg, Christoph , Ulrich und Raimund Fugger den Conrad Mayr als einen Verwandten („die eruesten Comat Mair vnnd Marx Walther , baid burger zu Augspurg , vnnscr liebe scliAVägcr vnnd vetter “ ). Strieder , Studien z. Gesch . kapitalist . Organisât ions form en . 33 3 504 Nachträge. not gezwungen, trotz der damaligen scharfen Monopolbekämpfung durch Reichstage , durch Landtage und durch die herrschenden ge¬ sellschaftlichen Anschauungen der Zeit seine Zustimmung gab , ist schließlich an dem Widerstand der sächsischen Zinngew'erken ge¬ scheitert . Man erzählte sich damals mit einer gewissen Schaden¬ freude in Sachsen , daß die Augsburger durch den Fehlschlag des Planes eine Tonne Goldes verloren hätten . Nach anderen gleich¬ zeitigen Quellen, die etwas genauere Angaben machen , wmrde- der Schaden des Konrad Mayr auf etwa 100 000 Gulden geschätzt 1. Tatsächlich müssen die Kapitaleinbußen bei dem Monopolisierungs¬ versuch noch bedeutend höher gewesen sein. Die Akten des FuggerArchivs 2 geben hierüber Aufschluß . Darnach hat Anton Fugger , der von 1526-—1560 die Leitung der Firma Fugger in der Hand hielt , nicht weniger als 600 000 Gulden in das böhmische Zinnmonopolunternehmen gesteckt . Den größeren Teil davon wohl auf Nimmerwiedersehen t Das bedeutete selbst für ein so kapitalkräftiges Handelshaus wie die Fugger eine ganz gewaltige Kapitaleinbuße . An Edelmetallgehalt dürfen wir den rheinischen Goldgulden, um den es sich hier handelt, für die damalige Zeit auf annähernd 8 Goldmark ansetzen . Dabei ist jedoch die viel höhere Kaufkraft dieses Goldquantums noch nicht in Rechnung gestellt. Einen ungefähren Begriff von der Größe dieser Kapitalinvestierung gewinnen wir auch durch folgende Erwägungen . Noch um 1527 hatte nach Ausweis der Fugger -Inventur dieses Jahres das gesamte Geschäfts¬ kapital der Firma Fugger etwa 2 Millionen rheinische Gulden betragen. Für die Mitte des 16. Jahrhunderts , die Zeit, in der das Projekt der Kartellierung der böhmischen und sächsischen Zinnproduktion spielte, schätzt Richard Ehrenberg das Handlungskapital der Firma Anton Fugger und Brüder -Söhne auf runcl 5 Millionen rheinische Gulden. Es w7ar das höchste , was sie je besessen hat und zweifellos auch das größte , welches zu jener Zeit bei einem Handlungshause vereinigt wrar L Offenbar hat Anton Fugger das Geheimnis der rechtlichen Form, in der das Kapital seiner Firma an dem großen Zinnspeku¬ lationsunternehmen teilnahm , nur mit Konrad Mayr geteilt . Seinem Wahlspruch „ Stillschweigen steht wohl an" entsprechend und ge¬ mäß der von Jakob Fugger begründeten , von seinem Neffen Anton aufrechterhaltenen absolutistischen Art der Fuggerschen Handelsge¬ sellschaftsleitung waren von dem reichen Handelsherrn nicht einmal die nächsten Blutsverwandten ins Vertrauen gezogen worden . So 1 Siehe oben S. 278. 2 Besonders Fugger -Archiv 17, 3, 12. Fugger -Archiv 32, 3 bietet nichts Neues gegenüber dem Fasz . 17, 3, 12. 3 Richard Ehrenberg, Das Zeitalter der Fugger , Geld¬ kapital und Kreditverkehr im 16. Jahrhundert , 2 Bde. Jena 1896. Seitdem mehrere anastatische Neudrucke . I. Bd . S. 149. Zweiter Nachtrag. 505 konnte es geschehen, daß erst 4 Jahre nach dem Tode von Anton Fugger dessen Söhne und Nachfolger in der Leitung der Firma , Marx und Hans Fugger , die obenerwähnten 600 000 Gulden von Konrad Mayr zurückforderten . Die Art , wie das geschah, ist charakteristisch für die unsichere Kenntnis , die die Anton Fuggerschen Erben von der Sachlage hatten . Am 16. September 1564 schickten Marx und Hans Fugger ihren Handelsangestellten und Stiftungsverwalter Georg Stegmann mitsamt einem Notar in die Schreibstube des greisen Kon¬ rad Mayr und forderten die Rückgabe von 30 000 thr . und 50 000 fl. laut vorgewiesenen Schuldverschreibungen 1. Kurze Zeit darauf be¬ riefen sich die Fugger auf weitere 15 Schuldverschreibungen Konrad Mayrs , die mit den obenerwähnten zwei zusammen eine Verpflichtung Mayrs von zirka 600 000 rheinischen Gulden bestätigten. Erst nach und nach waren wohl diese Dokumente ans Licht ge¬ zogen worden . Möglicherweise war es aber auch berechnende Absicht, daß die Fugger dem Konrad Mayr zunächst nur 2 der 17 Schuld¬ verschreibungen präsentierten . Wie dem auch sei, jedenfalls kam in den 17 Schuldscheinen das eine klar zum Ausdruck , daß der ehe¬ malige Faktor des Welthandelshauses Beträge in der Höhe von ins¬ gesamt 600 000 rheinischen Gulden in bar von Anton Fugger emp¬ fangen und in dem Zinnmonopolunternehmen investiert hatte. Vorhanden sind, soviel ich sehe, im Fugger -Archiv noch folgende Schuldverschreibungen Konrad Mayrs gegen Anton Fugger: 1. 1550 Juli 1. Konrad Mayr bekennt von Anton Fugger 48 000 Gulden („in Münz à 15 Batzen “) in bar erhalten zu haben : „ zu meinem gethonen zinkhauff vnd handl zu gebrauchen “ . Kopie im Fugger -Archiv 17, 3, 12. Dortselbst auch die zwei folgenden Schuldurkunden. 2. 1552 November 1. Konrad Mayr bekennt von Anton Fugger 30 000 Taler (jeden zu 17 Batzen gemeiner Landeswährung ) in bar auf drei Jahre erhalten zu haben : „ zu meinem gethonen zinkauff zu gebrauchen vnd stilligen zu lassen “ . 3. 1553 Juni 20. Konrad Mayr bekennt von Anton Fugger 50 000 rheinische Gulden (in Münz gemeiner Landeswährung ) auf drei Jahre in bar erhalten zu haben : „zu meinem gethonen zinkauf vnd handel zu gebrauchen vnd stilligen zu lassen 2“. Den Empfang der gewaltigen Kapitalsumme von mehr als 600 000 Gulden leugnete nun Konrad Mayr auch gar nicht . Weder vor den Augsburger Stadtpflegern und dem geheimen Rat (Gütever¬ fahren vor drei „ Einigungsherren “) noch vor dem Stadtgericht der 1 F.-A. 17, 3, 12. Bl. 1 ff. 2 Die Schuldverschreibungen stimmen textlich fast völlig überein. Die letztgenannte ist von mir am Schluß dieses zweiten Nachtrags in wörtlichem Abdruck beigefügt. 33* 4 506 Nachträge. alten Reichsstadt 1, an das schließlich die Angelegenheit gelangte. Was Mayr behauptete , war nur das Folgende : Es könne keine Rede davon sein, daß er das Geld als Darlehen („ anlehensweiss vnd ex causa mutui “) erhalten habe , wie Marx und Hans Fugger aussagten. Wäre das wirklich geschehen, dann hätte sich Anton Fugger , so argu¬ mentierte Konrad Mayr , doch sicherlich auch seinerseits nach dem Fehlschlagcn des Monopolversuchs schon bemüht , die hcrgcliehcnen Summen wenigstens zum Teil und nötigenfalls auf dem Gerichtswege von ihm zurückzuerlangen 2. Daraus , daß das nicht geschehen sei und aus anderen Gründen , auf die ich gleich noch zu sprechen kommen werde, erhelle, daß Anton Fugger ihm (Mayr) das Geld nicht geliehen, sondern zu dem Zwecke ausgehändigt habe , um damit das böhmische Zinnmonopolunternehmen , das scheinbar ihm (Mayr), in Wahrheit Anton Fugger gehört habe , zu finanzieren (zu „ verlegen ", wie die Ausdrucksweise des 16. Jahrhunderts lautete ). Dementsprechend seien die fraglichen Urkunden keine Schuldverschreibungen , sondern „allein solliche instrumenta , dardurch der empfang des verlaggelts erwisen" . Diesen Ausführungen gegenüber blieben Hans und Marx Fugger bei ihrer Behauptung , Mayr habe von ihrem Vater die 600 000 Gulden als echtes und reines Darlehen erhalten (anlehensweis vnd ex causa mutui ). Wenn Mayr das Geld für den Zinnmonopolverlag verwendet habe , so hätte er das auf eigene Rechnung und Gefahr getan 3. Auch dem Kaiser gegenüber habe er (Mayr) ja beteuert , daß das Zinnunter¬ nehmen ihm gehöre. Demgemäß habe er auch alle Verschreibungen mit Seiner Majestät gemacht . 1 „Stadtvogt , Burggraf , Ober- und andere gemeine Richter des Stadtgerichts ." 2 Darüber äußerte sich Conrad Mayr den drei Einigungsherren gegenüber wie folgt : „ Ich lass . . . ain jeden verstendigen bedenckhen, wann herr Anthoni Fugger seliger ob den 600 000 guldin sambt dem interesse an mich zu forderen vnd er nit gewißt , das dise sach ain andere maynung , gestalt , verstanndt vnd gelegenhait gehabt , ob er auch die sach wurde haben ersitzen lassen vnnd seine söne darzue so lang stillgeschwigen, das diser 17 brief halben inn 15 jarn nichts an mich gesuecht worden .“ Fugger -Archiv 17, 3, 12. Bl. 13—14. Demgegenüber behaup¬ teten allerdings die Fugger , ihr Vater Anton habe doch versucht , die Summen zurückzuerhalten . Fugger -Archiv 17, 3, 12. Bl. 30, 35, 36. 3 „Ob er nun dieselbige volgendts zum Verlag' seines damalen mit der vorigen kayserlichen majestät hochlöblichister gedechtnus habenden contract vnd zirthandls angewanndt , so hat ers doch inn solchem respect als nun sein aigen vnd ime selbst gehörig gelt zum Verlag obuermelter zinhandlung gebraucht .“ Fugger -Archiv 17, 3, 12. Bl. 10. 4 Ebenda Blatt 29. Zweiter Nachtrag. 507 Auf solche Behauptungen hin fühlte sich Konrad Mayr veranlaßt, die Stellung Anton Fuggers zu und innerhalb des böhmischen Zinn¬ monopolunternehmens noch deutlicher als es schon geschehen war, zu umgrenzen . Der Text der sog. Schuldurkunden , so erklärte er vor Gericht , lasse deutlich erkennen , daß die von Anton Fugger her¬ gegebenen Geldsummen lediglich und restlos zum „Verlag“ des ge¬ nannten Monopolunternehmens bestimmt waren . Eine anderweitige Verwendung sei ausgeschlossen gewesen und tatsächlich nicht vor¬ gekommen . Sie wurde ja übrigens auch nicht von seinen Prozeßgegnern behauptet . Die Hergabe der Kapitalien nur zu dem einen, fest¬ bestimmten Zwecke erhelle auch daraus , daß Anton Fugger als „Ver¬ leger der Zinnhandlung “ ihm (Mayr) den geringsten Teil des Geldes persönlich eingehändigt habe . Die Weitläufigkeit und Größe des Unter¬ nehmens hätten es vielmehr mit sich gebracht , daß er (Mayr) oft gar nicht dabei gewesen wäre, wenn „ das Geld erlegt“ wurde 1. Nach Konrad Mayrs Aussagen war Anton Fugger auch insofern die eigentliche Seele des ganzen Unternehmens , als er die Taktik der Monopolausnutzung angab 2. Gegen seinen (Mayrs) Rat habe Anton Fugger zunächst die Zurückhaltung des Zinns anbefohlen , dann frei¬ lich, als der Preis trotzdem noch gesunken sei, habe Anton Fugger zum allzu raschen Verkauf auch ohne Nutzen gedrängt . Der Erlös 1 Fugger-Archiv 17, 3, 12. Bl. 8b. 2 „ So ... hab ich seinem beuelch gemess den handl , wie billich fuern vnd sein willen volziehen, auch anfenglich die zin (vber das ichs aus beweglichen vrsachen widerrathen ) verhalten muessen . Da zwischen sindt sie aus allerlai furgefalnen vrsachen in abfal khomen. Dieweil aber ain grosse merckhliche barschafft daran verlegen vnd er letzlicli haben wollen, ich solle verkhauffen , damit wider gelt in die handt kheme , hab ichs gleichwol seinem beuelch nach gethon vnd ist ime ain grosse merckhliche barschafft , mer dann 100,000 gülden , so aus den zinen erlöst worden , wider einkhomen . -Wie ich aber jeder zeit nit fur rathsamb oder thuenlich achten khunden die zin one nutz vnd mit schaden zu uerschleissen vnd er hergegen per fortza gewöllt man soll verkhauffen , sie gelten gleich wleuil sie wollen, auch mich darzue nit willig gefunden , da hat er die zin selbst zu handen zu nemen begert, wölches ich dann nit verwaigert . Hab ime in alle leger, wo ers nun haben wollen, brief vnd beuelch gegeben, das man im die zin, schulden vnd Schuldbrief vberantwurten soll. Wölches also allenthalben , wo ers nun begert , zuNurmberg , Franckfurt , Craca, Schlackhenwaldt , Prag vnd anderer ortten beschehen . Schuldbrief , schulden vnd zin, auch also den handl mit seinem vorrath hat er zu seinen handen genomen, sich also des handls selbst vnderfangen vnd volgendts darmit — meinethalben ferrer vngefragt — seins willens vnd gefallens vmbgangen, ich auch ime darin aus obbegriffnen vrsachen khain Verhinderung oder eintrag zugefuegt .“ Fugger -Archiv 17, 3, 12. Bl . 24. 508 Nachträge. aus dem so verkauften Zinn (mehr als 100 000 Gulden) sei an Anton Fugger abgeführt worden. Dementsprechend könne davon keine Rede sein, daß er (Mayr) als der wirkliche Herr des großen böhmischen Zinnhandelsunter¬ nehmens angesprochen würde . Er sei nur der Strohmann gewesen. Ihn , als alten Faktor , der seit dem Jahre 1515 im Dienste des Augs¬ burger Handelshauses gestanden , habe Anton Fugger benützt , um ein großes Kapital mit hohen Gewinnaussichten anzulegen , um ver¬ steckt ein Unternehmen zu beginnen , das er öffentlich nicht habe führen wollen, dem er aus „ allerlei bewegnussen den namen nit habe geben wollen“ 1. Das klingt für den Kenner der Antimonopolbewegung des 16. Jahr¬ hunderts nicht unglaubhaft 2. Auf den Reichs - und Landtagen des ganzen 16. Jahrhunderts , in den Schriften und Predigten der Sozial¬ reformer der Zeit, in den Anschauungen des ganz überwiegenden Teiles der damaligen Gesellschaft wurden Monopole und Kartelle auf das lebhafteste bekämpft 3. Die Reichstagsbeschlüsse bedrohten die Monopolisten mit hohen Strafen . Der Fiskal , der Reichsanwalt des Reichskammergerichts war , angewiesen, unnachsichtlich gegen sie vor¬ zugehen . Es war auch nicht bei Drohungen geblieben . Die hochan¬ gesehene, vornehme Firma Bartholomä Welser in Augsburg hatte es erleben müssen , daß im Jahre 1530 des heiligen römischen Reichs Kammergerichtsbote in ihrer Schreibstube erschien und dem Senior¬ chef des Flauses, dem greisen Bartholomä Welser, die Ladung vor das oberste Gericht des Reiches überreichte . Gleichzeitig war die Klage wegen Monopolvergehens der Firma am Rathaus öffentlich an¬ geschlagen worden . Man versteht es, wenn Anton Fugger Vorsorge getroffen hätte , daß ihm nichts Ähnliches passierte. Nun war ja allerdings zumeist ein weiter Weg von einem gegen kapitalkräftige Kauf - und Finanzleute gerichteten Reichstagsbeschluß des 16. Jahrhunderts bis zu seiner Ausführung . Der Kaiser war zu¬ meist finanziell derartig von den großen Kaufleuten abhängig , daß er sie in der Mehrzahl der Fälle selbst vor den Antimonopolgesetzen in Schutz nahm . Insbesondere hatten sich die Fugger generell und für einzelne Monopolunternehmungen noch speziell diesen Schutz ver¬ briefen lassen . Auch in dem Kontrakt , den Konrad Mayr mit Ferdi1 Fugger-Archiv 17, 3, 12. Bl. 18 b f. 2 Ganz abgesehen davon , daß die Fugger auch sonst in jenen Jahren besondere Unternehmungen unter Namen und Firma von zuverlässigen Angestellten gehen ließen . Vgl. Konrad Häbler, Die Geschichte der Fuggerschen Handlung in Spanien . Weimar 1897. S. 109. 8 Vgl . oben S. 71 ff. und sonst öfters in dem voranstehenden Werk. 1 Für dieses interessante Gegeneinanderarbeiten von Reichstag und Kaisertum vgl. oben S. 81 und sonst. 2 Zweiter Nachtrag. 509 nand I. zwecks Übernahme der gesamten böhmischen Zinnproduktion schloß, war ihm ausdrücklich der Schutz des Kaisers gegenüber einer Monopolanklage des Reichskammergerichts zugesagt worden l . Zweifellos wäre aucli Anton Fugger — falls er offen das böhmische Zinnmonopol hätte übernehmen wollen — dieser Schutz Ferdinands I. gewährt worden . Zum Äußersten , zu einem energischen Vorgehen des Rcichslcammergerichtes wäre es vielleicht den Fuggern gegenüber am wenigsten gekommen . Trotzdem ist es verständlich , wenn Anton Fugger bei der stark exponierten und vielfach angegriffenen Stellung gerade seiner Firma den Skandal und die rechtlichen und ge¬ sellschaftlichen Unannehmlichkeiten hätte vermeiden wollen, denen er sich mit der eigenen öffentlichen Leitung des böhmischen Monopol¬ unternehmens aussetzte . Dazu mag noch eine andere Erwägung Anton Fuggers gekommen sein. Das Zinnunternehmen mußte von Anfang an als ein höchst riskantes geschäftliches Wagnis angesehen werden . Es war schon naheliegend , daß Anton Fugger für den Fall eines Mißlingens den Ruf und Kredit seiner Firma nicht aufs Spiel setzen wollte. Nicht weniger plausibel wie das vorhin behandelte Argument Konrad Mayrs klingt ein anderes , das er in den Verhandlungen gegen die Berechtigung der von seiten Hans und Marx Fuggers gestellten Geldanforderungcn vorbrachte . Wer wollte leugnen , daß es im höchsten Grade unwahrscheinlich ist , daß Anton Fugger seinem Faktor eine Summe von der ganz gewaltigen Höhe von 600 000 rheinischen Gulden geliehen haben sollte ? Für einen solchen Kredit war das mobile und immobile Kapital , über das Konrad Mayr zu verfügen hatte , viel zu klein . Mayr gehörte keineswegs zu den reichsten Männern Augs¬ burgs . Er sagt wohl die Wahrheit , wenn er meinte , Anton Fugger würde ihm (Mayr) gegen Verpfändung seines gesamten Hab und Guts nicht 2000 oder 3000 geschweige denn 600000 Gulden geliehen haben. Hier könnte nun freilich eingeworfen werden , daß Anton Fugger, der die Summe von 600 000 fl. ja nicht auf einen Schlag, sondern in 17 Teilzahlungen gab , sich sehr widerseinen Willen zu immer erneuten Gcldhergaben an Konrad Mayr aus dem Grunde gezwungen gesellen habe , um womöglich durch die späteren das erste Darlchn zu retten. Tatsächlich hat ja Anton die geschäftliche Unklugheit begangen, immer erneut Geld in das Danaidenfaß des böhmischen Unternehmens zu schütten , aber daß er das tat , daß er nicht die so sehr angebrachte kühle Distanzierung dem Unternehmen gegenüber zu halten ver¬ mochte , zeigt m. E . deutlich die rechtliche Situation . So wie sich 1 Siehe oben S. 270. 2 Vgl . oben S. 265. Nach dem Fehlschlag des böhmischen Unter¬ nehmens zeigt laut den Steuerbüchern das Vermögen Mayrs noch immer eine starke Zunahme . Er hatte also offenbar keine nennenswerten Ver¬ luste in dem Zinnunternehmen erlitten. 510 Nachträge. Anton Fugger als Kapitalist dem böhmischen Zinnmonopol gegenüberstellte,, hätte er sich als Nur -Darlelinsgeber des Konrad Mayr nimmer¬ mehr gestellt . Dieses immer erneute und immer größer werdende Risiko nimmt man nur einem Unternehmen gegenüber auf sich, das im Grunde einem selbst gehört. Leider hat die Behandlung der Streitfrage vor dem Stadtgericht von Augsburg nicht Licht in den interessanten Fall zu bringen vermocht. Um so weniger, als nach dem baldigen Tode des greisen Konrad Mayr (Ende 1565 oder Anfang 1566) dessen Erben einen Vergleich mit den Söhnen Anton Fuggers eingingen. Man einigte sich dahin, , daß die Familie Mayr auf 5000 Gulden verzichtete , die Hans Jacob Fugger dem Konrad Mayr schuldete . Nach dem Tode der Witwe Konrad Mayrs sollte dazu noch das Gut Bergheim (bei Augsburg ), das Konrad Mayr für 5550 Gulden gekauft hatte , den Fugger zufallen. Vorläufig sollte aus Schonung für die Witwe Konrad Mayrs die Übergabe noch nicht stattfinden l. Vielleicht hätte auch ein längerer Prozeß mit weiteren Instanzen als das Augsburger Stadtgericht nicht größere Klärung in die Frage über die rechtliche Form des Zusaminenarbeitens von Anton Fugger und Konrad Mayr in dem böhmischen Zinnmonopolunternehmen ge¬ bracht . Behauptung stand gegen Behauptung , und Anton Fugger , der allein Endgültiges und Bestimmtes aussagen konnte , war tot . Wie es auch sei, wie immer der Fall auch rechtlich gelagert sein mag , die Tatsache steht nunmehr fest, daß eines der riskantesten und wohl das verlustreichste Spekulationsunternehmen des 16. Jahrhunderts mit Fuggerschem Kapital geführt worden ist . Die Fugger haben dabei eine für damalige Verhältnisse ganz enorme Summe eingebüßt . Kein anderes deutsches , wahrscheinlich sogar kein anderes europäisches Handelshaus hätte sich nach solchen Kapitaleinbußen aufrecht zu er¬ halten vermocht . Zwei Jahrzehnte früher war Ambrosius Höchstetter, einer der reichsten Augsburger der zwanziger Jahre des 16. Jahr¬ hunderts , an einem ähnlichen Unternehmen , an dem Versuch , durch Kartellierung der Idrianer und der Almadener Quecksilberproduktion ein Weltmonopol in dieser Ware aufzurichten , verblutet , obwohl es sich um bedeutend kleinere Kapitalien dabei handelte 2. Ein anderer Augsburger Kaufmann , Konrad Rott , ist in den siebziger Jahren des 16. Jahrhunderts geschäftlich an einem groß1 Die Erben waren Konrad Mayr jr ., Regina Bimmel, die Tochter Konrad Mayrs, die Gattin des bekannten Augsburger Großkaufmanns Anton Bimmel , Apollonia Han , ebenfalls Tochter von K. Mayr senior, die den Obervogt von Mindelheim, Dr . Joh . Jacob Han , zum Manne hatte und endlich Eufrosina Roch , ebenfalls Tochter des K . Mayr sr., Gattin des Peter Roch. 2 Vgl . oben S. 307 f. und vorher . Material darüber auch F .-A. 2, 2 , 1. 3 Zweiter Nachtrag. 511 artigen Pfeffermonopolisierungsversuch zugrunde gegangen 1. Die Bei¬ spiele felilgeschlagener großer Mon'opolversuelic des 16. Jahrhunderts ließen sich mehren . Manche bedeutende Firma ging darin zugrunde. Die Fugger hatten die ungleich stärkeren Verluste an dem böhmischen Monopolunternehmen nicht zu stürzen vermocht . Anton Fugger ver¬ stand es, den harten Schlag zu rechter Zeit zu parieren . Das eine freilich muß die Fugger -Forschung aus der vorangehenden Unter¬ suchung lernen . Die Auffassung , als ob die Schwierigkeiten , die die Firma Fugger schon unter Anton , mehr noch unter seinen Nachfolgern des öfteren erlebte , nur aus den fatalen Finanzgeschäften mit den Habsburgern und anderen öffentlichen Gewalten resultiert hätten 2, ist falsch . Auch im spekulativen Warengeschäfte lagen — wie aus dem voranstehenden ersichtlich sein dürfte — Todeskeime für die größten Firmen des 16. Jahrhunderts verborgen. Beilage 1. Konrad Mayr bekennt von Anton Fugger 50 000 rhei¬ nische Gulden für das böhmisches Zinnmonopolunternehmen auf 3 Jahre erhalten zu haben . Augsburg 20. Juni 1553. Kopie im FuggerArchiv 17, 3, 12 Bl. l b. „Ich Conradt Mair, burger zu Augspurg , bekhenn offenlich mit disem brief meiner aigen handtschrifft für mich vnnd meine erben, das ich aufrechter , redlicher schuldt schuldig worden bin vnnd gelten soll dem edlen herrn Anthonien Fugger , römischer kaiserlicher vnd kuniglicher maiestat rathe , benantlichen funffzig tausend gülden reinisch in müntz gemayner landtswerung , die ich auf heut dato ditz briefs von ermeltem herrn Anthonien Fugger , dieselben auf drey jar lang die negsten nacheinander volgende zu meinem gethonen zinkauf vnd handel zu gebrauchen vnnd stilligen zu lassen also bar eingenomen vnnd empfangen hab . Dauon sollen vnnd wollen ich oder meine erben ernanten herrn Anthoni Fugger oder seinen erben die gedachten drey jarlang vnd jedes derselben besonder für gewinung vnd verzinssung zehen gülden von hundert alhie oder in der statt Nürmberg aussrichten vnd bezallen vnd mit erster jarszinsbezallung von dato ditz briefs vber ain jar anfahen . Vnnd nach verscheynung der drey jar sollen herr Anthoni Fugger oder seine erben — zu was zeitten es inen ge¬ legen sein wurdet , kurtz oder lanng darnach — guet macht haben mir oder meinen erben die hauptsuma fünffzig tausent gülden reinisch in müntz gar oder halb , welches inen fueglich sein wurdet , abzukhunden . Vnnd nach der abkhunung vber ain jar sollen vnnd wollen ich oder meine erben vil ermeltem herrn Anthonien Fugger oder seinen erben den halben thail dess abgekhundten hauptguets vnnd den andern abgekhundten halben tail vber acht monat darnach sambt 1 Vgl . oben S. 107 und die dort genannte Literatur über Konrad Rott. 3 So besonders Ehrenberg in seinem Zeitalter der Fugger. 512 Nachträge. verfalner , vnbezalter verzinssung alhie zu Augspurg oder in der statt Nurmberg one allen iren costen vnnd schaden , solang vnd vil biss sie der gantzen haubtsumma sambt vnbezalter verfallner verzinssung entricht vnd bezalt sein worden , ausrichten vnnd bezailen. Vnnd souerr sich zutragen , das Ich ermelte suma gelts lenger oder weitter inn meiner handlung nit zu gebrauchen haben würdt —• es wer inner der dreyen jarn oder nach ausganng derselben — so sollen vnnd mögen Ich oder meine erben dem gedachten herrn Anthonien Fugger oder seinen erben die halb oder gantz suma, wie vns das gelegen sein wurdet , aufsagen vnd abkhunden . Vnnd nach verscheinung solcher abkhundung vber ain jar sollen vnd wollen ich oder meine erben herrn Anthonien Fugger oder seinen erben die abgekhundten suma gelts sambt verfallner vnbezalter verzinssung alhie zu Augspurg oder inn der Stadt Nurmberg one allen iren costen vnnd schaden inn ganghaffter gemainer landtswherung ausrichten vnnd bezailen vnder verpindung aller meiner haab vnnd guetter , ligender vnd varender , gegenwurtiger vnnd kunfftiger , die ich inen hiemit ein¬ gesetzt vnnd verpfenndt haben will, getreulich vnd on geuerde . Dess zu warem vrkhundt hab ich zu enndt diser schrifft mein petschafftring aufgedruckht . Beschehen zu Augspurg am 20. tag des monats Juny als man zalt nach der gehurt Cristi vnsers herrn im 1553. jare .“ 513 Dritter Nachtrag. Einige Ergänzungen zum fünften Kapitel des dritten Buches , betitelt : „Monopole und Kar¬ telle im Idrianer Quecksilberhandel des 16.Jahr¬ hunderts “. Wider Erwarten fanden sich im Fugger -Archiv und im Stadt¬ archiv zu Augsburg eine Anzahl Materialien zum obigen Thema . Der Vertrag , den der Hofkammerrat Hans Khisl zu Kaltenbrunn , oberster Erbtruchseß der gefürsteten Grafschaft Görz im Namen des Erz¬ herzogs Karl von Österreich unter dem 5. Mai 1571 (gültig vom 8. Ok¬ tober 1571 an) mit den Gewerken von Idria abschloß \ liegt im FuggerArchiv unter der Signatur 2, 4, 3 Blatt 481 f. Der Vertrag wird in der damals üblichen Terminologie ein „ Kauf “, speziell ein „ Queck¬ silberkauf “ genannt . In dem Kontrakte übernimmt Erzherzog Karl die gesamte Ausbeute des Idrianer Quecksilberbergwerks von den Gewerken (Handelsmonopol des Staates ). Er behält sich dabei vor, das Monopol an andere weitergeben zu dürfen . Der Kontrakt des Erzherzogs mit den Idrianer Gewerken sollte fünf Jahre währen. Letztere hatten in dieser Zeitspanne 7500 Zentner Quecksilber, den Zentner (Wiener Gewicht) um 24 Gulden zu liefern . Sollte der Erz¬ herzog statt Quecksilber die Lieferung von Zinnober wünschen , so waren für den Wiener Zentner Zinnober 28 Gulden zu zahlen . Die ge¬ lieferte Ware mußte bar in Villach beglichen werden unter Abzug freilich von 10 °/„ für „ Fron “ und daraufhin noch von 1/a der Rest¬ summe für „Wechsel“ 2. Es blieben demnach den Gewerken statt 180 000 Gulden (7500 X 24 Gulden) nur 141 750 Gulden als Ver¬ kaufspreis für 7500 Zentner Quecksilber . Diese Summe wurde ihnen in bestimmten , vertraglich festgesetzten Fristen gezahlt. Eine Lieferung an andere Personen als den Erzherzog bzw . an denjenigen , an den dieser das Monopol weitergab , war den Gewerken auf das strengste verboten . Nur für eigenen Gebrauch durften ihnen kleine Mengen von dem Verweser abgegeben werden . Auch wollte der Erzherzog dafür sorgen, daß die gewerblich tätigen Einwohner seiner Länder das Quecksilber und Zinnober zu einem „ ziemlichen“ Preis erhielten (Anfänge merkantilistischer Gewerbepolitik ). 1 Vgl. im voranstehenden Werke S. 350 oben. 2 Fron und Wechsel waren Abgaben der Gewerken an den Regal¬ herrn , d. h. den Landesherrn. 3 514 Nachträge. Welch einen gewaltigen Gewinn der Erzherzog bei diesem Ab¬ schluß machte , sieht man aus den Bedingungen , unter denen er den „ Quecksilberkauf “ weitergab . Auch in dieser Frage sehe ich heute, auf Grund neuer archivalischer Funde , deutlicher als bei der Heraus¬ gabe der ersten Auflage . Die früher von mir vergeblich im Wiener Finanzarchiv gesuchte Urkunde , datiert vom 8. Oktober 1571, worin der eben genannte zwischen den Idriancr Gewerken und Erzherzog Karl abgeschlossene Monopolkontrakt wiederum , wie schon im Jahre 1566, an die Augsburger Firma David Haug 1, Hans Langenauer und Mitverwandte vom Erzherzog übertragen wurde , findet sich in meh¬ reren Abschriften im Fugger -Archiv 2. Sie vermittelt uns folgende Erkenntnisse : Nach langen Unterhandlungen , wobei der Erzherzog mit Rücksicht auf die Konkurrenz und das Überangebot 8 auf dem Quecksilbermarkt angeblich seine Forderung sehr maßvoll gestaltete, wurden der Augsburger Firma folgende Übernahmepreise für das Monopol bewilligt . Für die ersten 5000 Zentner sollte sie pro Zentner Quecksilber 55 Gulden, pro Zentner Zinnober 59 Gulden zahlen . Für die restlichen 2500 Zentner pro Zentner Quecksilber 40 Gulden, pro Zentner Zinnober 44 Gulden. Tn Summa erhielt der Fürst also für 7500 Zentner Quecksilber 375 000 rheinische Gulden in Münz 4 (à 60 Kreuzer ). Da die Gewerken nur 141 750 Gulden bekamen , so blieb dem Erzherzog aus dem Quecksilbermonopol ein Gewinn von 233 250 Gulden . Natürlich erhielt der Fürst diese schöne Einnahme nicht auf einmal , sie verteilte sich vielmehr durch Ratenzahlungen auf 5 Jahre. Zahlungsort für die erzherzoglichen Forderungen war Augsburg , für die der Gewerken, die gleichfalls direkt durch die Firma David Haug, Hans Langenauer und Mitverwandte erfüllt werden sollten, Villach. Erzherzog Karl von Österreich hat mit der Übergabe des Idrianer Quecksilbermonopolkontrakts an die Gesellschaft David Haug , Hans Langenauer und Mitveivandle keinen glücklichen Griff getan . Im Jahre 1574 mußte die bekannte Augsburger Firma ihre Zahlungen, W'ie an ihre sonstigen Gläubiger, so auch an die Gewerken von Idria und deren Regalherrn einstellen . In der bisherigen Literatur wurde nach dem Vorgang von Richard Ehrenberg (Zeitalter der Fugger) 1 Der Seniorchef der Firma David Haug war schon im Februar 1570 gestorben . Fugger -Archiv 2, 4, 4. 2 Z. B. 2, 4, 3, Blatt 484 ff. auch Bl. 558 ff., ferner 2, 4, 5 im letzten Fünftel des starken , nichtpaginierten Bandes. 3 Die Haug und Kompanie behaupteten , die Vorräte , die ihre Vorgänger im Idrianer Quecksilberhandelsmonopol (die Herwart in Augsburg ) noch in der Fland gehabt hätten , sowie die starken Förde¬ rungen anderer Bergwerke ließen das Idrianer Monopol infolge Absatz¬ stockung und Preisrückgang problematisch erscheinen. 4 Für den Unterschied zwischen rheinischen Gulden in „ Gold“ und in „Münz“ vgl. Strieder, Fugger -Inventur S. 27f. Dritter Nachtrag. 515 angenommen , daß es im wesentlichen die unrentablen Kapitalinve¬ stierungen Unserer Firma in den englischen Bergbau gewesen seien, die sie zu Fall gebracht hätten . In der ersten Auflage habe ich mich im wesentlichen noch dieser Ansicht angeschlossen . Heute möchte ich glauben , daß auch das Idrianer Unternehmen stark mit zum Sturz dieser Handelsgesellschaft beigetragen hat . Seit die genannte Augs¬ burger Firma im Jahre 1571 zum zweiten Male das Idrianer Monopol übernahm , gestaltete sich die Aufnahmefähigkeit des Weltmarktes für Quecksilber nicht besser — wie man gehofft hatte —, sondern im Gegenteil immer schlechter . Die Hauptursache dafür scheint mir im folgenden zu liegen. Hatten bisher die beiden wichtigsten euro¬ päischen Gewinnungsgebiete (Almaden in Spanien und Idria in Krain ) auch die spanischen Edelmetallbergwerke Amerikas mit dem für das Amalgamationsverfahren nötigen Quecksilber versehen 1, so trat in dem seit etwa 1565 in Betrieb befindlichen peruanischen GuancaVelica (am Ostabhang der Anden ) ein starker Konkurrent für die europäische Quecksilberausfuhr in die neue Welt auf 2. Offenbar ist in dieser Tatsache ein Hauptgrund für das Sinken des Quecksilber¬ preises, für Absatzschwierigkeiten unserer Firma und schließlich für ihren Bankerott zu suchen. Schon im Jahre 1566 muß sich die Handelsgesellschaft David Haug , Hans Langenauer und Mitverwandte mit ihrer eigenen Kapital¬ kraft dem Idrianer Unternehmen nicht mehr ganz gewachsen ge¬ fühlt haben . Bereits in dem Idrianer Quecksilbermonopolkontrakt, den die genannten Augsburger im Jahre 1566 auf fünf Jahre ab¬ schlossen, war der Passus enthalten , daß es der Firma erlaubt sein solle, den Kontrakt ganz oder teilweise an einen oder mehrere Kapi¬ talisten weiter zu übertragen 3. Denselben interessanten Vorbehalt, der uns zeigt, wie weit der Gedanke der Versachlichung , der Kommer¬ zialisierung der Unternehmungen schon fortgeschritten war , bringt auch der Vertrag von 1571. In der ersten Auflage des vorliegenden Werkes mußte ich es dahingestellt sein lassen, ob sich die Augsburger Firma David Haug , Hans Langenauer und Mitverwandte die Über¬ tragungsklausel nur für einen möglicherweise eintretenden Notfall bewilligen ließ, oder ob sie eine bestimmte Beteiligung bereits im Auge hatte , als der Kontrakt von 1566 abgeschlossen wurde . Jetzt be¬ stätigt neues Material , das ich zu dieser Frage im Fugger -Archiv und im Archiv der Stadt Augsburg fand , die letztere Annahme 4. In einem Vertrag vom 16. November 1566 traten David Haug , Hans Langenauer und Mitverwandte die Hälfte des Idrianer Quecksilbermonopols an 1 Vgl. oben S. 319 f. 2 Werner Sombart, Der moderne Kapitalismus . 2. Aufl. Bd . S. 575. 3 Siehe oben S. 348 f. 4 Fugger-Archiv 2, 4, 5, Fol . 9. 516 Nachträge. die Augsburger Firma Michael und Abraham Katzbeckh Gebrüder, Matthias Manlich und Mitverwandte ab. Durch einen glücklichen Zu¬ fall fand ich den interessanten Kontrakt , den ich lange im FuggerArchiv vergeblich suchte , im Archiv der Stadt Augsburg 1. Er stellt insofern ein wichtiges Dokument frühkapitalistischer Wirtschafts¬ weise dar , als er zeigt, daß dieselbe Entwicklung , die wir aus der Finanzgeschichte des Mittelalters bereits kannten , sich auch in den anderen Gebieten des Geschäftslebens Geltung verschafft hat . Wie die italienischen Finanzmänner des Mittelalters gewisse besonders ris¬ kante und große fürstliche Anleihen nicht allein, sondern in Konsortien übernahmen , so suchte auch unsere Firma in einem Geschäftszweig, in dem sich Handel , Industrie und Finanzoperation in charakteri¬ stischer Weise vermischen , die Konsortialbeteiligung sich zunutze zu machen 2. Die handelsrechtliche Form , in der das geschah, war die folgende : Die Firma David Haug , Hans Langenauer und Mitver¬ wandte schloß zum Zwecke der Beteiligung der Firma Michael und Abraham Katzbeckh Gebrüder , Matthias Manlich und Mitverwandte an dem Idrianer Quecksilbermonopol mit letzterer eine Gelegenheits¬ gesellschaft ab . Während die Haug , Langenauer und Co. allein diesel Gelegenheitsgesellschaft den Namen und die Geschäftsführung 3gaben, teilten sich die zwei assoziierten Firmen in die Kapitalaufbringung , in das Risiko und auch in den Gewinn an dem Monopol. Die Kapital¬ aufbringung wurde so geleistet , daß die Katzbeckh , Manlich und Co. jeweils die Hälfte der Zahlungen übernahmen , die in bestimmten Terminen , wie wir oben gesehen haben , an die Idrianer Gewerken und an den Erzherzog zu leisten waren . Auch die Hälfte der Unkosten bei Versendung und Verkauf des Quecksilbers war von der Firma Katzbeckh , Manlich und Co. zu bestreiten . Selbstverständlich geschah das gegen genaue Rechnungslegung von seiten der geschäftsführenden David Haug , Langenauer und Co. Das aus dem Verkauf des Queck¬ silbers gelöste Geld war ohne Verzug zur Hälfte den Katzbeckh , Man1 Der betreffende Aktenfaszikel ist dort niedergelegt im Schrank der Handelssachen sub Nr . 17. 2 In einem der vielen juristischen Gutachten über die Gelegen¬ heitsgesellschaft der Haug , Langenauer und Co. und der Katzbeckh Manlich und Co. (Augsburger Stadtarchiv , Schrank der Handels¬ sachen Nr . 17) ist darauf hingewiesen, daß auch Augustinus de Ghisiis (Agostino Chigi?) und Co. das Alaunmonopol von Tolfa, das er mit der Camera Apostolica abgeschlossen hatte , nicht allein behielt , sondern den Ambrosius de Spanochi zu sich in den Kontrakt nahm . Über das Tolfaer Alaunmonopol vgl. oben S. 181 ff. 3 Heute nennt man den oder die Konsorten die beauftragt oder befugt sind, die zur Durchführung des Geschäftes erforderlichen Rechtsakte im eigenen Namen , aber für Rechnung aller Beteiligten vorzunehmen , die Konsortialleiter. 41* Dritter Nachtrag. 517 lieh und Co. zuzustellen . Ohne Vorwissen ihrer Gesellschafter Katzbeckh , Manlich und Co. durften die Haug , Manlich und Co. kein anderes Quecksilber kaufen als Idrianer Monopolfabrikat . Dieselbe Verpflichtung übernahmen die Katzbeckh , Manlich und Co. Auch für die Zukunft sollte gegebenenfalls das Konsortium auf¬ recht gehalten werden . Sollten nämlich nach Ablauf des jetzigen Monopols die Haug , Langenauer und Co. mit Erzherzog Karl einen neuen „ Quecksilberkauf “ abschließen , so verpflichteten sie sich, die Firma Katzbeckh , Manlich und Co. oder deren Erben wiederum — falls es von ihnen gewünscht wurde — zum Halbpartner zu machen. Sollte dagegen die Handelsgesellschaft Haug , Langenauer und Co. die Erneuerung des Idrianer Monopolkontraktes nicht wünschen , oder sollten ihre diesbezüglichen Unterhandlungen mit dem Erzherzog von Österreich ohne Ergebnis verlaufen , so durften die Katzbeckh , Man¬ lich und Co. das Idrianer Quecksilbermonopol übernehmen . Jedoch mußten dann auch sie ihrerseits der Firma Haug , Langenauer und Co. oder deren Erben auf ihren Wunsch hin Halbpart zugestehen. Über die Auflösung des Konsortiums wurden ' folgende Bestim¬ mungen getroffen . Nach Ablauf des fünfjährigen Monopolkontrakts der Firma David Haug , Hans Langenauer .und Mitverwandte mit dem Erzherzog Karl sollte die Gelegenheilsgesellschaft nicht ohne weiteres ihr Ende finden . Das sollte erst dann geschehen, wenn alles Queck¬ silber, das aus diesem Kontrakt herrührte , verkauft war und wenn alle Schulden , die damit zusammenhingen , einkassiert waren. Auf dieser Grundlage ist das Konsortium 1566 gegründet , 1571 erneuert worden . Seine Auflösung hat es erst mit dem Bankerott der Firma David Haug , Hans Langenauer und Mitverwandte gefunden L 1 Nach einer anderen Quelle wären die Matthias Manlichschen und Abraham Katzbeckhschen Erben mit 3/e an dem Monopol beteiligt gewesen. Da ich nicht annehmen kann , daß diese Erben eine andere Firma darstellen als Michael und Abraham Katzbeckh Gebrüder, Matthias Manlich und Mitverwandte , die möglicherweise von den 4/s der Haug , Langenauer und Companie noch 3/g übernahmen , so denke ich an einen Irrtum der Quellenstelle, die also lautet : „Wahr und beweisslich sein, dass Dauit Haug , Hanns Langnawer vnd mitverwanten vber den quecksilber - vnd zinober-khauff , den sie von der fürstl . durchl . ertzhertzogen , Carl in Oesterreich , vnserm gnedigsten herrn verschiner jarn erlangt vnd gehapt mit ermelten Matthias Manlichschen und Abraham Katzbeckhschen erben den 16. Nouembris verschinen 66 isten jars ain gesellschafft vfgericht vnd sie vmb drey achtail in solchen contract jure societatis vf vnd zu sich genomen haben mit gedingen vnd pactionibus in nebenligendem vidimiertem exemplo mit A signiert aussgefuerdt vnd begrüffen ." Fugger -Archiv 2, 4, 5, Folio 9. Leider fehlt das mit A signierte , zuletzt genannte Schrift¬ stück in den Archivbeständen. 518 Nachträge. Beilage. Die Firma David Flaug, Hans Langenauer und Mit¬ verwandte schließt mit der Firma Michael und Abraham Katzbeckh Gebrüder, Mathias Manlich und Mitverwandte einen Gesellschafts¬ vertrag ab. Darin übernimmt die zuletzt genannte Firma zur Hälfte das Idrianer Quecksilbermonopol . Augsburg, 16. November 1566. — Stadtarchiv Augsburg im Schrank der Flandelssachen Nr. 17. „Wir hernachbenanten Dauidt Haug, Hanns Lanngnaur vnnd mituerwanndten der ainen, Michel vnnd Abraham Katzbeckhen gebrueder, Mathias Männlich vnnd mitverwanndten der anndern, bürger zue Augspurg bekhennen offenntlich für vns, aller erben vnnd thuen khundt allmenigclich mit dem brief: Nachdem der durchleuchtigist, hochgeboren furst vnd herr herr Carl, ertzhertzog zu Österreich hertzog zu Burgundi, Steir, Kärnndtn, Grain vnnd Wirttemberg, Graf zu Tyrol vnnd Görtzu. s. w., vnnser genedigister herr kurtz verruckhter weiln mit vns obgemelten Dauidt Haugen, Hanns Lanngnaur vnnd mituerwandten ainen kauff vnnd contract vfgericht vnd beschlossen alls nemblich vmb funff tausend Centner (wienisch gewichts) queckhsilber vnnd zinober vff funff jar lang zu libern mit merlay capitlen vnnd conditionen. Wie dan darüber ain contractbrief vonn höc hst g'emelter fürstl. durchl. verfertigt vnd aufgericht wordenn. Welcher am anfanng: Wir, Carl von Gottes genaden, ertzhertzog zu Öster¬ reich, hertzog zu Burgundi, Steir, Kärnndtn, Crain vnnd Wirttem¬ berg, Graf zu Tiroll vnnd Görtz u. s. w. bekhennen u. s. w. vnnd am dattum, der geben ist den achten tag Octobris nach Christi vnnsers lieben herrn gebuert im funfftzehenhundert vnnd im sechs vnnd sechtzigisten jare u. s. w. weisennde. Vnnd wir die ermelten, Haug, Lanngnawr vnnd mituerwanndten obgedachte Michel vnnd Abraham die Katzbeckhen vnnd mituerwandten aus guetter wolmainung vnd freundtschafft inn solchen angeregten gantzen queckhsilber- vnnd zinoberkhauff vnnd vertrag vmb ainen halben thail durchaus zu gewin vnnd Verlust (wie es Gott der allmechtig verleihen vnd fuegenn wirdet) auff- vnnd angenomen. Das wir vnns darauff zu beden thaillen fur vnns vnnd vnsere erben ainer geselschafft vnnd verwanndtnus mit ainander verainigt vnnd verglichen, auch das zue halten vnnd zue laisten bey ehren, trawenn vnd glauben, ainander zuegesagt vnnd versprochenn. Tliuen auch solches hiemit wissentlich inn crafft diß briefs nemlich dergestalt vnnd also: 1. Das wir, obgemelte Dauidt Haug, Hanns Lanngnawr vnd mit¬ uerwanndten inn diser geselschafft vnd hanndlung den namen haben vnnd fueren sollen vnnd wir, die Katzbeckh, Männlich vnnd mituer¬ wandten, oder vnsere erben zu vnderhaltung vnsers gebuerenden thails inen, Dauidt Haugen, Hanns Lanngnawrn vnd mituerwanndten, oder iren erben zu jeder bezallungfrist (wie die in abgeredtem kauff vnnd vertrag bestimbt vnnd begriffen) den halben thail an denselben ortten der bezallung, auch in golt oder muntz, wie der vertrag an- 519 Dritter Nachtrag. zaigt , jedes mais vnnd alwegen, zu rechter zeit vnnd one fehl, mangcl oder vfzug richtig vnd gewisslichen erlegen vnnd bezallen. 2. Gleicher gestalt sollen vnnd wellen auch wir, die Katzbeckh, Männlich vnnd mituerwanndten , oder vnsere erbenn inen, Dauidt Haugen , Hannsen Lanngnawer vnnd mituerwanndten , oder iren erben den halben thail alles gebuerlichen vflauffennden vncostens (wie derselbig mit verschickhen vnnd verfertigen der queckhsilber vnnd zinober zu wasser vnnd lanndt vnd in alle andere wege sein vnnd sich befinden wirdet ) auff ir, oder irer erben anzaigen vnnd guete rechnung auch jederzeit vnauffzüglich darlegen vnnd darsehiessen, also inn suma nit allain an dem kauffgelt , sonnder aucli allem vnnd jegclichem anderm vncosten vnd außgaben vmb ainen halben thail an dem ganntzen vertrag vnnd nit weitter hiemit verobligiert vnd verbunden sein. 3. Doch haben wir Haug , Lanngnawr vnd mituerwandten vnns bewilligt , das wir von denen orten , da wir dits queckhsilber vnnd zinober verschleissen wurden vnnd anderer vnserer hanndtierung halber daselbst diener vnnd factor halten muessen , fur vnns bede partheyen nit mer als ainen per cento fur vncosten - vnd factorey -gelt der diener vereheren wollen. 4. Hingegen so sollen vnnd wollen wir, die erstgemeltenn Dauidt Haug , Hanns Lanngnaur vnnd mituerwandten vnd vnsere erben, idisen obangedeuten queckhsilber - vnnd zinober-khauffvertrag mit zuthuen , rat vnnd hilff gedachter Katzbeckhen , Männlich vnd mitiierwandten vnserm bessten vleiss vnnd guetbedunckhen nach ge¬ treulich verwaltenn vnnd regiern, das queckhsilber vnd zinober widerumb auffs höchst , besst vnnd nutzlichest , als wir weg vnnd mitel finden mugen , verkhauffenn vnnd verschleissen, daruon vffrechte , erbarc , clare vnd guette rechnung halten , inen von allen hanndlungvnd monnat -zetlen abschrifft vnnd copeyen verfolgen lassen, auch innsonderhait wir Haug , Lanngnaur vnd mituerwanndten , oder vnsere erben den gedachten Michelnn vnnd Abraham Katzbeckhen vnnd mituerwandten , oder iren erben jeder zeit, so offt vnd wan aus solchen queckhsilbern vnnd zinobern gelt erlösst , gefallenn vnnd eingebracht wirdet , iren gebuerennden halben thail durchaus jedcsmals getreulichen zuethailen , auch ohne Verzug zuestellen vnnd erlegen. . 5. Vnnd auch zu aussganng vnd enndt beruerts queckhsilberynnd zinober -khauffs vnd Vertrags, auch dartzwischen (so offt es die ‘notturfft erfordert ) inen oder iren erbenn von aller des hanndels gelegenhait freundlichs annzaigen bericht , auch darzwischen , souill imer der gelegenhait nach sein khan , doch vngeuerlich inn jars zeitten, .erbare vnnd genuegsame rechnung vnnd raittung geben vnd zuestellen, vnnd also an haubtguet , gewinung oder Verlust, inen jeder zeit denn halben thail verfolgen vnnd zuestehen lassen. . Hinwider was auch wir, obgemelte Katzbeckhen , Manlich vnnd mituerwanndten dises hanndels halben inn erfarung bringen , sy die Strieder , Studien z. Gesch . kapitalist . Organisationsformen . 34 520 Nachträge. Haugen , Lanngnawr vnd mituerwanndten auch berichten vnd verstendigen. 6. Vnd ob dan zu aussganng des Vertrags zeit der funff jar an queckhsilber vnnd zinober, noch ichtzit , wenig oder vill vberbliben verhannden , dergleichen was alssdan an vneingebrachtenn schulden noch ausstenndig sein wurde , denselben vorstanndt vnnd recht ann queckhsilber vnnd zinober , schulden vnnd gelt sollen vnnd wollen wir, obgemelte Dauidt Haug , Hanns Lanngnawr vnnd mituerwanndten, oder vnsere erben alsdan ferrer mit gemelten Michel vnnd Abraham Katzbeckhen , Mathias Männlich vnnd mituerwanndten guetten ratt vorwissenn vnd willen auch verkhauffenn , verhanndlen vnnd die aussteenden schulden zum getreulichisten einziehen vnnd einbringen. Zuuor auch dise geselschafft ir enndtschafft nit haben soi. 7. Wir, offt gemelte Haug , Lanngnawr vnd mitverwanndten habenn auch innsonnderhait bewilligt inn zeit dises queckhsilber - vnnd zinoberkhauffs sonnst ferrer daneben ad partem , oder innsonderhait khain queckhsilber oder zinober, vber die suma so vnns , oder vnnsern erben in werendem vertrag sollen gelibert werden , ohne ir, vnserer obgemeltet gesellschaffter vorwissen vnnd bewilligen zu khauffen. Wie dan sie, die Katzbeckhen , Männlich vnnd mituerwanndten , oder ire erben gleicher gestalt auch thuen sollen. 8. Dessgleichen, ob wir oder vnsere erben , nach disem queckhsilberkhauff vnnd zu aussganng desselben ainen ferrem kauff vnnd hanndlung vmb queckhsilber vnd zinober thuen wolten oder wurden, so sollen vnnd wollen wir den gedachten Katzbeckhen , Manlich vnnd mitverwanndten oder iren erben alssdann (soferr es inen gelegenn vnnd gefellig) in derselben hanndlung aber den halben thail , wie in diser zue rechnen vnnd mitlassen. 9. Wan wir aber mit höchst gedachter fürstl . durchl . ertzhertzog Carln, oder wer von irer fürstl . durchl . wegen das queckhsilber vnnd zinober inn hannden haben wirdet , ferrer nit hanndien wolten , oder vnns mit irer fürstl . durchl . aines kauffs nit vergleichen kündten, mögen sie, die Katzbeckh , Männlich vnnd mituerwandten alssdan zu irer gelegenhait mit irer fürstl . durchl . ainen kauff für sich selbs beschliessen. Doch das sy vnns , oder vnsem erben volgents auch aift halben thail darinnen (so vns der anzunemen gefällig) mit lassen. 10. So sollen auch offt ermelte Katzbeckhen , Männlich vnd mit¬ verwanndten , oder ire erbenn vnns , den gedachten Dauidt Haugen, Hannsen Lanngnawr vnd mitverwanndten , oder vnsem erben gegen yeder gelterlegung allweg gebuerlich vrkhundt vnnd bekhanndtnus vnnd insonnderhait zu enndt der vertragszeit vnd vnser enndtlichen haubtrechnung , vberantwurttung vnnd zuestellung gebuerliche vnnd gnuegsame juratquittung fertigenn vnnd gebenn . Wie auch gleicher gestalt wir, oder vnnser erben inen , den Katzbeckhen , Männlich vnd mituerwanndten , oder iren erben gegen yedem iren gelterlegung auch Dritter Nachtrag. 521 gebuerlich vrkhundt vnnd ledstlichea auch freundtlich quittungbrief zuestellen sollen vnnd wollen. 11. Vnnd beschliesslich haben wir bede, oben bekhennende parteyen vnnd geselschaffter vnns ferrer dessen mit ainander verainigt: Wan sich in zeit diser werenden geselschafft begebenn vnnd des hanndels notturfft erhaischen wirdet , das wir an frembde ausslenndische ort verraissen muessten , sollen wir zu beden thaillen zugleich darmit verbunden sein, vnnd sich khainer dauon absonndern , sonnder in dem, wie in allem andern , ainander getreulich beystehen vnd der¬ massen erzaigen ails getreuen , redlichen geselschafftern wol zuestet vnnd gebuert . Alles vnnd jegclichs recht vnnd redtlich sonnder argKsst vnd gefehrde. Vnnd des zu warem vrkhundt sein diser Verschreibungen zwo gleichs lauts gemacht vnnd aufgericht , deren jeder thaill aine zuhannden genomen . Vnnd von vnns , obgemelten Dauidt Haugen, Hannsen Lanngnawr vnnd mituerwanndten mit vnser der Haugen vnnd Lanngnawrs nngebomen innsigeln vnnd selbs vnderzaichneten hanndschrifften , dessgleichen von vnserer , obgenanter Michel vnd Abraham Katzbeckhen , Mathes Männlich vnd mituerwandten wegen mit mein , gemelts Abraham Katzbeckhen vnd Anthpni Hörmans angebornen innsigeln, dabey wir vnns auch mit aignen hannden vnderschriben , verfertigt worden. Geben vnd beschechen zu Augspurg , den 16. tag des monnats Nouembris nach Christi vnsers lieben herrn , ainigen erlösers vnd seligmacbs gebürt funffzehenhundert vnd im sechs vnnd sechtzigisten jare. Dauidt Haug manu propria . Hanns Lanngnawr manu propria. Abraham Katzbeckh manu propria . Anthoni Hörman manu propria. 34* Orts= und Personenregister der Naditräge. Adria 494, 497. Afrika 499. Almaden 510, 515. Amerika 515. Amsterdam 494f. Ancona 497. Antwerpen (Anntorff ) 493—497, 499, 500. Fugger , Hans 505 f., 509 f. —, Hans Jacob 503, 510. —, Jacob 499, 501 f., 504. —, Marx 505 f., 509. —, Raimund 503. —, Ulrich 503. Furtenbach , Bonaventura 490. Genua 497. Augsburg 489 « ., 499 - 501, 503 ff., 508 Ghysiis , Augustinus de (= Agostino bis 518, 521. Chigi ?) 516. Goldküste 499. Baumgartner 498. Görz 513, 518. Belgien 499. Guanca -Velica 515. Bergheim 510. Bimmel , Anton 510. Han , Apollonia 510. —, Regina 510. —, Dr . Johann Jakob 510. Böhmen 492, 500, 503 « . Haug , David 514—521. Brabant 501. Herwart 514. Breyner , Philipp 501. Hobereckh , Melchior von 501. Breslau 494 f. Hochkirch 499. Burgund 501 , 518. Höchstetter 498 , 501 f., 510. Hörman , Anton 521. Cornwallis 503. Holzschuher , Veit 490. Dalmatien 501. Idria 510, 513- 521. Danzig (Tanizka ) 494 f. Innsbruck 498. Demschwam , Hans 491, 495. Italien 494, 497 f., 501. Donau 499 , 502. Kärnten 498 , 518. Ebner , Hans 490. Karl , Erzherzog von Österreich 513 f., Elisabeth , Königin von England 503. 517 f„ 520. Katzbeckh 516 - 521. England 503, 515. Khisl zu Kaltenbrunn , Hans 513. Ferdinand I., deutscher Kaiser 490 bis Kleinasien 491. 493, 495, 497, 500 f„ 503, 508 f. Köln 503. Frankfurt a . M. 507. Konstantinopel 491. Frankreich 494, 496 f. Krain 515, 518. Fugger 489—512. Krakau 491 , 494 f., 507. —, Anton 489 f., 492—495, 500f ., 503 Kroatien 501. bis 512. Langenauer , Hans 514—521. —, Christoph 503. Levante 497. —, Georg 503. Orts-' und Personenregister der Nachträge. Mayr , Konrad sr . 503—512. —, — jr . 510. Maltitz , Hans 498. Manlich , Matthias oder Mattheus bis -502, 516 —521. Marseille 497. Maximilian I., deutscher Kaiser 501. Mindelheim 510. Neusohl 489 - 491, 498 f., 500. Niederlande 494 ff., 498 f., 502. Nürnberg 490, 507, 511 f. Österreich 498 , 501, 518. Ohrdruf 499. Polen 495 f. Portugal 494, 496, 499. Prag 507. Preußen 495 f. Roch , Euphrosina 510. —, Peter 510. Rott , Konrad 510 f. Sachsen 503 f. Sauerzapf , Sebastian 491. Schlackenwald 507. Schlesien 495 f. Schwarz , Matthäus 503. 489 Scliwaz 493, 495 , 498, 501 f. Spanien 494—496, 515. Spanochi , Ambrosius de 516. 499, 502. 498, St . Georgenthal Stegnuyin , Georg 505. Steyr 518. Teschen 491. Thorn 494 f. Thüringen 498 f. Tirol 489 , 491 f., 497 f., 500, 518. Tolfa 516. Türken 489 f. Ungarn 489 ff., 494 f., 497 ff., 501 f. Venedig 498. Villach 513 f. Walther , Marx 503. Welser , Bartholomae 508. Wien 493, 501, 513 f. Württemberg 518. Zeller , M. 501. 523
https://openalex.org/W4241951625	http://hrcak.srce.hr/file/215936	English	null	Evaluation of Practical Application of Plant Simulation in the Form of Experiments	Naše more	2,015	cc-by	4,174	Evaluation of Practical Application of Plant Simulation in the Form of Experiments Procjena praktične primjene simulacije postrojenja u obliku eksperimenata Lukáš Kamenický Technical University of Kosice Slovakia e-mail: lukas.kamenicky@tuke.sk Michal Hovanec Technical University of Kosice Slovakia e-mail: michal.hovanec@tuke.sk Juraj Sinay Technical University of Kosice Slovakia e-mail: juraj.sinay@tuke.sk Hrozek František Technical University of Kosice Slovakia e-mail: frantisek.hrozek@tuke.sk Hrozek František Technical University of Kosice Slovakia e-mail: frantisek.hrozek@tuke.sk Petr Skřehot Technical University of Kosice Slovakia e-mail: p.skrehot@seznam.cz DOI 10.17818/NM/2015/SI19 UDK 338.36 Review / Pregledni rad Paper accepted / Rukopis primljen: 27. 4. 2015. DOI 10.17818/NM/2015/SI19 UDK 338.36 Review / Pregledni rad Paper accepted / Rukopis primljen: 27. 4. 2015. Summary KEY WORDS product lifecycle managment plant Simulation quality production digital plant digitalization This contribution deals with the Simulation of manufacturing systems. Plant simulation is a modern instrument for optimizing factory production; however, the initial investment is high, especially for human resources, since the creation of a simulation model requires a lot of data from manufacturing, which needs to be analyzed and used as input into the simulation so that the simulation model is as close as possible to the real manufacturing system. Simulation enables changes to first be carried out in the simulation model and the defining of possible problems, through which in this case it is possible to avoid when introducing changes into an operation. The measure of effectiveness of using inputs is the productivity of a manufacturing system, and the aim of organization management is to monitor, analyze and improve a manufacturing system through optimizing for the purpose of increasing its efficiency, ensuring higher quality and a higher volume of production, shortening supply time in the scope of transportation, improving logistics and increasing the profitability of the given organization. KLJUČNE RIJEČI vijek trajanja proizvoda menadžment simulacija postrojenja kvalitetna proizvodnja digitalno postrojenje digitalizacija Mjera učinkovitosti upotrebe ulaznih podataka je produktivnost proizvodnog sustava, a cilj organizacijskog menadžmenta je pratiti, analizirati i poboljšati proizvodni sustav optimizacijom kako bi se povećala učinkovitost, osigurala viša kvaliteta i veća proizvodnja, smanjilo vrijeme nabave tijekom transporta, poboljšala logistika i povećala profitabilnost date organizacije. vijek trajanja proizvoda menadžment simulacija postrojenja kvalitetna proizvodnja digitalno postrojenje digitalizacija Sažetak U ovom radu govori se o simulaciji proizvodnih sustava. Simulacija postrojenja je moderni instrument za optimizaciju tvorničke proizvodnje; međutm, početno ulaganje je veliko, posebice u ljudstvu, budući da pravljenje simulacijskog modela zahtjeva veliku količinu podataka iz proizvodnje, koje treba analizirati i upotrebiti u simulaciji tako da simulacijski model bude što vjeriniji stvarnom proizvodnom sustavu. Simulacija omogućuje da se promjene prije svega provedu u simulacijskom modelu i definiraju mogući problemi, a time je moguće izbjeći iste probleme kada se promjene uvedu u rad. Experiment 1 – shortening the period for the setting of the lacquer equipment; therefore, the work environment in which the process of manufacture took place was selected as the area of optimization in the organization. Optimization related in particular to the shortening of certain times for processing the sheets, transport and logical links, in which time reserves were identified. These were suitable for using and thus increasing the productivity of the entire manufacturing system. The process of setting in the model is represented by the object Buffer, with a processing time of 24 hours. The proposed solution in practice represents placing a drying oven into the manufacturing system between the processes “pressing” and “striking of CC”. The period for setting of the lacquer would in this way be shortened from the original 24 hours to an anticipated 4 hours (2 hours for setting of the lacquer, 2 hours for ventilating the ovens). By creating the experimental model, in the scope of which the period of processing in the object Buffer (see Fig.1) is defined as 4 hours, the influence of these changes will be verified in the total production of the manufacturing system and the impact on the capacity of loading of the object Buffer. The tables below shows the words “KU” and “sluzba” in software as a service and a CC. INTRODUCTIONf into production and verifying the correctness of technological proposals before actually introducing them into a real manufacturing system. Predicting the effects of different changes in the life cycle of a product can be achieved through use of a digital factory as software support, namely by simulation programs such as, for example, plant simulation, which enable predicting the course of the process of planning, transportation, logistics and manufacture in a short time. The use of this tool enables the performing of experiments without direct interference In the scope of conducting the experiments the process of lacquering and pressing of metal sheets and the process of manufacturing crown caps were described [1]. The processing of metal sheets itself runs on automated production 193 “Naše more” 62(3)/2015., pp. 193-199 Experiment 1 – shortening the period for the setting of the lacquer Experiment 2 – shortening the duration of the change of lacquer and colour the change of lacquer/colour), the number of processed entities for the output Service grew by 14.55%, Each change of lacquer and colour lasts 30 minutes; however, it is essential in the scope of carrying out individual orders, because the type of lacquer and the number of colours are specific for each order (see Fig.4). The proposed improvement consists in optimizing the process of changing the lacquer and colours such that the duration of this process is minimized. In the experimental model a proposed shortening of the time needed for a change of the lacquer of object LTG2 and the colour in object LTG1 was introduced from the original 30 minutes to an assumed 25 minutes. The effect of this change was monitored by comparing the statistical output from the experimental model versus the output from the basic model. -- the number of entities processed by lines LTG2 and LTG1 for the purpose of manufacture of CC grew; however, due to the loading of the object Buffer, this was manifested only in the collective in-process production. TOOLS AND METHOD In the following sections of the contribution 5 experiments are carried out in which the independent and common effects of the proposed improvements in the manufacturing process organization using the simulation instrument PLANT SIMULATION will be tested. These are the proposals for improvement: From the above-presented image in comparison with Fig. 2 and Fig. 3 the following effect of the simulated change follows: 1. Shortening of the time necessary for the setting of the lacquer by introducing a drying oven into the process of production between the process of lacquering and the process of manufacture of the CC. -- the loading of object Buffer dropped by 10%, -- the loading of object Buffer dropped by 10%, -- the period of processing of objects SACMI1 and SACMI2 grew by 2.8% for each piece of equipment, which had as a consequence growth in the volume of processed entities for the output of CC by 3.015%. -- the period of processing of objects SACMI1 and SACMI2 grew by 2.8% for each piece of equipment, which had as a consequence growth in the volume of processed entities for the output of CC by 3.015%. 2. Shortening of the time necessary for a change of lacquer and colour on the LTG lines. 3. Introducing of a third series SACMI line for the manufacture of CC, (SACMI - ejectors line, LTG - printing line, CC - Crown Caps . M. Hovanec et al: Evaluation of Practical Application of Plant... Figure 1 Statistical output from the model of experiment 1 Figure 2 Use of the operation time of equipment before the change Figure 1 Statistical output from the model of experiment 1 Figure 2 Use of the operation time of equipment before the change Figure 2 Use of the operation time of equipment before the change Figure 2 Use of the operation time of equipment before the change Figure 2 Use of the operation time of equipment before the change M. Hovanec et al: Evaluation of Practical Application of Plant... Figure 3 Statistical output of the basic simulation model before the change Figure 4 Statistical output of the basic simulation model Experiment 2 – shortening the duration of the change of lacquer and colour Experiment 3 – increasing the production capacity in the manufacture of CC At present the process of CC manufacturing is represented by 2 series of SACMI production lines. The loading capacity of the object Buffer (see Fig. 2) was evaluated as a space for introducing a third series of the SACMI production line. The proposed change was simulated in experiment model 3 by adding object SACMI3. On the basis of the statistical output from this model the effect of the simulated change in the loading of the object Buffer was monitored, as were the amount of processed entities and the loading capacity of objects SACMI1, SACMI2 and SACMI3. From the above-mentioned image, in comparison with Fig.3., the following effect of the simulated change results: -- the average period of processing 1 entity as a service dropped by 5 minutes (represents the time-savings during Figure 5 Statistical output from the model of experiment 3 Figure 6 Loading of the equipment in the model of experiment 3 Figure 6 Loading of the equipment in the model of experiment 3 195 “Naše more” 62(3)/2015., pp. 193-199 -- the share of processed entities in the output Service dropped by 21.74%, From the above-mentioned images, in comparison with Fig. 2 and Fig. 3., the following effect of the simulated changes results: -- the share of processed entities in the output CC grew by 54.27%, -- the volume of the processed entities for the output of CC grew by 6.78%, -- the relative loading of the object Buffer dropped significantly, -- the period of processing SACMI objects dropped to 66.3%, i.e. a reserve for further processing was created, -- the period of processing of the SACMI objects grew by 2.8%. -- the period of processing of the SACMI objects grew by 2.8%. -- the loading capacity of the object Buffer remained unchanged. Experiment 4 – simulation of the combination of Experiment 5 represents the simulation of common introducing of the proposed changes simulated in experiments 2 and 3. The change simulated in the experiment was excluded on the basis of the fact that the achieved benefit (higher volume of processed entities by 3% in the output of CC) is not considered as sufficient with respect to the financial demand of carrying out this change in practice (investment in the drying oven). experiments 1, 2 and 3 In the individual experiments 1, 2 and 3 the effect of the simulated changed was assessed individually in comparison with the basic simulation model of the real manufacturing system. In experiment 4 their common effect on the overall productivity of the system was simulated; thus all proposed changes were implemented in the scope of a single model. On the basis of statistical outputs the effect of the simulated changes on the loading capacity of the SACMI objects, the object Buffer and the amount of processed entities for outputs of CC and Service was monitored. In this experiment the introducing of a third series of the SACMI line and shortening of the time for changing lacquer and colour on the LTG lines and their common effect on productivity in the simulated manufacturing system was simulated. On the basis of statistical outputs the volume of the processed entities in the outputs Service and CC were monitored, as was the loading of the individual objects. From a comparison of the above-mentioned images with Fig. 2 and Fig. 3., the following effect of the simulated changes in experiment 4 results: -- production of objects LTG2 and LTG1 grew by 8.63%, Figure 7 Statistical output from the model of experiment 4 Figure 8 Loading of the equipment in the model of experiment 4 Figure 7 Statistical output from the model of experiment 4 gure 7 Statistical output from the model of experiment 4 Figure 8 Loading of the equipment in the model of experiment 4 Figure 8 Loading of the equipment in the model of experiment 4 M. Hovanec et al: Evaluation of Practical Application of Plant... Experiment 4 – simulation of the combination of Figure 9 Statistical output from the model of experiment 5 Figure 10 Loading of the equipment in the model of experiment 5 Figure 9 Statistical output from the model of experiment 5 Figure 9 Statistical output from the model of experiment 5 Figure 10 Loading of the equipment in the model of experiment 5 0 Loading of the equipment in the model of experiment On the basis of a comparison of the facts presented in Fig. 10 and Fig. 9 and with the statistical outputs from the basic model (Fig. 2 and Fig. 3) the following effect of the simulated changes result: to the object Buffer, 59% of entities head to the output Service). Placement of a third series of SACMI lines creates the space for a change of this ratio in favour of entities further processed for the purpose of CC manufacture. -- the volume of processed entities in the output Service grew by 10.03%, In the second part of experiment 5, the ratio of 100% of the processed entities was divided in the output from object LTG1 in the simulation model as follows: -- the volume of processed entities in the output CC grew by 6.53%, -- 60% head to the object Buffer (entities processed for the purpose of CC manufacture), -- the relative loading capacity of the object Buffer remained unchanged in comparison with the basic model, -- 40% head to the output Service (entities processed as a service). -- a decline in the overall period of processing the SACMI objects to 65.8% of the total operation time. Likewise, the capacity of the object Buffer was changed to 22 entities for the purpose of effective use of the operation time of the SACMI equipment, which (while the previously defined capacity of the object Buffer of 15 entities) expected 34.2% of its total operation time for the input to the process. The reason for the decline in overall processing period for the SACMI objects is the insufficient capacity of the object Buffer (15 entities) and likewise the simultaneous division of the flow of material in the output from object LTG1 (41% of entities head RESULTS, DISCUSSION AND CONCLUSIONSf -- 478 entities in the output Service (a decline by 20.07% in comparisons with the basic model, a decline by 27.36% in comparison with the original model of experiment 5), -- 597 entities in the output CC (growth by 50% in comparison with the basic model, growth by 40.8% in comparisons with the original model of experiment 5).f In the following table the anticipated effect of the proposed improvements on the basic simulation of experiments 1-5 is described. The number of processed entities was monitored for a period of 30 days overall and for the individual outputs Service and CC. The weights of financial demand of the introduced changes in practice (1-low, 2-medium, 3-high) were assigned to the individual experiments, and the generated outputs were recalculated for their possible financial evaluation, because the processed entities in the outputs Service and CC have different selling prices. The selling price presented is inclusive of the price of the initial input – the sheet metal. In experiments 1-3 each of the proposals for improvement was simulated independently, and in experiments 4 and 5 the combination of proposals for improvement was simulated; therefore experiments 1-3 and experiments 4 and 5 will be evaluated separately. The change of the selected evaluations is depicted as a percentage with respect to the basic model (a decline by red, growth by green). Experiments 1 and 3, unlike experiment 2, did not lead to any overall growth of production, but only to a shift of part of the processed sheets in favour of the process of CC manufacture, which is financially more interesting for the organization. For the mentioned reason, experiments 4 and 5, in which a combination of introducing all three of the proposed improvements, were carried out. From the results depicted in Tab. 1 it is evident that experiment 4 achieved better results, which is related to the volume of production of the simulated system; however, after taking the necessary investment costs into account, experiment 5, in which the manufacture system was simulated after introducing of proposals 2 and 3, was selected as the most suitable. The benefit of the experimental Table 1 Comparison of the results of individual experiments Experiment Description Number of processed entities Weight of fin. demand Value of output in EUR Fin. RESULTS, DISCUSSION AND CONCLUSIONSf RESULTS, DISCUSSION AND CONCLUSIONS From experiments 1-3 proposal number 2, the effect of which was simulated in experiment 2, was selected as the most suitable proposal for improvement for introduction into the real manufacturing system. The feasibility of improvement of number two appears in the assumed low costs (in comparison with the costs necessary for carrying out the other proposals) and was the only one that generated growth of production of the simulated manufacturing system; however, only for the output of sheets processed as a service. For the process of CC manufacture this fact means potential for re-evaluating the current capacities of CC manufacture, since CC manufacture brings the organization 65.1% higher financial evaluation than the service itself – lacquering and colour printing of sheet metal. Experiments 1 and 3, unlike experiment 2, did not lead to any overall growth of production, but only to a shift of part of the processed sheets in favour of the process of CC manufacture, which is financially more interesting for the organization. For the mentioned reason, experiments 4 and 5, in which a combination of introducing all three of the proposed improvements, were carried out. From the results depicted in Tab. 1 it is evident that experiment 4 achieved better results, which is related to the volume of production of the simulated system; however, after taking the necessary investment costs into account, experiment 5, in which the manufacture system was simulated after introducing of proposals 2 and 3, was selected as the most suitable. The benefit of the experimental From experiments 1-3 proposal number 2, the effect of which was simulated in experiment 2, was selected as the most suitable proposal for improvement for introduction into the real manufacturing system. The feasibility of improvement of number two appears in the assumed low costs (in comparison with the costs necessary for carrying out the other proposals) and was the only one that generated growth of production of the simulated manufacturing system; however, only for the output of sheets processed as a service. For the process of CC manufacture this fact means potential for re-evaluating the current capacities of CC manufacture, since CC manufacture brings the organization 65.1% higher financial evaluation than the service itself – lacquering and colour printing of sheet metal. Figure 11 Statistical output 2 from the model of experiment 5 Figure 12 Loading of the equipment 2 in the model of experiment 5 Figure 12 Loading of the equipment 2 in the model of experiment 5 “Naše more” 62(3)/2015., pp. 193-199 The most suitable variant is in the table indicated by the pink colour. Furthermore, the change versus all of the previous experiments consisted in a change of capacity of the object Buffer to 22 entities and a change of the ratio of the divided processed entities in the output from object LTG1. This change was shown to be effective, because the total period of processing the SACMI objects grew to 93% from the original 65.8%. This is also significantly manifested in the number of processed entities for the individual outputs: RESULTS, DISCUSSION AND CONCLUSIONSf contribution in EUR CC Service Total CC Service Total 0 basic model 398 (100%) 598 (100%) 996 (100%) - 1321887 1201980 2523867 (100%) - 1 shortening the period of setting of lacquer 410 (+3.02%) 587 (-1.84%) 997 (-) 3 1361742 1179870 2541612 17745 (+0.7%) 2 shortening the period of change of lacquer/colour 398 (-) 685 (+14.55%) 1083 (+8.73%) 1 1321887 1376850 2698736 174869 (+6.93%) 3 increasing prod. capacity of SACMI lines 425 (+6.78%) 570 (-4.68%) 995 (-) 2 1411562 1145700 2557262 33395 (+1.32%) 4 combination of exp. 1,2,3 614 (+54.27%) 468 (-21.74%) 1082 (+8.63%) 1+2+3 2039292 940680 2979972 456105 (+18.07%) 5 combination of exp. 2,3 597 (+50%) 478 (-20.07%) 1075 (+7.93%) 1+2 1982830 960780 2943610 419743 (+16.63%) Table 1 Comparison of the results of individual experiments M. Hovanec et al: Evaluation of Practical Application of Plant... 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https://openalex.org/W3108844232	http://www.ppmj.org.pk/index.php/ppmj/article/download/289/202	English	null	Gender Specific Distribution Of different patterns of Diabetic Retinopathy In Patients With Type 2 Diabetes Mellitus. A Cross-Sectional Study From a tertiary care specialized center in Lahore, Pakistan	Pakistan postgraduate medical institute	2,020	cc-by	2,397	ORIGINAL ARTICLE ORIGINAL ARTICLE ABSTRACT Background: The patients of diabetic retinopathy are not aware of early retinal vascular dysfunction, which is a feature of diabetic retinopathy. Objective: This study aimed to detect the different patterns of diabetic retinopathy and distribution in different genders, in a tertiary care specialized center. Methods: Consecutive patients attending the Diabetes Endocrine and Metabolic Center (DEMC) were consented and examined for a period of six months. Direct and indirect ophthalmoscopes were used by an ophthalmologist for fundus examination. Grading of the retinopathy patterns was done by International Clinical Diabetic Retinopathy Disease Severity Scale (ICDRDSS). The data was analyzed using SPSS version 23. Results: A total number of 250 patients were examined during the six months period of study. The frequency of diabetic retinopathy was more in females as compared to males. The incidence of non-proliferative diabetic retinopathy was more in males as compared to females, and the prevalence of proliferative diabetic retinopathy was same in both genders. Greater number of females had clinically significant macular edema and advance diabetic retinopathy was observed in only males. Conclusion: Diabetic retinopathy (DR) is common in our setup, with different genders having different prevalence of eye problems. A screening strategy system is needed to develop for the early detection and treatment of preventable cause of blindness in patients with type2 diabetes mellitus. Key Words: Distribution, Diabetes Mellitus, Retinopathy, Gender How to cite this article: Khan MIH, Waqar A, Riaz A, Azhar U, Tariq A, Ismail I, et al. Gender Specific Distribution Of different patterns of Diabetic Retinopathy In Patients With Type 2 Diabetes Mellitus. A Cross- Sectional Study From a tertiary care specialized center in Lahore, Pakistan. Pak Postgrad Med J 2019;30(1): 32-35 How to cite this article: Khan MIH, Waqar A, Riaz A, Azhar U, Tariq A, Ismail I, et al. Gender Specific Distribution Of different patterns of Diabetic Retinopathy In Patients With Type 2 Diabetes Mellitus. A Cross- Sectional Study From a tertiary care specialized center in Lahore, Pakistan. Pak Postgrad Med J 2019;30(1): 32-35 GENDER SPECIFIC DISTRIBUTION OF DIFFERENT PATTERNS OF DIABETIC RETINOPATHY IN PATIENTS WITH TYPE 2 DIABETES MELLITUS. A RETROSPECTIVE CROSS-SECTIONAL STUDY FROM A TERTIARY CARE SPECIALIZED CENTER IN LAHORE, PAKISTAN MUHAMMAD IMRAN HASAN KHAN, AMMARA WAQAR, AYESHA RIAZ, USAMA AZHAR, AEMIN TARIQ, IRSA ISMAIL, FIZZA ZUBAIR P t d t M di l I tit t / A d Di M di l C ll L h Q Postgraduate Medical Institute / Ameer ud Din Medical College, Lahore MUHAMMAD IMRAN HASAN KHAN, AMMARA WAQAR, AYESHA RIAZ et al MUHAMMAD IMRAN HASAN KHAN, AMMARA WAQAR, AYESHA RIAZ et al retinopathy (PDR) was depicted by formation of new vessels originating from the retina and optic disc which is known as neo-vascularization, in patients with uncontrolled blood sugar levels for a long period of time in diabetes mellitus.6Diabetic macular edema (DME) was a thickening of the macula, an area slightly lateral to the center of retina, which led to loss of vision. The most hellacious form of DME was Clinically Significant Macular Edema (CSME).7One of the concerns of this study was cost effectiveness of screening programs regarding diabetes related eye diseases.8 study. On a routine, 100 to 150 patients attend DEMC on a day, and their fundus was checked on first visit, unless there was a contraindication. An optometrist was available six days in week, with a non mydriatic fundus photograph camera, for examination of fundus. Direct and indirect ophthalmoscopes were used and this fundus camera, by an optometrist. International Clinical Diabetic Retinopathy Disease Severity Scale (ICDRDSS) was used for grading the retinopathy. The data was analyzed using SPSS version 23. After careful examination, only those patients between the ages of 15 to 90 were selected, from both genders. Only those having retinopathy findings, were included, and those without them, were not. It meant that a large portion without retinal findings was excluded. Presence of eye complication, restricting fundus photo were also excluded. All those with any other complication were included. A study was done at Aminu Kano Teaching Hospital, Kano, Nigeria in 2012 which showed the distribution of diabetic retinopathy among males and females. Total number of patients were 214. 88 males and 126 females (M:F=1:1.43) were included in that study. However, total number of subjects was small in this study. 9 Lin J-C et al10 conducted study in Taiwan; on 2926 cases of patients with DR among 63,582 patients with diabetes mellitus type 2. They described the changing trends through these years and examined whether the gender specific variation existed. Our study is from a totally different part of the world, where socio economic condition and provision of health services are compromised. Also lack of education, non availability of independent resources, lack of reflection of health in annual government budget and no access to specialized centers play pivotal role. This became all the more important to conduct this study in our circumstances. RESULTS The total number of patients included in the study was 250, as they had positive findings, during the study period. Diabetic retinopathy was found more commonly in females as compared to males (Figure 1 and 2). The non-proliferative diabetic retinopathy was more in males as compared to females, but the proliferative diabetic retinopathy remained same in both genders. However, greater number of females had clinically significant macular edema and advance diabetic retinopathy was observed in only 4 males. In Pakistan diabetic retinopathy is affecting a large number of diabetic patients but evidence is lacking locally, as very few number of researches has been carried out, and the data available is not sufficient and not properly documented, specially from a specialized center where resources were available. We conducted this study to determine distribution of diabetic retinopathy according to gender on a large group of patients. This study would help us to know the different patterns of retinopathy in patients attending Diabetes Endocrine and Metabolic Center (DEMC) which included NPDR, PDR, CSME, ADVDR and distribution of the disease in different genders. This could also help the health care providers to establish screening programs and providing effective care to the diabetic patients to decrease the incidence of preventable causes of blindness which was cost effective. Figure 1: Figure 2: Figure 1: Figure 1: Figure 2: INTRODUCTION retinopathy, nephropathy, neuropathy cardiovascular and stroke.1 Diabetes mellitus is known as a chronic metabolic disorder which is depicted by persistently raised blood glucose levels. According to a study by World Health Organization (WHO), diabetes mellitus had prevailed from 4.7% to 8.55% from 1980 to 2014 in population over 18 years of age. The persistent increase in blood glucose level led to the development of complications of diabetes mellitus which included both micro-vascular and macro-vascular degenerative disorders such as Diabetic retinopathy (DR) is of great importance and concern because the current studies showed that prevalence and incidence of diabetes mellitus Type 2 was anticipated to rise during the next decades.2 Due to step-up advances in early detection and prevention of diabetic retinopathy, there was a raised demand for DR treatment services.3 Patients of DR were generally incognizant of the developing early retinal vascular damage and changes which was also a characteristics of DR. Floaters or hazy vision were visual symptoms of DR that often transpire in more advanced stages, such as proliferative DR or clinically significant macular edema(CSME).4 Early stage of diabetic retinopathy was Non-proliferative diabetic retinopathy(NPDR). Automatic detection of NPDR is significant for clinical diagnosis, early screening and serial progression of disease in patients with DM.5Proliferative diabetic Correspondence to: Muhammad Imran Hasan Khan, Postgraduate Medical Institute / Ameer ud Din Medical College, Lahore, E-mail: mimranhkan@hotmail.com …………………………………………………………………. Received: Nov 07, 2019; Revised: Mar 02, 2020; Accepted: Jul 14, 2020 Correspondence to: Muhammad Imran Hasan Khan, Postgraduate Medical Institute / Ameer ud Din Medical College, Lahore, Pak Postgrad Med J Jan. – Mar. 2019 Vol. 30 No. 1 www.ppmj.org.pk 32 DISCUSSION totally different ethnicity and in that part of world with limited health care resources. This data might help to identify settings and subgroups of populations where DR is going in higher trends and help to set screening priorities and programs for prevention, early detection, optimized resources allocation and orient future to fill knowledge gaps. Diabetic retinopathy is one of the long-term consequences and complications of the ocular manifestation of diabetes mellitus. It is a vision threatening disease particularly with long disease duration and poor control of blood sugar levels. Luckily salutary and beneficial advances have occurred regarding the prevention and treatment of the disease by understanding the risk factors, underlying pathology and functional disability caused by diabetic retinopathy. LIMITATIONS: There were certain limitations in our study. First, it just included a very small number of patients. Results could have been different if larger number of patients were screened. Second, it only assessed the gender distribution. Endurance of Diabetes, Control, and either other complications exist or not could have given us a better idea about the factors contributing to these findings. Third, if we had included the frequency of routine screening in these patients, it would have been very helpful in drawing conclusion on that also. DR is of particular concern because of increasing incidence and advances in early detection, making it a preventable cause of blindness. Because of rapidly changing trends of DM and DR, there is strong rationale to update the current patterns of DR in sex specified population. Most of the patients attending the diabetic, metabolic and endocrine clinic are not aware, that the visual symptoms they are suffering from are due to an isolate eye disease or long-term consequences of diabetes. METHODS Consecutive patients who were attending DEMC and consented, for a period of six months, from January 2017 to June 2017, were selected and examined for the Figure 2: Figure 2: Pak Postgrad Med J Jan. – Mar. 2019 Vol. 30 No. 1 www.ppmj.org.pk 33 CONCLUSION Alba Aguada et-al did a study that multi-morbidity has great influence on health care provision in patients with type2 diabetes mellitus. A multi-morbidity network in the general population was constructed in distinction to age and gender. These networks were showing strong communication between T2DM and retinopathy, nephropathy, neuropathy and pancreas cancer.11Ourstudy did not address any of these features. However, it was similar to this study in respect to emphasizing the importance of getting a structured screening program for DR. Diabetic retinopathy is one of the leading cause of blindness in our setup particularly with long term subsistence of DM. Diabetic retinopathy is avert cause of blindness thus outgrowth of screening programs is in demand for the early detection of the disease, and to offer better treatment. ACKNOWLEDGEMENTS Authors would like to thank doctors working in DEMC and Medical Unit 3 for permission and help in carrying out this study. They would also like to thank Fred Hollows, and the ophthalmology department for permission and sharing data for this study. In 2012, Aminu Kano Teaching Hospital, Kano, Nigeria carried out a study which showed the distribution of diabetic retinopathy among males and females. Total number of patients were 214. Males and females were in ratio of M:F= 1:1.43 with number of 88 males and 126 females. However, total number was too small included in this study.12Our Study is similar to this study, as it shows patterns in both sex, with almost similar number of patients. However, it is more informative, as it further splits different types of DR, helping us in understanding gender distribution of advanced retinopathy. ETHICAL APPROVAL: The study was approved from Ethical Review Committee of Postgraduate Medical Institute, Lahore, Pakistan. The study was approved from Ethical Review Committee of Postgraduate Medical Institute, Lahore, Pakistan. AUTHORS’ CONTRIBUTION: MIHK: Idea, discussion, over all supervision AW: Introduction, discussion, data collection RA: Methods, discussion, data collection UA: Results, statistical analysis AT, II: Introduction, data analysis and collection FZ: Introduction AUTHORS’ CONTRIBUTION: Lin J-C et al13 conducted study in Taiwan; out of 63,582 patients with T2DM attending diabetic clinic, 2926 were having DR. They described the changing trends through these years and examined whether the gender specific variation existed. Our study is much smaller than this study. but it also shows similar trends in gender, in a different geological environment. Our study analyzed gender based distribution of different patterns of diabetic retinopathy in patients with Pak Postgrad Med J Jan. – Mar. 2019 Vol. 30 No. 1 www.ppmj.org.pk MUHAMMAD IMRAN HASAN KHAN, AMMARA WAQAR, AYESHA RIAZ et al MUHAMMAD IMRAN HASAN KHAN, AMMARA WAQAR, AYESHA RIAZ et al 7. Optical coherence tomography (OCT) for detection of macular oedema in patients with diabetic retinopathy. - PubMed - NCBI. https://www.ncbi.nlm.nih.gov/pubmed/?term=Optical +coherence+tomography+(OCT)+for+detection+of+ macular+oedema+in+patients+with+diabetic+retinop athy.+Review+article+Virgili+G%2C+et+al.+Cochra ne+Database+Syst+Rev.+2015. Accessed February 23, 2019. REFERENCES 1. 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Prognostic prediction models for diabetic retinopathy progression: a systematic review. - PubMed - NCBI. https://www.ncbi.nlm.nih.gov/pubmed/?term=.+Prog nostic+prediction+models+for+diabetic+retinopathy +progression%3A+a+systematic+review.+Review+ar ticle+Haider+S%2C+et+al.+Eye+(Lond).+2019. Accessed February 23, 2019. 9. Lawan A, Mohammed TB. Pattern of diabetic retinopathy in Kano, Nigeria.Ann Afr Med. 2012;11(2):75-79. doi:10.4103/1596-3519.93528 10. Lin J-C, Shau W-Y, Lai M-S. Sex- and Age-Specific Prevalence and Incidence Rates of Sight-Threatening Diabetic Retinopathy in Taiwan.JAMA Ophthalmol. 2014;132(8):922. doi:10.1001/jamaophthalmol.2014.859 4. P R, H M, P A, et al. Pan-retinal Photocoagulation and Other Forms of Laser Treatment and Drug Therapies for Non-Proliferative Diabetic Retinopathy: Systematic Review and Economic Evaluation. Health technology assessment (Winchester, England).doi:10.3310/hta19510 11. MorbiNet: multimorbidity networks in adult general population. Analysis of type 2 diabetes mellitus comorbidity AlbaAguado1, FerranMoratalla- Navarro, Flora López-Simarro& Victor Moreno.Published online 2020 Feb 12. doi: 10.1038/s41598-020-59336-1 g 5. Automatic non-proliferative diabetic retinopathy screening system based on color fundus image. - PubMed - NCBI. https://www.ncbi.nlm.nih.gov/pubmed/?term=1+Aut omatic+non- proliferative+diabetic+retinopathy+screening+system +based+on+color+fundus+image.+Xiao+Z%2C+et+a l.+Biomed+Eng+Online.+2017. Accessed February 23, 2019. 12. Lawan A, Mohammed TB. Pattern of diabetic retinopathy in Kano, Nigeria.Ann Afr Med. 2012;11(2):75-79. doi:10.4103/1596-3519.93528 13. Lin J-C, Shau W-Y, Lai M-S. Sex- and Age-Specific Prevalence and Incidence Rates of Sight-Threatening Diabetic Retinopathy in Taiwan.JAMA Ophthalmol. 2014;132(8):922. doi:10.1001/jamaophthalmol.2014.859 6. 6. SJ GK and B. Management of proliferative diabetic retinopathy. - PubMed - NCBI. https://www.ncbi.nlm.nih.gov/pubmed/?term=Manag ement+of+proliferative+diabetic+retinopathy.+Revie w+article+G%C3%BCnd%C3%BCz+K%2C+et+al. Accessed February 23, 2019. Pak Postgrad Med J Jan. – Mar. 2019 Vol. 30 No. 1 www.ppmj.org.pk 35
https://openalex.org/W3216622513	https://elar.urfu.ru/bitstream/10995/112042/1/2-s2.0-85121562013.pdf	English	null	Conjugate heat transfer numerical study of the ejector by means of SU2 solver	Journal of physics. Conference series	2,021	cc-by	4,366	Journal of Physics: Conference Series Journal of Physics: Conference Series You may also like Thermodynamic Analysis of Two-Phase Ejector as Expansion Device with Dual Evaporator Temperatures on Split Type Air Conditioning Systems M E Arsana, I G B Wijaya Kusuma, M Sucipta et al. - A thermodynamic investigation and optimization of an ejector refrigeration system using R1233zd(E) as a working fluid A Mwesigye, A Kiamari and S B Dworkin - An analysis of the performance of an ejector refrigeration cycle working with R134a F Memet and A Preda - PAPER • OPEN ACCESS PAPER • OPEN ACCESS You may also like dvbrezgin@urfu.ru Abstract. In this paper, the test supersonic ejector with conjugate heat transfer in solid bodies has been studied numerically. An extensive numerical campaign by means of open-source SU2 solver is performed to analyze the fluid dynamics of the ejector flowfield accounting for the heat conduction in solids. The fluid domain simulation is carried out by employing compressible RANS treatment whilst the heat distribution in solids is predicted by simultaneous solving the steady heat conduction equation. The working fluid is R245fa and all simulations are performed accounting for real gas properties of the refrigerant. Experimental data against numerical results comparison showed close agreement both in terms mass flow rates and static pressure distribution along the walls. Within the CFD trials, the most valuable flow parameters at a wall vicinity are compared: distribution across the boundary layer of the temperature and the turbulent kinetic energy specific dissipation rate, boundary layer displacement and momentum thicknesses. A comprehensive analysis of the simulation results cases with adiabatic walls against cases with heat permeable walls revealed the actual differences of the flow properties in the wall vicinity. However, the ejector performance has not changed noticeably while accounting for the heat conduction in solids. Conjugate heat transfer numerical study of the ejector by means of SU2 solver To cite this article: D V Brezgin et al 2021 J. Phys.: Conf. Ser. 2088 012004 View the article online for updates and enhancements. An analysis of the performance of an ejector refrigeration cycle working with R134a F Memet and A Preda - This content was downloaded from IP address 212.193.94.28 on 13/04/2022 at 14:39 This content was downloaded from IP address 212.193.94.28 on 13/04/2022 at 14:39 2088 (2021) 012004 IOP Publishing doi:10.1088/1742-6596/2088/1/012004 HMTHSF 2021 Journal of Physics: Conference Series HMTHSF 2021 Journal of Physics: Conference Series 2088 (2021) 012004 doi:10.1088/1742-6596/2088/1/012004 Conjugate heat transfer numerical study of the ejector by means of SU2 solver D V Brezgin 1, K E Aronson 1, F Mazzelli 2, A Milazzo 2 1 Ural Federal University - UrFU, Mira str., 19, Ekaterinburg, 620002, Russia 2 University of Florence, S. Marco str., 4, Florence, 50121, Italy D V Brezgin 1, K E Aronson 1, F Mazzelli 2, A Milazzo 2 1 Ural Federal University - UrFU, Mira str., 19, Ekaterinburg, 620002, Russia 2 University of Florence, S. Marco str., 4, Florence, 50121, Italy 1 Ural Federal University - UrFU, Mira str., 19, Ekaterinburg, 620002, Russia 2 University of Florence, S. Marco str., 4, Florence, 50121, Italy Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. Published under licence by IOP Publishing Ltd 1 1. Introduction Ejectors are widely used in refrigeration since their ability to utilize low temperature heat to produce cooling. As any jet pump, ejector’s design consists of two main parts: motive or primary nozzle and diffuser, which combines the mixing chamber and diffuser section. In motive nozzle, the potential energy of the primary flow is converted into kinetic energy of the jet (momentum). Further downstream in the ejector’s mixing chamber, the supersonic primary flow involves a static secondary flow through a turbulent interaction in the free shear layer. The bulk momentum of the mixed flows determines the backpressure upper limit value, which the ejector is able to overcome without loss in performance. In general, the performance of an ejector is determined as the Entrainment Ratio ‘Er’ (the secondary to the primary mass flow rates ratio) and, in “on-design” mode, it doesn’t depend on the discharge pressure value while it’s below the critical one. In recent years, an interest to the ejectors simulations by employing various commercial and open- source CFD solvers is greatly increased. However, most of the studies are carried out by assuming adiabatic walls, thereby limiting both the numerical results accuracy and the power of applicability of any CFD tool. The latter outcome stems from the fact that no other kind of research method lets predict forced heat transfer with such a little effort as a CFD modeling. Study in Ref [1] is an exception from 1 HMTHSF 2021 Journal of Physics: Conference Series 2088 (2021) 012004 IOP Publishing doi:10.1088/1742-6596/2088/1/012004 HMTHSF 2021 2088 (2021) 012004 doi:10.1088/1742-6596/2088/1/012004 Journal of Physics: Conference Series the general trend, where authors investigate the heat transfer process within the CO2 two-phase ejector. The numerical simulations presented in Ref [1] were carried out with the non-adiabatic inner walls and the insulated outer walls of the ejector. The numerical results comparison had led to significant decrease in Entrainment ratio (about 15%) for the non-adiabatic wall cases. In despite of such noticeable contradiction of numerical results for distinct wall model cases, no subsequent analysis was performed. Moreover, assuming insulated outer walls does not make sense in terms of conjugate heat transfer modeling because of the fact that such kind of steady state simulation will suffer either from convergence issues or will lead to the same adiabatic wall results by the end. the general trend, where authors investigate the heat transfer process within the CO2 two-phase ejector. 1. Introduction The numerical simulations presented in Ref [1] were carried out with the non-adiabatic inner walls and the insulated outer walls of the ejector. The numerical results comparison had led to significant decrease in Entrainment ratio (about 15%) for the non-adiabatic wall cases. In despite of such noticeable contradiction of numerical results for distinct wall model cases, no subsequent analysis was performed. Moreover, assuming insulated outer walls does not make sense in terms of conjugate heat transfer modeling because of the fact that such kind of steady state simulation will suffer either from convergence issues or will lead to the same adiabatic wall results by the end. Fluid mechanics theory for compressible viscous flows unambiguously shows that components of the viscous stress tensor act like prevail heat sources within conservation energy equation in the boundary layer where the largest velocity gradients take place. As a result, in some particular high Mach number flows, the enthalpy difference within boundary layer between adiabatic and real (non-adiabatic) walls may cause significant deviation. At that, in a coupled iterative solution it leads to all conservative quantities alteration and changes in numerical results accuracy. Therefore, accurate prediction of the high-speed flows requires imposing heat permeable walls where conjugate heat transfer through the strong boundaries is preferable solution because of CFD solution set-up flexibility. The present research builds on work in Ref [2] and extends it in terms of conjugate heat transfer modeling by means of the open-source SU2 CFD solver. The major features and capabilities of the open- source SU2 tool are described in details in Ref [3] whilst the description of the test rig ejector chiller and experimental data acquisition is held in Ref [2]. Although most part of the study in Ref [2] is concerned to numerical simulations as well, the present research had not set a task to make a comparison of the distinct numerical solvers, approaches and the results. The present research aim is to compare the ejector‘s performance by means of two distinct numerical modeling approaches: adiabatic and heat permeable walls as well as evaluate fluid flow features in the vicinity of solid walls. 𝐸௧= 𝜌൫𝑒+ 0.5(𝑣௥ ଶ+ 𝑣௭ ଶ)൯. (2) 2. Numerical model The numerical fluid model in this study is based on compressible RANS solver whilst the single heat equation is utilized for solving temperature distribution in solid bodies. At that, the standard approach is used in which the heat fluxes from the fluid domain are exposed to the solid boundaries through prescribed interface pairs. The governing equation for conservation of mass, radial momentum, axial momentum and total energy of the fluid domain in 2D axisymmetric form can be written as, డఘ డ௧+ డఘ௩ೝ డ௥+ డఘ௩೥ డ௭= − ఘ௩ೝ ௥ 𝜕𝜌𝑣௥ 𝜕𝑡+ 𝜕𝜌𝑣௥ ଶ+ 𝑝 𝜕𝑟 −𝜕 𝜕𝑟൤𝜇௘௙௙൬2 𝜕𝑣௥ 𝜕𝑟−2 3 ൬𝜕𝑣௭ 𝜕𝑧+ 𝜕𝑣௥ 𝜕𝑟൰+ 2 3 𝜌𝑘൰൨+ 𝜕𝜌𝑣௥𝑣௭ 𝜕𝑧 −𝜕 𝜕𝑧൤𝜇௘௙௙൬𝜕𝑣௭ 𝜕𝑟+ 𝜕𝑣௥ 𝜕𝑧൰൨= − ఘ௩ೝమ ௥+ ସఓ೐೑೑ ଷ ቀ ଵ ௥ డ௩ೝ డ௥− ௩ೝ ௥మቁ 𝜕𝜌𝑣௭ 𝜕𝑡 + 𝜕𝜌𝑣௥𝑣௭ 𝜕𝑟 −𝜕 𝜕𝑟൤𝜇௘௙௙൬𝜕𝑣௭ 𝜕𝑟+ 𝜕𝑣௥ 𝜕𝑧൰൨+ 𝜕𝜌𝑣௭ ଶ+ 𝑝 𝜕𝑧 −𝜕 𝜕𝑧൤𝜇௘௙௙൬2 𝜕𝑣௭ 𝜕𝑧−2 3 ൬𝜕𝑣௭ 𝜕𝑧+ 𝜕𝑣௥ 𝜕𝑟൰+ 2 3 𝜌𝑘൰൨= − ఘ௩ೝ௩೥ ௥ + ఓ೐೑೑ ௥ ቀ ଵ ଷ డ௩ೝ డ௭− డ௩೥ డ௥ቁ (1) డா೟ డ௧+ డ డ௥൫𝑣௥(𝐸௧+ 𝑝)൯+ డ డ௭൫𝑣௭(𝐸௧+ 𝑝)൯ − డ డ௥ቄ𝑣௥ቂ𝜇௘௙௙ቀ2 డ௩ೝ డ௥− ଶ ଷቀ డ௩೥ డ௭+ డ௩ೝ డ௥ቁ+ ଶ ଷ𝜌𝑘ቁቃ+ 𝑣௭ቂ𝜇௘௙௙ቀ డ௩೥ డ௥+ డ௩ೝ డ௭ቁቃ−𝑘௘௙௙ డ் డ௥ቅ − డ డ௭ቄ𝑣௭ቂ𝜇௘௙௙ቀ2 డ௩೥ డ௭− ଶ ଷቀ డ௩೥ డ௭+ డ௩ೝ డ௥ቁ+ ଶ ଷ𝜌𝑘ቁቃ+ 𝑣௥ቂ𝜇௘௙௙ቀ డ௩೥ డ௥+ డ௩ೝ డ௭ቁቃ−𝑘௘௙௙ డ் డ௭ቅ = ଵ ௥ቂ−𝑣௥(𝐸௧+ 𝑝) + 𝜇௘௙௙ቀ𝑣௭ డ௩೥ డ௥+ ௩೥ ଷ డ௩ೝ డ௭− ସ௩ೝ ଷ డ௩೥ డ௭+ ଶ௩ೝ ଷ𝜌𝑘ቁ−𝑘௘௙௙ డ் డ௥ቃ (1) , where 𝜇௘௙௙ and 𝑘௘௙௙ stands for the effective viscosity and effective thermal conductivity, k is the turbulent kinetic energy, 𝜌 is the density, T is the temperature, 𝑝 is the pressure and the total energy per unit volume 𝐸௧ is calculated as , where 𝜇௘௙௙ and 𝑘௘௙௙ stands for the effective viscosity and effective thermal conductivity, k is the turbulent kinetic energy, 𝜌 is the density, T is the temperature, 𝑝 is the pressure and the total energy per unit volume 𝐸௧ is calculated as 𝐸௧= 𝜌൫𝑒+ 0.5(𝑣௥ ଶ+ 𝑣௭ ଶ)൯. (2) (2) 2 2088 (2021) 012004 IOP Publishing doi:10.1088/1742-6596/2088/1/012004 HMTHSF 2021 HMTHSF 2021 Journal of Physics: Conference Series 2088 (2021) 012004 doi:10.1088/1742-6596/2088/1/012004 Journal of Physics: Conference Series As can be seen, all conservative equations in (1) resembles the planar two-dimensional form while the right-hand side part distinguishes the extra terms which are concerned to axisymmetric form only. 2. Numerical model The eddy viscosity is obtained by employing the standard Menter’s k-ω SST turbulence model expressed in axisymmetric form as well. Throughout all the simulations, modified Peng-Robinson equation of state is used. In order to get more accurate and consistent with the R245fa refrigerant thermodynamic properties, authors altered the built-in implementation of the Peng-Robinson equation of state by deliberately getting rid of using the specific heat ratio within the SU2 solver code routine. At that, the implementation of the cubic pressure-explicit equation of state is extracted from open-source library CoolProp [4], wherein the exact formulation is presented in Ref [5]. The corresponding fixes in the SU2 solver code made it possible to use a spatially variable value of the specific heat capacities, which had a positive effect not only on the accuracy of determining the thermodynamic properties of the fluid but on the solution convergence as well. Laminar viscosity is defined by imposing Sutherland’s model (𝜇௥௘௙= 1.703𝑒−5, 𝑇௥௘௙= 490.7, 𝑆= 405), whilst constant Prandtl treatment (Pr = 0.71) for the thermal conductivity is employed in numerical solution procedures. Both of the transport models are quite well fitted within the possible temperature range throughout all the simulations. l b d di i f h fl id d i b i i l d Inlet boundary conditions of the fluid domain are set by assigning total pressures and temperatures, static pressure condition is set for the diffuser discharge outlet. Solid body’s boundaries, which are not in direct contact with the fluid domain, are set by imposing the proper heat fluxes towards the environment. Convective fluxes in (1) are solved by means of classic Roe upwind scheme in conjunction with limiting the proper minimum convective eigenvalue (entropy correction is 0.02). In order to achieve second order upwind accuracy, the MUSCL reconstruction is employed with Venkatakrishnan-Wang slope limiter to suppress spurious oscillations at the shock regions. Spatial gradient are solved by means of the Green-Gauss node based method, whilst the FGMRES linear solver with ILU preconditioner is utilized to solve overall Euler implicit time discretization. All the domains (fluid and solids) are discretized by means of structured grid and distinguished by imposing the proper interfaces between each other. 2. Numerical model At that, the grid properly resolves the boundary layer by adjusting the first cell layer height to make sure that the wall Y+ value < 1.0 along the solid walls and at least 30 cells are placed until boundary layer edge is reached. In order to get more insight into boundary layer development features, the comprehensive post- process analysis is carried out throughout the present research. In fact, boundary layer displacement and momentum thicknesses along the nozzle and diffuser walls are evaluated. In general, displacement thickness defines the mass flow rate deficit that the real flow undergoes because of viscosity and it measures the hypothetical wall shift along the surface normal that is required to compensate such mass flow rate deficit. Momentum thickness has the similar definition except the fact that is related to momentum flow rate. The exact formulations of these two tools are the following: 𝛿= ∫ቀ1 − ఘ௩ ఘ೓௩೓ቁ𝑑𝑦 ௛ ଴ , 𝜃= ∫ ఘ௩ ఘ೓௩೓ቀ1 − ௩ ௩೓ቁ𝑑𝑦 ௛ ଴ (3) (3) , where δ is the displacement thickness, θ is the momentum thickness, h denotes the distance from the surface to the boundary layer edge, 𝜌௛ and 𝑣௛ are the flow density and free stream velocity at the boundary layer edge respectively, while 𝜌 and 𝑣 are the flow density and velocity perpendicular to surface normal at the wall distance dy. In present study, definite integrals (3) are solved numerically and the boundary layer edge is determined by employing a threshold value of the gradient of the turbulent kinetic energy specific dissipation rate (omega). It should be noted, that the exact threshold magnitude of ‘omega gradient’ is case dependent and requires additional calculations in advance. 3. Results and discussion At first, ejector flowfield is simulated with adiabatic walls. After the proper convergence is reached and all the validation steps (mass flow rates comparison and static pressure distribution matching) against the experimental data are successfully met, the next simulation is launched by including the solid 3 HMTHSF 2021 Journal of Physics: Conference Series HMTHSF 2021 Journal of Physics: Conference Series 2088 (2021) 012004 IOP Publishing doi:10.1088/1742-6596/2088/1/012004 HMTHSF 2021 2088 (2021) 012004 doi:10.1088/1742-6596/2088/1/012004 Journal of Physics: Conference Series domains and imposing the heat transfer through contacting surfaces. Such kind of approach lets run conjugate heat transfer modeling task from the already converged case of the fluid simulation, which significantly reduces the overall solution time. domains and imposing the heat transfer through contacting surfaces. Such kind of approach lets run conjugate heat transfer modeling task from the already converged case of the fluid simulation, which significantly reduces the overall solution time. g y A detailed numerical post process campaign has been carried throughout the present research in terms of results comparison for adiabatic and heat permeable (conjugate heat transfer) ejector cases. The description and explanation of the major exposed tools and features are presented hereafter. However, it must be admitted that the performance of the investigated ejector practically does not change when the conjugate heat transfer through solid walls is taking into account. In fact, there is a decrease in the secondary mass flow rate by 0.1%, but such a tiny drop appears to contradict with the results given in Ref [1]. Figure 1. Temperature distribution comparison across the suction and mixing chambers Figure 1. Temperature distribution comparison across the suction and mixing chambers Figure 1. Temperature distribution comparison across the suction and g The most remarkable qualitative results are presented in Figure 1, 2 in which the temperature field distribution of the adiabatic walls case (uppermost part from the ejector centerline) and conjugate heat transfer case is compared. The integer values on the Figure 1 indicate the fluid domain (1), the solid nozzle domain (2) and the solid diffuser domain (3). All three domains are simultaneously solved by multizone SU2 iterative solution. One may notice that the inlet part of the solid diffuser (#3 in Figure 1) is heated a little bit relatively to the secondary fluid flow (#2 in Figure 1). 3. Results and discussion Averaged temperature and TKE specific dissipation rate across the boundary layer Figure 4 represents the distribution of the boundary layer displacement and momentum thicknesses along the nozzle wall for two distinct cases: adiabatic walls and conjugate heat transfer model (CHT). The boundary layer analysis along the nozzle wall revealed that mass flow deficit, which is concerned to displacement thickness, decreases onto 1% in CHT case, whilst the momentum flux deficit increases up to 2%. Such contradicting results have physical explanation. A decrease in the mass flux deficit in the CHT case is largely associated with the density augmentation near the solid walls, while a velocity drop across the boundary layer causes an increase in the momentum deficit. Anyway, the overall differences between both of cases (adiabatic and CHT) are too negligible to have an impact on downstream flows mixing or ejector performance. Figure 4. Boundary layer displacement and momentum thicknesses along the nozzle wall Figure 5. Boundary layer displacement and momentum thicknesses along the diffuser wall in the shock vicinity Boundary layer along the diffuser wall is evaluated throughout present numerical research as well. It is noticeable that averaged temperature across the boundary layer height in the diffuser suction chamber is greater on 5 degrees for the CHT case, while in the mixing chamber the temperatures as well as displacement and momentum thicknesses are almost identical downstream of both the CHT and adiabatic cases. However, there is a remarkable boundary layer feature along the diffuser wall in the secondary shock vicinity. The shock is caused by the adverse pressure gradient and induces the boundary layer separation with the subsequent flow recirculation zone behind the shock. Figure 5 represents the boundary layer displacement and momentum thicknesses along the diffuser wall in the shock vicinity for two distinct cases: CHT and adiabatic walls. As can be seen from the Fig.5, the boundary layer separation onset is shifted upstream and it literally means that momentum loss caused by the viscous 0.00E+00 1.00E-05 2.00E-05 3.00E-05 4.00E-05 5.00E-05 6.00E-05 7.00E-05 8.00E-05 9.00E-05 0.03 0.035 0.04 0.045 0.05 0.055 0.06 0.065 0.07 0.075 0.08 0.085 0.09 Displacement thickness (adiabatic) Dispacement thickness (CHT) Momentum thickness (adiabatic) Momentum thickness (CHT) -2.00E-04 -1.50E-04 -1.00E-04 -5.00E-05 0.00E+00 5.00E-05 1.00E-04 1.50E-04 2.00E-04 2.50E-04 0.44 0.442 0.444 0.446 0.448 0.45 0.452 0.454 0.456 0.458 0.46 Displacement thickness (CHT) Displacement thickness (adiabatic) Momentum thickness (CHT) Momentum thickness (Adiabatic) Figure 5. 3. Results and discussion This phenomenon can be explained by the heat conduction and this process is evident while analyzing Figure 2, where the temperature of the metal in the shock vicinity and the flow temperature just behind the shock are identical. However, the flow temperature ahead of the shock deviates from the solid body temperature at the same cross section, where the solid diffuser is much hotter because of heat flux directed backward. Figure 2. Temperature distribution comparison across the entire ejector: uppermost - adiabatic walls, bottom – conjugate heat transfer Figure 2. Temperature distribution comparison across the entire ejector: uppermost - adiabatic walls, bottom – conjugate heat transfer Figure 3 represents the comparison of temperature and turbulent kinetic energy specific dissipation rate (Omega) distribution along the nozzle wall and averaged by integration across the boundary layer height. As can be seen from the Fig.3, the averaged boundary layer temperature is slightly displaced downward in CHT modeling case. In general, these temperature differences have not exceed 2 degrees downstream of the nozzle throat, however, the temperature differences on the solid surfaces itself may exceed 18 degrees. 4 HMTHSF 2021 2088 (2021) 012004 IOP Publishing doi:10.1088/1742-6596/2088/1/012004 2088 (2021) 012004 g doi:10.1088/1742-6596/2088/1/012004 Journal of Physics: Conference Series Figure 3. Averaged temperature and TKE specific dissipation rate across the boundary layer Figure 4 represents the distribution of the boundary layer displacement and momentum thicknesses along the nozzle wall for two distinct cases: adiabatic walls and conjugate heat transfer model (CHT). The boundary layer analysis along the nozzle wall revealed that mass flow deficit, which is concerned to displacement thickness, decreases onto 1% in CHT case, whilst the momentum flux deficit increases up to 2%. Such contradicting results have physical explanation. A decrease in the mass flux deficit in the CHT case is largely associated with the density augmentation near the solid walls, while a velocity drop across the boundary layer causes an increase in the momentum deficit. Anyway, the overall differences between both of cases (adiabatic and CHT) are too negligible to have an impact on downstream flows mixing or ejector performance. Figure 3. Averaged temperature and TKE specific dissipation rate across the boundary layer Figure 3. 3. Results and discussion Boundary layer displacement and momentum thicknesses along the diffuser wall in the shock vicinity -2.00E-04 -1.50E-04 -1.00E-04 -5.00E-05 0.00E+00 5.00E-05 1.00E-04 1.50E-04 2.00E-04 2.50E-04 0.44 0.442 0.444 0.446 0.448 0.45 0.452 0.454 0.456 0.458 0.46 Displacement thickness (CHT) Displacement thickness (adiabatic) Momentum thickness (CHT) Momentum thickness (Adiabatic) Figure 4. Boundary layer displacement and momentum thicknesses along the nozzle wall 0.00E+00 1.00E-05 2.00E-05 3.00E-05 4.00E-05 5.00E-05 6.00E-05 7.00E-05 8.00E-05 9.00E-05 0.03 0.035 0.04 0.045 0.05 0.055 0.06 0.065 0.07 0.075 0.08 0.085 0.09 Displacement thickness (adiabatic) Dispacement thickness (CHT) Momentum thickness (adiabatic) Momentum thickness (CHT) Figure 4. Boundary layer displacement and momentum thicknesses along the nozzle wall 0.03 0.035 0.04 0.045 0.05 0.055 0.06 0.065 0.07 0.075 0.08 0.085 0.09 Figure 4. Boundary layer displacement and momentum thicknesses along the nozzle wall 0.03 0.035 0.04 0.045 0.05 0.055 0.06 0.065 0.07 0.075 0.08 0.085 0.09 Figure 5. Boundary layer displacement and momentum thicknesses along the diffuser wall in the shock vicinity Boundary layer along the diffuser wall is evaluated throughout present numerical research as well. It is noticeable that averaged temperature across the boundary layer height in the diffuser suction chamber is greater on 5 degrees for the CHT case, while in the mixing chamber the temperatures as well as displacement and momentum thicknesses are almost identical downstream of both the CHT and adiabatic cases. However, there is a remarkable boundary layer feature along the diffuser wall in the secondary shock vicinity. The shock is caused by the adverse pressure gradient and induces the boundary layer separation with the subsequent flow recirculation zone behind the shock. Figure 5 represents the boundary layer displacement and momentum thicknesses along the diffuser wall in the shock vicinity for two distinct cases: CHT and adiabatic walls. As can be seen from the Fig.5, the boundary layer separation onset is shifted upstream and it literally means that momentum loss caused by the viscous Boundary layer along the diffuser wall is evaluated throughout present numerical research as well. It is noticeable that averaged temperature across the boundary layer height in the diffuser suction chamber is greater on 5 degrees for the CHT case, while in the mixing chamber the temperatures as well as displacement and momentum thicknesses are almost identical downstream of both the CHT and adiabatic cases. However, there is a remarkable boundary layer feature along the diffuser wall in the secondary shock vicinity. [1] Haida M et al. 2018 Heat transfer process within the R744 two-phase ejector: numerical and experimental study. Proc. 17th Int. Refrigeration and Air Conditioning Conf. (Purdue: Perdue University) [2] Mazzelli F, Milazzo A 2015 Int J Refrig 49 79-92 [3] Economon T D, Palacios F, Copeland S R, Lukaczyk T W and Alonso J J 2016 AIAA Journal 54(3) 828-846 [4] Bell I H, Wronski J, Quoilin S and Lemort V 2014 Ind Eng Chem Res 53(6) 2498-2508 [5] Bell I H and Jäger A 2016 J. Res. Nat. Inst. Stand. Technol 121 238 3. Results and discussion The shock is caused by the adverse pressure gradient and induces the boundary layer separation with the subsequent flow recirculation zone behind the shock. Figure 5 represents the boundary layer displacement and momentum thicknesses along the diffuser wall in the shock vicinity for two distinct cases: CHT and adiabatic walls. As can be seen from the Fig.5, the boundary layer separation onset is shifted upstream and it literally means that momentum loss caused by the viscous 5 2088 (2021) 012004 IOP Publishing doi:10.1088/1742-6596/2088/1/012004 HMTHSF 2021 HMTHSF 2021 Journal of Physics: Conference Series Journal of Physics: Conference Series Journal of Physics: Conference Series forces is greater for the CHT modeling case. At the same time, the actual differences in flow behavior between distinguished CHT and adiabatic walls models is negligible. 4. Conclusions: The study presents the numerical results comparison obtained on supersonic ejector for refrigeration with two distinct models: adiabatic walls and conjugate heat transfer. All the simulation cases are carried out by means of open-source SU2 solver and it’s turned out that taking into account the heat transfer through solid wall have negligible impact on overall ejector performance. Moreover, the general tools developed and implemented in SU2 code throughout the present study to estimate local boundary layer flow features (boundary layer displacement and momentum thicknesses) revealed that there is no major differences between both of distinct cases. Definitely, this outcome cannot be carried over to all ejector designs and operation modes. Thus, it is likely that at higher speed flows (with a higher degree of flow expansion in the primary nozzles), viscous stresses in the boundary layer will prevail, and in such a case, it will cause the ejector performance alteration. However, it’s well known that flows interaction within the mixing layer as well as the shock train located downstream and caused by the adverse pressure gradient are the main sources of ejector’s performance losses. Thereby, all the CFD treatments and efforts within ejector in the scope of the boundary layer model calibrating will fail until the most important domain regions (away from the solid surfaces) is not fully consistent with the actual fluid dynamics. References: 6 6
https://openalex.org/W4280603894	https://www.frontiersin.org/articles/10.3389/fpubh.2022.843845/pdf	English	null	The Mediating Role of Worker-Occupation Fit in the Relationship Between Occupational Stress and Depression Symptoms in 1988 Medical Workers: A Cross-Sectional Study	Frontiers in public health	2,022	cc-by	7,885	The Mediating Role of Worker-Occupation Fit in the Relationship Between Occupational Stress and Depression Symptoms in 1988 Medical Workers: A Cross-Sectional Study Ruican Sun 1, Keyao Lv 2, Zirui He 1, Liang Liao 1, Hongping Wang 1 and Yajia Lan 2* 1 Department of Preventive Medicine, School of Public Health, Chengdu Medical College, Chengdu, China, 2 Department of Environmental Health and Occupational Medicine, West China School of Public Health and West China Fourth Hospital, Sichuan University, Chengdu, China Objective: Occupational stress is generally acknowledged as a global phenomenon with signiﬁcant health and economic consequences. The medical worker is a vulnerable group at a high-level risk for depression symptoms. This study aimed to examine the mediating effect of worker-occupation ﬁt (WOF) in relation to occupational stress and depression symptoms among 1988 medical workers in China. Keywords: depression symptoms, occupational stress, worker occupation ﬁt, medical workers, mediate effect Edited by: Kai Zhang, Anhui Medical University, China Reviewed by: Lijia Chang, Chiba University, Japan Shu Cui, Chaohu Hospital of Anhui Medical University, China Correspondence: Yajia Lan yajialan501@126.com Edited by: Kai Zhang, Anhui Medical University, China Methods: A multi-center cross-sectional study was conducted during June and October 2020 in Henan Province, China. The participants were medical workers from four targeted hospitals (included one general and three specialized hospitals). The Depression, Anxiety, and Stress Scale (DASS-21 Scale), Worker-Occupation Fit Inventory (WOFI), as well as questions about demographic and occupational information were administered in questionnaires distributed to 1988 medical workers. Hierarchical linear regression analysis was used to examine the mediating role of worker occupation ﬁt. Reviewed by: Lijia Chang, Chiba University, Japan Shu Cui, Chaohu Hospital of Anhui Medical University, China Correspondence: Yajia Lan yajialan501@126.com Results: In this study, there are 43.5% (n = 864) of medical workers experienced depression symptoms. The mean score of WOF was 31.6 ± 7.1, characteristic ﬁt, need supply ﬁt and demand ability ﬁt were 11.3 ± 2.5, 10.1 ± 2.7, 12.9 ± 2.2, respectively. The occupational stress was negatively related to worker occupation ﬁt (r = −0.395, P < 0.001), characteristic ﬁt (r = −0.529, P < 0.001), need supply ﬁt (r = −0.500, P < 0.001), and demand ability ﬁt (r = −0.345, P < 0.001). The occupational stress and depression symptoms have a positive relationship (r = 0.798, P < 0.001). The proportion of worker occupation ﬁt mediation was 6.5% of total effect for depression symptoms. Specialty section: This article was submitted to Occupational Health and Safety, a section of the journal Frontiers in Public Health Received: 27 December 2021 Accepted: 11 April 2022 Published: 17 May 2022 ORIGINAL RESEARCH published: 17 May 2022 doi: 10.3389/fpubh.2022.843845 ORIGINAL RESEARCH published: 17 May 2022 doi: 10.3389/fpubh.2022.843845 INTRODUCTION are not only direct eﬀects but also mediating eﬀects between occupational stress and depression symptoms. Occupational stress involves all workplaces, which is generally acknowledged as a global phenomenon with signiﬁcant health and economic consequences in both developed and developing countries (1). The current work style and intensity of society will continue to increase the occurrence and development of occupational stress, and may even induce a variety of stress- related diseases. Occupational stress is associated with increased risk of mental disorders, [i.e., depression (2), burnout (3)], somatic diseases [i.e., insomnia (4), hypertension (5)], and organizational outcomes [i.e., job satisfaction (6), wellness (7)]. In recent decades, occupational stress represents a large, complex and costly phenomenon in the workplace worldwide (8). Worker-occupation ﬁt (WOF) was deﬁned as the ﬁt level of the worker’s characteristic, need, and ability with the occupational environment’s culture, supply, and demand (25). In our early research, we constructed a theoretical model of the WOF eﬀects on occupational stress and related disorders (26). According to the deﬁnition of WOF, it was divided into three types, namely, characteristic ﬁt (CF), need supply ﬁt (NSF) and demand ability ﬁt (DAF). WOF was derived from the theory of personal environment ﬁt. Since the 1980s, the study of person-environment ﬁt has begun to emerge as a research hot-spot in organizational behaviors and organizational management, with most studies focusing on the relationship between organizational outcomes and personal environment ﬁt (27–29). Many empirical studies were conducted in this area to ﬁnd a better ﬁt level to explore the positive eﬀects on job attitudes (30), organizational citizenship behaviors (31), and job performance (32). The better ﬁt level between individual and occupational environment, which can improve the higher levels of organizational citizenship behavior, led to fewer work errors and more harmonious interpersonal relationships. Therefore, the ﬁt of the individual (i.e., needs, values, abilities, and personal characteristics) with the occupational environment (i.e., values, rewards, job requirements, culture, and physical environment) is an important factor inﬂuencing the worker’s attitudes and behaviors in their workplaces (33). At present, there has few studies focuses on the association of WOF and mental disorders for medical workers. Medical workers is widely regarded as one of the most challenging professions. Medical workers often receive strict and long-term training to achieve their professional goals (9). Therefore, medical workers is a vulnerable group at a high- level risk for mental illnesses (10). INTRODUCTION Studies reported that a total of forty-six nurses suicide induced by anxiety/depression in China from 2007 to 2016 (11), as were a total of ﬁfty-one physicians suicide induced by anxiety/depression from 2008 to 2016 (12). The National Institute for Occupational Safety and Health suggested that medical workers have the common stressors in hospital settings consist of inadequate staﬃng levels, long work hours, shift work, role ambiguity, and exposure to infectious diseases (13). Another national research reported that higher staﬀturnover, more absences due to illness, decreased performance, and more complaints and grievances were signs of stress (14). The above-mentioned reports, medical workers have a higher risk of mental disorders, and many countries have begun to pay attention to the prevention of mental disorders in the occupational environment. Furthermore, WOF is most consistent with the theoretical implications of occupational stress, which is measured as, a negative physical, psychological, and behavioral response that occurs when a worker’s resources, skills, and needs do not match the job demands (34, 35). Our preliminary ﬁndings showed a signiﬁcant negative association between WOF and occupational stress, that is, higher levels of WOF lead to lower levels of occupational stress (25). Based on our preliminary study, it would be valuable to further explore the eﬀect of WOF on depression symptoms and to further explore whether there is a mediating role between them. Depression is a major mental health issue in the global world, which has long been a topic attracting much interest across a wide range of research ﬁelds. The relationship between occupational stress and depression symptoms is also an ongoing research issue. There are plenty of studies in various populations suggested that psychological capital (15, 16), social support (17), coping style (18), work demand (19) and work-family conﬂict (20) are considered as the mediating role on occupational stress and mental disorders. Previous reports highlighted that occupational stress aﬀects depression symptoms, and these vulnerable individuals need more support. Li et al. examined psychological capital signiﬁcantly mediated the associations of occupational stress and depression symptoms among Chinese physicians (21). Prerna Varma et al. found that poor sleep quality, lower levels of resilience, younger age and loneliness signiﬁcantly mediated the links between stress and depression among young adults (22). Wang et al. Citation: Sun R, Lv K, He Z, Liao L, Wang H and Lan Y (2022) The Mediating Role of Worker-Occupation Fit in the Relationship Between Occupational Stress and Depression Symptoms in 1988 Medical Workers: A Cross-Sectional Study. Front. Public Health 10:843845. doi: 10.3389/fpubh.2022.843845 Conclusion: Occupational stress has been identiﬁed as a risk factor for depression symptoms. Practical strategies for improving medical workers’ WOF level would help them better cope with various work-related stressors to reduce depression symptoms. Hospital administrators could reduce medical workers’ depression symptoms by taking comprehensive measures to improve the WOF. May 2022 \| Volume 10 \| Article 843845 Frontiers in Public Health \| www.frontiersin.org 1 WOF, Occupational Stress and Depression Sun et al. Frontiers in Public Health \| www.frontiersin.org INTRODUCTION (23) explored the mediating role of resilience between occupational stress and depression in female nurses, and the results suggested that mental elasticity has an indirect eﬀect on occupational stress and depression. The mediating eﬀect of mental elasticity accounted for 16.08% of the total eﬀect. Batalla et al. (24) examined the correlation between spirituality and depression and found the moderating eﬀect of occupational stress in registered nurses. Taken together, there To sum up, whether there is a correlation between WOF and depression symptoms, and whether WOF plays a mediating role between occupational stress and depression symptoms that it worth to conﬁrm. In short, this study aimed to examine the mediating eﬀect of WOF on occupational stress and depression symptoms in 1988 medical workers to provide scientiﬁc evidence for developing eﬀective interventions to improve depression symptoms. Participants and Procedures A cross-sectional study was conducted in Henan Province by multi-center survey from June to October, 2020. The participants were medical workers from four targeted hospitals (included a comprehensive grade 3A hospital, a psychiatric hospital, a children’s hospital and an infectious hospital). Based on the May 2022 \| Volume 10 \| Article 843845 2 WOF, Occupational Stress and Depression Sun et al. study design, the sample size calculated by Fisher’s method (36). The sample size was estimated to be 502 participants. Considering there are many research variables in this study, a design coeﬃcient of 1.3 was added into the ﬁnal sample size. Therefore, the ﬁnal sample size calculated was 653 participants. or female), age, marital status (single, cohabiting married, divorced and living alone, widowed and living alone), education (diploma or less, bachelor’s degree or higher), monthly income (<3,000 CNY, 3,000–3,999 CNY, 4,000–4,999 CNY, 5,000–5,999 CNY, ≥6,000 CNY). The occupational characteristics included work experience, department, professional title, night shift per week, and work hours per week. The following survey instruments were used to collect data: The inclusion criteria of this study were as follows: (1) individuals who had no family history of and were not taking medications for a mental disorder; (2) individuals who were regular employees of the targeted hospitals; and (3) individuals who were willing to participate in this survey. The exclusion criteria were as follows: (1) participants who were absent during the survey and (2) participants whose questionnaires were ineligible or were <80% completed. After written informed consent was obtained to conduct this study, a total of 2050 questionnaires were distributed. Nine participants did not agree to participate in the survey, thirty-two participants were absent from the hospital during the survey, and twenty-one participants returned incomplete (<80%) questionnaires. Finally, 1988 questionnaires were considered valid, corresponding to a response rate of 97.0% (1988/2050). The study ﬂowchart is presented in Figure 1. Measurement of Occupational Stress and Depression Symptoms Depression, Anxiety, and Stress Scale (DASS-21), which was developed by Lovibond et al. (37) and has been proven to be a valid measure to assess stress in the Chinese population (38), which was used to measurement of occupational stress and depression symptoms. The DASS-21 is composed of 21 items, which are rated using a four-point Likert scale to assess the level of depression, anxiety, and stress. Each item was scored from 0 (it does not apply to me at all in the last week) to 3 (it applies to me perfectly in the last week). There are three sub-scales, included stress (seven items: 1, 6, 8, 11, 12, 14, and 18), depression (seven items: 3, 5, 10, 13, 16, 17, and 21), anxiety (seven items: 2, 4, 7, 9, 15, 19, and 20), respectively. In this study, we mainly used the stress sub-scale and depression sub-scale. DASS-21 scores were multiplied by two to calculate the ﬁnal subscale scores, yielding a maximum of 42 points. Participants with a total score of 15 were rated as having occupational stress, the score of 10 was rated as having depression symptoms (37). We emphasized in the questionnaire that each response in the stress was related to work factors in hospitals. Measurements Researchers conducted a cross-sectional survey of medical workers at a general hospital and three specialty hospitals to evaluate the medical workers’ WOF level, occupational stress and depression symptoms. The collection of basic information by self-administered basic information questionnaire was divided into demographic and occupational characteristics for each participant. The demographic characteristics included sex (male FIGURE 1 \| Study ﬂowchart. FIGURE 1 \| Study ﬂowchart. FIGURE 1 \| Study ﬂowchart. Frontiers in Public Health \| www.frontiersin.org May 2022 \| Volume 10 \| Article 843845 3 WOF, Occupational Stress and Depression Sun et al. TABLE 1 \| Demographic and occupational characteristics of participants (n = 1988) and comparisons on the positive case of depression symptoms. TABLE 1 \| Demographic and occupational characteristics of participants (n = 1988) and comparisons on the positive case of depression symptoms. Variable n Positive P cases % Occupation 0.097 Physician 525 212 40.4 Nurse 1,463 652 44.6 Sex 0.006 Male 214 112 52.3 Female 1,774 752 42.4 Marital status 0.216 Single 511 207 40.5 Cohabit 19 115 7.9 Married 1,423 631 44.3 Divorce and live alone 31 12 38.7 Widowhood and live alone 4 3 75.0 Education <0.001 Bachelor 469 172 36.7 Master 1,452 653 45.0 Doctor 67 39 58.2 Income 0.005 <3,000 CNY 334 119 35.6 3,000–3,999 CNY 686 290 42.3 4,000–4,999 CNY 450 204 45.3 5,000–5,999 CNY 315 150 47.6 ≥6,000 CNY 203 101 49.8 Work experience <0.001 <1 year 150 42 28.0 1–3 years 297 117 39.4 4–10 years 757 355 46.9 11–19 years 512 242 47.3 ≥20 years 272 108 39.7 Department of 0.003 Internal Medicine 355 171 48.2 Surgery 325 148 45.5 Obstetrics and Gynecology 237 91 38.4 Pediatric Surgery 83 44 53.0 Psychiatry 248 108 43.6 Infectious Disease 111 34 30.6 Emergency 140 72 51.4 ICU 136 62 45.6 Outpatient Clinic 79 29 36.7 Others 274 105 38.3 Title 0.019 Primary 1,125 458 40.7 Intermediate 756 356 47.1 Senior 107 50 46.7 Night shift per week <0.001 No 691 256 37.1 ≥2 times 194 77 39.7 ≥4 times 175 82 46.9 ≥6 times 928 449 48.4 Work hour per week <0.001 <40 hours 534 196 36.7 40–49 hours 940 396 42.1 50–59 hours 300 142 47.3 ≥60 hours 214 130 60.8 May 2022 \| Volume 10 \| Article 843845 FIGURE 2 \| The conceptional model on the mediating role of WOF on the association between occupational stress and depression symptoms. Measurements WOF, worker occupation ﬁt. FIGURE 2 \| The conceptional model on the mediating role of WOF on the association between occupational stress and depression symptoms. WOF, worker occupation ﬁt. Measurement of the Worker-Occupation Fit WOF is an emerging and important factor for occupational health, which is still neglected in occupational stress research. The Worker-Occupation Fit Inventory (WOFI) is based on the questionnaire by Cable et al. (39), with some items and descriptions modiﬁed according to the Chinese culture and thinking to facilitate understanding by Chinese participants. The WOFI consists of three sections totalling nine items: (1) Characteristic Fit, for example, “Do you think your job style ﬁts your job?”; (2) Need-Supply Fit, for example, “Do you think the job provides what you expect?”; and (3) Demand-Ability Fit, for example, “Do you think that your educational background meets the job demands?”. Each item is rated on a 5-point Likert scale (extremely unﬁt/ﬁt, range 1–5). The higher the score is, the better the worker-occupation ﬁt. Items used in this section of the survey are described in Supplementary Table S1. In this study, Cronbach’s alpha coeﬃcient of the total scale was 0.888. Cronbach’s alpha coeﬃcients of CF, NSF, and DAF were 0.790, 0.848, and 0.895, respectively. The result of test-retest reliability is 0.897. May 2022 \| Volume 10 \| Article 843845 Frontiers in Public Health \| www.frontiersin.org The Participants Characteristic The results for the direct and indirect eﬀects of occupational stress on depression symptoms with WOF and WOF types as mediators are presented in Table 4. The standardized estimates of the path coeﬃcients for each variable are shown in Figure 4. SEM revealed signiﬁcant regression or correlation paths, and all beta path coeﬃcients were statistically signiﬁcant (P < 0.001). SEM revealed signiﬁcant regression or correlation paths, and all beta path coeﬃcients were statistically signiﬁcant (P < 0.01). The WOF to be a mediator, occupational stress had a direct eﬀect and an indirect eﬀect on depression symptoms with a path coeﬃcient of 0.677 (P < 0.01) and 0.047 (P < 0.01), respectively. The CF to be a mediator, occupational stress had a direct eﬀect and an indirect eﬀect on depression symptoms with a path coeﬃcient of 0.636 (P < 0.01) and 0.089 (P < 0.01), respectively. The NSF to be a mediator, occupational stress had a direct eﬀect and an indirect eﬀect on depression symptoms with a path coeﬃcient of 0.650 (P < 0.01) and 0.075 (P < 0.01), respectively. In addition, the DAF to be a mediator, occupational stress had a direct eﬀect and an indirect eﬀect on depression symptoms with a path coeﬃcient of 0.690 (P < 0.01) and 0.035 Demographic and occupational characteristics of the positive cases of depression symptoms in categorical variables are shown in Table 1. In this study, the average age of participants was 32.7 (SD = 7.8). There were a total of 1988 medical workers, 43.5% (n = 864) of the medical workers experienced depression symptoms. The mean score of WOF was 31.6 ± 7.1, CF, NSF and DAF were 11.3 ± 2.5, 10.1 ± 2.7, 12.9 ± 2.2, respectively. Data Analysis The demographic and occupational characteristics of the medical workers are described by the frequency and percentage. The categorical variables are expressed as frequencies (%) and were compared by Chi-square tests among all participants. Descriptive statistics for the level of occupational stress, depression symptoms and WOF were showed with mean, standard deviation (SD). The correlation between occupational stress, depression symptoms and WOF was used by Pearson’s correlation analysis. Before regression analyses, all the continuous variables in the models were centralized. The conceptual framework of the study is shown in Figure 2, the eﬀect of occupational stress May 2022 \| Volume 10 \| Article 843845 Frontiers in Public Health \| www.frontiersin.org 4 WOF, Occupational Stress and Depression Sun et al. on depression symptoms would have two path hypotheses. Hypothesis 1, the aim was to verify the direct eﬀect of occupational stress on depression symptoms (the c-path) after adjusting covariates; the hypothesis 2, the aim was to verify the mediating role of WOF between occupational stress and depression symptoms. We performed a hierarchical linear analysis for each of the WOF and the WOF types to test the mediating eﬀects on depression symptoms. At the ﬁrst step, the variables have a signiﬁcant association with depression symptoms by univariate analysis that those of them as the control variable in block 1. At the second step, occupational stress was added as an independent variable and a mediating variable in block 2. At the third step, WOF and WOF types (CF, NSF and DAF) entered into block 3, respectively. Besides, the bootstrap estimate was based on 2,000 bootstrap samples. A bias-corrected and accelerated 95% conﬁdence interval (BCa 95% CI) was calculated for each a×b product, and a BCa 95% CI excluding 0 signiﬁcantly manifested mediation. All statistical analyses were carried out using R Statistics (version 3.5.0), with a two-tailed probability value of <0.05 considered to be statistically signiﬁcant. between occupational stress and WOF (CF, NSF, and DAF) (path a). The WOF was correlated with depression symptoms (path b), whereas occupational stress was correlated with depression symptoms (path c). Mediation Effect Test The result of the hierarchical regression of depression symptoms is shown in Table 3. Based on the Table 3’s block 3-model 1, Figure 3 shows the WOF mediates the eﬀect of occupational stress on depression symptoms. Each step of the independent variables made a signiﬁcant contribution to variance in depression symptoms. In step 1, the occupational categories, sex, age, and work experience contributed to 4.6% of the variance in depression symptoms. The dimensions of occupational stress accounted for 64.4% of the variance in depression symptoms in step 2. The results supported that occupational stress was positively related to depression symptoms (β = 0.805, P < 0.001). In block 3 and model 1, WOF was negatively related to depression symptoms (β = −0.290, P < 0.001, R2 = 0.684). In block 3 and model 2, when the WOF types into the model, the results shown that WOF types were negatively related to depression symptoms, the regression coeﬃcient were −0.114, −0.114, −0.048 and −0.034, respectively (R2 = 0.692, P < 0.001). Frontiers in Public Health \| www.frontiersin.org Correlation of Occupational Stress, Depression Symptoms and WOF The results of the Pearson’s correlation analyses are showed in Table 2. Occupational stress, depression symptoms, and WOF were signiﬁcant correlated. The occupational stress was negatively related to WOF (r = −0.395, P < 0.001), CF (r = −0.529, P < 0.001), NSF (r = −0.500, P < 0.001), and DAF (r = −0.345, P < 0.001). The occupational stress and depression symptoms have a positive relationship (r = 0.798, P < 0.001). Based on these results, the paths were drawn to examine the mediating eﬀect (Figure 2). And there was a correlation TABLE 2 \| The correlations of depression symptoms, occupational stress, WOF and WOF types. Variable 1 2 3 4 5 6 1. WOF 1 2. CF 0.593 1 3. NSF 0.580 0.690 1 4. DAF 0.496 0.520 0.373 1 5. Depression symptoms −0.395 −0.529 −0.500 -0.345 1 6. Occupational stress −0.259 −0.431 −0.428 -0.258 0.798 1 WOF, worker-occupation ﬁt; CF, Characteristic ﬁt; NSF, need supply ﬁt; DAF, demand ability ﬁt. Correlation is signiﬁcant at the 0.001 level (two-tailed). TABLE 2 \| The correlations of depression symptoms, occupational stress, WOF and WOF types. 6 May 2022 \| Volume 10 \| Article 843845 Frontiers in Public Health \| www.frontiersin.org 5 WOF, Occupational Stress and Depression Sun et al. TABLE 3 \| Hierarchical multiple regression analysis of the association of occupational stress and WOF with depression symptoms. Variable Block 1 Block 2 Block 3 Model 1 Model 2 β VIF β VIF β VIF β VIF Occupational categories 0.088 1.22 −0.062 1.26 0.136 2.23 0.021 4.28 Sex (female) −0.036 1.08 −0.008 1.08 −0.006 1.08 −0.007 1.08 Age (year) −0.080 4.58 0.042 4.61 0.039 4.61 0.044 4.63 Work experience (year) 0.157 4.53 −0.009 4.57 −0.019 4.58 −0.022 4.58 Occupational stress 0.805 1.08 0.702 1.35 0.688 1.39 WOF −0.290 2.10 −0.114 7.03 CF −0.114 2.86 NSF −0.048 2.65 DAF −0.034 1.82 Wald χ2 116.94 2,864.27 3,627.30 3,944.34 R2 0.046 0.644 0.684 0.692 1R2 0.041 0.642 0.683 0.690 MSE 7.290 4.451 4.193 4.146 WOF, worker occupation ﬁt; CF, Characteristic ﬁt; NSF, need supply ﬁt; DAF, demand ability ﬁt. and depression symptoms and whether the WOF is a mediator into this relationship. In this study, there are 43.5% (864/1,988) medical workers with a positive rate of depression symptoms, and the average score of WOF was 31.6 ± 7.1. Correlation of Occupational Stress, Depression Symptoms and WOF The WOF to be a mediator, occupational stress had a direct eﬀect and an indirect eﬀect on depression symptoms with a path coeﬃcient of 0.677 and 0.047. The mediating eﬀect of WOF, CF, NSF, and DAF accounted for 6.5, 12.3, 10.3 and 4.8%, respectively. FIGURE 3 \| WOF moderates the effect of occupational stress on depression symptoms based on the Block 3-Model 1. According to the World Health Organization (WHO), nearly 300 million people of diﬀerent ages suﬀer from depression worldwide. In China, medical workers have reported high rates of depression (50%), anxiety (45%), and insomnia (34%) (40). The prevalence of depression increased by more than 18% from 2005 to 2015 (41). Among emergency medical service workers at the New York City Fire Service (FDNY), the prevalence of depression symptoms was 16.7% after 12 years of occupational exposure (42). Therefore, depression and anxiety have become a global consensus for medical workers. Medical workers are one of the highest risk groups for developing depression (43). We try to ﬁnd eﬀective ways to reduce the risk of depression among medical workers. In this sense, when medical workers experience depression symptoms in an occupational environment, improve the WOF level is the regulation process against depression, and can be an eﬀective factor in maintaining mental health. In our study, the participants were selected from one general hospital and three specialized hospitals located in Henan province, with a detection rate of depression symptoms were 43.5%. Compared to that of medical students (44), ﬁreﬁghters (45), economy staﬀ(46), medical workers have a higher detection rate of depression symptoms. FIGURE 3 \| WOF moderates the effect of occupational stress on depression symptoms based on the Block 3-Model 1. (P < 0.01), respectively. We also calculated that the total eﬀect of occupational stress on depression symptoms was 93.4% and the indirect eﬀect accounted for 6.5% of the total eﬀect. As shown in Table 4, the bootstrap estimate presented in our study was based upon 2,000 bootstrap samples. For each independent variable, the bias-corrected 95% CI all excluded 0, which indicated that the mediating role of WOF was statistically signiﬁcant. Frontiers in Public Health \| www.frontiersin.org DISCUSSION Practical strategies for improving medical workers’ WOF would help them better cope with various worker-related stressors to reduce depression symptoms. In our study, the structural model analysis indicated that occupational stress had both direct and indirect eﬀects on depression symptoms with WOF considering as mediators. In other words, occupational stress indirectly aﬀected depression symptoms by decreasing WOF, it was implied that WOF could mediate the association between occupational stress and depression symptoms. These results suggested that the WOF level are crucial for the mental and physical health of medical workers. In addition, results from block 3 (model 1 and model 2) by hierarchical regression, based on the absolute value of β, CF, NSF and DAF accounted for the observed variance in WOF. It is noteworthy that CF is more strongly associated with WOF than with NSF, DAF. The results suggest that hospital administrators assign work tasks based on the personal characteristics (i.e., personality or work style) of medical staﬀ, which can reduce the risk of depression symptoms. In a systematic review, the study indicated a high prevalence of depression among medical staﬀ and the consistency between the estimated prevalence (27.2% students and 28.8% residents) showed that depression coping was an important issue in all levels of medical training (47). the association between occupational stress and depression symptoms. The most important goal of this study was to examine the relationships between depression symptoms, occupational stress, WOF. Our results show that the WOF types (CF, NSF, and DAF) were negatively correlated with depression symptoms, and the occupational stress and depression symptoms was a positive relationship with 1988 medical workers. Practical strategies for improving medical workers’ WOF would help them better cope with various worker-related stressors to reduce depression symptoms. In our study, the structural model analysis indicated that occupational stress had both direct and indirect eﬀects on depression symptoms with WOF considering as mediators. In other words, occupational stress indirectly aﬀected depression symptoms by decreasing WOF, it was implied that WOF could mediate the association between occupational stress and depression symptoms. These results suggested that the WOF level are crucial for the mental and physical health of medical workers. In addition, results from block 3 (model 1 and model 2) by hierarchical regression, based on the absolute value of β, CF, NSF and DAF accounted for the observed variance in WOF. Frontiers in Public Health \| www.frontiersin.org DISCUSSION Occupational stress has been identiﬁed as a risk factor for poor mental health and depression symptoms. To develop eﬀective interventions, the roles of positive predictors and mediators should be explored to clarify the mechanism behind Improving WOF level was considered as a new approach to prevention of depression symptoms in workplace. This study aims to examine the relationship between occupational stress Frontiers in Public Health \| www.frontiersin.org May 2022 \| Volume 10 \| Article 843845 6 WOF, Occupational Stress and Depression Sun et al. TABLE 4 \| Mediating role of WOF or WOF types on the associations between occupational stress and depression symptoms. Mediators Model pathways Effect types β(std.) Bootstrap Bias-corrected 95% Lower bounds Upper bounds WOF Occupational stress→Depression symptoms Total effects 0.725 0.692 0.753 Indirect effects 0.047 0.039 0.056 Direct effects 0.677 0.648 0.707 CF Occupational stress→Depression symptoms Total effects 0.725 0.695 0.754 Indirect effects 0.089 0.074 0.104 Direct effects 0.636 0.603 0.668 NSF Occupational stress→Depression symptoms Total effects 0.725 0.696 0.754 Indirect effects 0.075 0.062 0.089 Direct effects 0.650 0.617 0.682 DAF Occupational stress→Depression symptoms Total effects 0.725 0.695 0.755 Indirect effects 0.035 0.026 0.044 Direct effects 0.690 0.658 0.722 WOF, worker occupation ﬁt; CF, Characteristic ﬁt; NSF, need supply ﬁt; DAF, demand ability ﬁt. The mediating effect of WOF, CF, NSF, and DAF accounted for 6.5, 12.3, 10.3 and 4.8%, respectively. WOF, worker occupation ﬁt; CF, Characteristic ﬁt; NSF, need supply ﬁt; DAF, demand ability ﬁt. The mediating effect of WOF, CF, NSF, and DAF accounted for 6.5, 12.3, 10.3 and 4.8%, respectively. In this sense, medical workers with depression should be given an early warning. The hospital administrators can reduce medical workers’ depression symptoms by taking comprehensive measures to improve the WOF such as establishing training sessions to improve the ability of medical workers to meet the hospital demands, promoting the balance between demands and rewards (48). It can also improve WOF level by creating a better ﬁt between workers’ characters and hospitals’ climate, workers’ ability and hospitals’ demands, workers’ needs and hospitals’ supply. the association between occupational stress and depression symptoms. The most important goal of this study was to examine the relationships between depression symptoms, occupational stress, WOF. Our results show that the WOF types (CF, NSF, and DAF) were negatively correlated with depression symptoms, and the occupational stress and depression symptoms was a positive relationship with 1988 medical workers. CONCLUSION hospital managers tend to use bonuses to incentivize their employees, which is usually linked to work outputs rather than work experience. The low income of health workers has been a serious challenge in many developing countries (53). For medical workers, it is important to note that strategies to enhance individuals’ WOF seems to be valuable to improve depression symptoms and promote the mental health of Chinese medical workers. In conclusion, WOF was found to be negatively associated with depression symptoms and partially mediated the relationship between occupational stress and depression symptoms. These results could help to further explore the relationship between occupational stress, depression symptoms and WOF, as well as a new perspective for the intervention of depression symptoms. Hospital managers can help medical workers coping with various worker-related stressors to reduce depression symptoms. To our knowledge, this is one of the few studies to explore the eﬀects of occupational stress, WOF on depression symptoms, to further examined the mediating roles of WOF among Chinese medical workers. It is provided with a new perspective for exploring the relationship between occupational stress and depression symptoms. However, there are two limitations: ﬁrstly, a cross-sectional study was conducted. The causal relationship between occupational stress, depression symptoms and WOF cannot be identiﬁed. Secondly, all of the medical workers were from one general hospital and three specialized hospitals in Henan Province, and the sample was not randomized, which suggested the generalization of results to other areas of China would be limited. DATA AVAILABILITY STATEMENT The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. DISCUSSION It is noteworthy that CF is more strongly associated with WOF than with NSF, DAF. The results suggest that hospital administrators assign work tasks based on the personal characteristics (i.e., personality or work style) of medical staﬀ, which can reduce the risk of depression symptoms. In a systematic review, the study indicated a high prevalence of depression among medical staﬀ and the consistency between the estimated prevalence (27.2% students and 28.8% residents) showed that depression coping was an important issue in all levels of medical training (47). Previous studies of organizational behavior and organizational management showed that high-level ﬁt of worker and occupational environment can improve work attitude (49), innovation behaviors (50), and organizational commitment (51), and turnover rate (52). It has a positive eﬀect on improving the optimization of the organizational outcome. The results of our study suggested WOF, occupational stress and depression symptoms have a signiﬁcant correlation, and WOF can explain 6.5% of the eﬀect of occupational stress on depression symptoms. A better WOF level can promote medical workers’ characters and habits more suitable for their job style in hospitals, and workers’ medical service skills can skillfully complete the tasks assigned by their managers, which would reduction of the medical workers’ occupational stress, thereby reducing the depression symptoms. The current study is one of the few to investigate WOF among Chinese medical workers, and to examine the correlation between occupational stress and depression symptoms, and it is the ﬁrst study to explore the mediating role of WOF in these relationships. In China, basic salary levels are generally low, May 2022 \| Volume 10 \| Article 843845 7 Sun et al. WOF, Occupational Stress and Depression FIGURE 4 \| The structural equation model on the relationship between occupational stress, depressive symptoms, WOF and WOF types among 1988 medical workers. Stress, occupational stress; Depression, depression symptoms. WOF, worker occupation ﬁt; CF, Characteristic ﬁt; NSF, need supply ﬁt; DAF, demand ability ﬁt. FIGURE 4 \| The structural equation model on the relationship between occupational stress, depressive symptoms, WOF and WOF types among 1988 medical workers. Stress, occupational stress; Depression, depression symptoms. 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Impact of personality trait and professional identity on work-related depression, anxiety and irritation among Chinese nurses. ETHICS STATEMENT The studies involving human participants were reviewed and approved by the Ethics Committees of West China School of Public Health and West China Fourth Hospital. The Frontiers in Public Health \| www.frontiersin.org May 2022 \| Volume 10 \| Article 843845 8 WOF, Occupational Stress and Depression Sun et al. Foundation of Chengdu Medical Collage (Grant Number: CYZYB21-32). The funders have no role in the analysis, or interpretation of the data, writing of the article, or the decision to submit the paper for publication. patients/participants provided their written informed consent to participate in this study. ACKNOWLEDGMENTS RS: study design, data analysis, data interpretation, and ﬁnished the manuscript. RS, ZH, LL, and HW: data acquisition. RS, KL, and YL: data analysis. YL: supervision of study. 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https://openalex.org/W3120051138	https://www.researchsquare.com/article/rs-120884/latest.pdf	English	null	Changes in the essential oil content and composition of <i>Salvia limbata</i> C.A. Mey at different growth stages and altitudes	Biomedical chromatography/BMC. Biomedical chromatography	2,021	cc-by	6,719	Changes in Essential Oil Content and Composition of Salvia Limbata C.A. Mey at Different Growth Stages and Altitudes Maryam Saffariha (  m.saffariha@gmail.com ) University of Tehran Hossein Azarnivand University of Tehran Mohammad Ali Zare Chahouki University of Tehran Ali Tavili University of Tehran Samad Nejad Ebrahimi Shahid Beheshti University Daniel Potter University of California, Davis Reza Jahani Shahid Beheshti University of Medical Sciences 1. Introduction Salvia is the largest and prominent genus of the Lamiaceae family, which includes more than 900 medicinal and ornamental species distributed in the world [1]. This genus is found in Central and South America, Western Asia and Eastern Asia [2]. Fifty- eight species of genus Salvia are found in Iran which seventeen species are endemic [3,4]. Salvia limbata C.A. Mey, a native plant of Iran, is a perennial, herbaceous and aromatic plant (30-60 cm tall) with thick, rounded and bright green leaves. The distribution of this plant within Iran is in Azerbaijan, Lorestan, Shiraz, Kermanshah, Semnan, and Damavand [5]. The genus Salvia has always been noticeable in doing research around the world for diverse biological activities and compounds in its essential oil [6,7]. Since the species of genus Salvia contain substantial amounts of essential oils, people have been applied for thousands of years in folk medicine to improve health and treat diseases [8,9]. Modern science illustrates that Salvia essential oils improve memory and could be effective in treating Alzheimer's in the future [10]. Salvia has also been used for treating coughs, colds and wounds and it has been considered as spasmolytic, antiseptic, astringent, and liver protective [11]. Moreover, the phenolic compounds of plants belonging to this genus have shown antiviral, antibacterial, antifungal, antioxidant, antitumor, antidiabetic, anxiolytic, sedative, and anti-inflammatory activities [12,13,,14,15,16,17,18]. Active compounds such as hydrocarbon monoterpenes, hydrogenated monoterpenes, oxygenated monoterpenes, di-terpenes, hydrocarbon sesquiterpenes and oxygenated sesquiterpenes have been obtained from Salvia species [19]. The contents of these compounds are influenced by various factors such as planting conditions, harvest time, organ used and growth stage. It seems that ecological factors and genetic are the main significant factors that influence the content of the plant active compounds [20]. To the best of our knowledge, few investigations have been done in the field of autecology and phytochemistry of S. limbata. Since the global approach has been perusing the use of medicinal herbs and natural compounds in the pharmaceutical, cosmetic and food industries, there is a strong need to delve more into the issue and do further research to understand how to increase the yield of active ingredients in varied ecological conditions. This could be economically important for the food, cosmetic and pharmaceutical industries. 1. Introduction Although phytochemistry of different species of this genus has already been studied in different ecological conditions (mostly in flowering stage), for the first time the effect of different phenological stages and altitudes on the essential oil content and composition of S. limbata was investigated in this paper. Discussion The obtained results showed that the content and constituents of S. limbata essential oil strongly depend on the growth stage and altitude. Our findings revealed that the vegetative stage at 1500 m is the optimal harvest time to obtain the highest content of oil yield. Results of the current study helps to find the optimum situation to gain the highest content of S. limbata essential oil but more researches are needed. Research Article Page 1/18 Background The present study investigates the effect of growth stage (vegetative, flowering and seed ripening) and altitude (1500, 2000 and 2500 m above sea level) on the content and chemical composition of S. limbata essential oil which belongs to Lamiaceae family. Results Results According to the oil analysis, 28 components representing 96.5% to 99.7% of the total volatile oil composition were characterized. The main compounds of S. limbata oils were α-pinene (14.7-38.7%), β-pinene (12.5-26.2%), allo-aromadendrene (9.2-21.7%), germacrene D (4.2-8.3%), bicyclogermacrene (6.5-14.5 %), and spathulenol (7.5-25.4 %). 2.5. Statistical analysis The statistical analyses were performed using Graph Pad Prism software (San Diego, CA; version 5.0). Data are presented as mean ± standard deviation (SD) in 5 randomized replicates. One way analysis of variance (ANOVA) and Tukey post-test were used to analyze obtained results. P-value < 0.05 was considered as statistically significant difference. 2.4. Identification of compounds The constituents of the essential oils were recognized by calculation of retention indices for all the components, using retention times of C6-C24 n-alkenes series as standards under the same chromatographic conditions. Identification of individual compounds were performed by comparison of their retention indices and mass spectral fragmentation patterns with those reported in the literature, Wiley library (New York, NY, USA) or published mass spectra [8,21]. The relative percentage of essential oil constituents were obtained from the GC-FID peak areas in the chromatogram without the use of correction factors. 2. Materials And Methods 2.1. Plant material 2.1. Plant material 2.1. Plant material Page 2/18 Aerial parts of S. limbata at several developmental stages (vegetative, flowering and seed ripening) were harvested in three replicates from its wild habitat at altitudes of 1500, 2000 and 2500 m above sea level from Taleghan rangeland (semi-humid) in Alborz province, Iran (36° 5' 19" N to 36° 19' 19" N and 50° 36' 43" E to 50° 53' 20" E) (Fig. 1). Taleghan is one of S. limbata’s main sites with mean relative humidity about 12%, and the average annual temperature of 11.4oC. In this area, there are 150 freezing days and annual precipitation is about 446 mm. Harvested plant materials were dried in the shade and then ground in a grinder (2mm mesh size). A voucher herbarium specimen (MP-300) was lodged at the Herbarium of Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, Tehran, Iran. Aerial parts of S. limbata at several developmental stages (vegetative, flowering and seed ripening) were harvested in three replicates from its wild habitat at altitudes of 1500, 2000 and 2500 m above sea level from Taleghan rangeland (semi-humid) in Alborz province, Iran (36° 5' 19" N to 36° 19' 19" N and 50° 36' 43" E to 50° 53' 20" E) (Fig. 1). Taleghan is one of S. limbata’s main sites with mean relative humidity about 12%, and the average annual temperature of 11.4oC. In this area, there are 150 freezing days and annual precipitation is about 446 mm. Harvested plant materials were dried in the shade and then ground in a grinder (2mm mesh size). A voucher herbarium specimen (MP-300) was lodged at the Herbarium of Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, Tehran, Iran. 2.2. Isolation of the essential oils 2.2. Isolation of the essential oils Samples (100 g) of the air-dried aerial parts of S. limbata were subjected to hydro distillation using a Clevenger-type apparatus for 4h, according to the method recommended in British Pharmacopoeia. The distilled oils were dried over anhydrous sodium sulfate and stored at 4oC in tightly closed dark vials to be analyzed. 2.3. Chemical composition of the essential oils For characterization of the volatile oil constituents, samples were subjected to gas chromatography-flame ionization detector (GC- FID) and gas chromatography-mass spectrometry (GC-MS). GC-FID analysis was performed by a Shimadzu 15 A gas chromatograph (Dyson Instruments, Newcastle, U.K) equipped with a split/splitless injector (250°C), a DB-5 (60 m length × 0.25 mm internal diameter, 0.25 μm film thickness) capillary column and flame ionization detector (250°C). The helium gas was used as the carrier gas (1 mL/min) and the oven temperature was 60-280°C at a rate of 5°C/min. The injector temperature was 250°C and 1μL of oil sample was injected in the split mode with the split ratio of 100:1. GC-MS analysis was carried out using a Thermo-Trace GC-MS system (Thermo Electron; San José, CA, USA). This device was equipped with a DB-5 silica column (60 m length × 0.25 mm internal diameter, 0.25 μm film thickness) containing 5%-phenyl 95%- methyl polysiloxane and a mass spectrometer detector (MS). The carrier gas was helium at a flow rate of 1 ml/ min. The column temperature was kept at 60°C for 3 min and then programmed to 250°C at a rate of 5°C/min. The injector and GC/MS interface temperatures were 290°C and 300°C, respectively. The mass spectra were taken at 70 eV in the scan range of m/z 50-550. 2.4. Identification of compounds 3. Results And Discussion As shown in Figure 2, comparison of the essential oil yield among different samples revealed that the highest content of essential oil belongs to the harvested aerial parts of S. limbata in the vegetative stage at an altitude of 1500 m (0.86% v/w) while no significant difference was observed in the essential oil content among other groups. In a published study authors observed the significant impact of different altitudes and phenological stages on the essential oil yield [22,23]. It was revealed that plant performance is strongly influenced by various factors such as altitude, climate, soil, developmental stages, extraction and analysis methods, genetic factors, abiotic stresses, and slope and modeling techniques can predict these factors in other areas [24,25,26,27, 28]. The results of the current study were compatible with other studies that found the highest content of essential Page 3/18 Page 3/18 oil of Origanum majorana in the vegetative stage, so they proposed the vegetative stage as the best stage to harvest Origanum majorana [29,30, 31,32, 33,34,35,36].Similar results were also found byon the essential oil content of Nepeta kotschyr [37]. Moreover, according to a study conducted by the highest yield of essential oil in Teucrium polium L. was obtained in the vegetative stage [38]. These results are in agreement with our findings. The accumulation of essential oil in vegetative stage could be due to the fact that plant protection is supplied by phenolic components which are in high amount in this stage [39]. oil of Origanum majorana in the vegetative stage, so they proposed the vegetative stage as the best stage to harvest Origanum majorana [29,30, 31,32, 33,34,35,36].Similar results were also found byon the essential oil content of Nepeta kotschyr [37]. Moreover, according to a study conducted by the highest yield of essential oil in Teucrium polium L. was obtained in the vegetative stage [38]. These results are in agreement with our findings. The accumulation of essential oil in vegetative stage could be due to the fact that plant protection is supplied by phenolic components which are in high amount in this stage [39]. On the contrary, it was reported bythat the highest essential oil in Satureja mutica is acquired in the flowering stage[40]. In another study [41] determined that the highest value of essential oil of Mentha pieperata in flowering stage, which contradicts our findings. 3. Results And Discussion Also, the percentage of essential oils in vegetative stage in Thymus vulgaris was the lowest and it rose in flowering stage [42]. One explanation for the increase in essential oil content in the flowering stage is the maintenance of the reproductive stage [43,44] and to attract insects for pollination [35]. Nevertheless, other researches have illustrated that the lowest amount of essential oils in the vegetative stage could be due to the lower activity of some enzymes in synthesizing phenolic compounds in this stage [45]. Since photosynthetic products accumulate in the endosperm during plant growth, it leads to a decrease in the amount of essential oil [46]. It is clear that phenological stages have a great impact on the essential oil metabolism, enzymatic activity and finally essential oil content [45]. As illustrated in Tables 1, 2 and 3, twenty-eight components were identified in the S. limbata essential oil by means of GC-FID and GC-MS analysis which represented about 96.5% to 99.7% of the total composition of the obtained essential oil. In the current study, the main identified compounds were α-pinene (14.7-38.7%), β-pinene (12.5-26.2%), allo-aromadendrene (9.2-21.7%), germacrene D (4.2-8.3%), bicyclogermacrene (6.5-14.5 %), and spathulenol (7.5-25.4 %). The molecular structures of the main identified compounds from S. limbata essential oil are presented in the Figure 3. Comparing the results of the current study to others, α-pinene (23.7 %), β-pinene (18.7%), sabinene (14.5%), 1, 8-cineole (9.9%) and β-caryophyllene (7.1%) as the major components of S. limbata essential oil in the flowering stage [47]. In another research, following GC-MS analysis of the aerial parts of S. limbata obtained from Turkey, 42 components were characterized representing 95.6% to 98.1% of the compounds including α-pinene (11.2-24.3%), β-pinene (10.0-20.9%) and sabinene (14.6-17.4%) as the major constituents of the essential oil [48]. [48]. Comparing of the monoterpenes and sesquiterpenes contents of the S. limbata essential oil at different altitudes and phenological stages in Figure 4, the amount of monoterpenes has decreased from vegetative stage to seed ripening stage; however, the obtained results for sesquiterpenes were reverse. These findings for Artemisia herba-alba essential oil were previously observed in another study [49]. Moreover, during the developing plants the amount of sesquiterpenes increased in Cannabis sativa L. which are in line with our results[50]. 3. Results And Discussion As we found out in our research, the highest amount of monoterpenes was related to the vegetative period at 2000 m, while the highest amount of sesquiterpenes was obtained in seed ripening stage at altitudes of 1500 and 2500 m. [51] 2002 in a research on Thymus vulgaris at different growth stages confirmed that the highest content of the monoterpene was related to the vegetative stage. As shown in Figure 5, the content of α-pinene, β-pinene, alloaromadendrene, germacrene D, bicyclogermacrene, and spathulenol illustrates some changes in different altitudes and developmental stages. The highest percentage of monoterpenes including α- pinene (‎41.3%) and β-pinene (30.1%) was obtained in the vegetative stage at 2000 m. The contents of α-pinene and β-pinene were decreased to the lowest values in the ripening stage. Moreover, the highest content for alloaromadendrene was measured 20.6% and 20.7% in the ripening stage at 1500 m and 2500 m, respectively without any significant difference between them. However, the lowest quantity was obtained 3.5% for the vegetative stage at 2000 m. The most abundant germacrene D reached in ripening stage at 2500 m (8.3%) and the lowest amount (1.2%) achieved at 2000 m in the vegetative stage. Moreover, high value of bicyclogermacrene was attained in the ripening stage at 1000 m and 2000m (14.5% and 14.3%, respectively) while no significant difference was observed between the mentioned altitudes. On the contrary, the lowest amount was attained in the vegetative stage at 1500 m (4.2%). Furthermore, the highest and the lowest contents of spathulenol (25.4% and 7.2%) were gained in the ripening stage at 2500 m and vegetative stage at 2000 m, respectively. Variation in the percentage of compounds at different stages of phenology and altitudes could be due to the high or low synthesis of compounds by enzymes, which leads to different percentages of compounds in essential oils. mical compositions of Salvia limbata essential oil in the vegetative stage at different altitudes Table 1. The percentage of chemical compositions of Salvia limbata essential oil in the vegetative stage at different altitudes able 1. The percentage of chemical compositions of Salvia limbata essential oil in the vegeta Page 4/18 Page 4/18 No. 3. Results And Discussion Compounds RI 1500 m 2000 m 2500 m 1 α-Thujene 922 Tr Tr Tr 2 α-Pinene 938 30.4±0.1 41.3±0.2 28.5±0.8 3 Camphene 952 1.2±0.1 1.4±0.2 0.5±0.0 4 Sabinene 975 2.4±0.1 1.9±0.1 4.1±0.1 5 β-Pinene 980 25.4±0.3 30.1±0.1 24.2±0.2 6 Myrcene 985 Tr Tr Tr 7 p-Cymene 1025 Tr Tr Tr 8 Limonene 1029 0.9±0.1 0.6±0.04 0.4±0.0 9 Z-β-Ocimene 1035 Tr Tr Tr 10 Linalool 1085 Tr Tr Tr 11 α-Campholenal 1103 Tr Tr Tr 12 Trans-Pinocarveol 1125 Tr 0.2±0.0 Tr 13 Trans-Verbenol 1162 Tr 0.3±0.0 0.2±0.01 14 Borneol 1186 Tr 0.2±0.0 Tr 15 Terpine-4-ol 1203 Tr 0.4±0.0 0.2±0.0 16 Myrtenal 1216 Tr 0.4±0.0 0.2±0.0 17 Verbenone 1239 0.3±0.1 0.2±0.0 0.3±0.0 18 Bornyl-acetate 1316 1±0.1 Tr Tr 19 Eugenol 1340 Tr 0.9±0.1 1.4±0.2 20 β-Caryophyllene 1426 Tr Tr 0.2±0.0 21 Allo-Aromadendrene 1482 9.5±0.1 3.5±0.2 12.6±0.3 22 γ-Muurolene 1485 Tr 0.6±0.0 Tr 23 Germacrene D 1498 3.7±0.2 1.2±0.1 4.1±0.2 24 Bicyclogermacrene 1505 4.6±0.2 5.5±0.3 8.5±0.9 25 Eugenol-acetate 1521 Tr 0.6±0.0 Tr 26 Spathulenol 1575 12.6±0.1 7.2±0.2 13.8±0.2 27 Caryophyllene oxide 1580 4.3±0.1 Tr Tr 28 Sclareol 2200 1.9±0.1 0.2±0.0 0.5±0.0 Monoterpene hydrocarbons 60.3 75.3 57.7 Oxygenated monoterpenes 1.3 2.6 2.3 Sesquiterpene hydrocarbons 17.8 10.8 25.4 Oxygenated 18 8 8 0 14 3 Data are presented as mean‎±SD; RI indicates retention indices relative to C6-C24 n-alkanes; Tr indicates trace (<0.1%) Data are presented as mean‎±SD; RI indicates retention indices relative to C6-C24 n-alkanes; Tr indicates trace (<0.1%) Table 2. The percentage of chemical compositions of Salvia limbata essential oil in the flowering stage at different altitudes Table 2. The percentage of chemical compositions of Salvia limbata essential oil in the flowering stage at different altitudes Page 6/18 No. mean‎±SD; RI indicates retention indices relative to C6-C24 n-alkanes; Tr indicates trace (<0.1%) 3. Results And Discussion Compounds RI 1500 m 2000 m 2500 m 1 α-Thujene 922 Tr Tr Tr 2 α-Pinene 938 21.2±0.2 29.5±0.4 21.7±0.6 3 Camphene 952 1.3±0.1 1.4±0.2 1.8±0.2 4 Sabinene 975 1.7±0.1 3.3±0.1 1.8±0.1 5 β-Pinene 980 17.3±0.1 26.2±0.2 20.1±0.2 6 Myrcene 985 Tr Tr Tr 7 p-Cymene 1025 Tr Tr Tr 8 Limonene 1029 1±0.1 1±0.1 0.8±0.1 9 Z-β-Ocimene 1035 Tr 1.2±0.1 Tr 10 Linalool 1085 Tr Tr Tr 11 α-Campholenal 1103 Tr Tr Tr 12 Trans-Pinocarveol 1125 0.3±0.0 Tr 0.4±0.0 13 Trans-Verbenol 1162 Tr Tr 0.3±0.0 14 Borneol 1186 Tr Tr 0.3±0.0 15 Terpine-4-ol 1203 0.6±0.0 0.6±0.0 Tr 16 Myrtenal 1216 0.6±0.0 0.6±0.1 Tr 17 Verbenone 1239 0.2±0.0 0.3±0.0 Tr 18 Bornyl-acetate 1316 Tr 0.2±0.0 Tr 19 Eugenol 1340 1.2±0.1 0.4±0.0 0.5±0.0 20 β-Caryophyllene 1426 Tr Tr Tr 21 Allo-Aromadendrene 1482 15.2±0.2 9.2±0.1 14.6±0.2 22 γ-Muurolene 1485 0.5±0.0 3.2±0.1 0.3±0.0 23 Germacrene D 1498 5.7±0.2 4.4±0.1 4.2±0.2 24 Bicyclogermacrene 1505 10.7±0.2 6.5±0.1 13.6±0.2 25 Eugenol-acetate 1521 Tr Tr Tr 26 Spathulenol 1575 18.6±0.3 7.5±0.1 15.5±0.3 27 Caryophyllene oxide 1580 1±0.1 2.1±0.2 Tr 28 Sclareol 2200 1.7±0.2 Tr 0.4±0.0 Monoterpene hydrocarbons 42.5 62.6 46.2 Oxygenated monoterpenes 2.9 2.1 1.5 Sesquiterpene hydrocarbons 32.1 23.3 32.7 Oxygenated sesquiterpenes 21.3 9.6 15.9 Total 98 8 97 6 96 3 Page 7/18 The percentage of chemical compositions of Salvia limbata essential oil in the ripening stage at different altitudes No. 3. Results And Discussion Compounds RI 1500 m 2000 m 2500 m 1 α-Thujene 922 Tr Tr Tr 2 α-Pinene 938 14.9±0.2 19.5±0.2 14.7±0.1 3 Camphene 952 0.9±0.0 1.6±0.2 0.6±0.0 4 Sabinene 975 1.3±0.2 2.2±0.2 1.1±0.1 5 β-Pinene 980 14.2±0.3 17.6±0.3 12.5±0.1 6 Myrcene 985 Tr Tr Tr 7 p-Cymene 1025 Tr Tr Tr 8 Limonene 1029 0.5±0.0 0.7±0.0 0.4±0.0 9 Z-β-Ocimene 1035 Tr Tr Tr 10 Linalool 1085 Tr Tr Tr 11 α-Campholenal 1103 Tr Tr Tr 12 Trans-Pinocarveol 1125 Tr Tr Tr 13 Trans-Verbenol 1162 Tr 0.2±0.0 Tr 14 Borneol 1186 Tr Tr Tr 15 Terpine-4-ol 1203 0.3±0.0 0.5±0.0 Tr 16 Myrtenal 1216 0.3±0.0 0.5±0.0 Tr 17 Verbenone 1239 0.2±0.0 0.2±0.0 Tr 18 Bornyl-acetate 1316 Tr Tr Tr 19 Eugenol 1340 0.6±0.0 Tr 1.5±0.2 20 β-Caryophyllene 1426 Tr 2.6±0.0 Tr 21 Allo-Aromadendrene 1482 20.6±0.2 16.3±0.2 21.7±0.2 22 γ-Muurolene 1485 0.3±0.0 0.5±0.0 Tr 23 Germacrene D 1498 7.6±0.2 6.3±0.2 8.3±0.3 24 Bicyclogermacrene 1505 14.5±0.0 14.3±0.2 12.4±0.1 25 Eugenol-acetate 1521 Tr 0.9±0.0 Tr 26 Spathulenol 1575 22.4±0.2 12.6±0.1 25.4±0.2 27 Caryophyllene oxide 1580 Tr Tr Tr 28 Sclareol 2200 0.7±0.0 Tr 0.4±0.0 Monoterpene hydrocarbons 31.8 41.6 29.3 Oxygenated monoterpenes 1.4 1.4 1.5 Sesquiterpene hydrocarbons 43.0 40.0 42.4 Oxygenated sesquiterpenes 23.1 13.5 25.8 Total 99.3 96.5 99.0 Data are presented as mean‎±SD; RI indicates retention indices relative to C6-C24 n-alkanes; Tr indicates trace 4. Conclusion The data presented in this paper confirmed that the essential oil yield and constituents of S. limbata were highly influenced by phenology and altitude. A significant difference in the composition of S. limbata essential oil at different growing stages and altitudes was observed. It is noteworthy that the dominant compounds of S. limbata essential oil were α-pinene, β-pinene, allo- aromadendrene, germacrene-D, bicyclogermacrene, and spathulenol. Since higher yield of essential oil was obtained in vegetative stage at 1500 m, this stage could be considered as the best stage to harvest plant. Moreover, it was revealed that the highest content of monoterpenes and sesquiterpenes could be obtained at the vegetative stage and ripening stage, respectively. This was the first research on the yield and constituents of S. limbata essential oil at different developmental stages and altitudes that could be economically beneficial for the food and pharmaceutical industries as well as other researchers to examine further investigations about this plant. Acknowledgment The authors wish to thank University of Tehran, Shahid Beheshti University and University of California, Davis to support this work. Declarations Ethic approval: Not applicable Ethic approval: Not applicable Consent for publication: Yes Availability of data and materials: The form of material is in Excel file. The location was in Taleghan rangeland. Competing interests: No Funding: No Funding: No Author's contribution: Hossein Azarnivand devised the project, the main conceptual ideas and proof outline. Dr Mohammad Ali Zare Chahouki and Dr Maryam Saffariha worked out almost all of the technical details, and performed the numerical calculations for the suggested experimented Ali Tavili and Dr Samad Nejad Ebrahimi verified the numerical results. Pr. Daniel Potter aided in interpreting the results and worked on the manuscript. Dr. Reza Jahani helped in analysing the data. All authors discussed the results and commented on the manuscript. References 1. Jash SK, Gorai D, Roy R (2016) Salvia genus and triterpenoids. International Journal Of Pharmaceutical Sciences And Research. 7(12):4710. 2. Jassbi AR, Asadollahi M, Masroor M, Schuman MC, Mehdizadeh Z, Soleimani M (2012) Chemical classification of the essential oils of the Iranian Salvia species in comparison with their botanical taxonomy. Chemistry & biodiversity. 9(7):1254- 71. 3. 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Essential Oil Composition at Different Plant Growth Development of Peppermint (Mentha x piperita L.) Under Water Deficit Stress. Journal of Essential Oil Bearing Plants. 22(2):431-40. 41. Abdi G, Shokrpour M, Salami SA. (2019). Essential Oil Composition at Different Plant Growth Development of Peppermint (Mentha x piperita L.) Under Water Deficit Stress. Journal of Essential Oil Bearing Plants. 22(2):431-40. 42. Al-Asmari AK, Athar MT, Al-Faraidy AA, Almuhaiza MS. (2017). Chemical composition of essential oil of Thymus vulgaris collected from Saudi Arabian market. Asian Pacific Journal of Tropical Biomedicine. 7(2):147-50. 42. Al-Asmari AK, Athar MT, Al-Faraidy AA, Almuhaiza MS. (2017). Chemical composition of essential oil of Thymus vulgaris collected from Saudi Arabian market. Asian Pacific Journal of Tropical Biomedicine. 7(2):147-50. 43. Fonseca LM, dos Santos Cruxen CE, Bruni GP, Fiorentini ÂM, da Rosa Zavareze E, Lim L-T.(2019). Development of antimicrobial and antioxidant electrospun soluble potato starch nanofibers loaded with carvacrol. International journal of biological macromolecules.139:1182-90. 43. Fonseca LM, dos Santos Cruxen CE, Bruni GP, Fiorentini ÂM, da Rosa Zavareze E, Lim L-T.(2019). Development of antimicrobial and antioxidant electrospun soluble potato starch nanofibers loaded with carvacrol. International journal of biological macromolecules.139:1182-90. antimicrobial and antioxidant electrospun soluble potato starch nanofibers loaded with carvacrol. International journal of biological macromolecules.139:1182-90. 44. Page 9/18 Sun X, Cameron RG, Bai J. (2020). Effect of spray-drying temperature on physicochemical, antioxidant and antimicrobial properties of pectin/sodium alginate microencapsulated carvacrol. Food Hydrocolloids. 100:105420. 44. Sun X, Cameron RG, Bai J. (2020). Effect of spray-drying temperature on physicochemical, antioxidant and antimicrobial properties of pectin/sodium alginate microencapsulated carvacrol. Food Hydrocolloids. 100:105420. 45. Sangwan N, Farooqi A, Shabih F, Sangwan R. (2001). Regulation of essential oil production in plants. Plant growth regulation 34(1):3-21. 45. Sangwan N, Farooqi A, Shabih F, Sangwan R. (2001). Regulation of essential oil production in plants. Plant growth regulation 34(1):3-21. Page 11/18 Page 11/18 46. Özcan MM, Chalchat JC. (2006). Effect of collection time on chemical composition of the essential oil of Foeniculum vulgare subsp. piperitum growing wild in Turkey. European Food Research and Technology. 224(2):279-81. 47. Rustaiyan A, Akhgar MR, Masoudi S, Nematollahi F.(2005). Chemical Composition of Essential Oils of Three Salvia Species Growing Wild in Iran: Salvia rhytidea Benth., S. limbata CA Mey and S. palaestina Benth. Journal of Essential Oil Research. 17(5):522-4. 48. Kürkçüoglu M, Demirci B, Baser K, Dirmenci T, Tümen G, Özgen U.(2005). The essential oil of Salvia limbata CA Meyer growing in Turkey. Journal of Essential Oil Research. 17(2):192-3. 49. Behtari B, Gholami F, Khalid KA, Tilaki GD, Bahari R. (2012). Effect of growth stages and altitude on Artemisia herba-alba Asso essential oil growing in Iran. Journal of Essential Oil Bearing Plants. 15(2):307-13. 49. Behtari B, Gholami F, Khalid KA, Tilaki GD, Bahari R. (2012). Effect of growth stages and altitude on Artemisia herba-alba Asso essential oil growing in Iran. Journal of Essential Oil Bearing Plants. 15(2):307-13. 50. Abdollahi M, Sefidkon F, Calagari M, Mousavi A, Mahomoodally MF.(2020). Impact of four hemp (Cannabis sativa L.) varieties and stage of plant growth on yield and composition of essential oils. Industrial Crops and Products. 155:112793. 50. Abdollahi M, Sefidkon F, Calagari M, Mousavi A, Mahomoodally MF.(2020). Impact of four hemp (Cannabis sativa L.) varieties and stage of plant growth on yield and composition of essential oils. Industrial Crops and Products. 155:112793. 51. Hudaib M, Speroni E, Di Pietra AM, Cavrini V. (2002). GC/MS evaluation of thyme (Thymus vulgaris L.) oil composition and variations during the vegetative cycle. Journal of pharmaceutical and biomedical analysis. 29(4):691-700. Page 12/18 g g y p y ( ) Figures Figure 1 Figures Page 12/18 Figure 1 Figure 1 Locations of the study area. Note: The designations employed and the presentation of the material on this map do not imply the expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors. Locations of the study area. Note: The designations employed and the presentation of the material on this map do not impl expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, ci area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the au Figure 1 Locations of the study area. Note: The designations employed and the presentation of the material on this map do not impl expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, ci area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the au Figure 2 The essential oil content of Salvia limbata at different phenological stages and different altitudes. Data are expressed as mean ± SD (n=5). * indicates P-value <0.001 compared to other groups Page 14/18 ( ) p g p Figure 2 The essential oil content of Salvia limbata at different phenological stages and different altitudes. Data are expressed as mean ± SD (n=5). * indicates P-value <0.001 compared to other groups Figure 1 Locations of the study area. Note: The designations employed and the presentation of the material on this map do not imply the expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors. Page 13/18 Figure 2 The essential oil content of Salvia limbata at different phenological stages and different altitudes. Data are expressed as mean ± SD (n=5). * indicates P-value <0.001 compared to other groups Figure 2 The essential oil content of Salvia limbata at different phenological stages and different altitudes. Data are expressed as mean ± SD (n=5). * indicates P-value <0.001 compared to other groups Page 14/18 Figure 3 The molecular structures of the main identified compounds from Salvia limbata essential oil Figure 3 Page 15/18 olecular structures of the main identified compounds from Salvia limbata essential oil 4 on in monoterpenes and sesquiterpenes of Salvia limbata essential oil at different altitudes and phenological stages 4 on in monoterpenes and sesquiterpenes of Salvia limbata essential oil at different altitudes and phenological stages The molecular structures of the main identified compounds from Salvia limbata essential o The molecular structures of the main identified compounds from Salvia limbata essential oil Figure 4 Variation in monoterpenes and sesquiterpenes of Salvia limbata essential oil at different altitudes and phenological stages Figure 4 Variation in monoterpenes and sesquiterpenes of Salvia limbata essential oil at different altitudes and phenological stages Variation in monoterpenes and sesquiterpenes of Salvia limbata essential oil at different altitudes and phenological stages Figure 4 Variation in monoterpenes and sesquiterpenes of Salvia limbata essential oil at different altitudes and phenological stages Figure 4 Variation in monoterpenes and sesquiterpenes of Salvia limbata essential oil at different altitudes and phenological stages Variation in monoterpenes and sesquiterpenes of Salvia limbata essential oil at different altitudes and phenological stages Page 16/18 Figure 5 Percentage of α-Pinene (A), β-Pinene (B), allo-Aromadendrene (C), germacrene D (D), bicyclogermacrene (E) a different altitudes and phenological stages. Different letters indicate significant difference among columns ( with the same letter are not statistically different. Figure 5 Percentage of α-Pinene (A), β-Pinene (B), allo-Aromadendrene (C), germacrene D (D), bicyclogermacrene (E) and spathulenol (F) at different altitudes and phenological stages. Different letters indicate significant difference among columns (P < 0.05). Columns with the same letter are not statistically different. Figure 5 Percentage of α-Pinene (A), β-Pinene (B), allo-Aromadendrene (C), germacrene D (D), bicyclogermacrene (E) and spathulenol (F) at different altitudes and phenological stages. Different letters indicate significant difference among columns (P < 0.05). Columns with the same letter are not statistically different. Percentage of α-Pinene (A), β-Pinene (B), allo-Aromadendrene (C), germacrene D (D), bicyclogermacrene (E) and spathulenol (F) at different altitudes and phenological stages. Different letters indicate significant difference among columns (P < 0.05). Columns with the same letter are not statistically different. Page 17/18 Figure 5 Percentage of α-Pinene (A), β-Pinene (B), allo-Aromadendrene (C), germacrene D (D), bicyclogermacrene (E) a different altitudes and phenological stages. Different letters indicate significant difference among columns ( with the same letter are not statistically different. Figure 5 Percentage of α-Pinene (A), β-Pinene (B), allo-Aromadendrene (C), germacrene D (D), bicyclogermacrene (E) and spathulenol (F) at different altitudes and phenological stages. Different letters indicate significant difference among columns (P < 0.05). Columns with the same letter are not statistically different. Figure 5 Percentage of α-Pinene (A), β-Pinene (B), allo-Aromadendrene (C), germacrene D (D), bicyclogermacrene (E) and spathulenol (F) at different altitudes and phenological stages. Different letters indicate significant difference among columns (P < 0.05). Columns with the same letter are not statistically different. Percentage of α-Pinene (A), β-Pinene (B), allo-Aromadendrene (C), germacrene D (D), bicyclogermacrene (E) and spathulenol (F) at different altitudes and phenological stages. Different letters indicate significant difference among columns (P < 0.05). Columns with the same letter are not statistically different. Page 18/18 Page 18/18
https://openalex.org/W4286411624	https://zenodo.org/record/3884763/files/MED2020__Junction_Detection_via_Unsupervised_Learning.pdf	English	null	Unsupervised Learning for Subterranean Junction Recognition Based on 2D Point Cloud	arXiv (Cornell University)	2,020	cc-by	4,817	Unsupervised Learning for Subterranean Junction Recognition Based on 2D Point Cloud ouri1, Farhad Pourkamali-Anaraki2, Miguel Casta˜no Arranz3, Ali-akbar Agha-mohammadi4, Joel Burdick5, and George Nikolakopoulos1 Sina Sharif Mansouri1, Farhad Pourkamali-Anaraki2, Miguel Casta˜no Arranz3, Ali-akbar Agha-mohammadi4, Joel Burdick5, and George Nikolakopoulos1 high-end 3D lidars can provide 3D point cloud, and it can be used for junction detection. However, we are trying to remove the dependency on high end and highly expensive sensors, replacing them with lower cost solutions. Visual sensors cannot provide sufﬁcient information regarding the complex geometry of subterranean environments due to lack of illumination. Furthermore, burdening a MAV with optical sensors in all directions and a bright light source will reduce the ﬂight time, and require a large computational resource to post-process the visual feedback. Abstract— This article proposes a novel unsupervised learn- ing framework for detecting the number of tunnel junctions in subterranean environments based on acquired 2D point clouds. The implementation of the framework provides valuable information for high level mission planners to navigate an aerial platform in unknown areas or robot homing missions. The framework utilizes spectral clustering, which is capable of uncovering hidden structures from connected data points lying on non-linear manifolds. The spectral clustering algorithm computes a spectral embedding of the original 2D point cloud by utilizing the eigen decomposition of a matrix that is derived from the pairwise similarities of these points. We validate the developed framework using multiple data-sets, collected from multiple realistic simulations, as well as from real ﬂights in underground environments, demonstrating the performance and merits of the proposed methodology. The 2D point cloud extracted from a 2D lidar is used in this paper to obtain important information about the number of junctions. The introduced method relies on a family of unsupervised learning techniques that compute a spectral embedding of the original data points by forming similarity graphs. To be more speciﬁc, spectral clustering algorithms typically consist of three steps [4]. The ﬁrst step involves forming a similarity graph that encodes information about all pairwise similarities among data points in the given 2D point cloud. A popular approach for deﬁning such similarities is to employ the Radial Basis Function (RBF) [5]. The next step is to compute the eigenvalue decomposition of the normalized Laplacian matrix that will provide a non-linear mapping of the original point cloud [6]. 5 Division of Engineering and Applied Sciences, California Institute of Technology, Pasadena, California, USA. I. INTRODUCTION Recent technological advances in the ﬁeld of robotics lead to the deployment of Micro Aerial Vehicles (MAVs) in challenging environments such as underground mine naviga- tion [1], search and rescue mission [2], large infrastructure inspections [3], to name a few. MAVs have the potential to navigate rapidly in complex, unpredictable, and diverse subterranean environments for collecting various data types or saving lives. Despite the broad spectrum of technological advances in the ﬁeld, autonomous navigation in subterranean envi- ronments is still an ongoing quest. Subterranean environ- ments face unique challenges, including irregular geological structures, complex geometry, lack of Global Positioning System (GPS) signal, the general absence of illumination, constrained passages, and unpredictable topology (see Fig- ure 1 for illustration). Complex geometry and the presence of multiple junctions hinder the navigation mission. Junction detection is a critical task to enable safer and more optimal overall mission execution. For this task, a 2D Lidar equipped on the MAVs is used for sensing the environment. The This work has been partially funded by the European Unions Horizon 2020 Research and Innovation Programme under the Grant Agreement No. 730302 SIMS. Funding from Vinnova in the project AI Factory for Railway is also acknowledged. Corresponding author’s e-mail: sinsha@ltu.se y gy 4 The author is with Jet Propulsion Laboratory California Institute of Technology Pasadena, CA, 91109. Unsupervised Learning for Subterranean Junction Recognition Based on 2D Point Cloud A key feature of our proposed framework is that the number of zero eigenvalues of the Laplacian reveals the number of connected components of the constructed similarity graph. The third step is to use the K-means clustering algorithm [7] to partition the 2D point cloud. Hence, our proposed method partitions the transformed 2D point cloud into connected components or clusters for identifying the number of junctions. 3 Department of Civil, Environmental and Natural Resources Engineering Lule˚a University of Technology, Lule˚a SE-97187, Sweden. 2 Department of Computer Science, University of Massachusetts Lowell, MA, USA 3 g p g 1 Robotics and AI Team, Department of Computer, Electrical and Space Engineering, Lule˚a University of Technology, Lule˚a SE-97187, Sweden B. Contributions Based on the state-of-the-art mentioned above, we list the main contributions of this work in the following. y Towards using visual feedback for junction detection, in [15], a Convolutional Neural Network (CNN) binary classiﬁer was developed for outdoor road junction detection. The method is experimentally evaluated on a commercially available MAV Bebop 2 from Parrot. In [16], a road in- tersection detection module has been proposed as binary classiﬁcation, using the Long-Term Recurrent Convolutional Network (LRCN) architecture to identify relative changes in outdoor features and eventually detecting intersections. In [17], the authors proposed an architecture that combines CNN, Bidirectional LSTM [16] and Siamese [18] style distance function learning for junction recognition in videos. In this work, the authors evaluated their approach on different data-sets spanning various experimental scenarios. Towards junction detection in underground mines, in [19], the authors proposed transfer learning with AlexNet [20]. The method is limited to identifying only three different junctions and relied on the looking forward camera heavily. Moreover, these methods mostly consider binary classiﬁers, while in real-life scenarios more complex types of junctions exist and the junction recognition should recognize the different types of junctions. Additionally, most of these works have been evaluated and tuned in out-door environments and with proper illumination, i.e., using rich data about the surrounding of the platforms. The ﬁrst contribution of this work is to develop a new framework for recognizing subterranean junctions using spectral clustering, which is a principled unsupervised learn- ing technique. The proposed method allows us to uncover intrinsic structures from the 2D point cloud extracted from a lidar. Unlike previous techniques, the proposed method applies to various settings without requiring any prior knowl- edge on the number of junctions and their topology. Fur- thermore, our method does not require any pre-training and work well when working with small sample sizes. Hence, our introduced framework is suitable for a wide variety of settings with complex geometry. In addition to our algorithmic contributions, the second contribution originates from a comprehensive evaluation of the proposed method in simulation environments with com- plex geometry and data-sets collected from the autonomous ﬂight of the MAV in real underground mines in Sweden. As we will see, the obtained results demonstrate the effective- ness of our proposed framework on situational awareness of real-world subterranean environments. The rest of the article is structured as follows. A. Background & Motivation Navigation based on visual sensors or 2D/3D lidar has received signiﬁcant attention in recent years in different application scenarios, e.g., [8], [9]. One of the essential ca- pabilities for successful autonomous navigation in unknown and uncertain environments is environmental awareness, such as identifying environment geometries, obstacles, junctions, dead-ends, etc. Several works have considered the use of visual sensors, lidar, and satellite images for detecting road intersections in uncertain environments [10]. For example, the previous work [11] developed an automatic road junction detection technique using airborne lidar data as well as the direction and width of road branches. Synthetic aperture radar systems are used for detecting L- and T- shape road junctions Fig. 1. Photos of an underground mine in Sweden illustrating complex geometry, irregular geological structure, etc. Fig. 1. Photos of an underground mine in Sweden illustrating complex geometry, irregular geological structure, etc. in [12] based on Markov optimization. The authors of [13] developed a machine learning technique based on the 3D point cloud generated by a laser rangeﬁnder to detect road junctions. The introduced method was evaluated on two different data-sets from Germany and Brazil roads. The studied frames were classiﬁed into two groups of “junctions” or “roads”. In [14], junction detection is used to improve map localization in areas with weak or erroneous GPS signals such as forests or urban canyon. The proposed method detected crossroads using the lidar data by identifying free spaces between obstacles. However, most of these methods cannot be used in mines, as the map of such environments is not readily available, and existing methods are only suitable for certain shapes of junctions. Therefore, the prior work on using the lidar data for identifying subterranean junctions has demonstrated only limited success. sion [21]. Many existing clustering methods such as K-means clustering learn hidden structures from data points that are connected within convex boundaries. However, in this work, our goal is to develop a framework for identifying junctions without posing restrictive assumptions. Thus, we propose to use a family of unsupervised learning methods known as spectral clustering [22]. The superior performance of spectral clustering stems from the ability to exploit non-linear pairwise similarities between data points. As a result, spectral clustering has found applications in many domains, including image segmentation and genomic data analysis [23], [24]. II. PROBLEM STATEMENT (1) (1) The next task is to compute a spectral embedding of the original data points x1, . . . , xn. Using the adjacency matrix W, we form the normalized Laplacian matrix L ∈Rn×n in the following form: The next task is to compute a spectral embedding of the original data points x1, . . . , xn. Using the adjacency matrix W, we form the normalized Laplacian matrix L ∈Rn×n in the following form: Fig. 2. Top-view concept image of a subterranean environment with multiple branches, while body ﬁxed frame of the MAV is depicted by B. L = I −D−1/2WD−1/2, (2) (2) where I is the identity matrix and D ∈Rn×n is a diagonal degree matrix, i.e., D(i, i) = Pn j=1 W(i, j). Hence, the matrix D−1/2 can be efﬁciently formed by computing the inverse square root of the entries of D on the main diagonal. The eigenvalue decomposition of L provides valuable insights about the structure of the similarity graph G. First, it is worth pointing out that every eigenvalue of the Laplacian matrix is non-negative, i.e., the matrix L is Positive Semi- Deﬁnite (PSD). Thus, we can represent all n eigenvalues of L in non-decreasing order as follows: 0 = λ1 ≤λ2 ≤. . . ≤λn. (3) (3) Furthermore, the number of connected components in the graph G is equal to the multiplicity of the 0 eigenvalue [27]. Hence, spectral clustering does not require any prior knowl- edge of the number of components. Fig. 2. Top-view concept image of a subterranean environment with multiple branches, while body ﬁxed frame of the MAV is depicted by B. B. Contributions Section II provides a brief overview of detecting junctions in sub- terranean environments. Section III presents our proposed methodology for identifying the number of intersections or junctions. Then, in Section IV, we evaluate the proposed method on various simulated and real-world data-sets. Fi- nally, Section V concludes this article by summarizing our ﬁndings and offering some future research directions to improve our framework further. In this work, we propose to identify the number of junctions in uncertain subterranean environments using an unsupervised learning framework. Cluster analysis is one of the most fundamental problems in machine learning for ﬁnding groups of similar data points without any supervi- II. PROBLEM STATEMENT II. PROBLEM STATEMENT utilize clustering techniques in order to identify the number of walls and their position. While one may attempt to use simple unsupervised learning techniques, such as K-means clustering, a challenge that we face in our problem of interest and it will be further analyzed in Section II, is to handle complex and non-linear structures. Hence, we propose to use spectral clustering techniques that will allow us to capture such complex structures. This article considers lightweight aerial scouts, which are disposable low-cost robots with the task of fast exploration of unknown subterranean environments for collecting data and provide preliminary feedback for higher level mission planner. These robots should be able to effectively navigate in such challenging environments based on the collected local information. It should be highlighted that most of the works investigating MAV navigation in subterranean environments consider high end platforms [25]. In fact, these methods rely on a global map or are evaluated in tunnels without multiple branches [26]. However, uncertainties and drift over time in localization or map will result in wrong directions, as the concept image depicts in Figure 2. In this case, the underground tunnel has multiple branches and existing methods have limited success. Thus, the objective of this article is to propose an effective software perspective to detect the number of junctions in man-made tunnels based on the local point cloud information which is obtained from 2D lidars. Thus, our method provides navigation capabilities with resource-constrained solutions. The point cloud is in a body ﬁxed frame of the MAV, and the MAV is centered of the point cloud. In the following, we explain the three main steps of spectral clustering in detail. Given the data matrix X, we ﬁrst construct a similarity graph. We denote the undirected similarity graph by G = (V, E, W), where we have a set of n nodes V that are interconnected by a set of edges E. The matrix W ∈Rn×n is the adjacency matrix of this graph such that W(i, j) ≥0 and W is symmetric. A common approach to construct the similarity graph is to use the RBF as a measure of similarity. For some user-deﬁned σ > 0, we compute the pairwise similarity between xi and xj (recall that xi refers to the i-th row of X): W(i, j) = exp(−σ∥xi −xj∥2 2), ∀i, j ∈{1, . . . , n}. III. JUNCTION DETECTION Let k be the estimated number of connected components using the above eigenvalue decomposition of the Laplacian. The subsequent task is to partition the original data points x1, . . . , xn into k clusters. To this end, we compute the eigenvectors associated with the k smallest eigenvalues of L. Next, we form a new matrix U ∈Rn×k by concatenating these k eigenvectors column-wise. Hence, we can view the i- th row of U, i.e., ui, as the spectral embedding of the original data point xi. We can thus look for the connected data points in the transformed k-dimensional data-set u1, . . . , un (instead of the original feature space R2). As mentioned before, this step is essential for many complex data-sets as the original data points may not be linearly separable [28]. In this section, we discuss our proposed methodology for identifying junctions in underground environments by utilizing the acquired 2D point clouds, extracted from a 2D lidar. Let X ∈Rn×2 be a data matrix comprising of n data points. Thus, each row of X corresponds to one data point in R2 from the given 2D point cloud. Without any prior information, our goal is to identify the number of junctions, which is equal to the number of walls. In this paper we target the junction recognition with the use of the point cloud generated by only one revolution of the lidar. A wall will be registered as a set of data points with geographical proximity. The goal of this paper is therefore to The last step is to perform the K-means clustering algo- rithm on the non-linearly transformed data points u1, . . . , un. The corresponding optimization problem has the following form: B. Experimental Data-sets , xn in R2 Parameter: σ > 0 Result: Number of junctions and clustering X 1 Form the adjacency matrix W ∈Rn×n using a non-linear similarity function, such that Wij = exp(−σ∥xi −xj∥2 2); 2 Form the normalized Laplacian matrix L = I −D−1/2WD−1/2; 3 Compute the eigenvalue decomposition of L and sort the eigenvalues in non-decreasing order; 4 Find the number of zero eigenvalues k; 5 Perform K-means++ clustering on the top k eigenvectors u1, . . . , un in Rk; Algorithm 1: Junction Detection via 2D Point Cloud Data: A set of n data points x1 x in R2 Algorithm 1: Junction Detection via 2D Point Cloud Result: Number of junctions and clustering X 1 Form the adjacency matrix W ∈Rn×n using a non-linear similarity function, such that Wij = exp(−σ∥xi −xj∥2 2); In the second case, the MAV autonomously navigates in an underground mine, however the width of the area is larger than the ﬁrst case and the walls have wet surfaces. The areas with three and four junctions are chosen for evaluating the proposed method as depicted in Figure 6. Conditions such as the large width and wet wall surfaces result to in a sparse point cloud from 2D lidar range measurements. As it can be seen in Figure 7, our proposed method detects the number of junctions and walls correctly. Future work should investigate the development of robust methodologies, as dust can be misclassiﬁed as junctions. However, in our case, the areas we studied have negligible amount of dust. 2 Form the normalized Laplacian matrix L = I −D−1/2WD−1/2; 3 Compute the eigenvalue decomposition of L and sort the eigenvalues in non-decreasing order; 3 Compute the eigenvalue decomposition of L and sort the eigenvalues in non-decreasing order; g g 4 Find the number of zero eigenvalues k; g g 4 Find the number of zero eigenvalues k; 5 Perform K-means++ clustering on the top k eigenvectors u1, . . . , un in Rk; B. Experimental Data-sets The data-sets have been collected from autonomous nav- igation of the MAV in two different underground mines, while the platform is equipped with a 2D lidar [1], [26]. The ﬁrst location is an underground tunnel with a few branches, and the second one is an underground mine with multiple branches. Both environments are located in Sweden. The width and height of the ﬁrst and second areas are approximately 3.5 m × 3 m and 6 m × 4 m respectively. min C f(C, U) = n X i=1 min c∈C ∥ui −c∥2 2, (4) (4) where C represents a set of k cluster centroids or repre- sentatives. Solving this optimization problem is known to be NP-hard and thus computationally intractable. However, there are approximation algorithms, such as the K-means++ algorithm, to solve this problem efﬁciently. As a result, we will ﬁnd the partitioning of the embedded data points u1, . . . , un into k clusters. Note that this assignment can be easily translated to the original data points x1, . . . , xn. The proposed algorithm is summarized in Algorithm 1. Figure 4 depicts the irregular geometry of the ﬁrst envi- ronment, while three speciﬁc areas are chosen to evaluate the proposed method, the ﬁrst and second areas are tunnels with unstructured walls, and the third area has three junctions. The point cloud extracted from 2D lidar is fed to our proposed method and Figure 5 shows the obtained results. The method detects the number of junctions correctly, even though the point cloud extracted from experimental data- set is sparse compare to simulation results. Additionally, it should be highlighted that the tunnel without any branch considers two junctions as the platform has two options to navigate. In the future work, the obtained junctions should be matched to avoid counting same one multiple times and the proposed method should be evaluated in the closed loop with navigation and mission planner modules. Algorithm 1: Junction Detection via 2D Point Cloud Data: A set of n data points x1, . . . A. Simulation Evaluations This article proposed a novel method for the problem of junction detection in subterranean environments. The pro- posed method uses a non-linear pairwise similarity function to form a graph and ﬁnd the number of connected com- ponents. The proposed method is computationally efﬁcient and detects the number of junctions and walls with average computation time of 0.2 s, while the detected walls can be used for wall following methods and number of junctions for local or high level planners. The method is successfully evaluated on multiple random generated 2D point clouds and extracted point clouds from autonomous navigation of the MAV in underground mines. Future works include matching the detected junctions for loop closure and to avoid counting same junctions multiple time, and evaluation of the proposed method in closed loop of navigation and planning components. The proposed unsupervised learning approach in this article will be also extended in the future work to handle 3D point clouds. It should be highlighted that access to subterranean envi- ronments is typically limited. Thus, initially for evaluating the proposed method with complex geometry, random range measurements from 2D lidars are generated in the MAT- LAB [29]. The 2D lidar is considered with maximum range of 15 m and with 360 measurements for each revolution. The obtained point cloud from 2D lidar measurements are fed to the proposed method, the number of junctions are indicated, and the walls are clustered and color coded. All simulations have been performed on a single core on a computer with an Intel Core i7-6600U CPU, 2.6GHz and 8GB RAM and σ = 1.5, while the average computation time for all scenarios is 0.2 s. Figure 3 depicts the obtained results, while the number of junctions are written above the ﬁgures, and the MAV is considered at the center of the point cloud. The proposed method successfully detects all junctions and walls when considering various types of complex geometry for the simulated 2D point clouds. Fig. 3. Simulation results for identifying the number of junctions in different scenarios using spectral clustering. Each wall is indicated with a different color and the number of junctions is written above each ﬁgure (best viewed in color). Fig. 3. Simulation results for identifying the number of junctions in different scenarios using spectral clustering. REFERENCES Intelligent Transportation Systems (ITSC). IEEE, 2011, pp. 2001– 2008. [15] S. Kumaar, S. Mannar, S. Omkar et al., “Juncnet: A deep neural network for road junction disambiguation for autonomous vehicles,” arXiv preprint arXiv:1809.01011, 2018. [1] S. S. Mansouri, P. Karvelis, C. Kanellakis, D. Kominiak, and G. Niko- lakopoulos, “Vision-based mav navigation in underground mine using convolutional neural network,” in IECON 2019-45th Annual Confer- ence of the IEEE Industrial Electronics Society, vol. 1. IEEE, 2019, pp. 750–755. [16] D. Bhatt, D. Sodhi, A. Pal, V. Balasubramanian, and M. Krishna, “Have i reached the intersection: A deep learning-based approach for intersection detection from monocular cameras,” in 2017 IEEE/RSJ International Conference on Intelligent Robots and Systems (IROS). IEEE, 2017, pp. 4495–4500. [2] T. Tomic, K. Schmid, P. Lutz, A. Domel, M. Kassecker, E. Mair, I. L. Grixa, F. Ruess, M. Suppa, and D. Burschka, “Toward a fully autonomous uav: Research platform for indoor and outdoor urban search and rescue,” IEEE robotics & automation magazine, vol. 19, no. 3, pp. 46–56, 2012. [17] A. Kumar, G. Gupta, A. Sharma, and K. M. Krishna, “Towards view- invariant intersection recognition from videos using deep network ensembles,” in 2018 IEEE/RSJ International Conference on Intelligent Robots and Systems (IROS). IEEE, 2018, pp. 1053–1060. [3] S. S. Mansouri, C. Kanellakis, E. Fresk, D. Kominiak, and G. Niko- lakopoulos, “Cooperative coverage path planning for visual inspec- tion,” Control Engineering Practice, vol. 74, pp. 118–131, 2018. [18] W.-t. Yih, K. Toutanova, J. C. Platt, and C. Meek, “Learning discrim- inative projections for text similarity measures,” in Proceedings of the ﬁfteenth conference on computational natural language learning. Association for Computational Linguistics, 2011, pp. 247–256. [4] U. Von Luxburg, “A tutorial on spectral clustering,” Statistics and computing, vol. 17, no. 4, pp. 395–416, 2007. ssociation for Computational Linguistics, 2011, pp. 247–256. [5] F. Pourkamali-Anaraki and S. Becker, “Improved ﬁxed-rank Nystr¨om approximation via QR decomposition: Practical and theoretical as- pects,” Neurocomputing, vol. 363, pp. 261–272, 2019. [19] S. S. Mansouri, P. Karvelis, C. Kanellakis, A. Koval, and G. Niko- lakopoulos, “Visual subterranean junction recognition for mavs based on convolutional neural networks,” in IECON 2019-45th Annual Conference of the IEEE Industrial Electronics Society, vol. 1. IEEE, 2019, pp. 192–197. [6] N. Tremblay and A. Loukas, “Approximating spectral clustering via sampling: a review,” in Sampling Techniques for Supervised or Unsupervised Tasks, 2020, pp. 129–183. [20] A. Krizhevsky, I. Sutskever, and G. E. REFERENCES Hinton, “Imagenet classiﬁcation with deep convolutional neural networks,” pp. 1097–1105, 2012. [Online]. Available: http://papers.nips.cc/paper/ 4824-imagenet-classiﬁcation-with-deep-convolutional-neural-networks. pdf [7] F. Pourkamali-Anaraki and S. Becker, “Preconditioned data sparsiﬁ- cation for big data with applications to PCA and K-means,” IEEE Transactions on Information Theory, vol. 63, no. 5, pp. 2954–2974, 2017. [8] C. Kanellakis and G. Nikolakopoulos, “Survey on computer vision for uavs: Current developments and trends,” Journal of Intelligent & Robotic Systems, pp. 1–28, 2017. p [21] P. Chen and L. Wu, “Revisiting spectral graph clustering with gener- ative community models,” in 2017 IEEE International Conference on Data Mining (ICDM), 2017, pp. 51–60. g pp [22] A. Ng, M. Jordan, and Y. Weiss, “On spectral clustering: Analysis and an algorithm,” in Advances in neural information processing systems, 2002, pp. 849–856. [9] T. D. Barfoot, C. McManus, S. Anderson, H. Dong, E. Beerepoot, C. H. Tong, P. Furgale, J. D. Gammell, and J. Enright, “Into dark- ness: Visual navigation based on a lidar-intensity-image pipeline,” in Robotics Research. Springer, 2016, pp. 487–504. [23] F. Tung, A. Wong, and D. Clausi, “Enabling scalable spectral cluster- ing for image segmentation,” Pattern Recognition, vol. 43, no. 12, pp. 4069–4076, 2010. [10] D. Chaudhuri, N. Kushwaha, and A. Samal, “Semi-automated road detection from high resolution satellite images by directional mor- phological enhancement and segmentation techniques,” IEEE journal of selected topics in applied earth observations and remote sensing, vol. 5, no. 5, pp. 1538–1544, 2012. [24] B. Wang, J. Zhu, E. Pierson, D. Ramazzotti, and S. Batzoglou, “Visualization and analysis of single-cell rna-seq data by kernel-based similarity learning,” Nature methods, vol. 14, no. 4, p. 414, 2017. [25] C. Papachristos, S. Khattak, F. Mascarich, and K. Alexis, “Au- tonomous navigation and mapping in underground mines using aerial robots,” in 2019 IEEE Aerospace Conference. IEEE, 2019, pp. 1–8. [11] Z. Chen, C. Liu, and H. Wu, “A higher-order tensor voting-based approach for road junction detection and delineation from airborne lidar data,” ISPRS journal of photogrammetry and remote sensing, vol. 150, pp. 91–114, 2019. [26] S. S. Mansouri, M. Casta˜no, C. Kanellakis, and G. Nikolakopoulos, “Autonomous mav navigation in underground mines using darkness contours detection,” in International Conference on Computer Vision Systems. Springer, 2019, pp. 164–174. [12] M. Negri, P. Gamba, G. Lisini, and F. Tupin, “Junction-aware ex- traction and regularization of urban road networks in high-resolution sar images,” IEEE Transactions on Geoscience and Remote Sensing, vol. 44, no. 10, pp. A. Simulation Evaluations Each wall is indicated with a different color and the number of junctions is written above each ﬁgure (best viewed in color). Fig. 4. The images of the MAV navigation in three different environments of an underground tunnel in Sweden. Fig. 4. The images of the MAV navigation in three different environments of an underground tunnel in Sweden. Fig. 5. The detected walls and junctions from a 2D point cloud utilizing the proposed framework, with experimental data-set from autonomous MAV navigation in an underground tunnel, while each wall is indicated with a different color. Fig. 5. The detected walls and junctions from a 2D point cloud utilizing the proposed framework, with experimental data-set from autonomous MAV navigation in an underground tunnel, while each wall is indicated with a different color. Fig. 6. The images of the MAV navigation in three different environments of an underground mine in Sweden. Fig. 6. The images of the MAV navigation in three different environments of an underground mine in Sweden. Fig. 7. The detected walls and junctions from 2D point cloud using the proposed method, with experimental data-set from autonomous MAV navigation in an underground mine, while each wall is indicated with a different color. Fig. 7. The detected walls and junctions from 2D point cloud using the proposed method, with experimental data-set from autonomous MAV navigation in an underground mine, while each wall is indicated with a different color. REFERENCES 2962–2971, 2006. y p g pp [27] A. Marsden, “Eigenvalues of the laplacian and their relationship to the connectedness of a graph,” University of Chicago, REU, 2013. [13] D. Habermann, C. E. Vido, F. S. Os´orio, and F. Ramos, “Road junction detection from 3d point clouds,” in 2016 International Joint Conference on Neural Networks (IJCNN). IEEE, 2016, pp. 4934– 4940. [28] R. Langone and J. Suykens, “Fast kernel spectral clustering,” Neuro- computing, vol. 268, pp. 27–33, 2017. [29] MATLAB, version 9.6. (R2019a). Natick, Massachusetts: The MathWorks Inc., 2019. [14] A. Mueller, M. Himmelsbach, T. Luettel, F. v. Hundelshausen, and H.- J. Wuensche, “Gis-based topological robot localization through lidar crossroad detection,” in 2011 14th International IEEE Conference on
https://openalex.org/W4362427040	https://aacr.figshare.com/articles/journal_contribution/Supplementary_Methods_from_The_Prognostic_Significance_of_Various_13q14_Deletions_in_Chronic_Lymphocytic_Leukemia/22443353/1/files/39894317.pdf	English	null	Supplementary Methods from The Prognostic Significance of Various 13q14 Deletions in Chronic Lymphocytic Leukemia	null	2,023	cc-by	569	Supplementary Methods and Results Supplementary Methods and Results The methods for algorithmic aCNA calling: Median-smoothened copy number data based on SNP 6.0 arrays were exported from dChipSNP and were transformed by raising all values to the 0.25 power to approximately stabilize the variance. Then, within each subject, the transformed normal DNA value was subtracted from the transformed tumor DNA value to create a subject- level difference summary. Using this difference summary, we then constructed running average and running variance statistics centered on each SNP, based on uniformly weighted windows of size 30 (for the average) and 120 (for the variance). We next constructed a pseudo Z-score as Z = square root (30) x A/square root (V), where A is the running average and V is the running variance. Initial lesion calls were based on the rule Z<-12 and A<-0.12 for losses (equivalent to ~CN estimates of ≤1.3) and a minimal lesion length of 25. These initial calls were then refined by bridging small gaps between consecutive lesion calls to mitigate against the effect of artificial lesion breaks due to array background fluctuations. Gaps between consecutive losses were bridged if they were up to 500 SNP positions in length, and the mean of the Z values within the gap was less than -2; thus only nominal statistical significance is required to bridge small gaps between lesions that had already been detected at high stringency thresholds. No further review or change to aCNA nominations were made and the aCNAs-losses nominated through this algorithm were finally combined with aCNA-gains (contributing to ~13% of all aCNA in CLL) previously identified through visual inspection of heatmaps as described and resulted in the complexity score-algo. Correlations of CLL FISH and SNP 6.0 array profiling results: Correlations of CLL FISH and SNP 6.0 array profiling results: Correlations of CLL FISH and SNP 6.0 array profiling results: To test our previously externally validated (see Methods) analytical approach for analysis of SNP 6.0 array data in our CLL cohort, we calculated the sensitivity and specificity for detection of conventional 13q14 deletions by SNP 6.0 profiling (13q14 deletions were selected because many are <1Mb or ~1Mb in length) as compared with CLL FISH and CLL-FISH-25 (FISH findings present in ≥25% of nuclei) data as the gold standard. Sensitivity and specificity for SNP 6.0 profiling were 89% and 95% using all CLL FISH results and 98% and 92% for CLL-FISH-25 results, respectively. We also calculated data for del17p (sensitivity and specificity for SNP 6.0 profiling were 92% and 99% using all CLL FISH results and 100% and 99% for CLL-FISH-25 results, respectively) and del11q (sensitivity and specificity for SNP 6.0 profiling were 73% and 99% using all CLL FISH results and 92% and 99% for CLL-FISH-25 results, respectively). These results confirm very high specificity of visual SNP 6.0 profiling- based aCNA calling as compared with FISH in CLL and also confirm that SNP 6.0 profiling is less sensitive for lesions present in <25% of the input DNA (range of % FISH positive nuclei for 11q lesions that were SNP 6.0 profiling negative was 9%-40%, mean=19%). We also detected relative (pseudo) gains at chromosome 7 at physical position 38.27-38.36 in all 255 CLL CD19+ samples compared with the averaged paired normal samples due to loss of chromosomal material at the T-cell receptor gamma- locus as a consequence of T-cell receptor gene rearrangements, thus providing additional high confidence in our analytical approach.
https://openalex.org/W3007921212	https://www.nature.com/articles/s41467-020-14904-x.pdf	English	null	Author Correction: Probing the physical limits of reliable DNA data retrieval	Nature communications	2,020	cc-by	238	© The Author(s) 2020 Author Correction: Probing the physical limits of reliable DNA data retrieval Lee Organick, Yuan-Jyue Chen, Siena Dumas Ang, Randolph Lopez, Xiaomeng Liu, Karin Strauss & Luis Ceze Correction to: Nature Communications https://doi.org/10.1038/s41467-020-14319-8, published online The original version of this Article omitted the following from the Acknowledgements: This research was supported by a sponsored research agreement and gifts from Microsoft and DARPA under the Molecular Informatics Program. This has now been corrected in both the PDF and HTML versions of the Article. This has now been corrected in both the PDF and HTML versions of the Article. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. NATURE COMMUNICATIONS \| (2020) 11:1080 \| https://doi.org/10.1038/s41467-020-14904-x \| www.nature.com/naturecommunications
https://openalex.org/W2357750051	https://europepmc.org/articles/pmc4893543?pdf=render	English	null	Predominance and high antibiotic resistance of the emerging <i>Clostridium difficile</i> genotypes NAP <sub>CR1</sub> and NAP9 in a Costa Rican hospital over a 2-year period without outbreaks	Emerging microbes & infections	2,016	cc-by	5,410	OPEN OPEN Emerging Microbes and Infections (2016) 5, e42; doi:10.1038/emi.2016.38 t / i Emerging Microbes and Infections (2016) 5, e42; doi:10.1038/emi.2016.38 www.nature.com/emi www.nature.com/emi ORIGINAL ARTICLE Laboratory of Research in Anaerobic Bacteriology and Research Center in Tropical Diseases, University of Costa Rica, San José 11501-2060, Costa Rica Correspondence: E Rodríguez-Cavallini E-mail: evelynrodricava@gmail.com Received 8 October 2015; revised 18 December 2015; accepted 7 January 2016 Predominance and high antibiotic resistance of the emerging Clostridium difﬁcile genotypes NAPCR1 and NAP9 in a Costa Rican hospital over a 2-year period without outbreaks Diana López-Ureña, Carlos Quesada-Gómez, Mónica Montoya-Ramírez, María del M Teresita Somogyi, César Rodríguez and Evelyn Rodríguez-Cavallini Diana López-Ureña, Carlos Quesada-Gómez, Mónica Montoya-Ramírez, María del Mar Gamboa-Coronado, Teresita Somogyi, César Rodríguez and Evelyn Rodríguez-Cavallini Clostridium difﬁcile is the major causative agent of nosocomial antibiotic-associated diarrhea. In a 2009 outbreak of C. difﬁcile- associated diarrhea that was recorded in a major Costa Rican hospital, the hypervirulent NAP1 strain (45%) predominated together with a local genotype variant (NAPCR1, 31%). Both strains were ﬂuoroquinolone-resistant and the NAPCR1 genotype, in addition, was resistant to clindamycin and rifampicin. We now report on the genotypes and antibiotic susceptibilities of 68 C. difﬁcile isolates from a major Costa Rican hospital over a 2-year period without outbreaks. In contrast to our previous ﬁndings, no NAP1 strains were detected, and for the ﬁrst time in a Costa Rican hospital, a signiﬁcant fraction of the isolates were NAP9 strains (n = 14, 21%). The local NAPCR1 genotype remained prevalent (n = 18, 26%) and coexisted with 14 strains (21%) of classic hospital NAP types (NAP2, NAP4, and NAP6), eight new genotypes (12%), four environmental strains classiﬁed as NAP10 or NAP11 (6%), three strains without NAP designation (4%) and seven non-toxigenic strains (10%). All 68 strains were resistant to ciproﬂoxacin, 88% were resistant to clindamycin and 50% were resistant to moxiﬂoxacin and rifampicin. Metronidazole and vancomycin susceptibilities were universal. The NAPCR1 and NAP9 strains, which have been associated with more severe clinical infections, were more resistant to antibiotics than the other strains. Altogether, our results conﬁrm that the epidemiology of C. difﬁcile infection is dynamic and that A−B+ strains from the NAP9 type are on the rise not only in the developed world. Moreover, our results reveal that the local NAPCR1 strains still circulate in the country without causing outbreaks but with equally high antibiotic-resistance rates and levels. q y g g Microbes and Infections (2016) 5, e42; doi:10.1038/emi.2016.38; published online 11 May 2016 Keywords: antibiotic resistance; Costa Rica; emerging Clostridium difﬁcile; NAPCR1; NAP9 INTRODUCTION Furthermore, variations in the combined repetitive oligopeptide domain of TcdB have been associated with increases in the virulence of epidemic strains.9 A minority of toxigenic C. difﬁcile strains also produce a third toxin, known as binary toxin Clostridium difﬁcile toxin (CDT), that is encoded by cdtA and cdtB at the CdtLoc locus and is separated from the PaLoc.10 CDT is composed of an ADP- ribosyltransferase that blocks actin polymerization and a binding component that is involved in toxin delivery.11 Although it is believed that CDT affects the cytoskeleton and enhances the adhesion and colonization of C. difﬁcile,10 its role in CDI remains controversial.12 remains controversial because, as Cartman et al.8 demonstrated, these deletions have little effect in signiﬁcantly increasing toxin production. Furthermore, variations in the combined repetitive oligopeptide domain of TcdB have been associated with increases in the virulence of epidemic strains.9 A minority of toxigenic C. difﬁcile strains also produce a third toxin, known as binary toxin Clostridium difﬁcile toxin (CDT), that is encoded by cdtA and cdtB at the CdtLoc locus and is separated from the PaLoc.10 CDT is composed of an ADP- ribosyltransferase that blocks actin polymerization and a binding component that is involved in toxin delivery.11 Although it is believed that CDT affects the cytoskeleton and enhances the adhesion and colonization of C. difﬁcile,10 its role in CDI remains controversial.12 Clostridium difﬁcile has become the leading cause of nosocomial diarrhea in adults.1 Clinical manifestations of C. difﬁcile infections (CDI) vary from asymptomatic to fulminant colitis, including pseudomembranous colitis (PMC) or antibiotic-associated diarrhea. There may be complications, such as toxic megacolon, colonic perforation and a few extraintestinal manifestations.2 Most disease-causing isolates of C. difﬁcile produce one or two toxins, i.e., TcdA and TcdB. These toxins enter intestinal epithelial cells and glycosylate various families of cytoplasmic GTPases,3 which leads to actin depolymerization with the loss of internal cell architecture, apoptosis, villus destruction and a mucosal inﬂammatory response.4,5 The genes encoding toxins A and B (tcdA and tcdB) are part of a so-called pathogenicity locus (PaLoc), which also includes tcdR (a sigma factor that promotes the transcription of both of the toxin genes), tcdE (a potential holin) and tcdC (a potential negative regulator of tcdA and tcdB).6 Although deletions in tcdC have been claimed to favor TcdA and TcdB hypersecretion,7 this conjecture Various methods have been used to type C. difﬁcile strains. INTRODUCTION remains controversial because, as Cartman et al.8 demonstrated, these deletions have little effect in signiﬁcantly increasing toxin production. Furthermore, variations in the combined repetitive oligopeptide domain of TcdB have been associated with increases in the virulence of epidemic strains.9 A minority of toxigenic C. difﬁcile strains also produce a third toxin, known as binary toxin Clostridium difﬁcile toxin (CDT), that is encoded by cdtA and cdtB at the CdtLoc locus and is separated from the PaLoc.10 CDT is composed of an ADP- ribosyltransferase that blocks actin polymerization and a binding component that is involved in toxin delivery.11 Although it is believed that CDT affects the cytoskeleton and enhances the adhesion and colonization of C. difﬁcile,10 its role in CDI remains controversial.12 Various methods have been used to type C. difﬁcile strains. While pulsed-ﬁeld gel electrophoresis (PFGE) is predominantly used in North America, ribotyping by PCR is most often used in Europe. PFGE NAP1 strains correspond to ribotype 027 and harbor toxin A, toxin B, CDT, a 18-bp mutation in tcdC and a point mutation in this gene at position 117. In contrast, most TcdA-negative and remains controversial because, as Cartman et al.8 demonstrated, these deletions have little effect in signiﬁcantly increasing toxin production. Furthermore, variations in the combined repetitive oligopeptide domain of TcdB have been associated with increases in the virulence of epidemic strains.9 A minority of toxigenic C. difﬁcile strains also produce a third toxin, known as binary toxin Clostridium difﬁcile toxin (CDT), that is encoded by cdtA and cdtB at the CdtLoc locus and is separated from the PaLoc.10 CDT is composed of an ADP- ribosyltransferase that blocks actin polymerization and a binding component that is involved in toxin delivery.11 Although it is believed that CDT affects the cytoskeleton and enhances the adhesion and colonization of C. difﬁcile,10 its role in CDI remains controversial.12 Various methods have been used to type C. difﬁcile strains. While pulsed-ﬁeld gel electrophoresis (PFGE) is predominantly used in North America, ribotyping by PCR is most often used in Europe. PFGE NAP1 strains correspond to ribotype 027 and harbor toxin A, toxin B, CDT, a 18-bp mutation in tcdC and a point mutation in this gene at position 117. In contrast, most TcdA-negative and remains controversial because, as Cartman et al.8 demonstrated, these deletions have little effect in signiﬁcantly increasing toxin production. naerobic Bacteriology and Research Center in Tropical Diseases, University of Costa Rica, San José 11501-2060, Costa Rica z-Cavallini RESULTS No outbreaks were reported from October 2010 to August 2012 in the hospital under study (Figure 1). Our genotyping procedure revealed that 28 isolates were positive for tcdA and tcdB, negative for cdtB and carried wild-type tcdC; these results were expected for classic hospital strains. Eighteen isolates exhibited the characteristic NAPCR1 pattern (i.e., tcdA+, tcdB+, cdtB−and tcdC with a deletion), 14 exhibited the A −B+ strain pattern (i.e., tcdA−, tcdB+, cdtB−and wild-type tcdC) and 1 isolate exhibited all 3 toxins and a deletion in tcdC. Seven isolates were non-toxigenic (Table 1). Isolates and bacteriological procedures difﬁcile (CD) toxins and were collected in a major hospital in Costa Rica from 2010 to 2012. The cases were diagnosed based on clinical evidence and toxin detection. Emerging Microbes and Infections Antibiotic susceptibility testing The minimum inhibitory concentrations (MIC) for clindamycin, ciproﬂoxacin, moxiﬂoxacin, rifampicin, metronidazole and vancomy- cin were determined using E-test strips (AB bioMérieux, Askim, Sweden) and Brucella agar plates containing 5% blood, 1 μg/mL vitamin K and 5 μg/mL hemin according to established guidelines.22 For susceptibility categorization, we used the resistance breakpoints recommended by the CLSI;23 i.e., 8 μg/mL for clindamycin, cipro- ﬂoxacin and moxiﬂoxacin and 32 μg/mL for metronidazole. For rifampicin and vancomycin, we adopted the breakpoints recom- mended in the document M100-S21 for Staphylococcus aureus because no values have been deﬁned for anaerobic bacteria; these values were 4 μg/mL for rifampicin and 16 μg/mL for vancomycin. Isolates and bacteriological procedures Isolates and bacteriological procedures so ates a d bacte o og ca p ocedu es This study included 68 C. difﬁcile isolates that were obtained from the diarrheal stools of non-pediatric patients who were admitted to a major hospital in Costa Rica with 633 beds between October 2010 and August 2012. All patients had been identiﬁed as having hospital- acquired CDI according to the criteria from the Infectious Diseases Society of America.20 Toxins A and B were detected in the stool samples by the hospital's clinical laboratory, and the samples with positive results were inoculated onto cefoxitin–cycloserine fructose agar plates (CCFA, Oxoid, Hampshire, UK). Yellow colonies on CCFA were cryopreserved at −80 °C in brain–heart infusion broth with 20% glycerol and sent to the Laboratory of Research in Anaerobic Bacteriology at the University of Costa Rica for further analysis and identiﬁcation. There, the strains were subcultured in selective C. difﬁcile moxalactam norﬂoxacin medium (Oxoid) and later on Brucella agar plates (BD Diagnostics, Franklin Lakes, NJ, USA) supplemented with 5% lysed horse blood (Oxoid) and 1 μg/mL vitamin K (Sigma-Aldrich, St. Louis, MO, USA) and (blood agar vitamin K) under an atmosphere composed of 90% N2, 5% H2 and 5% CO2 in an anaerobic chamber (Bactron II; ShellLab, Cornelius, OR, USA) at 37 °C for 48 h. The identities of the isolates were phenotypically conﬁrmed using selective media, the rapID 32A system (bioMériuex, Marcy-l'Étoile, France) and chartreuse ﬂuorescence on blood agar vitamin K under long-wave ultraviolet light and genoty- pically conﬁrmed through PCR-based detection of the C. difﬁcile marker tpi and molecular typing by PFGE.21 Although we observed a variety of genotypes (Figure 2 and Table 1), the local NAPCR1 genotype predominated (n = 18, 26%). The NAP9 genotype was the second-most prevalent genotype (n = 14, 21%), followed by the 14 isolates (21%) from the other traditional hospital pulsotypes of NAP2 (n = 2), NAP4 (n = 9), and NAP6 (n = 3). Environment- or community-associated pulsotypes, such as NAP10 and NAP11, were observed (n = 4, 6%) as were new pulsotypes (n = 8, 12%) and known pulsotypes with no NAP designations (100, 196 and 178; n = 3, 4%). Interestingly, no NAP1 strains were detected. Antimicrobial susceptibility tests revealed that 88% of the isolates were resistant to clindamycin with very high MICs (4256 μg/mL, Figure 1 Epidemic curves for the stool samples that were positive for C. INTRODUCTION While pulsed-ﬁeld gel electrophoresis (PFGE) is predominantly used in North America, ribotyping by PCR is most often used in Europe. PFGE NAP1 strains correspond to ribotype 027 and harbor toxin A, toxin B, CDT, a 18-bp mutation in tcdC and a point mutation in this gene at position 117. In contrast, most TcdA-negative and Antibiotic resistance of emerging C. difﬁcile D López-Ureña et al 2 TcdB-positive isolates belong to NAP9 and correspond to PCR ribotype 017.13 TcdB-positive isolates belong to NAP9 and correspond to PCR ribotype 017.13 C. difﬁcile strain ATCC 700057 (tcdA, tcdB, tcdC, cdtB) were used as controls. For the PFGE typing, we obtained chromosomal SmaI macrores- triction patterns with a published method19 and a CHEF-DRIII variable angle system. Gel pictures were analyzed with BioNumerics v4.6 (Applied Maths, Austin, TX, USA) and compared with the databases of the National Microbiology Laboratory of Public Health Agency of Canada. The effects, severity, complications, recurrence and even death rate of CDI have increased since 2003 in accordance with the increased isolation rates of hypervirulent strains, such as NAP1 and NAP9.14,15 The NAP1 strains have been associated with higher sporulation rates and greater resistance to antimicrobials, especially ﬂuoro- quinolones,14,16 whereas the NAP9 strains possess a TcdB that is capable of exerting a variant cytopathic effect.17 p g y p During a C. difﬁcile outbreak in a major Costa Rican hospital in 2009, the hypervirulent NAP1 strain (45%) and the NAPCR1 strains (31%) were the predominant genotypes. Both types of strains were resistant to ciproﬂoxacin, moxiﬂoxacin and levoﬂoxacin, and the NAPCR1 strains were also resistant to clindamycin and rifampicin. NAP9 and the other seven classical nosocomial strains were present but in minor proportions.18,19 Since this outbreak, the distribution of C. difﬁcile genotypes in other Costa Rican hospitals has not been reported. To determine whether the NAP1 and NAPCR1 genotypes were dominant in a non-pediatric hospital over a two-year period without C. difﬁcile outbreaks, 68 isolates from diarrheic patients were genotyped using PFGE and the antimicrobial susceptibilities of the isolates were tested. This information contributes to an understanding of CDI epidemiology worldwide and has the potential to guide local prevention efforts and treatment strategies. Molecular typing Genomic DNA from each strain was obtained from overnight cultures in brain–heart infusion broth (Oxoid) using the InstaGene reagent (Bio-Rad, Hercules, CA, USA). Fragments of tcdA, tcdB, cdtB and tcdC were ampliﬁed by PCR using known primers and conditions.21 A NAP1/027 strain (tcdA+, tcdB+, 18 bp-deletion in tcdC, cdtB+), a NAP7 strain (tcdA+, tcdB+, tcdC deletion418 pb, cdtB+), an A−B+ strain (tcdA−, tcdB+, wild-type tcdC, cdtB−) and the non-toxigenic Figure 1 Epidemic curves for the stool samples that were positive for C. difﬁcile (CD) toxins and were collected in a major hospital in Costa Rica from 2010 to 2012. The cases were diagnosed based on clinical evidence and toxin detection. Emerging Microbes and Infections Antibiotic resistance of emerging C. difﬁcile D López-Ureña et al 3 group of clinical isolates from another hospital and conﬁrmed the widespread distribution of these strains and their dominance even in the absence of outbreaks. Moreover, the identiﬁcation of NAPCR1 PFGE types reveals the ongoing evolution of this lineage and this species over a short time. Table 2). Half of the isolates were resistant to moxiﬂoxacin and rifampicin (MIC 432 μg/mL; Table 2). The MICs for metronidazole and vancomycin were rather low, and although all isolates were susceptible to both antibiotics, the MIC90 values were twice the MIC50 values (Table 2). p The worldwide prevalence of clinically signiﬁcant NAP9 strains seems to be increasing,26 particularly in Asian countries.27 Our results reinforce this view because this genotype was the second-most prevalent group. Many studies have found these strains in humans24,28 and in animals.29 NAP9 strains have been found once in Costa Rica18 and in Latin America,27 where they seem to be gradually replacing other circulating genotypes. As observed in many countries,30,31 our A−B+ strains were homogeneous, did not carry cdtAB, and harbored intact tcdC alleles. In contrast, in Australia, the tcdA−, tcdB+ strains are cdtB+.13 Furthermore, because our NAP9 strains were clindamycin-resistant, they may share a clonal origin with the strains that caused epidemics in Canada, the Netherlands, Ireland and Poland.13 We now know that these strains belong to the RT017 group (data not shown), but further studies are being performed to conﬁrm that their sequence type is indeed ST37 or ST86.32 All isolates from the two most common genotypes (i.e., NAPCR1 and NAP9) were resistant to clindamycin, moxiﬂoxacin and rifampi- cin (Table 1). Molecular typing Among all of the clindamycin-resistant strains, only those from the NAPCR1 and NAP9 genotypes exhibited MICs 4256 μg/mL. The remaining hospital, community and non- toxigenic isolates and the strains from the new genotypes exhibited low antibiotic-resistance levels (Table 1). aIncludes SmaI patterns 100, 196 and 178. DISCUSSION We detected a predominance of C. difﬁcile NAPCR1 and NAP9 strains in the diarrheal stool samples of patients admitted to a hospital in which no C. difﬁcile outbreaks had occurred during the period under study. This knowledge is relevant from the clinical perspective because both genotypes have been associated with more severe cases of CDI.19,24,25 Although NAP1 strains were previously isolated during a C. difﬁcile outbreak at another Costa Rican hospital, we only found a single tcdA+, tcdB+ and cdtB+ strain with a tcdC deletion in this study. This strain did not give rise to the 001 macrorestriction pattern associated with NAP1 in our previous reports18,19 but rather exhibited a PFGE pattern without a NAP designation (i.e., a 0196 macrorestriction pattern). Other NAP strains coexisted including common inhabitants of hospital environments, such as NAP2, NAP4 and NAP6 strains,33 and NAP10 and NAP11 strains with potential zoonotic or community origins.34 NAPCR1 strains have circulated in various Costa Rican hospitals since 2003 (López-Ureña D et al., 2003, unpublished data) and have had major roles in the 2009 C. difﬁcile outbreak in the San Juan de Dios Hospital.18 Here, we found NAPCR1 strains quite frequently in a Figure 2 Pulse ﬁeld gel electrophoresis results of representative C. difﬁcile genotypes that were isolated between October 2010 and August 2012 from a hospital without a history of outbreaks during the period under study. Figure 2 Pulse ﬁeld gel electrophoresis results of representative C. difﬁcile genotypes that were isolated between October 2010 and August 2012 from a hospital without a history of outbreaks during the period under study. Despite the marked increase in the recovery of clindamycin- resistant anaerobic strains in Costa Rica in the last decade,35,36 this antibiotic is still the ﬁrst-choice antibiotic for infections by anaerobic bacteria in Costa Rica and other geographic areas.37 The alarming clindamycin resistance level of C. difﬁcile observed in this study (88%) is slightly lower than the level recorded during a 2009 outbreak at another major hospital (97%)18 but is still much higher than the values reported from other latitudes.38,39 Almost half of the strains, all of which belonged to genotypes NAPCR1 and NAP9, had MICs 4256 μg/mL, whereas the remaining strains had MICs = 8 μg/mL. These ﬁndings indicate that only certain lineages acquire highly efﬁcient mechanisms of resistance to clindamycin. Figure 2 Pulse ﬁeld gel electrophoresis results of representative C. Emerging Microbes and Infections ACKNOWLEDGEMENTS difﬁcile (6%–39%), especially in multidrug-resistant strains.44,45 The reported MIC values of ⩽0.002 μg/mL for susceptible strains and 432 μg/mL for resistant strains largely match our ﬁndings.44,46 Up to 50% of our strains were rifampicin-resistant, particularly those from the NAPCR1 and NAP9 genotypes, which were also resistant to clindamycin, ciproﬂox- acin and moxiﬂoxacin. Interestingly, all of the isolates that were resistant to moxiﬂoxacin were also rifampicin-resistant, and this association should be explored further. 5 Chumbler NM, Farrow MA, Lapierre LA et al. Clostridium difﬁcile toxin B causes epithelial cell necrosis through an autoprocessing-independent mechanism. PLoS Pathog 2012; 8: e1003072. 6 Dingle KE, Elliott B, Robinson E et al. Evolutionary history of the Clostridium difﬁcile pathogenicity locus. Genome Biol Evol 2014; 6: 36–52. 7 Dupuy B, Govind R, Antunes A et al. Clostridium difﬁcile toxin synthesis is negatively regulated by TcdC. J Med Microbiol 2008; 57: 685–689. 8 Cartman ST, Kelly ML, Heeg D et al. Precise manipulation of the Clostridium difﬁcile chromosome reveals a lack of association between the tcdc genotype and toxin production. Appl Environ Microbiol 2012; 78: 4683–4690. p pp ; 9 Lanis JM, Heinlen LD, James JA et al. Clostridium difﬁcile 027/BI/NAP1 encodes a hypertoxic and antigenically variable form of TcdB. PLoS Pathog 2013; 9: e1003523. The antibiotic-resistance levels of the less abundant genotypes were rather low, but they supported the previously reported increased resistance to ﬂuoroquinolone of non-epidemic C. difﬁcile strains40 and the ciproﬂoxacin resistance that is present in virtually all strains of C. difﬁcile. 10 Gerding DN, Johnson S, Rupnik M et al. Clostridium difﬁcile binary toxin CDT: mechanism, epidemiology, and potential clinical importance. Gut Microbes 2014; 5: 15–27. 11 Schwan C, Stecher B, Tzivelekidis T et al. Clostridium difﬁcile toxin CDT induces formation of microtubule-based protrusions and increases adherence of bacteria. PLoS Pathog 2009; 5: e1000626. 12 Kuehne SA, Collery MM, Kelly ML et al. Importance of toxin A, toxin B, and CDT in virulence of an epidemic Clostridium difﬁcile strain. J Infect Dis 2014; 209: 83–86. All of the isolates were susceptible to metronidazole and vancomy- cin. In agreement with the known metronidazole MIC of C. difﬁcile,20 our MICs were invariably o2.0 μg/mL. However, a gradual change in the pattern of sensitivity to this antibiotic might be occurring within our strains because the MIC90 for metronidazole was twice the MIC50. ACKNOWLEDGEMENTS We thank Dr Michael R Mulvey, Dr George Golding and Tim Du (the National Microbiology Laboratory, Winnipeg, MB, Canada) for access to the NAP types database and technical assistance with the PFGE and ribotyping analyses. Pablo Vargas and Robin Cárdenas (the Laboratory of Research in Anaerobic Bacteriology, University of Costa Rica, San José, Costa Rica) are acknowledged for their technical assistance. This work was supported by the National Rector’s Council, Costa Rica (CONARE) and the Vice-Rectory for Research of the University of Costa Rica through grants VI-803-B1-654 and VI-803-B3-003. Abbreviation: minimum inhibitory concentration, MIC. in DNA gyrase genes.38–40 As described in 2010, all strains from this study were resistant to ciproﬂoxacin with MICs ⩾32 μg/mL.18 Moreover, half of the isolates were resistant to moxiﬂoxacin. All of the NAPCR1 and NAP9 strains were resistant to both quinolones. Older ﬂuoroquinolones, such as ciproﬂoxacin, exhibited moderate or poor activity against C. difﬁcile, and the third- and fourth-generation ﬂuoroquinolones, such as moxiﬂoxacin, were effective against these bacteria. However, recent studies indicate that the rates of resistance to moxiﬂoxacin in C. difﬁcile have increased dramatically in different countries.41–43 in DNA gyrase genes.38–40 As described in 2010, all strains from this study were resistant to ciproﬂoxacin with MICs ⩾32 μg/mL.18 Moreover, half of the isolates were resistant to moxiﬂoxacin. All of the NAPCR1 and NAP9 strains were resistant to both quinolones. Older ﬂuoroquinolones, such as ciproﬂoxacin, exhibited moderate or poor activity against C. difﬁcile, and the third- and fourth-generation ﬂuoroquinolones, such as moxiﬂoxacin, were effective against these bacteria. However, recent studies indicate that the rates of resistance to moxiﬂoxacin in C. difﬁcile have increased dramatically in different countries.41–43 2 Shivashankar R, Khanna S, Kammer PP et al. Clinical factors associated with development of severe-complicated Clostridium difﬁcile infection. Clin Gastroenterol Hepatol 2013; 11: 1466–1471. p 3 Aktories K. Bacterial protein toxins that modify host regulatory GTPases. Nat Rev Microbiol 2011; 9: 487–498. 4 Kim H, Kokkotou E, Na X et al. Clostridium difﬁcile toxin A-induced colonocyte apoptosis involves p53-dependent p21(WAF1/CIP1) induction via p38 mitogen- activated protein kinase. Gastroenterology 2005; 129: 1875–1888. Resistance to rifampicin is not unusual in C. ACKNOWLEDGEMENTS A similar situation might be occurring in terms of their vancomycin susceptibility because 12 strains (18%), six of which were classiﬁed as NAPCR1, exhibited reduced vancomycin susceptibility with MICs ⩾2 μg/mL. These data strongly support the presence of continuous monitoring programs both in clinics and the community. 13 Elliott B, Squire MM, Thean S et al. New types of toxin A-negative, toxin B-positive strains among clinical isolates of Clostridium difﬁcile in Australia. J Med Microbiol 2011; 60: 1108–1111. 14 Merrigan M, Venugopal A, Mallozzi M et al. Human hypervirulent Clostridium difﬁcile strains exhibit increased sporulation as well as robust toxin production. J Bacteriol 2010; 192: 4904–4911. 15 Kim J, Pai H, Seo M-R et al. Clinical and microbiologic characteristics of tcdA-negative variant Clostridium difﬁcile infections. BMC Infect Dis 2012; 12: 109. 16 Razavi B, Apisarnthanarak A, Mundy LM. Clostridium difﬁcile: emergence of hyperviru- lence and ﬂuoroquinolone resistance. Infection 2007; 35: 300–307. lence and ﬂuoroquinolone resistance. Infection 2007; 35: 300–30 17 Chaves-Olarte E, Freer E, Parra A et al. R-Ras glucosylation and transient RhoA activation determine the cytopathic effect produced by toxin B variants from toxin A-negative strains of Clostridium difﬁcile. J Biol Chem 2003; 278: 7956–7963. In this study, a local genotype of C. difﬁcile was the most prevalent strain in a set of 68 isolates that were recovered in a hospital over a 2-year period without outbreaks. This genotype has previously been found to be dominant in another hospital during an outbreak. The second-most prevalent genotype was NAP9, which is in line with its increased prevalence in the USA,31 Europe,24,31 Asia25 and Australia.13 The NAPCR1 and NAP9 genotypes exhibited high levels of antibiotic resistance, which reﬂects the use of antibiotics to which the strains have developed resistance and the association between CDIs and increased antibiotic use. The high levels of resistance to several antibiotics among the predominant genotypes could favor their 18 Quesada-Gómez C, Rodríguez C, Gamboa-Coronado MDM et al. Emergence of Clostridium difﬁcile NAP1 in Latin America. J Clin Microbiol 2010; 48: 669–670. 19 Quesada-Gómez C, López-Ureña D, Acuña-Amador L et al. Emergence of an outbreak- associated Clostridium difﬁcile variant with increased virulence. J Clin Microbiol 2015; 53: 1216–1226. 20 Cohen SH, Gerding DN, Johnson S et al. Clinical practice guidelines for Clostridium difﬁcile infection in adults: 2010 update by the society for healthcare epidemiology of America (SHEA) and the infectious diseases society of America (IDSA). 1 Slimings C, Riley T. Antibiotics and hospital-acquired Clostridium difﬁcile infection: Update of systematic review and meta-analysis. J Antimicrob Chemother 2014; 69: 881–891. DISCUSSION difﬁcile genotypes that were isolated between October 2010 and August 2012 from a hospital without a history of outbreaks during the period under study. Figure 2 Pulse ﬁeld gel electrophoresis results of representative C. difﬁcile genotypes that were isolated between October 2010 and August 2012 from a hospital without a history of outbreaks during the period under study. Fluoroquinolone resistance is increasing in epidemic strains of C. difﬁcile primarily due to the emergence of chromosomal mutations Table 1 Antibiotic resistances of various Clostridium difﬁcile genotypes recovered between October 2010 and August 2012 at a hospital without a history of outbreaks during the period under study Table 1 Antibiotic resistances of various Clostridium difﬁcile genotypes recovered between October 2010 and August 2012 at a hospital without a history of outbreaks during the period under study Resistance (% isolates) Genotype Number of isolates(%) Clindamycin Ciproﬂoxacin Moxiﬂoxacin Rifampicin Metronidazole Vancomycin NAPCR1/012 18 (26%) 100 100 100 100 0 0 NAP9/017 14 (21%) 100 100 100 100 0 0 NAP2, NAP4, NAP6 14 (21%) 93 100 7 0 0 0 NAP10, NAP11 4 (6%) 75 100 0 0 0 0 New genotypes 8 (12%) 88 100 12 8 0 0 No NAP designationa 3 (4%) 25 100 25 25 0 0 Non-toxigenic 7 (10%) 71 100 0 0 0 0 aIncludes SmaI patterns 100, 196 and 178. of various Clostridium difﬁcile genotypes recovered between October 2010 and August 2012 at a hospital during the period under study Antibiotic resistance of emerging C. difﬁcile D López-Ureña et al Table 2 MICs and resistances of 68 isolates of Clostridium difﬁcile recovered between October 2010 and August 2012 from a Costa Rican hospital without a history of outbreaks during the period under study Antibiotic MIC range (μg/mL) MIC50 (μg/mL) MIC90 (μg/mL) Resistance (% isolates) Clindamycin 1.5–4256 256 256 88 Ciproﬂoxacin 432 432 432 100 Moxiﬂoxacin 0.75–432 432 432 50 Rifampicin o0.002–432 432 432 50 Metronidazole 0.064–1.5 0.4 1 0 Vancomycin 0.38–4.0 1 2 0 Abbreviation: minimum inhibitory concentration, MIC. persistence in the hospital environments and dominance over other genotypes. Constant characterization of circulating C. difﬁcile isolates in terms of their population structures and antibiotic resistances not only improves our understanding of the epidemiology of CDI but also guides sanitary authorities and physicians in efforts to reduce the burden associated with this emerging pathogen. Emerging Microbes and Infections ACKNOWLEDGEMENTS Infect Control Hosp Epidemiol 2010; 31: 431–455. 21 Spigaglia P, Mastrantonio P. Comparative analysis of Clostridium difﬁcile clinical isolates belonging to different genetic lineages and time periods. J Med Microbiol 2004; 53: 1129–1136. 22 Letournel-Glomaud C, Houssaye S, Milhaiha L et al. E-test antibiotics susceptibility of strict anaerobic bacteria. Anaerobe 2003; 9: 281–284. Antibiotic resistance of emerging C. difﬁcile D López-Ureña et al 5 23 Clinical and Laboratory Standards Institute. 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Sequence variation in tcdA and tcdB of Clostridium difﬁcile: ST37 with truncated tcdA is a potential epidemic strain in China. J Clin Microbiol 2014; 52: 3264–3270. 39 Tickler IA, Goering RV, Whitmore JD et al. Strain types and antimicrobial resistance patterns of Clostridium difﬁcile isolates from the United States, 2011 to 2013. Antimicrob Agents Chemother 2014; 58: 4214–4218. 26 Collins D, Hawkey P, Riley T. Epidemiology of Clostridium difﬁcile infection in Asia. Antimicrob Resist Infect Control 2013; 2: 21. 40 Spigaglia P, Barbanti F, Louie T et al. Molecular analysis of the gyrA and gyrB quinolone resistance-determining regions of ﬂuoroquinolone-resistant Clostridium difﬁcile mutants selected in vitro. Antimicrob Agents Chemother 2009; 53: 2463–2468. 27 Goorhuis A, Legaria MC, van den Berg RJ et al. 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https://openalex.org/W2753973403	https://repository.escholarship.umassmed.edu/bitstream/20.500.14038/40995/2/s13059_019_1658_7.pdf	English	null	Measuring the reproducibility and quality of Hi-C data	bioRxiv (Cold Spring Harbor Laboratory)	2,017	cc-by	15,960	© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. RESEARCH p Measuring the reproducibility and quality of Hi-C data Galip Gürkan Yardımcı1, Hakan Ozadam2, Michael E. G. Sauria3, Oana Ursu4, Koon-Kiu Yan5, Tao Yang6, Abhijit Chakraborty7, Arya Kaul7, Bryan R. Lajoie2, Fan Song6, Ye Zhan8, Ferhat Ay7, Mark Gerstein9, Anshul Kundaje4,10, Qunhua Li11, James Taylor3,14, Feng Yue6,12, Job Dekker2,8,13* and William S. Noble1* Measuring the reproducibility and quality of Hi-C data Galip Gürkan Yardımcı1, Hakan Ozadam2, Michael E. G. Sauria3, Oana Ursu4, Koon-Kiu Yan5, Tao Yang6, Abhijit Chakraborty7, Arya Kaul7, Bryan R. Lajoie2, Fan Song6, Ye Zhan8, Ferhat Ay7, Mark Gerstein9, Anshul Kundaje4,10, Qunhua Li11, James Taylor3,14, Feng Yue6,12, Job Dekker2,8,13* and William S. Noble1* Abstract Background: Hi-C is currently the most widely used assay to investigate the 3D organization of the genome and to study its role in gene regulation, DNA replication, and disease. However, Hi-C experiments are costly to perform and involve multiple complex experimental steps; thus, accurate methods for measuring the quality and reproducibility of Hi-C data are essential to determine whether the output should be used further in a study. Results: Using real and simulated data, we profile the performance of several recently proposed methods for assessing reproducibility of population Hi-C data, including HiCRep, GenomeDISCO, HiC-Spector, and QuASAR-Rep. By explicitly controlling noise and sparsity through simulations, we demonstrate the deficiencies of performing simple correlation analysis on pairs of matrices, and we show that methods developed specifically for Hi-C data produce better measures of reproducibility. We also show how to use established measures, such as the ratio of intra- to interchromosomal interactions, and novel ones, such as QuASAR-QC, to identify low-quality experiments. Conclusions: In this work, we assess reproducibility and quality measures by varying sequencing depth, resolution and noise levels in Hi-C data from 13 cell lines, with two biological replicates each, as well as 176 simulated matrices. Through this extensive validation and benchmarking of Hi-C data, we describe best practices for reproducibility and quality assessment of Hi-C experiments. We make all software publicly available at http://github.com/kundajelab/3DChromatin_ReplicateQC to facilitate adoption in the community. Measuring the reproducibility and quality of Hi-C data Item Type Journal Article Authors Yardimci, Galip Gurkan; Ozadam, Hakan; Lajoie, Bryan R.; Zhan, Ye; Dekker, Job; Noble, William S. Citation <p>Genome Biol. 2019 Mar 19;20(1):57. doi: 10.1186/ s13059-019-1658-7. <a href="https://doi.org/10.1186/ s13059-019-1658-7">Link to article on publisher's site</a></p> DOI 10.1186/s13059-019-1658-7 Rights © The Author(s). 2019 Open Access: This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/ by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Download date 24/10/2024 05:46:27 Item License http://creativecommons.org/licenses/by/4.0/ Link to Item http://hdl.handle.net/20.500.14038/40995 Yardımcı et al. Genome Biology (2019) 20:57 https://doi.org/10.1186/s13059-019-1658-7 Open Access * Correspondence: job.dekker@umassmed.edu; william-noble@uw.edu 2Program in Systems Biology, University of Massachusetts Medical School, Worcester, USA 1Department of Genome Sciences, University of Washington, Seattle, USA Full list of author information is available at the end of the article Background loci, many such pairs are required to adequately characterize all pairwise interactions across a complete genome [11–13]. Consequently, the Hi-C assay can be costly to run. It is thus essential to have accurate and robust methods to evaluate the quality and reproduci- bility of Hi-C experiments, both to ensure the validity of scientific conclusions drawn from the data and to indicate when an experiment should be repeated or se- quenced more deeply. Reproducibility measures are also important for deciding whether two replicates can be pooled, a strategy that is frequently used to obtain a large number of Hi-C interactions [11]. The Hi-C assay couples chromosome conformation capture (3C) with next-generation sequencing, making it possible to profile the three-dimensional structure of chromatin in a genome-wide fashion [1]. Recently, application of the Hi-C assay has allowed researchers to profile the 3D genome during important biological pro- cesses such as cellular differentiation [2, 3], X inactivation [4–6], and cell division [7] and to identify hallmarks of 3D organization of chromatin, such as compartments [1], topologically associating domains (TADs) [8–10], and DNA loops [11]. Because the Hi-C assay measures the 3D conformation of a genome in the form of pairs of mapped reads (“interactions”) connecting different A rich collection of literature for assessing the qual- ity and reproducibility of a large collection of next- generation sequencing-based genomics assays, such as ChIP-seq [14] and DNase-seq [15], has been compiled over the past decade [16–18]. For these assays, enrich- ment of signal (“peaks”) at loci of interest [19] and assay-specific properties of sequencing fragments have 1Department of Genome Sciences, University of Washington, Seattle, USA Full list of author information is available at the end of the article Experimental and simulated Hi-C datasets for performance evaluation Experimental and simulated Hi-C datasets for performance evaluation Experimental and simulated Hi-C datasets for performance evaluation Experimental and simulated Hi-C datasets for performance evaluation We performed two replicate Hi-C experiments on cells from 13 immortalized human cancer cell lines from a variety of tissues and lineages using HindIII and DpnII restriction enzyme digestion (Additional file 1: Table S1). After aligning and filtering of paired end sequencing reads, we obtain 10 to 61 million paired reads per ex- periment for 11 cell types (generated using HinDIII) and more than 400 million paired reads for the remaining two deeply sequenced cell types (generated using DpnII). These Hi-C interactions serve as a readout of three-dimensional proximity of the corresponding genomic loci. The interac- tions are binned into fixed-sized bins, and a count of the number of Hi-C interactions that connect each pair of bins is stored in a Hi-C contact matrix. Unless otherwise noted, we used 40-kb bins because this value achieves reasonable sparsity of the Hi-C contact matrices, based on the depth of sequencing of the datasets used in our study. Also, this resolution has been adopted in multiple previous studies [7, 8]. We use the resulting Hi-C matrices as input to every reproducibility and quality control analysis in this study, except where indicated. A variety of methods have been used previously to measure the quality and reproducibility of Hi-C exper- iments. Ad hoc measures include using, for reproduci- bility, the Pearson or Spearman correlation coefficient [2, 25–27] and, for data quality, statistics that describe the properties of Hi-C fragment pairs [1, 28]. The drawbacks of using correlation as a reproducibility measure for genomics experiments, both because of its susceptibility to outliers and because it implicitly treats all elements of the Hi-C matrix as independent measurements, have been documented [16, 29]. In practice, because most of the Hi-C signal arises from interactions between loci less than 1 Mb apart [23, 24], the correlation coefficient will be dominated by these short-range interactions. To alleviate such problems, distance-based stratification [30] and dimensionality reduction of Hi-C signal [31], prior to measuring the correlation, have been proposed. Conversely, simple mapping statistics may be used to indicate a high or low percent of invalid or artefactual Hi-C fragments [24, 32], but such statistics reflect only the mapping stage of the analysis and cannot be immediately com- bined into a robust quality score. Experimental and simulated Hi-C datasets for performance evaluation p For use in assessing reproducibility and quality mea- sures for Hi-C data, we designed a model for simulating noisy Hi-C experiments (Fig. 1a). Our noise model aims to simulate a contact matrix from a Hi-C experiment performed on chromatin that lacks any high-order struc- ture, such as loops and topologically associating do- mains. For this purpose, our simulation models two main phenomena: the “genomic distance effect,” i.e., the higher prevalence of crosslinks between genomic loci that are close together along the genome [1], and ran- dom ligations generated by the Hi-C protocol [24]. For the first phenomenon, we use real Hi-C data, and we sample from the empirical marginal distribution of counts as a function of genomic distance. The second phenomenon, random ligation noise, is modeled by gen- erating Hi-C interactions between random bin pairs (see the “Methods” section for details). Counts generated by these two “noise” components of the model can be mixed with different proportions to produce simulated “pure noise” Hi-C matrices. We then mix the simulated contacts with experimental contact matrices in varying proportions to obtain noise-injected matrices. To overcome these problems, members of the ENCODE Consortium have recently developed methods for asses- sing both the quality and the reproducibility of the Hi-C assay [33–36]. In this study, we used large sets of real and simulated Hi-C data to assess and compare the perform- ance of methods for measuring the reproducibility of Hi-C data and evaluating Hi-C data quality. We generated multiple benchmarks for testing the performance of re- producibility measures and established that all of these methods can accurately measure the reproducibility of Hi-C data, whereas correlation coefficient cannot. Simi- larly, we have used real and simulated datasets to profile the performance of quality control methods and com- pared these methods to established statistics that have been used as indicators of high-quality Hi-C experiments. Here, we offer a thorough assessment of quality control and reproducibility methods and describe best practices for analyzing the quality and reproducibility of Hi-C data. In addition to noise, we tested the effects of sparsity and the resolution of Hi-C matrices on the performance of each method. We profiled the effects of sparsity expli- citly by downsampling real Hi-C matrices to contain a set of fixed total number of intrachromosomal Hi-C inter- actions. Page 2 of 19 Page 2 of 19 Page 2 of 19 Yardımcı et al. Genome Biology (2019) 20:57 Yardımcı et al. Genome Biology (2019) 20:57 Results been used as indicators of the quality of an experi- ment [16]. Correlation coefficient [20–22] and statis- tical methods such as the irreproducible discovery rate (IDR) [17] have been used to measure the re- producibility of such assays. However, all of these methods are designed to operate on data that is laid out in one dimension along the genome. Further- more, unlike other functional genomics assays, Hi-C data must be analyzed at an effective resolution de- termined by the user [13, 23, 24]. For these reasons, existing methods for assessing genomic data quality and reproducibility are not directly applicable to Hi-C data. Experimental and simulated Hi-C datasets for performance evaluation Binning resolution further controls the sparsity of a Hi-C matrix, at the same time dictating the scale of chromatin organization that can be observed in a Hi-C Page 3 of 19 Yardımcı et al. Genome Biology (2019) 20:57 (2019) 20:57 Yardımcı et al. Genome Biology A B A B A A B C Fig. 1 (See legend on next page.) B C Fig. 1 (See legend on next page.) C Page 4 of 19 Yardımcı et al. Genome Biology (2019) 20:57 Yardımcı et al. Genome Biology (See figure on previous page.) Fig. 1 Overview of the study. a Schematic showing the approach for generating noise-injected Hi-C matrices. In the upper panel, we generate two types of noise from real Hi-C data (center): random ligation noise (right) and genomic distance effect noise (left). The three matrices are then mixed to generate noisy datasets (lower panel). By changing the mixing proportions, we can create datasets with varying percentages of noise. b To benchmark the performance of various quality control and reproducibility measures, we compiled a large number of Hi-C replicates from 13 cell types and simulated noise-injected datasets from the original data. Real and simulated datasets binned at different resolutions and downsampled to different coverage levels are the inputs to reproducibility and quality control measures where each replicate pair and single replicate are assigned a score. Performance of each measure is evaluated on their ability to correctly rank real and simulated datasets. c Summary of the basic principles of the four reproducibility methods evaluated in this study noise levels. Intuitively, a good reproducibility measure should declare the least noisy replicate pair as most repro- ducible and the noisiest replicate pair as least reproducible. We paired a real Hi-C contact matrix with a noisier version of the same matrix using a wide range of simulated noise levels (5%, 10%, 15%, 20%, 30%, 40%, and 50%). This pro- cedure yielded seven pairs of replicates for each of 11 differ- ent cell types. We performed this approach using two different sets of randomly generated noise matrices, using one-third genomic distance noise and two-thirds random ligation noise or vice versa. Each replicate pair was assigned a reproducibility measure by HiCRep, GenomeDISCO, HiC-Spector, QuASAR-Rep, and Pearson correlation. matrix. Experimental and simulated Hi-C datasets for performance evaluation By binning deeply sequenced Hi-C datasets containing at least 400 million intrachromosomal Hi-C interactions from two cell types, we generated Hi-C matrices binned at high, mid, and low resolutions (10 kb, 40 kb, 500 kb) and used these to investigate the effect of reso- lution on each method as well (Additional file 1: Table S1). A schematic of the full range of datasets used in this study to validate each method is shown in Fig. 1b. Measures for quality and reproducibility of Hi-C data Four recently developed methods for measuring the quality of and reproducibility of Hi-C experiments were assessed in this study (Fig. 1c). HiCRep [34], GenomeDISCO [35], HiC-Spector [33], and QuASAR-Rep [36] measure repro- ducibility, and QuASAR-QC measures quality of Hi-C data. The four reproducibility methods we evaluate employ a var- iety of transformations of the Hi-C contact matrix. HiCRep stratifies a smoothed Hi-C contact matrix according to gen- omic distance and then measures the weighted similarity of two Hi-C contact matrices at each stratum. In this way, HiCRep explicitly corrects for the genomic distance effect and addresses the sparsity of contact matrices through stratification and smoothing, respectively. GenomeDISCO uses random walks on the network defined by the Hi-C contact map to perform data smoothing before computing similarity. The resulting score is sensitive to both differ- ences in 3D DNA structure and differences in the genomic distance effect [35] and makes it thus more challenging for two contact maps to be reproducible, as they have to satisfy both criteria to be deemed similar. HiC-Spector transforms the Hi-C contact map to a Laplacian matrix and then summarizes the Laplacian by matrix decomposition. QuA- SAR calculates the interaction correlation matrix, weighted by interaction enrichment. The two variants of QuA- SAR, QuASAR-QC and QuASAR-Rep, both assume that spatially close regions of the genome will establish similar contacts across the genome, and they measure quality and reproducibility, respectively, by testing the val- idity of this assumption for a single and pair of replicates. Our analysis showed that all reproducibility measures were able to correctly rank the simulated datasets. Aver- aged over 11 different cell types, we observed a mono- tonic trend for all of these measures (Fig. 2a). Indeed, for every cell type and every measure, increasing the noise level always led to a decrease in estimated repro- ducibility (Additional file 1: Figure S1). Qualitatively, the trends in Fig. Experimental and simulated Hi-C datasets for performance evaluation 2a suggest that QuASAR and HiCRep may be more robust to noise than the other reproducibility measures. Comparing the two noise models, we saw less consistent trends. HiC-Spector assigned higher reproducibility scores to matrices with 66% genomic distance noise and 33% random ligation noise. GenomeDISCO showed the oppos- ite behavior whereas QuASAR-Rep, HiCRep, and Pearson correlation gave similar scores regardless of the underlying noise proportions. This variability suggests that the vari- ous reproducibility measures exhibit different sensitivities to different sources of noise, thus potentially yielding complementary assessments of reproducibility. Assessment using real datasets reveals differences among reproducibility measures Inevitably, any simulation approach is only as good as its underlying assumptions; thus, we also analyzed the per- formance of the four reproducibility measures using real data. Specifically, we asked whether the reproducibility measures can discriminate between pairs of independent Hi-C experiments repeated on the same cell type versus pairs of experiments from different cell types. In this setup, we used three types of replicate pairs: a single pair Reproducibility measures correctly rank noise-injected datasets To assess the performance of the reproducibility mea- sures, we simulated pairs of Hi-C matrices with varying Page 5 of 19 Yardımcı et al. Genome Biology (2019) 20:57 Yardımcı et al. Genome Biology A B C D Fig. 2 Comparison of reproducibility measures. a Curves showing the mean reproducibility score assigned to 11 cell types at each noise injection level for 33% and 66% random ligation noise configurations. Vertical bars represent one standard deviation away from the mean. b Reproducibility scores assigned to biological replicate (blue), non-replicate (red), and pseudo-replicate (purple) pairs for each cell type. Coverage values are the mean number of interactions for each pair of replicates. c Reproducibility scores assigned to biological replicate (blue), non- replicate (red), and pseudo-replicate (purple) pairs from six cell types at seven different coverage levels. Dashed lines indicate the empirical threshold for distinguishing biological replicate pairs from non-replicate pairs. d Reproducibility scores assigned to biological replicate (blue) and non-replicate (red) pairs for clone-8 and S2 cells from Drosophila. Each panel shows the separation between two replicate pair types for each Hi-C reproducibility measure. Dashed lines correspond to the empirical thresholds inferred from human Hi-C data A A A B B C C D D rison of reproducibility measures. a Curves showing the mean reproducibility score assigned to 11 cell types at each noise and 66% random ligation noise configurations Vertical bars represent one standard deviation away from the mean b Fig. 2 Comparison of reproducibility measures. a Curves showing the mean reproducibility score assigned to 11 cell types at each noise injection level for 33% and 66% random ligation noise configurations. Vertical bars represent one standard deviation away from the mean. b Reproducibility scores assigned to biological replicate (blue), non-replicate (red), and pseudo-replicate (purple) pairs for each cell type. Coverage values are the mean number of interactions for each pair of replicates. c Reproducibility scores assigned to biological replicate (blue), non- replicate (red), and pseudo-replicate (purple) pairs from six cell types at seven different coverage levels. Dashed lines indicate the empirical threshold for distinguishing biological replicate pairs from non-replicate pairs. d Reproducibility scores assigned to biological replicate (blue) and non-replicate (red) pairs for clone-8 and S2 cells from Drosophila. Each panel shows the separation between two replicate pair types for each Hi-C reproducibility measure. Dashed lines correspond to the empirical thresholds inferred from human Hi-C data Fig. 2 Comparison of reproducibility measures. Reproducibility can be determined over a range of experimental coverage We limited this analysis to real data from six cell types with higher coverage, and we subsam- pled each replicate multiple times to contain 1 to 30 mil- lion total Hi-C interactions (see the “Methods” section for details). These datasets were used for testing the abil- ity of each method to distinguish among different repli- cate types at lower coverage levels and for explicitly profiling the dependence of reproducibility scores on coverage levels. Hi-C reproducibility measures retained their ability to distinguish between replicate types, even at extremely low coverage levels. Visualization of the reproducibility scores revealed that the HiCRep, HiC-Spector, and Geno- meDISCO measures successfully separate non-replicates from biological replicates even with only 5 million Hi-C interactions, a feat that Pearson correlation cannot achieve at even the highest coverage level (Fig. 2c). QuASAR-Rep can successfully separate biological replicates from non- replicates at 25 and 30 million interactions but fails to distinguish them when coverage is lower than 20 million interactions, consistent with the results from Fig. 2b. As before, pseudo-replicate pairs continue to serve as an upper bound for reproducibility measures. However, the separation between pseudo-replicates and biological repli- cates is reduced at lower coverage levels, and so is the sep- aration between biological replicates and non-replicates. Furthermore, this analysis suggests we can infer empirical thresholds for these reproducibility measures that can effectively separate all biological replicates from non-repli- cates at a given coverage level, as explained in the “Methods” section. These empirical thresholds, selected as the midpoint between the most reproducible non- replicate pair and the least reproducible replicate pair, are shown as dashed lines in Fig. 2c and can be found in Additional file 1: Table S2. Consistent with the trends observed in the analysis of real datasets, the reproducibility of downsampled repli- cate pairs exhibits a dependence on sequencing depth. We observe that reproducibility scores associated with biological replicates become significantly smaller as coverage decreases, according to a one-sided Wilcoxon signed rank test (P < 0.05, Additional file 1: Figure S5). The HiCRep, GenomeDISCO, QuASAR-Rep, and Pear- son correlation scores exhibit a statistically significant drop for every level of coverage. In contrast, reproduci- bility scores from HiC-Spector only start to significantly and consistently decay below 20 million interactions, exhibiting a lesser degree of dependence on the coverage Pseudo-replicate reproducibility scores provide an upper bound for each reproducibility measure. Reproducibility can be determined over a range of experimental coverage pseudo-replicate pairs (Fig. 2b). The reproducibility score of a replicate pair is the score obtained by averaging re- producibility scores assigned to each chromosome. All four reproducibility measures and the Pearson correl- ation can separate replicate pair types from each other (Additional file 1: Figure S2); however, the reproduci- bility measures generally achieved clearer separation between different replicate pair types. These differences are statistically significant according to a one-sided Kolmogorov-Smirnov test (P < 0.01). In addition to the Pearson correlation, we considered the rank-based Spear- man correlation as a potential method for assessing repro- ducibility. We also considered using either type of correlation in conjunction with ICE normalization. The re- sults (Additional file 1: Figure S3) show that none of these four methods successfully separates biological replicate from non-replicate pairs. Intuitively, we prefer a measure that separates non-replicates from biological replicates with a clear margin. By this measure, the HiC-Spector measure yields the largest separation, followed by HiCRep, QuASAR-Rep, and GenomeDISCO (Fig. 2b). Among them, HiC-Spector and HiCRep cor- rectly rank all replicate types for all 11 comparisons, with a clear separation between biological replicates and non-replicates. GenomeDISCO ranks a biological replicate lower than a non-replicate for a single case out of 11. The pair of biological replicates that GenomeDISCO ranks lower than non-replicates shows a marked difference in genomic distance effect (Additional file 1: Figure S4), to which this method is sensitive [35]. QuASAR-Rep is able to correctly rank biological replicates above non-replicates in 7 out of 11 cases. The cell types in which it fails have only 12 to 28 million interactions, suggesting that QuASAR-Rep does not perform well when coverage is low and the resolution is set to 40 kb. However, re-analysis of the same data suggests that switching to a larger resolution (120 kb) improves QuASAR-Rep’s performance, leading to separation between replicates and non-replicates for all cell lines but two (data not shown). As expected, the Pearson correlation performs worse than the Hi-C-specific measures, ranking non-repli- cates higher than biological replicates in 7 cases. To directly investigate the effects of the coverage of a Hi-C experiment on the reproducibility measures, we downsampled real Hi-C matrices to contain fewer inter- actions and examined the effects on the resulting repro- ducibility scores. Reproducibility measures correctly rank noise-injected datasets All four reproducibility measures and the Pearson correl- ation can separate replicate pair types from each other (Additional file 1: Figure S2); however, the reproduci- bility measures generally achieved clearer separation between different replicate pair types. These differences are statistically significant according to a one-sided Kolmogorov-Smirnov test (P < 0.01). In addition to the Pearson correlation, we considered the rank-based Spear- man correlation as a potential method for assessing repro- ducibility. We also considered using either type of correlation in conjunction with ICE normalization. The re- sults (Additional file 1: Figure S3) show that none of these four methods successfully separates biological replicate from non-replicate pairs. Intuitively, we prefer a measure that separates non-replicates from biological replicates with a clear margin. By this measure, the HiC-Spector measure yields the largest separation, followed by HiCRep, QuASAR-Rep, and GenomeDISCO (Fig. 2b). Among them, HiC-Spector and HiCRep cor- rectly rank all replicate types for all 11 comparisons, with a clear separation between biological replicates and non-replicates. GenomeDISCO ranks a biological replicate lower than a non-replicate for a single case out of 11. The pair of biological replicates that GenomeDISCO ranks lower than non-replicates shows a marked difference in genomic distance effect (Additional file 1: Figure S4), to which this method is sensitive [35]. QuASAR-Rep is able to correctly rank biological replicates above non-replicates in 7 out of 11 cases. The cell types in which it fails have only 12 to 28 million interactions, suggesting that QuASAR-Rep does not perform well when coverage is low and the resolution is set to 40 kb. However, re-analysis of the same data suggests that switching to a larger resolution (120 kb) improves QuASAR-Rep’s performance, leading to separation between replicates and non-replicates for all cell lines but two (data not shown). As expected, the Pearson correlation performs worse than the Hi-C-specific measures, ranking non-repli- cates higher than biological replicates in 7 cases. Reproducibility can be determined over a range of experimental coverage Reproducibility measures correctly rank noise-injected datasets a Curves showing the mean reproducibility score assigned to 11 cell types at each noise injection level for 33% and 66% random ligation noise configurations. Vertical bars represent one standard deviation away from the mean. b Reproducibility scores assigned to biological replicate (blue), non-replicate (red), and pseudo-replicate (purple) pairs for each cell type. Coverage values are the mean number of interactions for each pair of replicates. c Reproducibility scores assigned to biological replicate (blue), non- replicate (red), and pseudo-replicate (purple) pairs from six cell types at seven different coverage levels. Dashed lines indicate the empirical threshold for distinguishing biological replicate pairs from non-replicate pairs. d Reproducibility scores assigned to biological replicate (blue) and non-replicate (red) pairs for clone-8 and S2 cells from Drosophila. Each panel shows the separation between two replicate pair types for each Hi-C reproducibility measure. Dashed lines correspond to the empirical thresholds inferred from human Hi-C data sampling, with no biological (including distance effect) or technical variance. Therefore, we expect pseudo-replicates to exhibit the highest reproducibility. Conversely, non-repli- cate pairs are expected to have the lowest degree of reproducibility, because they contain all the experimental variation observed in biological replicates, as well as cell type-specific differences in 3D chromatin organization. of matrices from the same cell type (which we call “bio- logical replicates,” although each pair represents the same cells being prepped twice, rather than two different sets of cells), pairs of matrices from different cell types (non-replicates), and pairs of matrices sampled from combined biological replicates (pseudo-replicates, see the “Methods” section for details about the generation of pseudo-replicates) [34]. We assigned a reproducibility score to every matrix pair for each measure and asked if reproducibility scores differ among replicate pair types. In contrast to the simulation analysis, the analysis using real datasets showed distinct differences among the five methods. For each of the 11 cell types and each reproduci- bility measure, we assigned reproducibility scores to a single biological replicate pair, 20 non-replicate pairs, and 3 Because pseudo-replicates are generated from pooled bio- logical replicates, their variation solely stems from statistical Yardımcı et al. Genome Biology (2019) 20:57 Yardımcı et al. Genome Biology (2019) 20:57 Page 6 of 19 Page 6 of 19 Page 6 of 19 pseudo-replicate pairs (Fig. 2b). The reproducibility score of a replicate pair is the score obtained by averaging re- producibility scores assigned to each chromosome. Reproducibility can be determined over a range of experimental coverage In gen- eral, these scores show similar trends to those de- scribed above. For example, the Pearson correlation scores assigned to pseudo-replicates show a relatively wide separation from the rest of the scores, even though non-replicates and biological replicates are intermingled. On the other hand, GenomeDISCO, HiC-Spector, HiCRep, and QuASAR-Rep show the desired behavior: a high de- gree of separation between non-replicates and biological replicates, and a relatively small separation between bio- logical replicates and pseudo-replicates. Page 7 of 19 Page 7 of 19 Yardımcı et al. Genome Biology (2019) 20:57 Yardımcı et al. Genome Biology (2019) 20:57 Yardımcı et al. Genome Biology at both high and low resolutions. The four Hi-C-specific methods can correctly rank pseudo, biological, and non- replicate pairs at 10-kb, 40-kb and 500-kb resolutions (Fig. 3a) with a clear margin between biological replicate and non-replicate pairs. Surprisingly, we found that the Pearson correlation can correctly rank replicate types for these deeply sequenced datasets. Notably, the repro- ducibility scores from the four methods are largely independent of resolution. While GenomeDISCO and especially QuASAR-Rep exhibit some dependence of resolution, assigning lower reproducibility scores to replicates with lower coverage, they maintain a clear boundary with large margins between biological and non-replicates at all resolutions. However, the Pearson correlation exhibits a larger degree of dependence on resolution for all replicate pair types and maintains relatively smaller margins between non-replicate and biological replicate pairs. Simulated datasets further validate that reproducibility scores from each method decrease with increasing levels of noise at 10-kb, 40-kb and 500-kb resolution (Fig. 3b). level. This may be because the leading eigenvectors used by HiC-Spector tend to capture local or mesoscopic structures, which are less likely to be affected by cover- age. Despite varying levels of dependence on coverage, downsampling analysis convincingly shows that all mea- sures exhibit a dependence on coverage. Thus, coverage of different replicate pairs must be factored into repro- ducibility analyses, especially for comparative purposes. Noise reduces the consistency and the prevalence of higher order structures in Hi-C matrices Having investigated four different methods for evaluat- ing the reproducibility of a given pair of Hi-C matrices, we now focus on methods for evaluating the quality of a single Hi-C matrix. As before, we perform this evalu- ation by injecting noise into real Hi-C data, producing a collection of 88 matrices corresponding to 11 cell types and 8 different noise profiles (see the “Methods” section). Among our four Hi-C reproducibility measures, only one (QuASAR-QC) provides a variant to assess the quality of a single matrix. The procedure yields a single, bounded summary statistic indicative of homogeneity of the under- lying sample population and the signal-to-noise ratio of the interaction map. In addition to QuASAR-QC analysis, we profiled two well-known features of 3D organization: statistically significant long-range contacts [38, 39], which include DNA loops, and topologically associating domains (TADs). Intuitively, we expect that significant contacts and TADs should be harder to detect in noisy matrices and that such matrices should have a lower degree of consistency. We observed that the four reproducibility measures show variable trends in how reproducibility scores assigned to biological replicates decay with respect to in- creasing resolution (Additional file 1: Figure S6). For HiCRep, GenomeDISCO, and QuASAR-Rep, the HindIII replicates (A549, G410, and LNCaP) exhibit a decay in reproducibility scores, whereas the scores assigned to replicate pairs generated by DpnII (HepG2) are more ro- bust to changes in resolution. Notably, for these three reproducibility measures, the degree of decay also corre- lates with the sequence coverage of the data. For HiC-Spector, we do not observe consistent trends. These observations generally support the idea that deeply se- quenced replicates generated by a 4-cutter such as DpnII can support resolutions higher than 40 kb, whereas rela- tively shallow replicates (< 100 million read pairs) gener- ated using a 6-cutter are not suitable for binning resolutions higher than 40 kb. However, given the lack of a clear elbow or maximum in Additional file 1: Figure S6, we do not recommend using reproducibility scores to attempt to select an appropriate resolution. Our analysis suggests that QuASAR-QC is indeed sen- sitive to the noise and the coverage of a Hi-C matrix. For each simulated Hi-C matrix from 11 cell types, QuASAR-QC detects a perfectly monotonic relationship between the noise level and the consistency of the matrix (Fig. 4a). Reproducibility measures accurately quantify reproducibility of Hi-C data from non-human genomes million intrachromosomal interactions). For HiCRep, QuASAR-Rep, and GenomeDISCO, we observed that reproducibility scores tend to plateau at 240 million interactions at 10-kb and 40-kb resolutions, whereas reproducibility scores of 500-kb resolution matrices benefit little from higher coverage (Fig. 3c). Consistent with our previous observations, HiC-Spector exhibits a lower degree of dependence on coverage, with scores reaching maxima at 120 kb. Overall, the four Hi-C repro- ducibility measures exhibit robustness to coverage and resolution differences, as measured by their ability to dis- tinguish between replicate and non-replicate pairs. p y g We investigated whether the four Hi-C reproducibility measures can be applied to data derived from a non-hu- man genome. We wanted to investigate a genome that is markedly different from human, but replicate Hi-C experiments in organisms other than human and mice are rare. We used Hi-C data from Ramirez et al., which has two biological replicates from two cell types (clone-8 and S2) from the fruitfly Drosophila melanogaster [37]. The fruitfly genome is approximately 18 times smaller than the human genome. For this analysis, we binned the Hi-C matrices at 10 kb and compared the reproduci- bility of the four large, non-heterochromatic chromo- somes in Drosophila (chromosomes 2, 3, 4, and X). As before, we assigned reproducibility scores to each repli- cate pair and each non-replicate pair. The results show that biological replicate pairs are clearly separated from non-replicates for each measure in both cell types (Fig. 2d). Furthermore, for three out of the four reprodu- cibility measure, the empirical thresholds that we in- ferred from the human Hi-C data (shown as dashed lines in Fig. 2d) generalize to the fruitfly genome. Next, we tested whether the reproducibility measures can be used to select empirically the optimal resolution for a Hi-C dataset. Although resolution strongly influ- ences almost every downstream analysis of Hi-C data, this parameter is generally set in an ad hoc fashion. To explore the performance of the measures as a function of the resolution parameter, we binned four pairs of biological replicates at an increasingly high resolution ranging from 40 kb, 20 kb, 10 kb and 5 kb and asked if the reproducibility scores of biological replicates decay significantly at higher resolutions. We chose six samples performed using HindIII with coverage values ranging from 15 million to 60 million interactions and two sam- ples generated using DpnII and coverage of ~ 400 mil- lion interactions. Reproducibility measures are robust to changes in resolution The resolution of a Hi-C matrix effectively dictates the scale of 3D organization observable from the data: a low-resolution matrix can only reveal compartments and TADs [1, 8], whereas high-resolution matrices reveal additional finer scale structures like chromatin loops [11]. To investigate the effect of resolution on reproduci- bility, we used deeply sequenced Hi-C replicates with at least 400 million intrachromosomal interactions gener- ated from the HepG2 and HeLa cell lines. From these data, we generated real and simulated replicate pairs at 10-kb, 40-kb, and 500-kb resolution, and we measured the reproducibility of each replicate pair. Next, we used deeply sequenced datasets to further in- vestigate the effect of coverage on reproducibility scores of biological replicates at three resolution levels using a wider range of coverage values (30, 60, 120, 240, and 400 y HiCRep, GenomeDISCO, HiC-Spector, QuASAR-Rep, and Pearson correlation accurately measure reproducibility Fig. 3 Effects of resolution on reproducibility measures. a Reproducibility scores assigned to biological replicate (blue), non-replicate (red), and pseudo-replicate (purple) pairs from HepG2 and HeLa Hi-C datasets at 10-kb, 40-kb and 500-kb resolutions. b Reproducibility scores assigned to different cell types at different resolutions, plotted as a function of noise level. c Reproducibility scores assigned to downsampled biological replicate pairs at different resolutions. Both the HepG2 and HeLa datasets contain > 400 million read pairs Fig. 3 Effects of resolution on reproducibility measures. a Reproducibility scores assigned to biological replicate (blue), non-replicate (red), and pseudo-replicate (purple) pairs from HepG2 and HeLa Hi-C datasets at 10-kb, 40-kb and 500-kb resolutions. b Reproducibility scores assigned to different cell types at different resolutions, plotted as a function of noise level. c Reproducibility scores assigned to downsampled biological replicate pairs at different resolutions. Both the HepG2 and HeLa datasets contain > 400 million read pairs Yardımcı et al. Genome Biology (2019) 20:57 Yardımcı et al. Genome Biology (2019) 20:57 Page 8 of 19 Page 8 of 19 Reproducibility measures accurately quantify reproducibility of Hi-C data from non-human genomes Noise reduces the consistency and the prevalence of higher order structures in Hi-C matrices The same trend is observed in deeply sequenced HepG2 and HeLa cell types at 10-kb, 40-kb, and 500 kb resolutions (Additional file 1: Figure S8). Finally, we compared the run times of each reproducibil- ity measure, using a large number of pairs of chromosome 21 contact matrices binned at 40-kb resolution. As seen in Additional file 1: Figure S7, QuASAR-Rep achieves the fast- est median running time (0.82 s), followed by HiC-Spector (2.76 s), GenomeDISCO (5.77 s), and HiCRep (9.00 s). Page 9 of 19 (2019) 20:57 Yardımcı et al. Genome Biology (2019) 20:57 Yardımcı et al. Genome Biology Although the majority of noise-free combined repli- distances for each cell type revealed that the three cell Fig. 4 Quality measures. a QuASAR-QC scores assigned to noise-injected matrices from 11 cell types (b). Total number of significant contacts above a 5% FDR threshold from noise-injected matrices from 11 cell types. c Violin plots showing the distribution of TAD boundary distances between biological replicates and noise-injected replicates for T470 cells. There is no significant change in the distribution of TAD boundary distances at any given noise level. d QuASAR-QC scores assigned to downsampled replicates from six different cell types. e Total number of significant contacts above a 5% FDR threshold from downsampled replicates from six different cell types. f Violin plots showing the distribution of distances between domain boundaries in biological replicates and noise-injected replicates for T470 cells. In panels c and f, asterisks indicate that the distribution of boundary distances is significantly larger than the null distribution, which is obtained by comparing biological replicates Fig. 4 Quality measures. a QuASAR-QC scores assigned to noise-injected matrices from 11 cell types (b). Total number of significant contacts above a 5% FDR threshold from noise-injected matrices from 11 cell types. c Violin plots showing the distribution of TAD boundary distances between biological replicates and noise-injected replicates for T470 cells. There is no significant change in the distribution of TAD boundary distances at any given noise level. d QuASAR-QC scores assigned to downsampled replicates from six different cell types. e Total number of significant contacts above a 5% FDR threshold from downsampled replicates from six different cell types. f Violin plots showing the distribution of distances between domain boundaries in biological replicates and noise-injected replicates for T470 cells. Noise reduces the consistency and the prevalence of higher order structures in Hi-C matrices In panels c and f, asterisks indicate that the distribution of boundary distances is significantly larger than the null distribution, which is obtained by comparing biological replicates distances for each cell type revealed that the three cell types with lower QuASAR-QC scores have significantly higher contact probabilities at genomic distances larger than 50 Mb (Additional file 1: Figure S9). Because such long-range contacts are unlikely to occur due to the organization of chromatin, it is likely that such long-range contacts represent random ligation of uncrosslinked DNA fragments, which is a known source of noise in a Hi-C ex- periment [24]. Thus, the QuASAR-QC measure is poten- tially sensitive to both the level of simulated noise and the Although the majority of noise-free combined repli- cates are assigned a QuASAR-QC score ranging from 0.05 to 0.07, three cell types have strikingly lower QuASAR-QC scores ranging from 0.03 and 0.02. The Hi-C matrices from these three cell types (LNCaP, SKNDZ, SKNMC) contain fewer Hi-C interactions. Thus, the lower consistency scores are likely partially due to the sparsity that results from low experimental coverage (Additional file 1: Table S1). Furthermore, in- vestigation of contact probabilities at given genomic Although the majority of noise-free combined repli- cates are assigned a QuASAR-QC score ranging from 0.05 to 0.07, three cell types have strikingly lower QuASAR-QC scores ranging from 0.03 and 0.02. The Hi-C matrices from these three cell types (LNCaP, SKNDZ, SKNMC) contain fewer Hi-C interactions. Thus, the lower consistency scores are likely partially due to the sparsity that results from low experimental coverage (Additional file 1: Table S1). Furthermore, in- vestigation of contact probabilities at given genomic Page 10 of 19 Yardımcı et al. Genome Biology (2019) 20:57 Page 10 of 19 matrices at 10-kb and 40-kb resolutions; however, QuASAR-QC scores at 500 kb tend to benefit less from deeper coverage, likely because coarse resolutions do not require large numbers of Hi-C interactions (Add- itional file 1: Figure S14). Similarly, the number of statisti- cally significant long-range interactions also decreases as we reduce the number of total Hi-C interactions. However, the number of significant interactions decreases at a much higher rate: even at 15 million interactions, most cell lines lose the majority of significant interactions (Fig. 4e). Quality control measures are consistent with mapping statistics g g Surprisingly, we found that topologically associating domain detection is highly robust to noise. We identified TADs using the insulation score [5, 40] method for the 88 simulated matrices, and we characterized the changes in the total number of TADs and TAD size distribution and the changes to TAD boundaries with respect to the noise level. The total number of identified TADs and their size distribution are only altered at the highest level of noise injection (Additional file 1: Figures S11 and S12). In addition, TAD boundaries between the original repli- cate and noise-injected levels exhibit the same degree of variation between two biological replicates, further sup- porting the idea that TAD boundaries identified with the insulation score approach are highly robust to noise (Fig. 4c, Additional file 1: Figure S13). To further validate the performance of the quality con- trol measures at our disposal, we investigated the rela- tionship between the QuASAR-QC scores assigned to real Hi-C matrices and various read-mapping statistics that have been used previously to evaluate Hi-C data quality [24]. The four statistics we compared against are the percentages of fragment pairs that can be mapped uniquely to the genome (aligned pairs), fragment pairs from the same restriction fragments (invalid pairs), intrachromosomal interactions (intrachromosomal per- centage), and fragment pairs that are repeated in the dataset (PCR duplicate rate). Overall, we observe varying degrees of correlation between the quality control measures and the mapping statistics for biological replicates. The percentage of aligned pairs is corre- lated with higher quality experiments, consistent with what one would intuitively expect from high-quality sequencing li- braries (Fig. 5a). The percentage of invalid pairs is also weakly anti-correlated with QuASAR-QC scores, consistent with the fact that invalid pairs represent uninformative Hi-C interactions (Fig. 5b). However, we observed the highest de- gree of correlation between QuASAR-QC scores and intra- chromosomal percentage (Fig. 5c). In a typical Hi-C experiment, a portion of interchromosomal interactions re- sult from random ligation of non-crosslinked fragments; thus, a significant enrichment of interchromosomal interac- tions, which results in a depletion of intrachromosomal Noise reduces the consistency and the prevalence of higher order structures in Hi-C matrices Larger numbers of significant interactions are detected in deeply sequenced datasets, due to added statistical power, but a similar relationship between coverage and number of significant contacts is observed at both 10-kb and 40-kb resolutions (Additional file 1: Figure S15). Conversely, we found that TADs detected by insulation score are robust to low coverage levels. Using the same approach for noise- injected datasets, we found that the total number of TADs and their size distribution are not altered by lower coverage (Additional file 1: Figures S16 and S17). Indeed, the distances between TAD boundaries identified at lower coverage and original replicates only differ from the base- line distribution at 10 million or fewer interactions (Fig. 4f, Additional file 1: Figure S18). differences in the level of inherent noise that each com- bined replicate contains. p Statistically significant mid-range (50 kb–10 Mb) inter- actions are depleted in noisy Hi-C matrices. We identi- fied statistically significant Hi-C contacts using Fit-Hi-C [38] for each of the Hi-C matrices that make up our simulated dataset. Because robust identification of such contacts requires deeply sequenced datasets that contain large numbers of Hi-C interactions, we chose to use a somewhat liberal false discovery rate threshold of 0.05 to facilitate discovery of statistically significant contacts. For 11 cell types, we observed that 8 out of 11 cell types exhibit a perfect or near perfect anti-correlation between the injected noise percentage and the total number of significant interactions (Fig. 4b). For the other three cell lines (LNCaP, SKNDZ, SKNMC), Fit-Hi-C identifies al- most no significant contacts with or without any noise injection, further supporting the conclusion that these Hi-C datasets have low quality. These three cell lines are also the cell lines that have the lowest QuASAR-QC scores, corroborating the results between these two in- dependent analyses. For the deeply sequenced two data- sets (HepG2 and HeLa), we observed a similar trend at both 10-kb and 40-kb resolutions, with a higher number of significant mid-range contacts due to the higher coverage, as expected (Additional file 1: Figure S10). Quality control measures require different levels of experimental coverage Continuing our assessment of Hi-C quality measures, we used downsampled Hi-C matrices to investigate the relationship between experimental coverage and each QC measure using a similar setup as before (see the “Methods” section). Quality control metrics exhibit a predictable dependence on the coverage of Hi-C matrices. For each of the six cell types we downsampled, we observed that QuASAR-QC scores are lower for Hi-C matrices with fewer interactions (Fig. 4d). We observe the same trend for deeply sequenced Page 11 of 19 Yardımcı et al. Genome Biology (2019) 20:57 Yardımcı et al. Genome Biology Fig. 5 Comparison of QuASAR-QC to mapping statistics. Scatter plots of QuASAR-QC scores of biological replicates from 13 cell types plotted against quality statistics that describe percentages of a successful mapping, b artifactual Hi-C fragments, c intrachromosomal interactions, and d PCR duplicates. Dots correspond to low coverage Hi-C replicates from 11 cell types generated using HindIII, and triangles correspond to replicates from two deeply sequenced cell types generated by DpnII. Red dots correspond to a subset of samples with very similar total coverage (138–171 million read pairs). Each plot lists two Pearson correlation coefficients: the correlations between the given statistic and QuASAR-QC scores for only the 11 HinDIII cell types and for all 13 cell types Fig. 5 Comparison of QuASAR-QC to mapping statistics. Scatter plots of QuASAR-QC scores of biological replicates from 13 cell types plotted against quality statistics that describe percentages of a successful mapping, b artifactual Hi-C fragments, c intrachromosomal interactions, and d PCR duplicates. Dots correspond to low coverage Hi-C replicates from 11 cell types generated using HindIII, and triangles correspond to replicates from two deeply sequenced cell types generated by DpnII. Red dots correspond to a subset of samples with very similar total coverage (138–171 million read pairs). Each plot lists two Pearson correlation coefficients: the correlations between the given statistic and QuASAR-QC scores for only the 11 HinDIII cell types and for all 13 cell types interactions, indicates a low-quality Hi-C experiment. In par- ticular, six biological replicates with lower than 30% intra- chromosomal interactions have the lowest QuASAR-QC scores; these replicates are from the LNCaP, SKNDZ, and SKNMC cell types. Analysis of downsampled data shows that this effect is not simply due to the overall lower se- quencing depth of these three replicates (Additional file 1: Figure S19). Quality control measures require different levels of experimental coverage These replicates were also identified to have lower quality in our simulation studies (Fig. 4a) and are de- pleted for significant mid-range interactions, establishing the consistency of quality control measures overall. We note that this finding is consistent with the previously sug- gested range of 40–60% intrachromosomal interactions for high-quality experiments [24]. The PCR duplicate rate is uncorrelated with QuASAR-QC. Note that the PCR dupli- cate rate may be influenced by overall coverage, which we have not controlled for in this experiment. Nonetheless, even for sets of experiments with very similar coverage (red dots in Fig. 5d), we observe very little correlation. Using a rich set of Hi-C experiments from a variety of hu- man cell types, we tested whether these methods can identify reproducible and high-quality experiments. Fur- thermore, we generated Hi-C contact matrices with con- trolled levels of noise by designing a simulated noise injection process. Our analysis shows that these measures perform well and improve upon the shortcomings of using generic or qualitative approaches. The Hi-C reproducibility measures that we evaluated as- sess reproducibility more accurately than the Pearson or Spearman correlation for real and simulated datasets. In particular, measures specifically designed for Hi-C data can better distinguish subtle differences in the 3D organization of different cell types, because these methods directly ac- count for the special noise properties of this data type that are overlooked by traditional similarity scores. Selecting an appropriate reproducibility measure for a given study may depend in part upon the goals of the study. A scientist may be primarily interested in a meas- ure that distinguishes between biological replicates and non-replicates. Such a goal might be appropriate, for ex- ample, if the method will be used to check for sample swaps during large-scale experiments. In this setting, our Discussion and conclusions The QuASAR-QC measure provides an interpretable score that can accurately rank simulated datasets accord- ing to noise levels and distinguish low-quality real Hi-C experiments from high-quality ones (in submission). This measure correlates with previously established statistics that indicate high quality in a Hi-C experiment and have been used as qualitative indicators of quality. Each of these statistics captures different sources of error in a Hi-C assay. In contrast, QuASAR-QC offers a single score that allows direct ranking of multiple experiments. g p p Significant mid-range interactions, such as DNA loops, are also depleted in low-quality Hi-C experiments in both simulated and real datasets. Surprisingly, we found that TAD detection is fairly robust to all but high levels of noise, presumably because TAD detection only requires that a dataset contains a sufficient proportion of valid short-range Hi-C interactions and ignores mid- and long-range interactions. Unfortunately, it is challenging to convert the enrichment of such features into a quality control measure, due to other quality-independent bio- logical processes which can cause variation of these fea- tures. However, a near total depletion of these features, mid-range interactions in particular, may certainly indicate lower quality overall. The scores produced by all four reproducibility methods decrease in the presence of decreasing se- quencing depth and fixed resolution or in the presence of increasing resolution at a fixed sequencing depth. Nonetheless, three out of four methods (Genome- DISCO, HiCRep, and HiC-Spector) show robustness to increasing sparsity, as measured by their ability to distin- guish replicate from non-replicate pairs. Only QuASAR- Rep fails to measure reproducibility accurately for the most sparse datasets at high resolutions, though this effect is ameliorated if the data is analyzed using a lower reso- lution (data not shown). Thus, we hypothesize that one reason why GenomeDISCO and HiCRep perform well on low-coverage datasets is because they perform smoothing on the contact matrix. Overall, these results suggest that experimenters can assess whether a given set of samples are “reproducible enough” with as few as valid 5 million Hi-C interactions and then follow up with deeper sequen- cing. Among the four methods, HiC-Spector exhibits the least dependence on sequencing depth (Fig. 3c) or reso- lution (Additional file 1: Figure S6). These results are fur- ther consistent with the hypothesis that HiC-Spector focuses on local features of chromatin structure, which ex- plains HiC-Spector’s robustness to low coverage. Discussion and conclusions We evaluated the recently proposed methods for measur- ing the quality and reproducibility of Hi-C experiments. Page 12 of 19 Page 12 of 19 Page 12 of 19 Yardımcı et al. Genome Biology (2019) 20:57 Yardımcı et al. Genome Biology (2019) 20:57 Yardımcı et al. Genome Biology of reproducibility scores on data sparsity must be taken into account. For example, in our study, the SKMEL5 and SKNMC experiments differ in sequencing depth by a factor of 2. This difference could confound attempts to cluster or hierarchically organize cell types. In such a setting, all datasets should be randomly downsampled to a common sequencing depth prior to analysis. results show that HiC-Spector often had the best margin among all four measures (Fig. 2b and c). This is true even when we place all four measures on a similar scale by using the variance associated with non-replicate pairs (data not shown). On the other hand, simply discrimin- ating among replicates and non-replicates may not be sufficient in some contexts. If the study aims to use the reproducibility measure to quantify similarities among various experiments, then HiCRep has been shown pre- viously to discriminate well among cell types [34], whereas the other methods in this study have not been examined in this fashion. An important question is whether the methods and thresholds derived here will generalize to non-human genomes. Preliminary analysis (Fig. 2d) suggests that the empirical reproducibility thresholds derived for Geno- meDISCO, HiCRep, and HiC-Spector may generalize to the much smaller Drosophila genome, whereas the QuASAR-Rep measure does not. However, this result is preliminary due to the small number of currently available, replicated Hi-C experiments in non-human genomes. Furthermore, our analysis also suggests that the differ- ent reproducibility measures may be more sensitive to different types of noise, with GenomeDISCO showing more sensitivity to random ligation noise than to gen- omic distance noise, HiC-Spector showing the opposite behavior, and QuASAR-Rep and HiCRep showing simi- lar sensitivities to both types of noise (Fig. 2a). Because genomic distance noise preferentially affects short-range Hi-C interactions, this observation is consistent with the hypothesis that HiC-Spector largely focuses on local struc- tures which are detected by short-range Hi-C interactions. Overall, QuASAR-Rep and HiCRep appear to exhibit an overall lower sensitivity to varying noise levels than HiC-Spector and GenomeDISCO. Also, GenomeDISCO tends to be more sensitive to differences in genomic dis- tance effect between the samples compared [35]. Measures of reproducibility HiCRep This method assesses reproducibility by taking into ac- count two dominant spatial features of Hi-C data: distance dependence and domain structure. The method first smooths the given Hi-C matrices to help capture domain structures and reduce stochastic noise due to insufficient sampling. It then addresses the distance-dependence effect by stratifying Hi-C data according to genomic distance. Specifically, the method consists of two stages. An additional direction for future research is the devel- opment of alternative score functions that are designed to focus on particular aspects of chromatin architecture. For example, in the context of single-cell Hi-C analysis, mea- sures have been developed that focus entirely on the gen- omic distance effect, for use in segregating cells according to cell cycle stages [42]. Similarly, for bulk or single-cell Hi-C, researchers may wish to separately assess whether two cells or cell types exhibit similar chromosome territor- ies, compartment structure, domain structure, or patterns of looping interactions. Developing scores that separately assess these aspects of genome 3D architecture will facili- tate automated inference from growing Hi-C datasets. In the first stage, HiCRep smooths the Hi-C raw contact map using a 2D mean filter, which replaces the read count of each contact with the average counts of all contacts in its neighborhood. The neighborhood size is obtained from a deeply sequenced benchmark dataset using a training procedure. In this analysis, neighborhood size parameter of 20, 5, and 1 are used for the resolutions of 10 kb, 40 kb, and 500 kb, respectively. Smoothing improves the contigu- ity of regions with elevated interaction, consequently en- hancing the domain structures. We release a software package that incorporates the four reproducibility measures and the QuASAR-QC measure (https://github.com/kundajelab/3DChromatin_- ReplicateQC). Until recently, proven measures have been lacking, and currently, there is no standard for measur- ing for quality and reproducibility of Hi-C data. This tool will both greatly simplify the task of measuring both the quality and reproducibility of Hi-C datasets robustly by using the methods we show to be accurate in this study. We also propose a set of empirical quality and re- producibility thresholds for use at various coverage levels, which are built into the software package to make it easy to determine whether samples pass quality and reproducibility standards (Additional file 1: Table S2). In the second stage, HiCRep takes into account the distance-dependence effect by a stratification and aggrega- tion strategy. Discussion and conclusions We anticipate that the reproducibility measures we evaluated in this study may be applicable to data from recently developed single-cell Hi-C assays [41–43]. The primary challenge, in this setting, would be the extreme sparsity of single-cell data. Our experiments show that, even when we randomly downsample to 1 million inter- actions per cell, all four methods are capable of distin- guishing replicates from non-replicates (Fig. 2c), with the best separation provided by HiCRep. This difference may arise because HiCRep explicitly incorporates an ex- plicit smoothing procedure; in contrast, GenomeDISCO uses an implicit smoothing procedure and the other two methods do not perform smoothing at all. Note that Note that if the goal of a study is to quantify similar- ities among various experiments, then the dependence Page 13 of 19 Page 13 of 19 Page 13 of 19 Yardımcı et al. Genome Biology (2019) 20:57 Yardımcı et al. Genome Biology (2019) 20:57 Yardımcı et al. Genome Biology standard toolkit will help to improve the quality and re- producibility of Hi-C data generated in the future. these results do not fully resolve the question of whether the reproducibility measures will generalize to single-cell data, because in addition to higher sparsity, the variance and noise characteristics of single-cell data are expected to markedly differ from those of bulk Hi-C data. Hence, exploring the applicability of these methods to single-cell Hi-C data more fully is an important direction for future research. Measures of reproducibility HiCRep This stage consists of two steps. The algo- rithm first stratifies the contacts according to the genomic distances of the contacting loci and computes the correl- ation coefficients within each stratum. HiCRep then as- sesses the reproducibility of the Hi-C matrix by applying a novel stratum-adjusted correlation coefficient statistic (SCC) to aggregate the stratum-specific correlation coeffi- cients using a weighted average, with the weights derived from the Cochran-Mantel-Haenszel (CMH) statistic. The SCC has a range of [−1, 1] and is interpreted in a way similar to the standard correlation coefficient. While the methods we compared are tailored for Hi-C data, similar chromosome conformation capture assays such capture Hi-C [44] and ChIA-PET [45] are used to study three-dimensional interactions in the genome. These assays differ from Hi-C due to their targeted na- ture; however, they share many properties of Hi-C assay, such as the genomic distance effect, and can be repre- sented as a contact matrix similar to Hi-C [46, 47]. Re- producibility and quality measures of these assays are lacking in general, raising the possibility of adaptation of the methods we evaluate here to these assays. GenomeDISCO Thi h d f This method focuses on two key aspects of contact maps: the need for smoothing and the multiscale nature of these maps. The need for smoothing arises because contact maps are insufficiently sampled, especially at low sequen- cing depths. This means that a pair of genomic regions can exhibit a low count either from a lack of contact or from insufficient sampling. This problem is addressed by smoothing the data, essentially assuming that two contact maps are reproducible as long as they capture similar higher order structures, even if they differ in terms of indi- vidual contacts. GenomeDISCO investigates contact maps at multiple scales by comparing them at different levels of smoothing and computing a reproducibility score that takes all these comparisons into account. In summary, we show that the recently proposed Hi-C quality and reproducibility measures accurately measure these qualities on a large collection of real and simulated data. By profiling various parameters of Hi-C contact matrices, we describe best practices for applying and inter- preting these measures. We also make available a conveni- ent software tool that simplifies the application of these measures to Hi-C datasets. We hope that adoption of this Page 14 of 19 Page 14 of 19 Yardımcı et al. Genome Biology (2019) 20:57 Yardımcı et al. Genome Biology (2019) 20:57 The smoothing approach is based on random walks on networks. Each contact map is treated as a network, where each node is a genomic region and each edge is weighted by the Hi-C count matrix, following normalization. In this work, square root was used for normalization, but similar results were obtained by using alternative normalization methods, including simple row- and column-based normalization or Knight-Ruiz normalization [48] (data not shown). Random walks are performed on networks to smooth the data, asking for each pair of nodes what is the probability of reaching node i from node j, if t steps are allowed in a random walk biased by the edge weights. The smoothed data can be computed by raising the adjacency matrix of our weighted network to the power t. Lower values of t perform local smoothing of the data, revealing structures such as domains, while larger values of t emphasize compartments. This graph-based smoothing scheme aims to preserve sharp domain boundaries that 2D methods may dilute. which Dii ¼ P jW ij. Reproducibility ¼ 1−combined distance ð Þ This score is in the range [−1, 1], with higher scores representing higher reproducibility. This is because, for each node, the maximum L1 difference is 2, correspond- ing to the case when the node has mutually exclusive contacts in the two contact maps being compared. Thus, the combined distance lies in the range [0, 2], making the reproducibility score fall in the range [−1, 1]. Parameter optimization on an orthogonal dataset revealed the optimal t = {3} [34], which was used in this study. In all pairwise comparisons in this paper, the sample with higher coverage was downsampled to match the coverage of the other sample. GenomeDISCO Thi h d f The matrix L is further normalized by the transformation D−1/2LD−1/2, and its leading eigen- vectors are found. As in other commonly used dimen- sionality reduction procedures, the first few eigenvalues are of particular importance because they capture the basic structure of the matrix, whereas the latter eigen- values are essentially noise. Given two contact maps WA and WB, their corresponding Laplacian matrices LA and LB and corresponding eigenvectors are calculated. Let f λA 0 ; λA 1 ,…, λA n−1} and fλB 0; λB 1 ,…, λB n−1} be the spectra of LA and LB and fυA 0 ; υA 1 ,…, υA n−1} and fυB 0; υB 1 ,…, υB n−1} be their normalized eigenvectors. A distance metric is defined as: Sd A; B ð Þ ¼ Xr−1 i¼0 vA i −vB i : Here ‖‖ represents the Euclidean distance between the two vectors. The parameter r is the number of leading eigenvectors used. In general, Sd provides a metric to gauge the similarity between two contact maps. The dis- tance is then linearly rescaled to a reproducibility score ranging from 0 to 1. To obtain the GenomeDISCO reproducibility score, each contact map is separately smoothed across a range of t values. For each value of t, the L1 distance (i.e., the sum of the absolute values in the difference matrix) be- tween the two smoothed contact maps is computed and normalized by the average number of nodes with non-zero total counts across the two original contact maps compared. Afterward, a combined distance be- tween the two contact maps is obtained by computing the area under the curve of the L1 difference as a func- tion of t. This allows us to consider multiple levels of smoothing and thus multiple scales when computing our scores. Finally, this distance is converted into a re- producibility score as follows: QuASAR-Rep h l p The Quality Assessment of Spatial Arrangement Repro- ducibility (QuASAR) measure uses the concept that within a distance matrix, as the distance between two features approaches zero, the correlation between the rows corresponding to those two features approaches one. This relationship is exploited by calculating the interaction correlation matrix, weighted by interaction enrichment. To determine reproducibility across repli- cates, the correlation of weighted correlation matrices is calculated as follows. In every case, matrices are first fil- tered by removing intrachromosomal interaction matrix rows and columns such that all remaining rows and col- umns contain at least one non-zero entry within 100 bins up- or downstream of the diagonal. The background signal-distance relationship is estimated as the mean num- ber of reads for each inter-bin distance. The interaction correlation matrix is calculated across all pairwise sets of rows and columns within 100 bins of each other from the log-transformed enrichment matrix (non-filtered counts divided by background signal-distance values), excluding bins falling on the diagonal in either set. For a given pair of rows A and B, the correlation is calculated from all col- umns within 100 bins of both A and B, excluding filtered columns. The interaction matrix is then found by adding 1 to valid entries and taking the square root. The weighted correlation matrix is an element-wise product of the cor- relation matrix and the interaction matrix divided by the sum of all valid interaction matrix entries. The replication score is the correlation of weighted correlation matrices between two samples. Note that, to distinguish the use of Empirical reproducibility score thresholds To infer empirical thresholds for distinguishing non-repli- cates for biological replicate pairs for each method, we used the distribution of reproducibility scores assigned to non-replicate pairs and biological replicate pair at a given coverage level. Similar to the concept of a maximal margin hyperplane, the empirical threshold we inferred is the midpoint of the reproducibility score of the highest scor- ing non-replicate pair and the reproducibility score of the lowest scoring biological replicate pair. For each coverage level from 30 million Hi-C interactions to 5 million inter- actions, we inferred a single empirical threshold for each reproducibility metric. These thresholds are available in Additional file 1: Table S2. We adopted the same approach for investigating the effect on coverage on insulation score-identified TADs. For each of the six downsampled cell lines, we identified TADs using insulation score method and compared the total number of TADs, the size distribution of TADs, and the differences between TAD boundaries between the original replicate and downsampled replicates. We observe that the total number of TADs detected and TAD size distributions are similar at all coverage levels (Additional file 1: Figures S16 and S17). We calculated the distances between TAD boundaries identified from downsampled replicates against the TAD boundaries from original biological replicates, and we compared this distribution against the distances between biological rep- licates as a baseline. For five of the six cell types, down- sampling causes the TAD boundaries to shift away from the original boundaries significantly (Kolmogorov-Smir- nov test, P < 0.05) only 10 million and lower number of interactions, further supporting the idea that TAD boundary by insulation score detection is mostly robust to low coverage (Fig. 4f, Additional file 1: Figure S18). HiC-Spector The starting point of spectral analysis is the Laplacian matrix L, which is defined as L = D −W, where W is a symmetric and non-negative matrix representing a chromosomal contact map and D is a diagonal matrix in Page 15 of 19 Page 15 of 19 Yardımcı et al. Genome Biology (2019) 20:57 Yardımcı et al. Genome Biology (2019) 20:57 Yardımcı et al. Genome Biology QuASAR for assessing reproducibility versus data quality (described below), we refer in the main text to “QuA- SAR-Rep” and “QuASAR-QC.” QuASAR for assessing reproducibility versus data quality (described below), we refer in the main text to “QuA- SAR-Rep” and “QuASAR-QC.” insulation score method as described in the previous section. For noise-injected datasets, we found that the number of identified TADs across the genome is only al- tered at the highest noise levels: the number of total TADs increased only by 5% with 50% noise injection (Additional file 1: Figure S11). Consistent with the changes in the total number of TADs, the distribution of TAD sizes is only altered at high noise levels. For 7 out of 11 cell types, we detect a statistically signifi- cant reduction in the TAD size distribution (P < 0.01, Kolmogorov-Smirnov test) only at either 40% or 50% noise (Additional file 1: Figure S12). Furthermore, po- sitions of TAD boundaries are not altered with in- creasing noise levels. For 11 cell types, we calculated the distances between the TAD boundaries of the combined noise-free biological replicate and the TAD boundaries from noise-injected replicates. These distances were com- pared against the TAD boundary distances from biological replicate pairs, which serves as a baseline for how much the TAD boundaries fluctuate between different replicates from the same cell type. Again, we found that the boundary dis- tances are significantly larger than the baseline distribution (one-sided KS test, P < 0.05) only at the 50% noise level for four cell types and never larger for the remaining four cell types (Fig. 4c, Additional file 1: Figure S13). Processing of reproducibility scores All the reproducibility measures we use in this study as- sign a reproducibility score to a pair of Hi-C contact matrices. Due to the sparsity and noise nature of inter- chromosomal matrices, reproducibility scores are only calculated for intrachromosomal matrices. The final re- producibility score assigned to a pair of Hi-C experi- ments in this study is the mean of the reproducibility scores assigned to pairs of Hi-C contact matrices of each chromosome. TAD boundary calling and analysis TAD boundaries were identified using the insulation score [40]. This score captures the density of signal in the Hi-C contact matrix around the diagonal, as a func- tion of genomic position. Because the signal is weaker at the boundary of two TADs, minima in the insulation score profile correspond to TAD boundaries. We used the TAD calling software described in Giorgetti et al. [5], employing the previously used parameters (--ss 80000 --im iqrMean --is 480000 --ids 320000) for calculation of the insulation score and identification of minima. Measures of quality QuASAR-QC The sample quality measure from QuASAR (“QuA- SAR-QC”) uses the same transformation as described above for reproducibility. However, instead of looking at weighted correlation matrices between samples, the quality score is found by taking the weighted correlation mean across all chromosomes and then subtracting the unweighted correlation mean across all chromosomes. Mapping statistics g To generate the genomic distance noise matrix G, we sampled from empirical distributions derived from real Hi-C matrix. In this setting, the genomic distance D is de- fined as the number of bins that lie between a pair of bins i and k, i.e., ∣i −k ∣= D. For every value of D, we build a vector S by collecting the set of real Hi-C matrix entries Mik for which ∣i −k ∣= D. We then randomly select values from S for insertion into G, again considering only entries Gik for which ∣i −k ∣= D. This sampling strategy effectively shuffles the matrix entries in M at a fixed dis- tance, thus preserving the original genomic distance effect while disrupting other higher order structures. However, instead of uniformly sampling from S, we adopted a strati- fied sampling strategy to better model GC and mappabil- ity biases. Specifically, S was broken into multiple strata before sampling. The strata are determined by products of marginals, i.e., Mik is assigned to a certain stratum based on the product of the marginals of bin i and bin k. For a given value of D, we chose stratum size in such a way that each stratum contains 100 elements. When sampling the Gik, we sampled a value from the stratum that Mik belongs to. By repeating the stratified sampling for every value of D, the final matrix G is obtained. We have used three statistics to summarize alignment qual- ity, valid Hi-C fragment pairs, and the ratio of intrachromo- somal and interchromosomal Hi-C interactions. A thorough description of these statistics and their application is reviewed in Lajoie et al. [24]. The first statistic we use is the percentage of aligned pairs, which corresponds to the per- centage of Hi-C fragment pairs that uniquely map to the genome on both sides. Typically, single-sided and non-unique alignments are discarded in Hi-C pipelines [23, 24]. The second statistic is invalid pairs, which is the percent- age of aligned pairs that map against the same restriction fragment. These fragment pairs are non-informative since they do not correspond to a fragment between two different regions [24]. The third statistic is the percentage of intra- chromosomal valid pairs. Number of significant contacts For a given normalized Hi-C contact map, we computed the number of contacts that are deemed statistically significant using Fit-Hi-C [38]. Hi-C contact maps were binned at the 40-kb resolution and normalized using the Knight-Ruiz matrix balancing algorithm [42]. Deeply sequenced Hi-C data from two cell types were binned at To characterize the effects of noise and coverage on TAD boundary identification, we used noise-injected and downsampled datasets as explained before and used Page 16 of 19 Yardımcı et al. Genome Biology (2019) 20:57 Yardımcı et al. Genome Biology (2019) 20:57 10-kb and 40-kb resolutions for Fit-Hi-C analysis (Add- itional file 1: Table S2). Fit-Hi-C assigns a statistical signifi- cance to each contact between two bins by assigning a P value and a q value. For each experiment, we counted the number of contacts that are above a given q value threshold for every intrachromosomal interaction and aggregated them over all chromosomes and used this sum as the total number of significant contacts for a given experiment. 10-kb and 40-kb resolutions for Fit-Hi-C analysis (Add- itional file 1: Table S2). Fit-Hi-C assigns a statistical signifi- cance to each contact between two bins by assigning a P value and a q value. For each experiment, we counted the number of contacts that are above a given q value threshold for every intrachromosomal interaction and aggregated them over all chromosomes and used this sum as the total number of significant contacts for a given experiment. Additionally, the Hi-C assay is subject to the same biases that other next-generation sequencing assays suffer from. These biases’ results include a bias in favor of GC-rich regions and a bias against regions of low mapp- ability. During the generation of both types of noise matri- ces, we factored in such biases by using the sum of each row as a proxy for the overall bias of a bin. Coverage normalization of Hi-C matrices [1] similarly uses mar- ginals to counter such biases. Mapping statistics Random ligations are much more likely to result in interchromosomal fragments; thus, a high ratio of non-informative random ligation events results in an enrichment of interchromosomal interactions and a de- pletion of intrachromosomal interactions [24]. The fourth statistic is the percentage of PCR duplicates, which is esti- mated from the number of aligned pairs that map to the exact same coordinates as another aligned pair [24]. To generate the random ligation noise matrix R, we generated random Hi-C interactions and aggregated them to build a Hi-C contact matrix. We generated these interactions by randomly choosing two bins i and k and adding one to the matrix entry Rik in the random noise contact matrix. Instead of sampling the bins uni- formly, the probability of sampling a bin was set propor- tional to marginal of that bin, thus modeling the GC and mappability bias of each bin. The sampling process was repeated N times, where N is the total number of inter- actions in the original Hi-C contact matrix M, to gener- ate a random ligation noise matrix. Simulation of noisy Hi-C matrices To generate noise for Hi-C data in a realistic manner, we simulated two Hi-C contact matrices that would re- sult from two processes that are not dictated by the 3D organization of chromatin. These “pure noise” matrices are mixed with the real Hi-C contact matrix to generate the final, noisy Hi-C matrix. The first noise matrix models the genomic distance effect, namely the higher probability of observing a Hi-C interaction between two regions that are close along the one-dimensional length of a chromosome. Because such regions are constrained to be close to each other, they are more likely to interact compared to more distal regions, in the absence of any higher order structure. This effect has been documented early on and is generally corrected in Hi-C contact matrices to better visualize medium- and long-range in- teractions [1]. The second noise matrix models the ligation of non-crosslinked DNA fragments during the ligation step of the Hi-C protocol. Fragment pairs that result from random ligation are uninformative since they can link two regions independently of 3D organization. After both noise matrices are generated from the ori- ginal Hi-C matrix, these matrices were mixed in varying proportions to generate a series of noisy Hi-C matrices. Each such matrix is a mixture of three matrices: a real matrix, a genomic distance noise matrix, and a random ligation noise matrix. To generate a simulated matrix with c total counts from, we sampled counts uniformly at ran- dom from one real and two simulated matrices at a given target ratio. In practice, we varied the total proportion of noise from 0 to X%, and for each total noise level, we Page 17 of 19 Page 17 of 19 Yardımcı et al. Genome Biology (2019) 20:57 Yardımcı et al. Genome Biology assigned to biological replicate pairs from four cell types: A549, G410, LNCaP, and HepG2. Hi-C data from the first three cell types were generated by the HindIII restriction enzyme, whereas the HepG2 data was generated using DpnII. These samples also ex- hibit differing levels of coverage (Additional file 1: Table S1). In this analysis, we binned the contact matrix of each replicate at 40-kb, 20-kb, 10-kb, and 5-kb resolution and calculated the various reproduci- bility scores assigned to each biological replicate pair. For this analysis only, we limited the computa- tion of reproducibility scores to the contact matrices of chr22. Funding g G.G.Y and W.S.N are supported by awards NIH U41HG007000, U24HG009446. H.O. is supported by DK107980. B.R.J is supported by awards HG004592, HG003143 and J.D. is supported by awards HG004592, HG003143, DK107980. M.E.G.S. and J.T. is supported by awards NIH R24 DK106766, U41 HG006620. O.U is supported by Howard Hughes Medical Institute International Student Research Fellowship and a Gabilan Stanford Graduate Fellowship award and A.K. is supported by awards NIH DP2OD022870, U24HG009397, R01ES025009- 02S1. T.Y. is supported by NIH T32 GM102057 (CBIOS training program to The Pennsylvania State University), a Huck Graduate Research Innovation Grant and Q.L. is supported by award NIH R01GM109453. For the analysis of QC measures, we generated down- sampled biological replicates from the same six cell types to have fewer interactions (30 × 106, 25 × 106, 20 × 106, 15 × 106, 10 × 106, 5 × 106, 106), resulting in a set of 84 matrices. In addition, we applied the same setup to deeply sequenced datasets from two cell types at a wider range of coverage values (30 × 106, 60 × 106, 120 × 106, 240 × 106, 400 × 106), at multiple resolutions, resulting in 30 matrices. For each downsampled matrix, we calcu- lated QuASAR scores and identified statistically signifi- cant long-range contacts and TAD boundaries. Generation of pseudo-replicates Given two biological replicate experiments, we generated pseudo-replicates by aggregating the two replicates and downsampling from the combined matrix. Combination of two biological replicates is performed by summing the two Hi-C contact matrices of these replicates. Following combin- ation, the resulting combined Hi-C matrix is downsampled as described above to generate pseudo-replicates. We forced the pseudo-replicates to have the average of the total number of interactions of two seed biological replicates. Downsampling l d d Downsampled datasets were generated by converting an input Hi-C matrix into a set of pairwise individual intra- chromosomal interactions and uniformly sampling a given number of interactions from this set. Following downsampling, we re-binned the set of chosen interac- tions into a Hi-C matrix. Simulation of noisy Hi-C matrices consider two settings for the relative proportions of gen- omic distance noise random ligation noise: we either used one third of matrix G and two thirds of matrix R, or vice versa. We note that most analyses in this study were ro- bust to either scenario. The software for injecting noise into Hi-C contact matrices is available at https://github.com/gurkanyar- dimci/hic-noise-simulator. Availability of data and materials h C d d f The Hi-C data used for generating simulated hi-c matrices and the evaluation of quality control and reproducibility methods is publicly available at https:// www.encodeproject.org/. The accession code of each dataset is available in Additional file 1: Table S2. We also list the ENCODE cell line names and the corresponding cell type IDs in the following table. Biosample ENCODE sample IDs A549 ENCSR444WCZ CAKI2 ENCSR401TBQ G401 ENCSR079VIJ LNCaP ENCSR346DCU NCIH460 ENCSR489OCU PANC1 ENCSR440CTR RPMI7951 ENCSR862OG SKMEL5 ENCSR312KHQ SKNDZ ENCSR105KFX SKNMC ENCSR834DXR T47D ENCSR549MGQ HepG2 ENCSR194SRI HeLa ENCSR693GXU Additional file For analysis of reproducibility measures, we limited the analysis to real data from six cell types with replicates of at least 30 million interactions, and we downsampled each in- dividual replicate to have a wide range of total interactions (30 × 106, 25 × 106, 20 × 106, 15 × 106, 10 × 106, 5 × 106, 106). Using a single pseudo-replicate and a single biological replicate pair for each cell type and 15 non-replicates at each coverage level, we generated a total of 189 replicate pairs. These datasets were used for testing the ability of each method to distinguish among different replicate types at lower coverage levels and for explicitly profiling the de- pendence of reproducibility scores on coverage levels. 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https://openalex.org/W3119622399	https://hal.science/hal-03438369/file/msab160.pdf	English	null	Inferring long-term effective population size with Mutation-Selection models	bioRxiv (Cold Spring Harbor Laboratory)	2,021	cc-by	13,554	To cite this version: Thibault Latrille, Vincent Lanore, Nicolas Lartillot. Inferring Long-Term Effective Population Size with Mutation–Selection Models. Molecular Biology and Evolution, 2021, 38 (10), pp.4573-4587. 10.1093/molbev/msab160. hal-03438369 Abstract Mutation–selection phylogenetic codon models are grounded on population genetics ﬁrst principles and represent a principled approach for investigating the intricate interplay between mutation, selection, and drift. In their current form, mutation–selection codon models are entirely characterized by the collection of site-speciﬁc amino-acid ﬁtness proﬁles. However, thus far, they have relied on the assumption of a constant genetic drift, translating into a unique effective population size (Ne) across the phylogeny, clearly an unrealistic assumption. This assumption can be alleviated by introducing variation in Ne between lineages. In addition to Ne, the mutation rate (l) is susceptible to vary between lineages, and both should covary with life-history traits (LHTs). This suggests that the model should more globally account for the joint evolutionary process followed by all of these lineage-speciﬁc variables (Ne, l, and LHTs). In this direction, we introduce an extended mutation–selection model jointly reconstructing in a Bayesian Monte Carlo frame- work the ﬁtness landscape across sites and long-term trends in Ne, l, and LHTs along the phylogeny, from an alignment of DNA coding sequences and a matrix of observed LHTs in extant species. The model was tested against simulated data and applied to empirical data in mammals, isopods, and primates. The reconstructed history of Ne in these groups appears to correlate with LHTs or ecological variables in a way that suggests that the reconstruction is reasonable, at least in its global trends. On the other hand, the range of variation in Ne inferred across species is surprisingly narrow. This last point suggests that some of the assumptions of the model, in particular concerning the assumed absence of epistatic interactions between sites, are potentially problematic. Key words: phylogenetic, codon models, mutation–selection models, population genetic, population size, mutation rate, life-history traits. HAL Id: hal-03438369 https://hal.science/hal-03438369v1 Submitted on 22 Nov 2021 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Inferring Long-Term Effective Population Size with Mutation– Selection Models Thibault Latrille ,,1,2 Vincent Lanore,1 and Nicolas Lartillot1 1Laboratoire de Biometrie et Biologie Evolutive UMR 5558, Universite de Lyon, Universite Lyon 1, CNRS, Villeurbanne, France 2Ecole Normale Superieure de Lyon, Universite de Lyon, Universite Lyon 1, Lyon, France 1Laboratoire de Biometrie et Biologie Evolutive UMR 5558, Universite de Lyon, Universite Lyon 1, CNRS, Villeurbanne, France 2Ecole Normale Superieure de Lyon, Universite de Lyon, Universite Lyon 1, Lyon, France 1Laboratoire de Biometrie et Biologie Evolutive UMR 5558, Universite de Lyon, Universite 2Ecole Normale Superieure de Lyon, Universite de Lyon, Universite Lyon 1, Lyon, France Corresponding author: E-mail: thibault.latrille@ens-lyon.org. Associate editor: Rasmus Nielsen V C The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons. org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Open Access Mol. Biol. Evol. 38(10):4573–4587 doi:10.1093/molbev/msab160 Advance Access publication June 30, 2021 4573 Introduction underlying population-genetic parameters (mutation biases, selective landscapes, effective population size), suggesting the possibility to infer the past variation of these parameters over the phylogeny. Independently, ecological properties, such as phenotypic characters or life-history traits (LHTs) can be ob- served in extinct or in present-day species. Using the com- parative method (Felsenstein 1985), these traits can be reconstructed for the unobserved ancestral species. Combined together, genetic and phenotypic ancestral recon- structions can then be used to unravel the interplay between evolutionary and ecological mechanisms. Since the realization by Zuckerkandl and Pauling (1965) that genetic sequences are informative about the evolutionary history of the species, molecular phylogenetics has developed into a mature and very active field. A broad array of models and inference methods have been developed, using DNA sequences for reconstructing the phylogenetic relationships among species (Felsenstein 1981), for estimating divergence times (Thorne and Kishino 2002), or for reconstructing the genetic sequences of remote ancestors (Liberles 2007). However, genetic sequences might contain information about other aspects of the evolutionary history and, in par- ticular, about past population-genetic regimes. Practically, in order to disentangle mutation, selection and genetic drift, we need to classify individual substitutions into different categories, differing in the strength of mutation, se- lection or genetic drift. In protein-coding DNA sequences, the mutational process occurs at the nucleotide level. Assuming that synonymous mutations are selectively neutral and that selection mostly acts at the protein level, synonymous sub- stitutions can be used to infer the patterns of mutation, Interspecific divergence is the long-term outcome of population-genetic processes, in which point mutations at the level of individuals are then subjected to selection and genetic drift, leading to substitutions at the level of the pop- ulation. As a result, the substitution patterns that can be reconstructed along phylogenies are modulated by the V C The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons. org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Open Access Mol. Biol. Evol. 38(10):4573–4587 doi:10.1093/molbev/msab160 Advance Access publication June 30, 2021 4573 Open Access Concerning variation in dN=dS between lineages, and in a context mostly characterized by purifying selection, the nearly neutral theory predicts that changes in the global strength of selection (measured as dN=dS) is related to changes in the relative strength of genetic drift, which is in turn mediated by changes in effective population size (Ne) (Ohta 1992). Mechanistically, populations with high Ne are characterized by more efficient purifying selection against mildly deleterious mutations, resulting in lower dN=dS (Kimura 1979; Welch et al. 2008). Codon models allowing for variation in dN=dS across branches (Yang and Nielsen 1998; Yang 1998, 2007; Dutheil et al. 2012) have been used to empirically measure such changes in the efficacy of purifying selection along phyloge- nies. Alternatively, dN=dS can be modeled as a continuous trait, varying along the phylogeny as a stochastic process, splitting at each node of the tree into independent processes (Seo et al. 2004). Once empirical estimates of the variation in dN=dS between lineages or groups have been obtained, these can be compared with changes in Ne across lineages, so as to test the validity of the predictions of the nearly neutral theory. Independent empirical estimation of Ne is usually done vie proxies, such as the neutral diversity within species (Galtier 2016), or LHTs. For instance, animal species characterized by a large body size or an extended longevity are typically expected to also have a low Ne (Romiguier et al. 2014). Alternatively, a Bayesian integrative framework has been proposed (Lartillot and Poujol 2011), extending the approach of Seo et al. (2004), in which the joint variation in dS, dN=dS and in LHTs or other proxies of Ne is modeled as a multivariate Brownian process, with a variance–covariance matrix capturing the signal of their correlated evolution. Although not directly expressed in terms of this variable, the mutation–selection formalism induces an equilibrium dN=dS, which is theoretically lower than 1, thus explicitly modeling purifying selection (Dos Reis 2015; Spielman and Wilke 2015). As a result, the mutation–selection codon framework proved to be a valuable null (nearly neutral) model, against which to compare the observed dN=dS by classical codon models, so as to test for the presence of ad- aptation (Rodrigue and Lartillot 2017; Bloom 2017). p g However, these mutation–selection methods have so far assumed the strength of genetic drift, or equivalently Ne, to be constant across the phylogeny. Open Access 4573 MBE Latrille et al. . doi:10.1093/molbev/msab160 without any interference contributed by selection. Then, by comparing the nonsynonymous substitution rate relative to the synonymous substitution rate (the ratio dN=dS), one can estimate the global strength of selection acting on proteins. This idea was formalized using phylogenetic codon models (Goldman and Yang 1994; Muse and Gaut 1994). This led to a broad range of applications, either to detect proteins under adaptive selection (Kosiol et al. 2008), or to measure the modulations of the strength of purifying selection between sites (Echave et al. 2016), genes (Zhang and Yang 2015), or lineages (Lartillot and Poujol 2011). addressed in the context of a mechanistic modeling approach. pp A first attempt in this direction was proposed by Nielsen and Yang (2003), using a population-genetic argument to relate the distribution of dN=dS across sites with the under- lying distribution of fitness effects. This first approach assumes that all nonsynonymous mutations at a given site have the same selection coefficient. As a result of this assumption, there is a simple, one-to-one mapping between the dN=dS at a given site and the selection coefficient associated with all nonsynonymous mutations at that site. In practice, different nonsynonymous mutations are likely to have different fitness effects. In this direction, an alternative mutation–selection codon modeling approach originally proposed by Halpern and Bruno (1998) explicitly assigns a fitness parameter to each amino acid. As a result, the substitution rate between each pair of codons can be predicted, as the product of the mutation rate and the fixation probability of the new codon, which is in turn dependent on the fitness of the initial and the final codons. Since the strength of selection is typically not homogeneous along the protein sequence, and depends on the local physicochemical requirements (Echave et al. 2016; Goldstein and Pollock 2016, 2017), local changes in selective strength are usually taken into account by allowing for site- specific amino-acid fitness profiles. Site-specific amino-acid preferences are typically estimated either by penalized max- imum likelihood (Tamuri et al. 2012, 2014), or in a Bayesian context, using an infinite mixture based on a Dirichlet process prior (Rodrigue et al. 2010; Rodrigue and Lartillot 2014). This second approach is further considered below. New Approaches To address these questions, here we introduce a variant of the mutation–selection codon model, in which selection is mod- ulated along the sequence (using site-specific amino-acid profiles), whereas the mutation rate (l), the effective popu- lation size (Ne), and LHTs are allowed to vary along the phy- logeny (fig. 1). Methodologically, our model is fundamentally an integration between the Bayesian nonparametric version of the Halpern and Bruno (1998) mutation–selection model (Rodrigue and Lartillot 2014), and the molecular comparative framework modeling the joint evolution of life-history and molecular traits (Lartillot and Poujol 2011). In the model introduced here, Ne and l are allowed to vary between species (among branches) as a multivariate geomet- ric Brownian process, but are assumed constant along the DNA sequence. Conversely, amino-acid fitness profiles f are considered constant along the tree but are assumed to vary across sites, being modeled as independent and identically distributed random-effects from an unknown distribution estimated using a Dirichlet process prior. Of note, since Ne and f are confounded parameters (eq. 3), the effective pop- ulation size at the root is set to 1 for identifiability of the fitness profiles. As a result, all values of Ne along the phylogeny are relative to that of the root, with a value of Ne > 1 reflect- ing an increase in Ne along the branches (respectively a de- crease for Ne < 1) compared with the Ne at the root. Formally, the substitution rate (per unit of time) from codon i to j, denoted Qi;j, is equal to the total rate of mutation (per unit of time) at the level of the population (2Neli;j) multiplied by the probability of fixation of the mutation Pfixði; jÞ: This model was implemented in a Markov Chain Monte Carlo (MCMC) framework, allowing for joint inference of site- specific selection profiles and reconstruction of LHTs and population-genetic regimes along the phylogeny. After vali- dating our model and our inference framework against sim- ulated data, we apply it to several cases of interest across metazoans (placental mammals, primates and isopods), for which some proxies of Ne are available. Qi;j ¼ 2Neli;jPfixði; jÞ: (1) (1) In the case of synonymous mutations, which we assumed are neutral, the probability of fixation is independent of the original and target codon, and equals 1=2Ne, such that Qi;j simplifies to: A B FIG. 1. Model summary. Open Access This assumption is clearly not realistic, as attested by the empirically measured variation in dN=dS between lineages using classical codon models or, more directly, by the broad range of synonymous neutral diversity observed across species (Galtier 2016). The impact of this assumption on the estimation of the fitness landscape across sites (Rodrigue and Lartillot 2014; Tamuri et al. 2014), or on the tests for the presence of adaptation (Rodrigue and Lartillot 2017; Bloom 2017) is totally unknown. Relaxing this assumption of a constant Ne is thus necessary. Analyses using these approaches and these proxies of Ne have suggested a negative correlation between dN=dS and Ne (Popadin et al. 2007; Lanfear et al. 2010; Lartillot and Poujol 2011; Lartillot and Delsuc 2012; Romiguier et al. 2014; Figuet et al. 2017), thus confirming the theoretical prediction of the nearly neutral theory. However, the universality and robust- ness of the correlation between dN=dS and Ne is still debated (Nabholz et al. 2013; Lanfear et al. 2014; Figuet et al. 2016; Bolıvar et al. 2019), and further investigation might be re- quired. Moreover, these analyses do not explicitly formalize the quantitative relationship between Ne and dN=dS. This relation is in principle dependent on the underlying fitness landscape (Cherry 1998; Welch et al. 2008; Goldstein 2011), and can show complicated behavior due to nonequilibrium properties (Jones et al. 2017). These questions could be Conversely, since the mutation–selection formalism explic- itly incorporates Ne as a parameter of the model, extending the model so as to let Ne vary across lineages is relatively straight- forward,atleastconceptually.Thisideawas previouslyexplored in the context of two mechanistic models, relying on the dis- tribution of dN=dS across sites (Nielsen and Yang 2003) or accounting for selection on codon usage (Nielsen et al. 2007). 4574 g Long-Term Effective Population Size with Mutation–Selection Models . doi:10.1093/molbev/msab160 Qi;j ¼ li;j: (2) (2) Doing this in the context of mutation–selection models with site-specific amino-acid preferences would provide an occasion to address several important questions: Do we have enough signal in empirical sequence alignments, to estimate the evo- lutionary history of Ne along a phylogeny? Can we more gen- erally revisit the question of the empirical correlations between Ne and ecological LHTs (longevity, maturity, weight, size, ...), previously explored using classical dN=dS based models, but now in the context of this mechanistic framework? Open Access In the case of nonsynonymous mutations, the probability of fixation depends on the difference in fitness between the amino acid encoded by the initial and final codons: Qi;j ¼ li;j 4Ne fAðjÞ fAðiÞ 1 e 4Ne fAðiÞfAðjÞ ; (3) (3) where f is a 20-dimensional vector specifying the log-fitness for each amino acid, and AðiÞ is the amino acid encoded by codon i. Validation Using Simulations Validation Using Simulations The inference framework was first tested on independently simulated multiple sequence alignments (MSAs) (see Materials and Methods). With the aim of applying the infer- ence method to empirical data sets, the simulation parame- ters were chosen so as to match an empirically relevant empirical regime. Thus, the tree topology and the branch lengths were chosen based on a tree estimated on the mam- malian data set further considered below. The other aspects of the simulation model (fitness landscape, variation in Ne) were then varied along a gradient of increasing complexity, so as to test the inference framework under increasingly chal- lenging conditions. The finite population was now modeled explicitly, using a Wright–Fisher simulator, tracking the frequency of each allele at the gene level and at each generation along the phylogeny. No recombination was implemented within genes. These more complex simulation settings account for small popula- tion size effects, for hitchhiking of weakly deleterious muta- tions during selective sweep and for background selection due to linkage disequilibrium. In addition, the effective pop- ulation size Ne and the mutation rate were allowed to fluc- tuate continuously along the branches of the tree (changing by a small amount after each generation of the underlying Wright–Fisher process). Finally, short-term fluctuations of Ne, of the order of 20% per generation, were accounted for by adding a random noise to the Brownian process describing the long-term evolution of Ne. In spite of these deviations between the simulation and the inference models, branch lengths and branch-specific effective population sizes could again be robustly recovered by the inference framework (slope of 0.868, r2 ¼ 0:919, fig. 2B and E). A first series of simulations was meant to test the sound- ness of our inference framework, by simulating essentially under the model used for inference, although with an inde- pendently developed software. Thus, the mutation–selection approximation was assumed to be valid, and sites were sim- ulated under different fitness profiles empirically determined (Bloom 2017), and finally, Ne was assumed to undergo dis- crete shifts at the tree nodes but otherwise to remain con- stant along each branch. In this context, branch lengths and branch-specific values of Ne were accurately estimated by our inference method (fig. 2A and D). Concerning Ne, the slope of the linear regression between true and estimated branch- specific Ne is 0.794 (r2 ¼ 0:915). New Approaches (A) Our method requires a (given) rooted tree topology, an alignment of protein-coding DNA and (optionally) quan- titative life-history trait for the extant species. (B) Relying on a codon model based on the mutation–selection formalism, assuming an autocorre- lated geometric Brownian process for the variation through time in effective population size (Ne), mutation rate (l), and life-history traits, our Bayesian inference method estimates amino-acid ﬁtness proﬁlesacross sites, variation in mutation rateand effective population size along the tree, as well as the node ages and the nucleotide mutation rates. B A B FIG. 1. Model summary. (A) Our method requires a (given) rooted tree topology, an alignment of protein-coding DNA and (optionally) quan- titative life-history trait for the extant species. (B) Relying on a codon model based on the mutation–selection formalism, assuming an autocorre- lated geometric Brownian process for the variation through time in effective population size (Ne), mutation rate (l), and life-history traits, our Bayesian inference method estimates amino-acid ﬁtness proﬁlesacross sites, variation in mutation rateand effective population size along the tree, as well as the node ages and the nucleotide mutation rates. 4575 Latrille et al. . doi:10.1093/molbev/msab160 Results Ne is expected to undergo continuous changes along the lineages of the phylogeny. Second, the diffusion approxima- tion for the probability of fixation (eq. 3) may not hold in small finite populations. Third, assuming a separate substitu- tion process for each site is equivalent to assuming no linkage between sites (free recombination). In practice, however, there is limited recombination, at least within exons, and this could induce deviations from the mutation–selection approximation, due to Hill–Robertson effects. Validation Using Simulations When applied to data simulated using this model, our infer- ence framework could accurately recover the simulated branch lengths (fig. 2D). On the other hand, the distribution of Ne across the tree could not be accurately recovered (slope of 0.0196, r2 ¼ 0:0122, fig. 2F). In fact, no meaningful varia- tion in Ne is detected, and the little variation in Ne that is inferred shows no correlation with the true branch-specific mean Ne values. This effect can be explained by the predicted independence of dN=dS, and more generally of the scaled selection coefficients associated with nonsynonymous muta- tions, to changes in Ne in this specific model of protein sta- bility, as shown theoretically by Goldstein (2013). effective population size is likely to show substantial variation across lineages. This variation in Ne is expected to covary with LHTs, such that large-bodied species are expected to have smaller effective population sizes, compared with small- bodied species. For computational reasons, we restricted our analyses to a random set 18 of orthologous genes, which are then concatenated into a single MSA for analysis. Of note, the mutation–selection model considered here assumes that the fitness profiles do not change with time. In contrast, some genes might experience fluctuating fitness landscapes through time. Such fluctuations are in fact one main cause of ongoing adaptation (Mustonen and L€assig 2009; Rodrigue and Lartillot 2017). For that reason, genes for which positive selection was detected using a site codon model were ex- cluded from the analysis. To assess the reproducibility of our inference and check that the signal about variation in Ne is not driven by particular genes, we analyzed in total four different concatenated MSA each containing 18 randomly sampled genes. The different concatenated MSA showed sim- ilar trends in the change of l and Ne between pairs of rep- licates (see supplementary, Supplementary Material online). The reconstructed long-term changes in effective popula- tion size (Ne) is displayed in figure 3. We visually observe a global trend of increasing Ne throughout the tree around 90 and 60 My. We also observe Ne to be lower in some clades, such as Cetacea and Camelidae, whereas being higher in other clades, such as Rodentia and Pecora. In some cases, a decrease in Ne can be observed along an isolated branch of the tree, for example on the branches leading to the Alpaca (Vicugna pacos) or the cheetah (Acinonyx jubatus). Validation Using Simulations As an alternative model of epistasis between sites, a Fisher geometric model was also considered for the simulations (see supplementary, Supplementary Material online). The results under this model are intermediate between simulations with- out epistasis and simulations under the biophysically inspired model considered above. More specifically, under data simu- lated using Fisher’s geometric model, the true and estimated branch-specific Ne are strongly correlated with each other (r2 ¼ 0:73). On the other hand, the slope of the correlation is substantially <1 (0.571). In other words, the trends in Ne across the tree are correctly recovered, but the range of the variation in effective population size over the tree is substan- tially underestimated. As for the branch lengths, they are again correctly estimated. In summary, our simulation experi- ments show that our inference framework is reliable in the absence of model misspecification and is robust to violations concerning short- versus long-term variation in Ne or to the presence of empirically reasonable levels of Hill–Robertson interference. On the other hand, and very importantly, epis- tasis, which is ignored by the inference model, appears to lead to a general underestimation of the true variation in Ne, to an extent that depends on the exact epistatic model but can go as far as completely obliterating any signal about the true variation in Ne across the tree in the most extreme situations. p ) ( y j ) The estimated covariance matrix (table 1) gives a global synthetic picture of the patterns of covariation between the mutation rate per unit of time l, the effective population size Ne and the three LHTs. First, l covaries negatively with body mass, age at sexual maturity and longevity (table 1). These correlations, which were previously reported (Lartillot and Delsuc 2012; Nabholz et al. 2013) probably reflect generation time effects (Lanfear et al. 2010; Gao et al. 2016). Similarly, and more interestingly in the present context, Ne covaries nega- tively with LHTs (table 1). This is consistent with the expec- tation that small-sized and short-lived species tend to be characterized by larger effective population sizes (Romiguier et al. 2014). Of note, these results mirror previous findings, based on classical codon models, showing that dN=dS tends to be positively correlated with LHTs (Lartillot and Delsuc 2012; Nabholz et al. 2013; Figuet et al. 2017). Validation Using Simulations This positive correlation between dN=dS and LHTs was also recovered on the present data set, using a classical dN=dS based codon model (supplementary materials, Supplementary Material online). Interestingly, the correlation between dN=dS and LHTs is weaker than the correlation between our inferred Ne and LHTs, as expected if the variation in dN=dS indirectly (and imperfectly) reflects the underlying variation in Ne. Finally, Ne and l are positively correlated in their variation (q ¼ 0:44), which might simply reflect the fact that both covary negatively with LHTs. The partial-correlation coeffi- cients (see supplementary, Supplementary Material online) Validation Using Simulations https://academic.oup.com/mbe/article/38/10/4573/6311666 by guest on 11 October 2021 However, the assumptions made for this first round of simulations are almost certainly violated in practice. First, p g These results are encouraging. However, they still rely on the assumption of a site-independent fitness landscape, A B C D E F FIG. 2. (A–C) Branch lengths in expected number of substitutions per site. (D–F) Ne values across nodes (including the leaves) relative to Ne at the root. From left to right: Simulation under the mutation–selection approximation (A, D), under a Wright–Fisher model accounting for small population size effects (5,000 individuals at the root), site linkage and short term ﬂuctuation of Ne (B, E), and ﬁnally accounting for site epistasis in the context of selection for protein stability (C, F). The tree root is 150 My old, where the initial population starts with a mutation rate of 108 per site per generation and generation time of 10 years. These experiments conﬁrm that signal in the placental mammalian tree can allow to reliably infer the direction of change in Ne, even if linkage disequilibrium, short term ﬂuctuation of Ne and ﬁnite population size effects are not accounted for in the inference framework. However, the presence of epistasis between sites is a serious threat to the inference of Ne. B A B A C D F D D F E E FIG. 2. (A–C) Branch lengths in expected number of substitutions per site. (D–F) Ne values across nodes (including the leaves) relative to Ne at the root. From left to right: Simulation under the mutation–selection approximation (A, D), under a Wright–Fisher model accounting for small population size effects (5,000 individuals at the root), site linkage and short term ﬂuctuation of Ne (B, E), and ﬁnally accounting for site epistasis in the context of selection for protein stability (C, F). The tree root is 150 My old, where the initial population starts with a mutation rate of 108 per site per generation and generation time of 10 years. These experiments conﬁrm that signal in the placental mammalian tree can allow to reliably infer the direction of change in Ne, even if linkage disequilibrium, short term ﬂuctuation of Ne and ﬁnite population size effects are not accounted for in the inference framework. However, the presence of epistasis between sites is a serious threat to the inference of Ne. 4576 g Long-Term Effective Population Size with Mutation–Selection Models . Validation Using Simulations doi:10.1093/molbev/msab160 which is equivalent to assuming no epistasis. Yet this assump- tion is almost certainly violated in practice (Pollock and Goldstein 2014; Shah et al. 2015). Accordingly, we imple- mented a more complex, site-dependent fitness landscape accounting for the selective interactions between sites in- duced by the 3-dimensional structure of protein. In this model, the conformational stability of the protein determines its probability of being in the folded state, which is in turn taken as a proxy for fitness (Williams et al. 2006; Goldstein 2011; Pollock et al. 2012). Under this evolutionary model, and at any given time, the fitness landscape at a particular codon site is dependent on the amino acids that are currently pre- sent at those sites that are in the vicinity of the focal site in 3D space (see supplementary, Supplementary Material online). When applied to data simulated using this model, our infer- ence framework could accurately recover the simulated branch lengths (fig. 2D). On the other hand, the distribution of Ne across the tree could not be accurately recovered (slope of 0.0196, r2 ¼ 0:0122, fig. 2F). In fact, no meaningful varia- tion in Ne is detected, and the little variation in Ne that is inferred shows no correlation with the true branch-specific mean Ne values. This effect can be explained by the predicted independence of dN=dS, and more generally of the scaled selection coefficients associated with nonsynonymous muta- tions, to changes in Ne in this specific model of protein sta- bility, as shown theoretically by Goldstein (2013). which is equivalent to assuming no epistasis. Yet this assump- tion is almost certainly violated in practice (Pollock and Goldstein 2014; Shah et al. 2015). Accordingly, we imple- mented a more complex, site-dependent fitness landscape accounting for the selective interactions between sites in- duced by the 3-dimensional structure of protein. In this model, the conformational stability of the protein determines its probability of being in the folded state, which is in turn taken as a proxy for fitness (Williams et al. 2006; Goldstein 2011; Pollock et al. 2012). Under this evolutionary model, and at any given time, the fitness landscape at a particular codon site is dependent on the amino acids that are currently pre- sent at those sites that are in the vicinity of the focal site in 3D space (see supplementary, Supplementary Material online). Empirical Experiments We next applied our inference framework to a series of four empirical data sets spanning different taxonomic groups within metazoans. As a first empirical case, we considered a data set of 77 placental mammals, for which complete ge- nome sequences and information about LHTs is available. Placental mammals offer an interesting example, for which 4577 MBE Latrille et al. . doi:10.1093/molbev/msab160 FIG. 3. Inferred phylogenetic history of Ne (left) and l (right) across placental mammals (posterior mean estimate), based on an analysis of a concatenation of 18 genes randomly chosen among single-copy orthologs putatively under an exclusively purifying selection regime. Ne estimates are relative tothevalue ofNe atthe root.The scale forl is per nucleotidesite andper totaltree depth (i.e.,total time fromthe mostrecent common ancestor to the present). If we assume the root to be 105 My old (Meredith et al. 2011; Kumar et al. 2017), the rescaled mutation rate per site per year in extant species is between 1:1 1010 and 7:8 109. Icons are adapted from http://phylopic.org. FIG. 3. Inferred phylogenetic history of Ne (left) and l (right) across placental mammals (posterior mean estimate), based on an analysis of a concatenation of 18 genes randomly chosen among single-copy orthologs putatively under an exclusively purifying selection regime. Ne estimates are relative tothevalue ofNe atthe root.The scale forl is per nucleotidesite andper totaltree depth (i.e.,total time fromthe mostrecent common ancestor to the present). If we assume the root to be 105 My old (Meredith et al. 2011; Kumar et al. 2017), the rescaled mutation rate per site per year in extant species is between 1:1 1010 and 7:8 109. Icons are adapted from http://phylopic.org. Table 1. Correlation Coefﬁcients between Effective Population Size (Ne), Mutation Rate per Site per Unit of Time (l), and Life-History Traits (maximum longevity, adult weight, and female maturity). Correlation (q) Ne l Maximum Longevity Adult Weight Female Maturity Ne — 0.439* –0.525* –0.544* –0.47* l — — –0.832* –0.835* –0.833* Maximum longevity — — — 0.827* 0.845* Adult weight — — — — 0.809* Female maturity — — — — — NOTE.—Asterisks indicate strength of support of the posterior probability to be different than 0 (pp) as pp > 0:975. able 1. Correlation Coefﬁcients between Effective Population Size (Ne), Mutation Rate per Site per Unit of Time maximum longevity, adult weight, and female maturity). Discussion Mechanistic phylogenetic codon models express the substi- tution rates between codons as a function of the mutation rates at the nucleotide level, selection over amino-acid sequences and effective population size. Thus far, the devel- opment of mutation–selection models of the Halpern and Bruno (1998) family (Rodrigue et al. 2010; Tamuri et al. 2012) has mostly focused on the question of fully accounting for the fine-scale modulations of selection between amino-acids and across sites (Rodrigue et al. 2010; Tamuri et al. 2012). However, the issue of the variation in the global population-genetic regime between species has received much less attention. In particular, effective population size (Ne) is expected to vary substantially over the species of a given clade, yet current mutation–selection models all invari- ably assume Ne to be constant across the phylogeny. To assess the reproducibility of our inference, we analyzed in total six different concatenated MSA each containing 12 randomly sampled genes. The six different concatenated MSA showed similar trends in the change of l and Ne be- tween pairs of replicates (see supplementary, Supplementary Material online). A statistical analysis performed on the pooled estimation of Ne across the six different concatenated MSA exhibits a statistically significant reduction in Ne for underground or depigmented species, or for species with vi- sual impairment (see fig. 4). Of note, the species that did not undergo a transition to subterranean environments feature a relative Ne close to 1, meaning that Ne has not changed much along the lineages (since the root of the tree). Again, the total range of the inferred variation in Ne across the entire extant taxa is surprisingly narrow, with ratio of 3.3 at most between high and low Ne (see supplementary, Supplementary Material online). Here, we have introduced an extension of the mutation– selection model that accounts for this variation. When ap- plied to an alignment of protein coding sequences, this mech- anistic model returns an estimate of the modulations of amino-acid preferences across sites. Simultaneously, it recon- structs the joint evolution of LHTs and molecular and population-genetic parameters (mutation rate l and effective population size Ne) along the phylogeny, whereas estimating the correlation matrix between these variables, intrinsically accounting for phylogenetic inertia. ) Next, our empirical framework was also applied on a set of genes sampled across primates, taken from Perelman et al. (2011) and reanalyzed in Brevet and Lartillot (2021). Empirical Experiments een Effective Population Size (Ne), Mutation Rate per Site per Unit of Time (l), and Life-History Traits female maturity). NOTE.—Asterisks indicate strength of support of the posterior probability to be different than 0 (pp) as pp > 0:975. As another case study, we analyzed a group of isopod species that have made multiple independent transitions to subterranean environments. The transition from a terrestrial to a subterranean lifestyle is typically associated with a global life-history and ecological syndrome characterized by a loss of vision, longer generation times and, most interestingly, smaller population sizes, due to a lower carrying capacity of the subterranean environment (Capderrey et al. 2013). Protein coding DNA sequence alignments and qualitative LHTs, such as habitat (surface or underground), pigmentation (depigmented, partially depigmented or pigmented), and oc- ular structure (anophthalmia, microphthalmia, or ocular) are available for these species (Eme et al. 2013; Saclier et al. 2018). The assumption of a Brownian autocorrelated process for describing the changes in Ne along the tree may not be so between Ne and LHTs are not significantly different from 0. However, this might simply be due to the very strong corre- lation between the three LHTs considered here, such that controlling for any one of them removes most of the signal contributed by the empirically available variation between species. Thus, altogether, the inferred trends in Ne across species appear to be as expected, based on considerations about life- history evolution. On the other hand, the total range of the inferred variation in Ne across the entire extant taxa is sur- prisingly narrow, with one order of magnitude (9.2) at most between high and low Ne (see supplementary, Supplementary Material online). This almost certainly repre- sents an underestimate of the true range of variation across placental mammals. 4578 g Long-Term Effective Population Size with Mutation–Selection Models . doi:10.1093/molbev/msab160 finding of Brevet and Lartillot (2021) on the same data set based on dN=dS-based codon models, where the estimated Ne was found to span several orders of magnitude, and cor- related positively with pS. well adapted to the present case, since the changes in Ne associated with the transition to a subterranean environment are likely to correspond to relatively sudden shifts, rather than continuous variation, and the ecological correlate (subterra- nean vs. terrestrial) is not a quantitative trait. Empirical Experiments However, the data set considered here contains independent transitions to a subterranean lifestyle, thus offering an opportunity to test for a potential correlation between inferred Ne variation and terrestrial versus subterranean lifestyles over the terminal branches. Discussion In addi- tion to LHTs (mass, female maturity, generation time and longevity), information about nuclear synonymous diversity (pS) and nonsynonymous over synonymous diversity (pN=pS), are available for 10 species across the data set and are expected to correlate with Ne according to population genetics (Eyre-walker and Keightley 2007; Galtier 2016). However, the correlation coefficient between our inferred Ne and pS or pN=pS and LHTs are not statistically significant, nor with LHTs (see supplementary, Supplementary Material online). Again, the total range of the inferred variation in Ne across the entire tree is narrow, with ratio of 6.4 at most between high and low Ne. These results contrast with the Reliability of the Inference of the Phylogenetic History of Ne The reconstructions obtained on several empirical data sets, in particular in mammals and in isopods, suggest that the method is able to correctly infer the directional trends of the changes in Ne across species. In particular, in mammals, the inferred variation in Ne correlates negatively with body size and, more generally, with LHTs, as expected under the rea- sonable assumption that large-bodied mammals would tend A B C FIG. 4. Ne estimation for extant isopods species sorted according to their habitat (A), pigmentation (B), and ocular structure (C). Estimated Ne are pooled across six different multiple sequence alignments (MSA) each containing 12 randomly sampled genes in isopods species. All three qualitative traits statistically correlate with changes in Ne in the terminal branches. A A B B C C FIG. 4. Ne estimation for extant isopods species sorted according to their habitat (A), pigmentation (B), and ocular structure (C). Estimated Ne are pooled across six different multiple sequence alignments (MSA) each containing 12 randomly sampled genes in isopods species. All three qualitative traits statistically correlate with changes in Ne in the terminal branches. 4579 MBE Latrille et al. . doi:10.1093/molbev/msab160 and more generally the substitution process is virtually insen- sitive to Ne. This is confirmed by our experiments, showing that the mutation–selection approach explored here cannot infer the true variation in Ne under this model. to have smaller effective population sizes (Popadin et al. 2007; Lartillot and Delsuc 2012; Nabholz et al. 2013; Figuet et al. 2017). Similarly, in isopods, smaller effective population sizes are inferred in subterranean species, again, as expected (Capderrey et al. 2013). e A less extreme outcome is obtained under an alternative model also implementing epistatic interactions between sites via Fisher’s geometric model (Tenaillon 2014; Blanquart and Bataillon 2016). Interestingly, under this model, our inference framework is able to infer the correct trends of Ne, although with a substantially underestimated range of inferred varia- tion, thus mirroring the results obtained on placental mam- mals. Of note, these results do not necessarily imply that models based on biophysics are empirically less relevant than Fisher’s geometric model. Instead, they might just betray that the response of the substitution process to changes in Ne may be sensitive to the exact quantitative details of the un- derlying fitness landscape. More work is probably needed here to characterize these exact conditions. Reliability of the Inference of the Phylogenetic History of Ne Nevertheless, our sim- ulation experiments suggest a global pattern: Epistatic inter- actions induce a buffering of the response of the substitution process to changes in Ne. The meaningful correlation patterns observed with LHTs in the case of placental mammals suggest that this buffering is not complete. Nevertheless, ignoring epistatic interactions at the inference level appears to result in a substantial underestimation of the range over which Ne varies across species. However, although the changes in Ne are in the expected direction (negative correlation with body size, weight, and maturity) (Romiguier et al. 2014), the magnitude of the changes inferred across the phylogeny is surprisingly narrow (at most a factor 9.2 in mammals). This range does not match independent empirical estimates of the variation in mam- mals, where synonymous diversity varies by a factor at least 10 between species (Galtier 2016). In animals, the synony- mous diversity roughly spans two orders of magnitude, whereas Ne varies considerably more across species, by a fac- tor of 103 (Galtier and Rousselle 2020). For instance, effective population sizes estimated based on population genomic data are of the order of 10,000 in humans (Li and Durbin 2011), and 100,000 in mice (Geraldes et al. 2008). Thus, clearly, our approach underestimates the true variation. Different mechanisms not accounted for by the model could explain this result. First, genetic hitchhiking, Hill–Robertson interference, and short-term fluctuations of Ne could generate this effect. However, inference conducted on alignments simulated un- der a Wright–Fisher model accounting for linkage and for short-term variation in Ne suggests that empirically reason- able levels of Hill–Robertson interferences are not strong enough to explain this observation, at least in the regimes explored. Second, l and Ne could also be fluctuating along the genome (Ellegren et al. 2003; Gossmann et al. 2011; Eyre- Walker and Eyre-Walker 2014). This assumption needs to be tested, though we expect that relaxing this assumption would not change drastically the magnitude of inferred Ne since some of this fluctuation should be absorbed by the inferred site-specific fitness profiles. Third, the DNA sequences could also be misaligned at some sites. However, we observe the same magnitude of inferred Ne for different sets of genes indicating this might not be the primary reason. Fourth, the genes selected in our alignments could be under adaptive evolution, or their function could have changed. Reliability of the Inference of the Phylogenetic History of Ne However, at least in mammals, the impact of this potential problem was minimized by the use of genes for which no positive selection was detected using standard phylogenetic codon site models. Interestingly, the magnitude of the inferred range of Ne variation is similar for the placental and the primate data sets (with a 9-fold and 6-fold variation in mammals and primates, respectively), whereas one would have expected a much larger range of variation over the broader phylogenetic scale of placental mammals, compared with primates. An explana- tion could be that the effects of epistasis are more apparent at longer time-scales. Indeed, the total number of substitutions from root to leaves is greater, and as a result, the local envi- ronment, and therefore the fitness landscape at the level of each site, has been less stable across the phylogeny. Although modeling epistasis in an inference framework is a complex biological, mathematical and computational prob- lem, our work points to a potential signal of epistasis that could be retrieved in a phylogenetic context. More specifi- cally, since the slope of the response of the substitution pro- cess to changes in Ne appears to be informative about the epistatic regime, then, conversely, by relying on independent estimates of Ne (e.g., using polymorphism), this effect could be used to leverage a quantitative estimate of the statistical distribution of epistatic effects. g p y g Finally, one key assumption of the mutation–selection model that is likely to be violated in practice is the assump- tion of site-independence. In reality, epistasis might be prev- alent in protein coding sequence evolution (Pollock and Goldstein 2014; Shah et al. 2015). Our simulations under an epistatic landscape point to epistasis being a major factor to be investigated. Indeed, Ne could not be appropriately esti- mated under these simulation settings, although the out- come more specifically depends on the exact model for the fitness landscape. An extreme case is obtained using a bio- physically inspired model, assuming purifying selection for conformational stability. This model was previously explored using simulations and theoretical developments Goldstein (2013), and it was shown that, under this model, dN=dS Other methods have recently been developed to recon- struct phylogenetic changes in Ne. For example, a method recently developed uses polymorphism and generation time for some present-day species to reconstruct Ne along the phylogeny, based on a classical (dN=dS-based) codon model (Brevet and Lartillot 2021). Reliability of the Inference of the Phylogenetic History of Ne This method implicitly relies on a nearly neutral model, assuming a fixed and gamma-shaped distribution of fitness effects across nonsynonymous muta- tions. The approach is calibrated using fossils, and as a result, returns estimates of the absolute value of Ne and of its phy- logenetic variation. Here, in contrast, our method requires 4580 g Long-Term Effective Population Size with Mutation–Selection Models . doi:10.1093/molbev/msab160 strength of drift, in a context where, ultimately, the signal about the intensity of drift comes from the rate of nonsynon- ymous substitutions relative to that of synonymous substitu- tions. However, this purely phylogenetic approach does not leverage a second aspect of Ne at the population level, namely, the fact that Ne also determines the levels of neutral genetic diversity that can be maintained (p ¼ 4Neu, where u is the mutation rate per generation). Hence, neutral diversity yields an independent empirical estimate of Ne. In principle, our mechanistic model could be extended so as to incorpo- rate polymorphism data within species at the tips of the phylogeny. A similar method has been previously pioneered in the case of three species and using a distribution of fitness effect (Wilson et al. 2011). More generally, the nearly neutral theory of evolution defines a long-term Ne, which might be different from the short-term definition of Ne (Platt et al. 2018). Thus we could ask if empirical independent estima- tions of Ne from within species (based on genetic diversity) and between species (based on the substitution process) are congruent, and if not, what are the mechanisms responsible for this discrepancy. neither generation times nor polymorphism data, and the fitness effects are not constrained to a specific distribution. On the other hand, the inferred effective population sizes are only relative. Potential Applications and Future Developments Apart from reconstructing the phylogenetic history of Ne and investigating its causes and covariates, another potentially interesting application of our approach is in detecting adap- tation. In this direction, mutation–selection models represent a useful null nearly neutral model, explicitly modeling the background of purifying selection acting over protein coding genes. Adaptation can then be detected by measuring the deviation from this null model (Rodrigue and Lartillot 2017; Bloom 2017). However, by assuming a constant Ne along a phylogeny, the statistical power of this approach to detect sites under adaptive evolution may not be optimal. Reliability of the Inference of the Phylogenetic History of Ne In particular, the site- specific fitness profiles inferred by the model are averaged along the phylogeny and are seemingly more diffuse than those estimated profiles under our present framework (see supplementary materials, Supplementary Material online). Thus, our method should provide a better null model of purifying selection against which to test for the presence of adaptive evolution. Notwithstanding theoretical considerations on the nearly neutral theory of evolution, empirical clues about the long- term trends in the modulations of the intensity of genetic drift opens up a large diversity of ecological and evolutionary questions. Spatial and temporal changes of genetic drift along ecological niches and events can now be investigated, so as to disentangle the underlying evolutionary and ecological pressures. This approach can be further extended in other directions. First, the mutation rate (l) is considered site-invariant, an assumption which could be relaxed by introducing site- specific mutation rate to account for variation in mutation rate along the sequence. Second, currently, our model also assumes no selection on codon usage. In the case of primates or placental mammals, this assumption is probably reasonable (Yang and Nielsen 2008), although it is more questionable for other groups, in particular Drosophila (Duret and Mouchiroud 1999; Plotkin and Kudla 2011). In principle, this assumption can be relaxed by implementing selective codon preferences that are shared across all sites (Nielsen et al. 2007). Such an implementation would provide the advantage of estimating codon usage biases, whereas simultaneously accounting for its confound- ing effect when estimating selection on amino-acids and in- terspecific variation in Ne. Materials and Methods In the model presented here, Ne and l and quantitative traits are allowed to vary between species (among branches) as a multivariate geometric Brownian process, but assumed con- stant along the DNA sequence. Conversely, amino-acid fitness profiles are assumed to vary across sites, but are considered constant along the tree. The model makes several assump- tions about the evolutionary process generating the observed alignment. First, the species tree topology is supposed to be known, and each gene should match the species tree, mean- ing genes are strict orthologs (no paralogs and no horizontal transfers). Second, there is no epistasis (interaction between sites), such that any position of the sequence has its own independent evolutionary process and a substitution at one position does not affect the substitution process at other positions. Third, from a population genetics perspective, we assumed sites of the protein to be unlinked, or equivalently the mutation rate is low enough such that there is neither Hill–Robertson interference nor genetic hitchhiking. Fourth, polymorphism is ignored in extant species. Third, providing a computationally more efficient imple- mentation of the model would be important for broader application. Currently, running the program on an MSA of 18 mammals genes (77 extant species, and on the order of 15,000 nucleotide sites) for 4,000 iterations of the chain (1,000 are left as burn-in) takes approximately 2–4weeks of compu- tations, which is quite long although still accessible for rea- sonably small data sets. Increasing the computational efficiency could be achieved by several means: First, parallel- izing the program could be achieved by dispatching genes over multiple cores. Second, a large fraction of the computing time is spent in updating the fitness profiles, and thus, fixing them to empirical values or using pre-estimated profiles un- der a constant Ne would lead to a substantial acceleration. The parameterization of the models is described as a Bayesian hierarchical model, including the prior distributions and the parameters of the model. This hierarchical model is formally represented as directed acyclic graph, depicted in figure 5. Finally, estimating Ne in a mutation–selection phyloge- netic model relies on the relation between Ne and the relative 4581 MBE Latrille et al. . doi:10.1093/molbev/msab160 FIG. 5. Directed acyclic graph (DAG) of dependencies between variables. Nodes of the directed acyclic graph are the variables, and edges are the functions. Materials and Methods Hyper-parameters are depicted in red circles, random variables in blue circles, and transformed variables in black. Blue dashed line denotes a drawing from a random distribution, and black solid lines denote a function. All the nodes pointing toward a given node (upstream) are its dependencies which determine its distribution. The other way around, following the arrows in the DAG (downstream), simple prior distributions are combined together to form more complex joint prior distribution which ultimately deﬁnes the prior distribution of the model. FIG. 5. Directed acyclic graph (DAG) of dependencies between variables. Nodes of the directed acyclic graph are the variables, and edges are the functions. Hyper-parameters are depicted in red circles, random variables in blue circles, and transformed variables in black. Blue dashed line denotes a drawing from a random distribution, and black solid lines denote a function. All the nodes pointing toward a given node (upstream) are its dependencies which determine its distribution. The other way around, following the arrows in the DAG (downstream), simple prior distributions are combined together to form more complex joint prior distribution which ultimately deﬁnes the prior distribution of the model. The general time-reversible nucleotide matrix is normal- ized such that the total flow equals to 1: Nucleotide Mutation Rates The generalized time-reversible nucleotide mutation rate ma- trix R is a function of the nucleotide frequencies r and the symmetric exchangeability rates q (Tavare 1986). r ¼ ðrA; rC; rG; rTÞ is the equilibrium base frequency vector, giving the frequency at which each base occurs at each site. q ¼ ð qAC; qAG; qAT; qCG; qCT; qGTÞ is the vector of exchangeabil- ities between nucleotides. Altogether, the rate matrix is: X a2fA;C;G;Tg raRa;a ¼ 1: (8) (8) Site-Dependent Selection R ¼ A C G T qACrC qAGrG qATrT qACrA qCGrG qCTrT qAGrA qCGrC qGTrT qATrA qCTrC qGTrG 0 B B B B B B B B @ 1 C C C C C C C C A : (4) Site-specific amino-acid fitness profiles are assumed i.i.d. from a mixture model, itself endowed with a truncated Dirichlet process prior. Specifically, the mixture has K components (K ¼ 50 by default). The prior on component weights (h) is modeled using a stick-breaking process, truncated at K and of parameter b: (4) h eStickBreakingðK; bÞ () hk ¼ wk Y k1 a¼1 ð1 waÞ; k 2 f1; ... ; Kg; By definition, the sum of the entries in each row of the nucleotide rate matrix Ris equalto 0, giving the diagonalentries: Ra;a ¼ X b6¼a;b2fA;C;G;Tg Ra;b: (5) (5) (9) where wk are i.i.d. from a beta distributioN The prior on the exchangeabilities q is a uniform Dirichlet distribution of dimension 6: wk Betað1; bÞ; k 2 f1; ... ; Kg: (10) (10) q Dirð1; 1; 1; 1; 1; 1Þ: (6) (6) Of note, the weights decrease geometrically in expectation, at rate b, such that lower values of b induce more heteroge- neous distributions of weights. The prior on the equilibrium base frequencies r is a uni- form Dirichlet distribution of dimension 4: Each component of the mixture defines a 20-dimensional fitness profile /ðkÞ (summing to 1), for k 2 f1; . .. ; Kg. These fitness profiles are i.i.d. from a Dirichlet of center c and con- centration a: r Dirð1; 1; 1; 1Þ: (7) (7) 4582 g Long-Term Effective Population Size with Mutation–Selection Models . doi:10.1093/molbev/msab160 /ðkÞ Dirðc; aÞ; k 2 f1; . .. ; Kg: (11) Along a branch b 2 f1; . .. (11) Site allocations to the mixture components jðzÞ 2 f1; ... ; Kg, for z 2 f1; .. . ; Zg running over the Z sites of the alignment, are i.i.d. multinomial of parameter h: Site allocations to the mixture components jðzÞ 2 f1; ... ; Kg, for z 2 f1; .. . ; Zg running over the Z sites of the alignment, are i.i.d. multinomial of parameter h: m MultinomialðhÞ; (12) (12) m MultinomialðhÞ; (12) wheremk ¼ X z2f1; ...; Zg 1jðzÞ¼k: (13) wheremk ¼ X z2f1; ...; Zg 1jðzÞ¼k: (13) (13) Bðb#ÞeN ðBðb"Þ; DTðbÞRÞ; b 2 f1; .. . ; 2P 2g: (17) (17) For a given parameter configuration for the mixture, the Malthusian fitness selection coefficients f ðzÞ at site z, is obtained by taking the logarithm of the fitness profile assigned to this site: The Brownian process at the root of the tree is uniformly distributed, except for the first component fixed to 0 for identifiability (see above). The prior on the covariance matrix is an inverse Wishart distribution, parameterized by j¼ 1 and with q ¼ L þ 1 degrees of freedom: fðzÞ ¼ lnð/ jðzÞÞÞ; z 2 f1; ... ; Zg: (14) (14) R Wishart1ðjI; qÞ: (18) (18) We are interested in approximating the expected substi- tution rates between codons over the branch. Ideally, under the Brownian process just described, the rates of substitution between codons are continuously changing through time. Also, even conditional on the value of Ne at both ends, the Brownian path along the branch entails a random compo- nent, leading to complicated integral expressions for substi- tution rates (Horvilleur and Lartillot 2014). Here, a branchwise approximation is used (Lartillot and Poujol 2011), which con- sists of first deriving an approximation for the mean Ne along the branch, conditional on the values of Ne at both ends, and then using this mean branchwise Ne to define the codon substitution rates. Dated Tree The topology of the rooted phylogenetic tree is supposed to be known and is not estimated by the model. The model estimates the dates at which branches split, thus the dated tree requires P 2 internal node ages that are free parame- ters, where P is the number of extant taxa (leaves of the tree). By definition, leaf ages are all set to 0. The root age is set arbitrarily to 1, but if fossils data are also available the dated tree can be rescaled into absolute time using cross- multiplication. A uniform prior is assumed over internal node ages TðnÞ; n 2 fP þ 1; . .. ; 2P 2g. g ; f ; ; g The duration DTðbÞ represented by a given branch b, for b 2 f1; .. . ; 2P 2g is defined as the difference in ages be- tween the oldest node at the tip of the branch Tðb"Þ, and the youngest node Tðb#Þ: In the case of geometric Brownian process, the most likely path (or geodesic) from Bðb"Þ to Bðb#Þ is the straight line, and therefore, it would make sense to take the mean value of eBðnÞ along this geodesic. We then have NðbÞ e and lðbÞ for each branch b 2 f1; . .. ; 2P 2g of the tree: DTðbÞ ¼ Tðb"Þ Tðb#Þ: (15) (15) NðbÞ e ¼ eBðb#Þ 1 eBðb"Þ 1 Bðb#Þ 1 Bðb"Þ 1 ; lðbÞ ¼ eBðb#Þ 2 eBðb"Þ 2 Bðb#Þ 2 Bðb"Þ 2 : 8 > > > > > < > > > > > : (19) Branch Dependent Traits Nucleotide Mutation Rates ; 2P 2g of the tree, a geo- metric Brownian process starts at the oldest node at the tip of the branch (b"), and ends at the youngest node (b#). The rate of change of the geometric Brownian process per unit of time is constant and determined by the positive semidefinite and symmetric covariance matrix R. Thus the distribution at node b# of Bðb#Þ is multivariate Gaussian, with mean equals to the Brownian process sampled at the oldest node Bðb"Þ, and var- iance DTðbÞR: (11) Branch Dependent Traits Branch Dependent Traits The effective population size Ne and mutation rate per unit of time l are assumed to evolve along the phylogeny, and to be correlated. If quantitative LHTs are also available for some nodes of the tree (leaves and/or internal nodes), they are also assumed to evolve along the phylogeny and to be cor- related between them, and with Ne and l. The total number of traits is noted L, when counting Ne, l and all user-defined LHT (denoted X). Their variation through time is modeled by an L-dimensional geometric Brownian process B. By conven- tion, the first component of the log-Brownian corresponds to Ne, and the second component to l. Thus: (19) Codon Substitution Rates The mutation rate between codons i and j, denoted li;j depends on the underlying nucleotide change between the codons. First, if codons i and j are not nearest-neighbors, li;j is equal to 0. Second, if codons i and j are only one mutation away, Mði; jÞ denotes the nucleotide change (e.g., MðAAT; AAGÞ ¼ TG), and li;j is given by the underlying nucleotide relative rate (RMði;jÞ) scaled by the mutation rate per time (l). Technically, the 4-dimensional nucleotide relative rate matrix (R) is normalized such that we expect one substitution per unit of time, hence the scaling by l. B1ðtÞ ¼ lnNeðtÞ; B2ðtÞ ¼ lnlðtÞ; Bkþ2ðtÞ ¼ lnXkðtÞ; k 2 f1; .. . ; Lg: 8 > > < > > : (16) (16) The effective population size at the root is set to 1 for identifiability of the fitness profiles. 4583 4583 MBE Latrille et al. . doi:10.1093/molbev/msab160 then switching to the uniformization approach of Rodrigue et al. if the first round has failed). For a given branch b and a given site z, the codon substi- tution rate (per unit of branch lenght) matrix Qðb;zÞ is given by: Then, we write down the probability of H given the parameters, and finally, we collect all factors that depend on some parameter of interest and make some simplifica- tions. This ultimately leads to relatively compact sufficient statistics (see supplementary, Supplementary Material online) allowing for fast numerical evaluation of the likelihood (Irvahn and Minin 2014; Davydov et al. 2017). Bayesian Implementation To test the robustness of the model, four parameterized simulators were developed: SimuDiv, SimuPoly, SimuFold, & SimuGeo. All four simulators use a geometric Brownian mul- tivariate process to model the changes in the mutation rate per generation, the generation time and Ne along the lineages. SimuDiv, SimuFold, & SimuGeo all simulate point substitu- tions along the phylogenetic tree. In our simulations, the tree is composed of 77 species (see supplementary, Supplementary Material online), the tree root is 150 million years old, the initial mutation rate is 108 per site per gener- ation and the initial generation time is 10years. The simulator starts from an initial sequence at equilibrium, composed of 15,000 codon sites. The change in fitness is computed for all possible mutations, hence computing all strictly positive sub- stitution rates. At each point, the next substitution is chosen proportional to these rates using in Gillespie’s algorithm (Gillespie 1977). At each node, the process is split, and finally stopped at the leaves of the tree. SimuPoly simulates explicitly each generation along the phylogeny under a Wright–Fisher population, consisting of three steps: Mutation, selection and genetic drift of currently segregating alleles. Mutations are drawn based on a user-defined nucleotide matrix, where our simulations used a symmetric time-reversible mutation matrix. Drift is induced by the multinomial resampling of the currently segregating alleles. We assume that the DNA Bayesian inference was conducted using MCMC. Most phy- logenetic MCMC samplers target the distribution over the model parameters given the sequence alignment, which means that they have to repeatedly invoke the pruning algo- rithm to recalculate the likelihood which is most often the limiting step of the MCMC. An alternative, which is used here, is to do the MCMC conditionally on the detailed substitution history H, thus doing the MCMC over the augmented con- figuration (H, D), under the target distribution obtained by combining the mapping-based likelihood with the prior over model parameters. The key idea that makes this strategy efficient is that the mapping-based likelihood depends on compact summary statistics of H, leading to very fast evaluation of the likelihood. On the other hand, this requires to implement more complex MCMC procedures that have to alternate between: (1) sampling H conditionally on the data and the current parameter configuration. (2) resampling the parameters conditionally on H. Branch Dependent Traits ; Lg can be obtained from the covariance matrix R: Together, the probability of transition between codons for a given branch b and site z is: Together, the probability of transition between codons for a given branch b and site z is: qa;b ¼ Ra;b ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ Ra;aRb;b p : (23) (23) Pðb;zÞ ¼ elðbÞQðb;zÞ; (22) (22) This correlation coefficient is then averaged over the pos- terior distribution, and statistical support is assessed based on the posterior probability of having a positive (or negative) value for the coefficient. which are the matrices necessary to compute the likelihood of the data (D) given the parameters of the model using the pruning algorithm. Branch Dependent Traits As an example, making an MCMC move on the Ne at a given node of the tree is faster since only the mapping-based likelihood (using path sufficient statistics) at the neighboring branches of the node is necessary, instead of computing the likelihood for the entire tree. Qðb;zÞ i;j ¼ 0 if codons i and j are not nearest neighbors; Qðb;zÞ i;j ¼ RMði;jÞ if codons i and j are synonymous; Qðb;zÞ i;j ¼ RMði;jÞ 4NðbÞ e ðfðzÞ AðjÞ fðzÞ AðiÞÞ 1 e4NðbÞ e ðfðzÞ AðiÞfðzÞ AðjÞÞ if i and j are nonsynonymous Qðb;zÞ i;i ¼ X 61 j6¼i;j¼1 Qðb;zÞ i;j : 8 > > > > > > > > > > > > > < > > > > > > > > > > > > > : ( ) Qðb;zÞ i;j ¼ RMði;jÞ if codons i and j are synonymous; Qðb;zÞ i;j ¼ RMði;jÞ 4NðbÞ e ðfðzÞ AðjÞ fðzÞ AðiÞÞ 1 e4NðbÞ e ðfðzÞ AðiÞfðzÞ AðjÞÞ if i and j are nonsynonymous > > > > < > > > > Qðb;zÞ i;i ¼ X 61 j6¼i;j¼1 Qðb;zÞ i;j : (20) MCMC are run for 4,000 points and the first 1,000 points are discarded as burn-in. Convergence is then assessed (see supplementary, Supplementary Material online) by compar- ing two independent chains, checking that both site-specific fitness and branch Ne have the same posterior mean. We see from this equation that, f and Ne are confounded, such that increasing the effective population size whereas decreasing the fitnesses by the same factor leads to the same substitution rate. The branch lengths lðbÞ are defined as the expected num- ber of neutral substitutions per DNA site along a branch: Correlation between Traits The correlation between trait a and trait b 2 f1; .. . ; Lg can be obtained from the covariance matrix R: Correlation between Traits The correlation between trait a and trait b 2 f1; .. . ; Lg can be obtained from the covariance matrix R: Correlation between Traits Correlation between Traits lðbÞ ¼ lðbÞDTðbÞ: (21) (21) The correlation between trait a and trait b 2 f1; .. . Acknowledgments De Magalh~aes JP, Costa J. 2009. A database of vertebrate longevity records and their relation to other life-history traits. J Evol Biol. 22(8):1770–1774. This study was supported by French National Research Agency (ANR-15-CE12-0010-01/DASIRE). We wish to thank Tristan Lefebure for sharing the isopods phylogeny, align- ments, and life-history traits. We also thank Philippe Veber for insightful discussion on mutation–selection models and software development. We gratefully also acknowledge the help of Nicolas Rodrigue, Laurent Gueguen, Benoit Nahbolz, Laurent Duret, Richard Goldstein, Carina Farah Mugal, and Julien Yann Dutheil for their advice and review concerning this manuscript. We also thank the editor and three anony- mous reviewers for their constructive comments, which helped us to improve the manuscript and the analysis. This study was performed using the computing facilities of the CC LBBE/PRABI. Dos Reis M. 2015. How to calculate the non-synonymous to synony- mous rate ratio of protein-coding genes under the Fisher–Wright mutation–selection framework. Biol Lett. 11(4):20141031. Duret L, Mouchiroud D. 1999. Expression pattern and, surprisingly, gene length shape codon usage in Caenorhabditis, Drosophila, and Arabidopsis. Proc Natl Acad Sci USA. 96(8):4482–4487. Dutheil JY, Galtier N, Romiguier J, Douzery EJ, Ranwez V, Boussau B. 2012. Efficient selection of branch-specific models of sequence evolution. Mol Biol Evol. 29(7):1861–1874. Echave J, Spielman SJ, Wilke CO. 2016. Causes of evolutionary rate var- iation among protein sites. Nat Rev Genet. 17(2):109–121. Ellegren H, Smith NG, Webster MT. 2003. Mutation rate variation in the mammalian genome. Curr Opin Genet Dev. 13(6):562–568. Eme D, Malard F, Konecny-Dupre L, Lefebure T, Douady CJ. 2013. Bayesian phylogeographic inferences reveal contrasting colonization dynamics among European groundwater isopods. Mol Ecol. 22(22):5685–5699. Data Availability The data underlying this article are available in Github, at https://github.com/ThibaultLatrille/MutationSelectionDrift (last accessed June 06, 2021), as well as scripts and instructions necessary to reproduce the simulated and empirical experi- ments. The Bayesian inference model, written in Cþþ in the component based (Lanore 2019) software BayesCode, is avail- able at https://github.com/ThibaultLatrille/bayescode (last accessed June 06, 2021). The simulators written in Cþþ are available at https://github.com/ThibaultLatrille/SimuEvol (last accessed June 06, 2021). Supplementary materials de- scribing additional analysis are available online. Bayesian Implementation To implement the mapping-based MCMC sampling strat- egy, we first sample the detailed substitution history H for all sites along the tree. Several methods exist for doing this (Nielsen 2002; Rodrigue et al. 2008), which are used here in combination (first trying the accept-reject method of Nielsen, 4584 g Long-Term Effective Population Size with Mutation–Selection Models . doi:10.1093/molbev/msab160 sequence is composed of exons, with no linkage between exons, and total linkage of sites within an exon. Moreover, in SimuPoly, the instant value of log-Ne can also be modeled as a sum of a geometric Brownian process and an Ornstein– Uhlenbeck process. The geometric Brownian motion accounts for long-term fluctuations, whereas the Ornstein– Uhlenbeck introduces short-term fluctuations. In SimuDiv and SimuPoly, each codon site contributes independently to the fitness depending on the encoded amino acids, through site-specific amino-acid fitness profiles experimen- tally determined (Bloom 2017). In SimuFold, the fitness of a sequence is computed as the probability of the protein to be in the folded state. 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https://openalex.org/W2599875584	https://europepmc.org/articles/pmc5434185?pdf=render	English	null	Olfaction in <i>Parkin</i> carriers in Chinese patients with Parkinson disease	Brain and behavior	2,017	cc-by	5,587	K E Y W O R D S gene, olfaction, Parkin, Parkinson disease, Sniffin’ Sticks test Ying Wang1,* \| Jian-Jun Wu1,2,* \| Feng-Tao Liu1 \| Kui Chen1 \| Chen Chen1 \| Su-Shan Luo1 \| Yi-Xuan Wang1 \| Da-ke Li1 \| Rong-Yuan Guan1 \| Yu-Jie Yang1 \| Yu An3 \| Jian Wang1 \| Yi-Min Sun1 Ying Wang1,* \| Jian-Jun Wu1,2,* \| Feng-Tao Liu1 \| Kui Chen1 \| Chen Chen1 \| Su-Shan Luo1 \| Yi-Xuan Wang1 \| Da-ke Li1 \| Rong-Yuan Guan1 \| Yu-Jie Yang1 \| Yu An3 \| Jian Wang1 \| Yi-Min Sun1 1Department and Institute of Neurology, Huashan Hospital, Fudan University, Shanghai, China 2Department of Neurology, Jing’an District Center Hospital of Shanghai, Shanghai, China 3Institute of Biomedical Sciences, Medical School, Fudan University, Shanghai, China Funding information Funding information National Natural Science Foundation of China, Grant/Award Number: 81571232, 81401048 and 81371413; Project from Ministry of Science and technology of China, Grant/ Award Number: 2016YFC1306500; Project from Science and Technology Commission of Shanghai Municipality, Grant/Award Number: 15ZR1435800 Results: There were 68 patients found having a negative result in PD genetic testing and 43 patients carrying homozygous or compound heterozygous Parkin mutations. Among them, 49 PD panel negative patients and 33 PD-Parkin patients had results of olfactory assessment. PD-Parkin patients performed significantly better on the Sniffin’ Sticks tests than panel negative patients (8.0 ± 1.7 vs. 5.7 ± 1.9, p < .001), but still worse compared to healthy controls (9.4 ± 1.5, p = .003). These differences persisted after adjusting for confounders. Conclusions: Among Chinese population, PD-Parkin patients had relatively preserved olfaction compared to PD panel negative patients after eliminating the interference of other PD related genes, but were still worse than healthy controls. Received: 9 January 2017 \| Revised: 10 February 2017 \| Accepted: 13 February 2017 Received: 9 January 2017 \| Revised: 10 February 2017 \| Accepted: 13 February 2017 Received: 9 January 2017 \| Revised: 10 February 2017 \| Accepted: 13 February 2017 DOI: 10.1002/brb3.680 DOI: 10.1002/brb3.680 Abstract Abstract Background: Olfactory identification was reported to be better among PD (Parkinson disease) patients with Parkin mutations, but previous studies didn’t eliminate the inter- ference of other PD related genes on olfaction, and whether olfaction of Parkin muta- tions patients was better in Chinese population was still unknown. Objective: To assess olfaction function among PD patients with Parkin mutations in Chinese population. Background: Olfactory identification was reported to be better among PD (Parkinson disease) patients with Parkin mutations, but previous studies didn’t eliminate the inter- ference of other PD related genes on olfaction, and whether olfaction of Parkin muta- tions patients was better in Chinese population was still unknown. ference of other PD related genes on olfaction, and whether olfaction of Parkin muta tions patients was better in Chinese population was still unknown. Objective: To assess olfaction function among PD patients with Parkin mutations in Chinese population. Objective: To assess olfaction function among PD patients with Parkin mutations in Chinese population. Correspondence Yi-Min Sun and Jian Wang, Department and Institute of Neurology, Huashan Hospital, Shanghai, China. E il 2504@ i Correspondence Yi-Min Sun and Jian Wang, Department and Institute of Neurology, Huashan Hospital, Shanghai, China. Emails: ys2504@sina.com; wangjian336@hotmail.com Materials and Methods: A total of 226 PD patients with a positive family history or an early-onset age (<50 years) were enrolled for genetic testing of PD related genes by target sequencing and multiple ligation-dependent probe amplification. The clinical data including olfactory function test were investigated. Linear regression was per- formed to adjust for the covariates between all groups. (Daniel & Lees, 1993). The written informed consent was obtained from each subject after the aims and protocol was fully explained. The project was approved by the Huashan Hospital Institutional Review Board. were associated with the presence of incidental Lewy bodies in the brains of decedents without Parkinsonism or dementia during life (Ross et al., 2006). Parkinsonism associated with Parkin gene mutation was one of the most common familial forms of PD, which was characterized by early onset of symptoms (mainly before age 50), slow progression, elective dopaminergic neuronal loss and the absence of Lewy bodies (Gouider-Khouja et al., 2003; Lucking et al., 2000; Mizuno, Hattori, & Matsumine, 1998). Based on the special clinical and pathological fea- tures, Parkin mutation causing Parkinsonism has been postulated as a different disease entity compared to idiopathic PD (Doherty et al., 2013; Khan et al., 2004). While impaired olfaction was frequently associated with PD, olfactory identification was reported to be bet- ter among patients with Parkin mutations (Alcalay et al., 2011; Malek et al., 2016). This was in line with pathologic findings in PD patients that Parkin mutation carriers showed less extensive distribution of Lewy bodies, which might not follow Braak staging and spares olfac- tory structures (Doherty et al., 2013; Gouider-Khouja et al., 2003; Hayashi et al., 2000). 2.3 \| Variant analysis The variants on the exonic and splicing sites (within 100 bp of a splice junction) were analyzed. Mutation reads <5 and mutation frequency <30% were filtered out. Synonymous variant and the variants whose allele frequencies were higher than 1% in 1000Genome, ESP6500 and inhouse databases were excluded. HGMD professional (RRID: SCR_001888) and Clinvar (http://www.ncbi.nlm.nih.gov/clinvar) da- tabase (RRID: SCR_006169) were used to detect the pathogenic level of the variants. The variants not included in these two databases were further classified by the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG) Standards using the terms of ‘pathogenic’, ‘likely pathogenic’, ‘uncertain significance (VUS)’, ‘likely benign’ and ‘benign’ (Richards et al., 2015). 2.1 \| Subject New and follow-up PD patients with a positive family history or an early-onset age (<50 years), were enrolled consecutively from the Movement Disorders Clinic at Huashan Hospital from June 1, 2014 to April 30, 2016 for genetic testing. The clinical materials of these patients were investigated retrospectively especially for the olfactory assessment. Thirty-four gender matched subjects were voluntarily recruited from the community as controls, who had no neurological disorders, psychiatric disorders or sinonasal diseases. All evaluations and tests were carried out in those PD patients after at least 12 hr withdrawal of dopaminergic medication. A detailed assess- ment of clinical materials, neurologic examination including Hoehn and Yahr scale, Unified Parkinson disease rating scale (UPDRS) motor examination were done as previously reported (Xiong et al., 2016). 2.2 \| Genetic testing and variant analysis A panel of 40 PD related genes including Parkin was designed (Table S1). The genetic analysis was carried out by target sequencing and multiple ligation-dependent probe amplification (MLPA) as previ- ously reported (Huang et al., 2013). Briefly, all exons and their cor- responding flanking regions of the genes in the panel were selected as target regions. DNA (Deoxyribonucleic Acid) from the peripheral blood of the patients was prepared as an Illumina sequencing library which were enriched for these target regions. The captured librar- ies were then sequenced using IlluminaNextSeq500 Sequencer with a sequencing depth of 200 × . The raw data was then compared to the reference sequence provided by Mygenostics Inc by standardized procedures (Huang et al., 2013). Khan and colleagues first assessed whether olfactory function differed in Parkin related PD patients compared with early onset PD patients (EOPD) in 2004, and found that olfaction scores of Parkin group were better than EOPD group (Khan et al., 2004). Alcalay and colleagues further compared olfaction between Parkin single het- erozygotes and compound heterozygotes. Among PD patients, com- pound heterozygotes had higher University of Pennsylvania Smell Identification Test scores (UPSIT) than heterozygotes or noncarriers (Alcalay et al., 2011). Until now, whether Parkin mutation carriers in Chinese population had the same olfaction manifestation as Caucasian populations was still unknown. Previous studies about olfaction func- tion in patients of PD with Parkin mutations only excluded several spe- cial genes’ interference (Alcalay et al., 2011; Khan et al., 2004; Malek et al., 2016). Whether the patients carried other PD related genes that might influence olfaction was not clear. In addition, their sample size was relatively small. In order to assess olfaction function in Parkin re- lated PD patients among Chinese population, we evaluated olfactory function in a cohort of Chinese PD patients with Parkin mutations by a gene panel containing all known PD related genes. The variants on all suspected variants were further confirmed by sanger sequencing using standardized procedure. The MLPA was carried out by the kit of SALSA MLPA probemix P051-D1/P052-D2 Parkinson (MRC-Holland) according to the proto- col provided by the manufacture. 1 \| INTRODUCTION License, which permits use, distribution and reproduction in any medium, diagnosis by at least 4 years (Ross et al., 2008). Olfaction test has been proposed as a useful tool for screening those who have a higher risk for development of PD later (Chaudhuri, Healy, & Schapira, 2006; Ross et al., 2008). Neuropathological advances suggested that the ol- factory system was among the earliest brain regions involved in PD (Del Tredici, Rub, De Vos, Bohl, & Braak, 2002) and olfactory deficits Olfactory impairment is one of the earliest manifestations of id- iopathic Parkinson disease (PD), which could predate a clinical Brain and Behavior. 2017;7:e00680. \| 1 of 7 https://doi.org/10.1002/brb3.680 wileyonlinelibrary.com/journal/brb3 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2017 The Authors. Brain and Behavior published by Wiley Periodicals, Inc. (Del Tredici, Rub, De Vos, Bohl, & Braak, 2002) and olfactory deficits Ying Wang and Jian-Jun Wu contributed equally to this work. \| 1 of 7 wileyonlinelibrary.com/journal/brb3 2 of 7 WANG et al. Gudziol, & Mackay-Sim, 2007). The test was conducted on bilateral nostrils. Each stick was held approximately 2 cm in front of the nostrils for 2–3 s, with an interval of 20–30 s between each stick. The patient identified a smell by selecting one out of four possible answers from multiple choice standard cards as previously reported (Huang et al., 2016). Test scores ranged from 0 to 12 and higher scores indicate bet- ter olfaction. All examiners were unaware of the genetic status of the participants at the time of recruitment and thereafter. Gudziol, & Mackay-Sim, 2007). The test was conducted on bilateral nostrils. Each stick was held approximately 2 cm in front of the nostrils for 2–3 s, with an interval of 20–30 s between each stick. The patient identified a smell by selecting one out of four possible answers from multiple choice standard cards as previously reported (Huang et al., 2016). Test scores ranged from 0 to 12 and higher scores indicate bet- ter olfaction. All examiners were unaware of the genetic status of the participants at the time of recruitment and thereafter. to group II. And the 34 controls from community were included in group III. The detailed procedure was shown in Figure 1. The clinical details of patients in group I, group II and group III were shown in Table 1. Age at onset was significantly earlier and disease du- ration was significantly longer in group II. There were no statistic dif- ference in UPDRS III (p = .818), Hoehn and Yahr stage (p = .193), LEDD (p = .100), gender (p = .898), family history (p = .342), MMSE (p = .952) and education (p = .631) between group I and group II. Olfactory examination was conducted at least 1 hr after the last cigarette, meal or beverage. Patients with sinonasal disorders or a cold were not tested. 2.6 \| Statistical analysis Demographics and clinical characteristics were compared among the PD-Parkin patients and PD panel negative patients. Student t test was used for continuous value, and χ2 test for categorical data as ap- propriate. Age and olfaction performance were compared among the PD-Parkin patients, PD panel negative patients and healthy controls using one way ANOVA, and the post hoc tests were conducted by Bonferroni multiple comparison tests. Multiple forward linear re- gression analyses were used to adjust for the covariates between all the groups, including current age, education, Mini-Mental State Examination (MMSE), and levodopa equivalent daily dose (LEDD). p < .05 was considered significant. All analyses were performed with SPSS version 22.0 statistical software (RRID: SCR_002865). Patients in group II were further categorized by compound het- erozygotes and homozygotes, but we didn’t find any significant differ- ence in olfaction scores between them (p = .222). Though statistical significance was not found between the Parkin single heterozygotes and Parkin homozygotes/compound heterozygotes due to the small number of heterozygotes (n = 6), the mean olfaction score of hetero- zygotes was lower than that of the patients in group II (5.3 vs. 8.0). 3.3 \| Olfaction assessment in PD patients and controls Group II performed significantly better (8.0 ± 1.7) than group I (5.7 ± 1.9) on the Sniffin’ Sticks test scores (p < .001), despite longer disease duration (Figure 2 and Table 1). In a linear regression model including all PD patients, the PD-Parkin patients were associated with higher olfaction scores when compared with the PD panel negative patients (p = .005), after adjustment for age, education, MMSE and LEDD. In addition, the olfaction test scores were still significantly lower in the PD-Parkin patients compared with healthy controls (p = .003), and the differences persisted after adjustment for age (p = .001) (Figure 2 and Table 1). 4 \| DISCUSSION The average sequencing depth of the target region was 417.8× and the mean percentage of the target region covered at least 20× or 10× was 92.45% or 96.57% respectively. 625 exons were sequenced with 5 (0.8%) exons having reads depth below 10× (Table S2). These areas were mainly in repetitive or GC rich area. Parkin related PD had clinical and pathologic features that might show varying degrees of difference with idiopathic PD (Doherty et al., 2013). Young onset age, more symmetric involvement, dystonia at presenta- tion, brisk deep tendon reflexes, a good response to levodopa therapy (Lohmann et al., 2003; Lucking et al., 2000), and more symmetric and more marked reduction of dopamine uptake on 123I FP-CIT SPECT scan (DaTSCAN) were found in Parkin-related disease (McNeill et al., 2013). Parkin mutation carriers (homozygotes or compound heterozy- gotes) also showed better cognitive and motor performance than non- carriers, suggesting a slower disease progression (Alcalay et al., 2014). Our study found that the PD-Parkin group had relatively preserved olfaction compared with the PD panel negative group in Chinese PD patients after eliminating the interference of other PD related genes, which further supported the hypothesis that Parkin-related PD was a different clinicopathologic entity to idiopathic PD. 2.5 Olfactory function test was performed with a 12-item Sniffin’ Sticks tests, according to the manufacturer’s instructions (Hummel, Kobal, The diagnosis of PD was according to the United Kingdom Parkinson’s Disease Society Brain Bank criteria for idiopathic PD WANG et al. 3 of 7 3.2 \| Sample characteristics Statistical significance was not found between the Parkin heterozygotes and Parkin homozygotes/ compound heterozygotes due to the small number of heterozygotes, but the mean olfaction score of heterozygotes was lower than that of the patients in group II, which was consistent with previous reports that heterozygous Parkin mutations is regard as risk factors of PD (Alcalay et al., 2011; Malek et al., 2016). Previous study found that the mean UPSIT score in Parkin patients with early-onset Parkinsonism was higher than the Parkin-negative group (Khan et al., 2004). Later, a US study showed that olfaction in young onset PD was related to Parkin mutation status, and those with compound heterozygous mutations had preserved olfactory function, unlike those with single heterozygous mutations and non- carriers (Alcalay et al., 2011). However, all the previous studies only detected and excluded certain genes. Whether other undetected PD related genes had an influence on the olfaction manifestation was not clear. In our study, we compared olfactory function among patients with PD grouped by the Parkin mutation status and found that olfac- tory function was better in the patients harboring 2 Parkin mutations compared to those PD panel negative patients, but still worse than healthy controls. According to our findings, the mean onset age and disease duration was significantly longer in the PD-Parkin group than the PD-panel negative group. However, olfactory test scores were in- dependent of the disease stage and duration (Doty, Deems, & Stellar, 1988). Thus, the different total olfaction scores between the two groups were not due to the different onset age and disease duration. In our study, the genes were detected by the target sequencing and MPLA, which could identify both sequence and dosage mutations in patients with PD. Our control group of Parkin negative patients re- ferred to those with no mutations or variants ranked as pathogenic, likely pathogenic or VUS detected by the panel, thus eliminated the interference of other genes on olfaction (Marras et al., 2011; Saunders-Pullman et al., 2011). This was the most significant differ- ence from previous studies (Alcalay et al., 2011; Malek et al., 2016). Limitations of our study included its sample size and heterozy- gote number, which did not allow us to analyze the olfaction in the single heterozygotes and carriers with 2 Parkin mutations, separately. Another limitation of our study was that the clinical materials of the patients were investigated retrospectively. 3.2 \| Sample characteristics A total of 226 PD patients met the criteria of age at onset <50 or positive family history and enrolled in the current study. According to the genetic testing, 68 patients had no mutations or variants ranked as pathogenic, likely pathogenic or VUS detected by the panel. 43 pa- tients carried homozygous and compound heterozygous Parkin muta- tions only. The other 115 patients were with 1) other causative PD mutations or 2) risk variants (including two patients with both Parkin single heterozygous mutation and other PD-related risk variants, six patients with single heterozygous mutation) or 3) variants ranked as likely pathogenic or VUS or 4) homozygous Parkin mutations plus one other PD-related risk variant (one patient). Olfaction had been the subject of few prior studies with detailed Parkin mutation analysis in patients with PD (Alcalay et al., 2011; Malek et al., 2016). Assessing olfactory function in Parkin related PD was important for a better understanding of the underlying patho- physiology and for the clinical recognition of the Parkin phenotype. Among all the PD panel negative and PD-Parkin patients, 49 (group I) and 35 had results of olfactory assessment. Two PD-Parkin patients were excluded for the history of rhinopolyp. The rest 33 were assigned 4 of 7 \| WANG et al. FIGURE 1 Enrollment scheme of the study WANG et al. WANG et al. 4 of 7 FIGURE 1 Enrollment scheme of the study We also had olfaction scores of healthy people as an internal control group. Meanwhile, this was the first study about olfaction in a cohort of Chinese patients with Parkin-linked PD with a large sample size. Thus, our data strongly supported that the preserved olfaction in the PD patients harboring 2 Parkin mutations compared to idiopathic PD was one component of a wider pattern of distinguishing features. We didn’t find any significant difference in the olfaction scores between the compound heterozygotes and homozygotes (p = .222). This was consistent with previous reports that harboring 2 Parkin mutations typically lead to juvenile-onset or early-onset forms of PD (Abbas et al., 1999; Lucking et al., 2000). 3.2 \| Sample characteristics Group II) p-value (Group II vs. Group III) p-value (Group I vs. Group III) Olfaction score 5.7 ± 1.9 (2–9) 8.0 ± 1.7 (5–11) 9.4 ± 1.5 (6–12) <.001a <.001b .003b <.001b Current age (years) 50.3 ± 10.9 (17–73) 35.1 ± 7.4 (24–49) 62.7 ± 3.7 (57–73) <.001a <.001b <.001b <.001b Age at onset 45.4 ± 10.7 (13–66) 25.1 ± 5.7 (14–41) – – <.001 – – Disease duration (months) 60.7 ± 40.5 (9–193) 119.1 ± 71.3 (19–280) – – <.001 – – Male sex (% male) 35 (71) 24 (73) 24 (71) .981 .898 .846 .934 Family history (%) 23 (47) 12 (36) – – .342 – – Education (years) 11.6 ± 3.4 (5–19) 12.0.±3.3 (6–17) – – .631 – – Hoehn & Yahr stage 2.2 ± 0.7 (1–4) 2.4 ± 0.6 (1–4) – – .193 – – UPDRS–III (“off” medication) 34.2 ± 13.2 (6–64) 34.9 ± 12.6 (14–68) – – .818 – – LEDD (mg/day) 479.3 ± 400.6 (0–2251.2) 348.4 ± 252.6 (0–1100) – –– .100 – – MMSE score 27.9 ± 2.3 (19–30) 28.0 ± 2.0 (23–30) – – .952 – – UPDRS, Unified Parkinson’s Disease Rating Scale; LEDD, levodopa equivalent daily dose; PD, Parkinson disease. 2 UPDRS, Unified Parkinson’s Disease Rating Scale; LEDD, levodopa equivalent daily dose; PD, Parkinson disease. Data were shown as mean ± SD (range) except where indicated. All statistical analyses were done by t tests for continuous variables and by χ2 test for categorical variables as appropriate unless stat aOne way ANOVA bPost hoc tests were conducted by Bonferroni multiple comparison test nt daily dose; PD, Parkinson disease. l analyses were done by t tests for continuous variables and by χ2 test for categorical variables as appropriate unless comparison test FIGURE 2 Scatter diagram of olfaction scores in Parkinson disease (PD) panel negative group, PD-Parkin group and healthy controls. p < .01, p < .001 Better performance on the olfaction scores might reflect a dif- ferent distribution of pathology among the Parkin positive patients compared with the negative patients. In PD, degeneration of the olfactory nucleus with impairment of smell starts early in Braak stageI(Braak et al., 2003) and was thought to be specific for Lewy body-type neurodegeneration. 3.2 \| Sample characteristics Though the patients were collected continuously, the olfaction data was not, which meant not all the PD patients had an olfactory examination. For the data analysis in our study, we chose the patients with results of both genetic test and olfactory assessment, retrospectively. Prospective studies were needed to confirm our results in the future. \| 5 of 7 FIGURE 2 Scatter diagram of olfaction scores in Parkinson disease (PD) panel negative group, PD-Parkin group and healthy controls. p < .01, **p < .001 WANG et al. 5 of 7 UPDRS, Unified Parkinson’s Disease Rating Scale; LEDD, levodopa equivalent daily dose; PD, Parkinson disease. Data were shown as mean ± SD (range) except where indicated. All statistical analyses were done by t tests for continuous variables and by χ2 test for categorical variables as appropriate unless stated otherwise aOne way ANOVA bPost hoc tests were conducted by Bonferroni multiple comparison test Better ferent dis compared olfactory stageI(Bra body-type extensive not follow et al., 201 preserved Parkin mu formation familial-lin synphilin- (Chung et patients w anism for only neuro .9 ± 2.3 (19–30) 28.0 ± 2.0 (23–30) – – .952 – – ting Scale; LEDD, levodopa equivalent daily dose; PD, Parkinson disease. except where indicated. All statistical analyses were done by t tests for continuous variables and by χ2 test for categorical variables as appropriate unless stated otherwise e conducted by Bonferroni multiple comparison test. FIGURE disease (P controls. f f f s a ease Rating Scale; LEDD, levodopa equivalent daily dose; PD, Parkinson disease. (range) except where indicated. All statistical analyses were done by t tests for continuous variables and by χ2 test for categorical variables as appropriate unless stated otherwise sts were conducted by Bonferroni multiple comparison test. RS, Unified Parkinson’s Disease Rating Scale; LEDD, levodopa equivalent daily dose; PD, Parkinson disease. were shown as mean ± SD (range) except where indicated. All statistical analyses were done by t tests for continuous variables and by χ2 test for categorical variables as appropriate unless stated otherwise way ANOVA. bPost hoc tests were conducted by Bonferroni multiple comparison test. TABLE 1 Demographics, disease characteristics and olfaction performance of PD panel negative group, PD-Parkin group and healthy controls PD panel negative group (Group I) (n = 49) PD-Parkin group (Group II) (n = 33) Healthy controls (Group III) (n = 34) p-value (Three groups) p-value (Group I vs. REFERENCES Lim, K. L., Chew, K. C., Tan, J. M., Wang, C., Chung, K. K., Zhang, Y., … Dawson, T. 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Olfaction in Parkin carriers in Chinese patients with Parkinson disease. Brain Behav. 2017;7:e00680. https://doi. org/10.1002/brb3.680 How to cite this article: Wang Y, Wu J-J, Liu F-T, et al. Olfaction in Parkin carriers in Chinese patients with Parkinson disease. Brain Behav. 2017;7:e00680. https://doi. org/10.1002/brb3.680 How to cite this article: Wang Y, Wu J-J, Liu F-T, et al. Olfaction in Parkin carriers in Chinese patients with Parkinson disease. Brain Behav. 2017;7:e00680. https://doi. org/10.1002/brb3.680 Xiong, W. X., Sun, Y. M., Guan, R. Y., Luo, S. S., Chen, C., An, Y., … Wu, J. J. (2016). The heterozygous A53T mutation in the alpha-synuclein gene in a Chinese Han patient with Parkinson disease: Case report and liter- ature review. Journal of Neurology, 263(10), 1984–1992.
https://openalex.org/W4386826318	https://www.nature.com/articles/s41467-023-39984-3.pdf	English	null	Probing three-dimensional mesoscopic interfacial structures in a single view using multibeam X-ray coherent surface scattering and holography imaging	Nature communications	2,023	cc-by	16,356	Article https://doi.org/10.1038/s41467-023-39984-3 1X-ray Science Division, Argonne National Laboratory, Lemont, IL 60439, USA. 2Deutsches Elektronen-Synchrotron (DESY), Notkestr. 85, 22607 Hamburg, Germany. 3Present address: Department of Materials Science and Engineering, University of Virginia, Charlottesville, VA 22904, USA. e-mail: mqichu@anl.gov; wangj@anl.gov Article linear-accelerator-based X-ray free-electron laser facilities14, lensless X-ray coherent diffraction imaging (CDI) has become a popular microscopic method in materials and biological sciences11,15. For iso- lated objects such as single nanocrystalline particles, the Bragg diffraction-based CDI (BCDI)16,17 can probe 3D strain distribution and dynamics during structural transformation18–20. Ptychographic X-ray computed tomography is effective in reconstructing extended sam- ples such as integrated circuits1,21–23 and neuronal reconstruction24. Thanks to the weak interaction between hard X-rays and most of the materials on nano, micro, and even macro scales, conventional trans- mission and Bragg CDI reconstruction methods can take fulladvantage of kinematic analysis (or BA), as the 3D structure can be resolved but requires scanning numerous projection angles. The reconstruction algorithm uses iterative Fourier transforms (FT) and inverse FTs, operated over the form factors of the subject in real and reciprocal spaces11. As a result, the X-ray coherent imaging methods, including theory, instrumentation, and applications, have been growing rapidly25–28. Speciﬁcally, speckles and their intensity distributions are signiﬁcantly distorted near the critical angle of the TER, which is, in turn, sensitive to the depth-dependent structural characteristics in 3D. With surface patterns minimally perturbing the electric ﬁeld intensity proﬁle in the in-plane direction, Yang and Sinha developed a rigorous algorithm to reconstruct the 3D sample structures using the DWBA approach44, where an in-plane homogeneous electric ﬁeld is assumed. On the other hand, in the CSSI measurements, the sizeof the coherent X-rayprobe is comparable with the sample dimension. The samples consisting of high-density materials can signiﬁcantly alter the electric ﬁeld dis- tribution in the off-the-plane direction and in the plane locally. As a result, the average lateral electric ﬁeld assumption in the conventional DWBA becomes insufﬁcient, even after considering the partial coher- ence effects45. Thus, the analysis assuming an in-plane homogeneous electric-ﬁeld proﬁle cannot treat the general coherent scattering cases. On the other hand, Lloyd’s-mirror interferences were observed in radio waves, lasers, and soft X-rays, with applications such as plasma density measurement and surface metrology46–49. Thus far, Lloyd’s mirror-based holography involves the inference with the incident (or primary) waves (beams) in the bright ﬁeld. Generally, conventional holography uses a plane wave or a known wavefront as the reference beam to generate the hologram and reconstruct the real-space images50. Article Due to the high coherence, sensitivities to chemical and magnetic properties, and easiness of wavefront manipulation, most of the holographic methods have been developed in soft X-ray and EUV regimes, and they rely on computationalmethods to retrieve the phase information to reconstruct 2D or 3D real-space images51–57. With hard X-rays, it is more challenging to generate small and high-ﬂux (and coherent) plane waves as the reference for studying micro- or nano- size samples. Thus far, most hard X-ray holography has used the interference of X-ray-induced ﬂuorescence to study single-crystal structures58–61. One of the recent examples is by Jiang et al. extending the ﬂuorescence holography from a simple X-ray mirror case62 to a one-dimensional X-ray waveguide, which resulted in characterizing nanoparticle diffusion with sub-nm resolution in real-time63. For probing surface/interface structures, the transmission scat- tering geometry will not be applicable. For decades, surface X-ray scattering techniques have been developed to interrogate substrate- supported structures, owing to their sensitivity to buried interfaces and the easiness of adapting in situ sample environments29,30. For example, X-ray reﬂectivity can reveal the electron density proﬁle in the direction perpendicular to a thin ﬁlm with a sub-nm resolution31,32. And grazing-incident small- and wide-angle X-ray scattering (GISAXS/ WAXS) provides thin ﬁlms’ in-plane and off-the-plane structural information in either crystalline or non-crystalline forms33,34. Taking advantage of high-coherence X-ray sources, the coherent surface scattering imaging (CSSI) technique35 provides ideal tools to recon- struct surface/interface structures in 3D using scattering data in the kinematic regime in grazing-incidence geometry. g g g g y In CDI and ptychography, specimens are either free of or isolated from substrates. To accommodate transmission measurement, substrate-supported samples, such as integrated circuits, must be prepared with intrusive micromachining processes. However, the supporting substrates of mesoscale planar structures are often an integral part of the sample for enabling functionalities, which can also be used to enhance the measurements, as demonstrated in this work. The grazing-incidence X-ray surface scattering offers distinct advan- tages by allowing more convenient and non-invasive sample prepara- tion than transmission electron and X-ray microscopy36. Unlike in transmission geometry, at surfaces and interfaces, the interference between the incident, scattered, and reﬂected X-rays can generate multiple scattering scenarios, such as standing waves37 and guided surface waves38–41, which can be harnessed to provide the depth sen- sitivity for revealing buried structures with nm or even sub-nm spatial resolution. Article However, such strong surface-induced multiple scattering, for example, total external reﬂection (TER), analogous to total internal reﬂection in optical ﬁbers, can make the surface scattering complex and render the conventional kinematic approximation ineffective. Fundamentally, quantitative analysis of X-ray (or radiations of other wavelengths) scattering at surfaces and interfaces for determining 3D mesostructures requires solving Maxwell’s equations with complex boundary conditions. To understand surface scattering in the con- ventional X-ray scattering regime, Sinha et al. developed distorted- wave Born approximation (DWBA) to solve surface roughness effects statistically, as reported in their 1988 seminal work, which has made an enormous impact over more than 30 years42. Jiang et al. extended the DWBA method to situations where the surface and interface structures signiﬁcantly alter the distribution of electric ﬁelds at surfaces43, in a model-dependent fashion for resolving the statistical structure infor- mation in the sample’s lateral directions. In the case of CSSI, the scattering and reﬂection waves near the TER in either or both incident and exit directions generate dominating multibeam scattering. In this work, to overcome these difﬁculties of gaining true 3D morphological information from coherent surface scattering, we develop a ﬁnite-element DWBA (FE-DWBA) approach using a 3D grid system to compute the scattering from highly heterogenous surface structures in a self-consistent and accurate fashion. The FE-DWBA approach is validated by experimental data from 3D patterns in both simple and complex forms–revealing a quantitative agreement between simulation and experiment results. The FE-DWBA simula- tions allow us to discover the holographic nature of surface scat- tering in grazing-incidence geometry, arising from interference between the two most prominent surface scattering paths. This resembles Lloyd’s mirror interference concept but differs dis- tinctively from previously proposed experimental themes. The holographic imaging demonstrated here is from the interference between the scattering wave from the 3D planar pattern at the sur- face and its reﬂection from the substrate. The interfering waves, traveling along the sample/substrate horizon and signiﬁcantly away from the primary and reﬂected X-ray beam directions, form dark- ﬁeld, high-contrast, and low-noise holographic patterns that contain all 3D information of the sample morphology in a single-view image. With the surface holography concept, we demonstrate that we can unlock the 3D information accurately with a ﬁrst-principles calcula- tion using the interference of the two scattering paths with a mod- iﬁed free-propagation approach. Probing three-dimensional mesoscopic interfacial structures in a single view using multibeam X-ray coherent surface scattering and holography imaging Miaoqi Chu 1 , Zhang Jiang1, Michael Wojcik1, Tao Sun 1,3, Michael Sprung 2 & Jin Wang 1 Miaoqi Chu 1 , Zhang Jiang1, Michael Wojcik1, Tao Sun 1,3, Michael Sprung 2 & Jin Wang 1 Visualizing surface-supported and buried planar mesoscale structures, such as nanoelectronics, ultrathin-ﬁlm quantum dots, photovoltaics, and hetero- geneous catalysts, often requires high-resolution X-ray imaging and scattering. Here, we discovered that multibeam scattering in grazing-incident reﬂection geometry is sensitive to three-dimensional (3D) structures in a single view, which is difﬁcult in conventional scattering or imaging approaches. We developed a 3D ﬁnite-element-based multibeam-scattering analysis to decode the heterogeneous electric-ﬁeld distribution and to faithfully reproduce the complex scattering and surface features. This approach further leads to the demonstration of hard-X-ray Lloyd’s mirror interference of scattering waves, resembling dark-ﬁeld, high-contrast surface holography under the grazing- angle scattering conditions. A ﬁrst-principles calculation of the single-view holographic images resolves the surface patterns’ 3D morphology with nan- ometer resolutions, which is critical for ultraﬁne nanocircuit metrology. The holographic method and simulations pave the way for single-shot structural characterization for visualizing irreversible and morphology-transforming physical and chemical processes in situ or operando. Surface/interface phenomena associated with mesoscale and low- dimensional materials are of great interest to scientists and engi- neers for establishing structure–function relationships. These sys- tems include but are not limited to, planar nanoelectronic circuits1, hierarchical mesoscale surface structures2, thin-ﬁlm-based quantum dots3 and photovoltaic4, heterogeneous catalysts5, biological mem- branes, and supramolecules6–8, where observation in situ, operando, and in real-time is important. In addition, scientiﬁc and technological applications of surface/interface fabrication and manipulation require precise determination of mesoscale 3D structures from tens micrometers down to sub-nanometers (sub-nm) in heterogeneous and non-periodical systems that control the functionality of the devices. Probing structures and dynamics with nanometer (nm) spatial resolution usually requires X-ray scattering and imaging techniques, thanks to the short wavelength. The Bragg diffraction from crystals9 and small-angle scattering10 from non-periodical materials are usually analyzed by assuming kinematic scattering using Born approximation (BA) under weak perturbations. This approach has been proven pow- erful in non-destructive structural characterization for vast scientiﬁc and technological applications11,12. With the advent of coherent X-ray sources provided by modern storage-ring-based synchrotron13 and Nature Communications\| (2023) 14:5795 Nature Communications\| (2023) 14:5795 Nature Communications\| (2023) 14:5795 1 https://doi.org/10.1038/s41467-023-39984-3 Article Nature Communications\| (2023) 14:5795 Results The coherent scat- tering image near the reﬂection spot (qx = 0 and qy = 0) resembles the kinematic scattering from a rectangular pattern in transmission geo- metry. As shown in Fig. S1c, d, the oscillatory intensity along qx and qy directions is the signature of the pattern length (70 µm) along and the width (4 µm) perpendicular to the X-ray beam, respectively. Although the single incident angle speckle pattern contains a limited qz con- tribution in the kinematic regime, the oversampling from data is insufﬁcient to reconstruct the sample structure in the z-direction. The kinematic scattering patterns taken at a series of incident angles are the base for reconstructing the surface structure in 3D, using the FT methods as demonstrated in ref. 35, which is not the subject of this work. Rather, the focus of this study is the large area in Fig. 1b (in αf 2θ space) where αf < 0.4°, where the momentum transfer in the z-direction cannot be deﬁned by Eqs. S1–S3 and a simple qz due to multibeam scattering and interference, or dynamical scattering effects. Note the intense scattering speckles in the region just above the horizon (αf slightly larger than 0) well below the Si and Au critical angles. This low exit-angle region below the critical angles, from 0 to 0.3° as highlighted in the box and magniﬁed in Fig. 1c, should not have contained intense scattering because of the optical reciprocity. Instead, the strong scattering near the sample/substrate horizon is linked to the surface waveguide phenomena41,64 due to the substrate- induced multibeam interference, analogous to standing waves gener- ated above an X-ray mirror below or near the critical angle, the focus of this work. Furthermore, high-intensity, high-contrast speckle fringes Article The analysis is tested with a simple but true 3D sample by varying thicknesses across the surface. The holographic calculation captures all the tell-tale features of the 3D micro-patterns with unprecedented nm or sub-nm spatial resolution. Reproducing these features by a ﬁnite-element-based numerical simulation may be difﬁcult, if not impossible, due to the computa- tional cost and simulation limitations. Nature Communications\| (2023) 14:5795 2 Article https://doi.org/10.1038/s41467-023-39984-3 Fig. 1 \| X-ray coherent surface scattering imaging (CSSI) of an ultrathin gold single micro-bar pattern supported by a ﬂat silicon substrate. a Reﬂective scattering geometry (drawn not to scale) showing the grazing-incident angle (αi) of the coherent X-ray beam impinging onto the surface pattern, the reﬂected and scattered X-rays at an exit angle (αf), and azimuthal scattering angle (2θ). The cartesian coordinate systems are ﬁxed at the sample surface: x along the X-ray beam direction, y parallel to the surface and perpendicular to the beam, and z perpendicular to the substrate surface (out-of-plane). b CSSI scattering pattern recorded by an EIGER X4M detector placed 5 m away from the sample. The pixels are converted linearly to the scattering angles (αf and 2θ), which are the para- meters or coordinators used in most of the simulations in this work. c Close-up of the scattering pattern in a low-αf and high-2θ region, where the intensity is dominated by multibeam scattering interference in the dashed-line box in panel b. Fig. 1 \| X-ray coherent surface scattering imaging (CSSI) of an ultrathin gold single micro-bar pattern supported by a ﬂat silicon substrate. a Reﬂective scattering geometry (drawn not to scale) showing the grazing-incident angle (αi) of the coherent X-ray beam impinging onto the surface pattern, the reﬂected and scattered X-rays at an exit angle (αf), and azimuthal scattering angle (2θ). The cartesian coordinate systems are ﬁxed at the sample surface: x along the X-ray beam direction, y parallel to the surface and perpendicular to the beam, and z perpendicular to the substrate surface (out-of-plane). b CSSI scattering pattern recorded by an EIGER X4M detector placed 5 m away from the sample. The pixels are converted linearly to the scattering angles (αf and 2θ), which are the para- meters or coordinators used in most of the simulations in this work. c Close-up of the scattering pattern in a low-αf and high-2θ region, where the intensity is dominated by multibeam scattering interference in the dashed-line box in panel b. Results the detector intercepts the Ewald sphere in the grazing-incidence reﬂection geometry35. The scattering pattern from the simple bar sample can be related to the sample form factors more easily when remapped from the (αf ,2θ) coordinates into the conventional reci- procal space or the qx – qy plane using Eqs. S1–S3. The coherent scat- tering image near the reﬂection spot (qx = 0 and qy = 0) resembles the kinematic scattering from a rectangular pattern in transmission geo- metry. As shown in Fig. S1c, d, the oscillatory intensity along qx and qy directions is the signature of the pattern length (70 µm) along and the width (4 µm) perpendicular to the X-ray beam, respectively. Although the single incident angle speckle pattern contains a limited qz con- tribution in the kinematic regime, the oversampling from data is insufﬁcient to reconstruct the sample structure in the z-direction. The kinematic scattering patterns taken at a series of incident angles are the base for reconstructing the surface structure in 3D, using the FT methods as demonstrated in ref. 35, which is not the subject of this work. Rather, the focus of this study is the large area in Fig. 1b (in αf 2θ space) where αf < 0.4°, where the momentum transfer in the z-direction cannot be deﬁned by Eqs. S1–S3 and a simple qz due to multibeam scattering and interference, or dynamical scattering effects. Note the intense scattering speckles in the region just above the horizon (αf slightly larger than 0) well below the Si and Au critical angles. This low exit-angle region below the critical angles, from 0 to 0.3° as highlighted in the box and magniﬁed in Fig. 1c, should not have contained intense scattering because of the optical reciprocity. Instead, the strong scattering near the sample/substrate horizon is linked to the surface waveguide phenomena41,64 due to the substrate- induced multibeam interference, analogous to standing waves gener- ated above an X-ray mirror below or near the critical angle, the focus of this work. Furthermore, high-intensity, high-contrast speckle fringes the detector intercepts the Ewald sphere in the grazing-incidence reﬂection geometry35. The scattering pattern from the simple bar sample can be related to the sample form factors more easily when remapped from the (αf ,2θ) coordinates into the conventional reci- procal space or the qx – qy plane using Eqs. S1–S3. Experiment setup To illustrate and use the dynamical scattering effect from the sample and substrate near the critical angle, we ﬁrst designed a simple sample so that all sample features and form factors are identiﬁable in a single scattering image at any grazing-incidence angle. The 3D surface pat- tern consists of an electron-beam-lithography patterned gold (Au) bar with dimensions of 4.0-µm width, 70-µm length, and 55-nm thickness, deposited onto polished silicon (Si) substrate with the 5-nm titanium (Ti) adhesive layer. The preparation of the samples is described in more detail in Methods. Coherent surface scattering imaging of this sample was performed at P10, a dedicated coherent scattering beam- line at the PETRA III synchrotron facility, using an X-ray photon energy, E, of 8.0 keV (wavelength λ = 0:155 nm). At this wavelength, the incre- ments of the refractive index of Si and Au are 7:67 × 106 and 4:77 × 105, resulting in critical angles of 0.22° and 0.56°, respectively. The monochromatic and coherent X-ray beam was focused to 1.7 × 3.5 (V × H, full-width-at-half-maximum, FWHM) µm2 using a set of com- pound refractive lenses. The detector is an EIGER X 4M with 75 × 75 µm2 pixels placed 5.0 m downstream from the sample. As shown in Fig. 1a, X-rays impinge on the sample at an incident angle of αi = 0:6, above both critical angles. The coordinate system is described in more detail in Methods and Supplementary Information (SI) #1. Figure 1b shows the measured scattered intensity distribution in logarithmic scales. Due to the high coherent ﬂux and coherence-preserving optics at the beamline, coherently scattered speckles are strong and visible on the entire detector, even with an exposure time of 1 s per image. In the higher exit angle (αf > 0:4) region, the scattering is mostly kinematic, as discussed in SI #1, where an overall interpretation of the coherent image is given. Brieﬂy, the arch-shaped scattering pattern is due to how Nature Communications\| (2023) 14:5795 3 https://doi.org/10.1038/s41467-023-39984-3 Article Fig. 2 \| Scattering intensity distribution simulated based on conventional distorted-wave Born approximation (DWBA). a Schematic of conventional DWBA scattering wave vector transfers in the reﬂection geometry (not to scale) with a layered approach developed for evaluating grazing-incidence small-angle scattering (GISAXS) using incoherent X-rays. Experiment setup In the DWBA scheme, the electron density in a layer is averaged in the x-y plane over the X-ray footprint, as highlighted in the shaded areas of the schematic. b Schematic of the four momentum-transfe terms in the DWBA theory, which constitute the components described in Eqs. (S14–S17) in an arbitrary single layer, e.g., the top layer in the schematic. The shading colors represent the lateral electron density proﬁle in the layer. The red arrows highlight the exit wave vector in each scattering path. c Surface scattering intensity, when all four terms are included in the simulation surface scattering intensity, fails to reproduce the experimental data when αf is between 0° and 0.3° out-of-plane direction. Figure 2b shows the most critical aspect of DWBA, the multibeam scattering paths, which account for four scat- tering components in layer l: (1) scattering of the incident beam by the layer, the same as BA (kinematic theory), (2) scattering from the sample then reﬂected at the layer bottom interface, (3) scattering from the reﬂected beam, and (4) reﬂected scattering from the reﬂected beam from the bottom interface. The notation describing the four scattering paths in the illustration are Ti,f l and Ri,f l , the incident and exit (superscripts i and f, respectively) complex transmissive (T) and reﬂective (R) coefﬁcients, ki,f l , incident and exit wave vectors in layer l, expressed in the coordinate system, rðx, y, zÞ, ﬁxed on the sample where z is normal to the layers. This method and an algorithm can be found in refs. 43,63, where GISAXS scattering patterns can be simu- lated perfectly for the dynamical scattering in the low-exit angle regime. In this experiment, the Au micropattern creates a strong in-plane inhomogeneity when its lateral dimension is comparable to the coherent X-ray beam size. Also, with high-Z material Au, the averaged electron density in the pattern layer can be higher than the substrate (silicon in this case). This renders the conventional DWBA approaches ineffective, shown in Fig. 2c, demonstrating that the dynamical scat- tering features in the experimental data cannot be reproduced by this layered DWBA approach. It fails to produce the intense interference fringes and the complex dynamical scattering patterns where 2θ is between 0 and 0.3° on the detector. Experiment setup Instead, the simulated pattern below 0.3° is relatively weak, implying that X-rays cannot penetrate or exit from the sample at the low angles because they are close to and below the critical angles of the substrate 0.22° and the sample layer (0.56°). Since the simulation does not yield the multibeam interference features in the low exit angle region (αf < 0.3°), a new method to implement the dynamical theory is required for rigorously modeling the coherent scattering from substrate-supported surface patterns quantitatively. Fig. 2 \| Scattering intensity distribution simulated based on conventional distorted-wave Born approximation (DWBA). a Schematic of conventional Fig. 2 \| Scattering intensity distribution simulated based on conventional Fig. 2 \| Scattering intensity distribution simulated based on conventional distorted-wave Born approximation (DWBA). a Schematic of conventional DWBA scattering wave vector transfers in the reﬂection geometry (not to scale) with a layered approach developed for evaluating grazing-incidence small-angle scattering (GISAXS) using incoherent X-rays. In the DWBA scheme, the electron density in a layer is averaged in the x-y plane over the X-ray footprint, as highlighted in the shaded areas of the schematic. b Schematic of the four momentum-transfer terms in the DWBA theory, which constitute the components described in Eqs. (S14–S17) in an arbitrary single layer, e.g., the top layer in the schematic. The shading colors represent the lateral electron density proﬁle in the layer. The red arrows highlight the exit wave vector in each scattering path. c Surface scattering intensity, when all four terms are included in the simulation surface scattering intensity, fails to reproduce the experimental data when αf is between 0° and 0.3°. g. \| Scatte g te s ty d st but o s u ated based o co e t o a distorted-wave Born approximation (DWBA). a Schematic of conventional DWBA scattering wave vector transfers in the reﬂection geometry (not to scale) with a layered approach developed for evaluating grazing-incidence small-angle scattering (GISAXS) using incoherent X-rays. In the DWBA scheme, the electron density in a layer is averaged in the x-y plane over the X-ray footprint, as highlighted in the shaded areas of the schematic. b Schematic of the four momentum-transfer terms in the DWBA theory, which constitute the components described in Eqs. (S14–S17) in an arbitrary single layer, e.g., the top layer in the schematic. The shading colors represent the lateral electron density proﬁle in the layer. Finite-element DWBA To tackle this problem, instead of just treating the sample as a layered structure, we further divide the sample and its surroundings into a 3D grid, not only in the out-of-plane direction as in conventional DWBA but alsoin the in-plane directions. The top and side views of the 3D grid are shown in Fig. 3a. In other words, our approach takes into account the sample’s 3D inhomogeneity inside the coherently illuminated volume. We ﬁrst estimate the region of the sample/substrate illumi- nated by X-rays using the measured FWHM of the X-ray probe and the incident angle. The areas outside the X-ray footprint do not contribute to the scattering process and are not included in the grid to accelerate the computation. The dimensions of a grid cell are deter- mined by the resolution of the measurement as deﬁned by the max- imal momentum transfer in each direction on the detector of more than 4 million pixels. In practice, we chose to use smaller 3D cells size of δx,y,z = (175 × 5 × 0.5) nm3 to avoid numerical artifacts. With this resolution-limited cell size, the in-plane electron density variation is not detectable inside each cell. Then, the electron density proﬁle in each vertical stack of cells, a column along the sample normal direc- tion, in the out-of-plane direction is sliced into layers, as illustrated in Fig. 3b, where the scattering terms or paths at the interface between the 3D adjacent elements are also schematically shown. The neigh- boring cells are labeled as subscripts a and b. It should be noted that the electron density of the surface sample pattern is no longer aver- aged over each horizontal layer for computing the electric ﬁeld and coefﬁcients. Rather, they are computed locally in each grid element. Next, the transmission and reﬂection in each layer are calculated by using Parratt’s recursive formalism to satisfy the boundary conditions for the electromagnetic ﬁeld in the out-of-plane direction. The eigen- states for the incident wave (ψi p) and the exit wave (ψf p) of the pth cell can be computed as shown in SI #3. are observed along and around the arc in Fig. 1c. The periodic scat- tering fringes along both αf and 2θ angles indicate that the 3D form factors of the surface pattern can be retrieved, as shown in the fol- lowing subsections. Experiment setup The red arrows highlight the exit wave vector in each scattering path. c Surface scattering intensity, when all four terms are included in the simulation surface scattering intensity, fails to reproduce the experimental data when αf is between 0° and 0.3°. Nature Communications\| (2023) 14:5795 Finite-element DWBA First, we compute the expected scattering pattern using the kinematic or BA approach (as described in SI #2), assuming the elec- trons in the sample scatter the unperturbed incident plane wave. Thus, the scattering intensity is given by the squared modulus of the FT of the sample’s electron density proﬁle. As expected, the BA fails to predict the scattering patterns satisfactorily (Fig. S2b). Starkly differ- ent from the experimental data, the simulated scattering pattern lacks all features in the low exit angle region, where dynamical scattering effects dominate. The DWBA is another powerful approach for computing surface scattering from ﬂat and rough surfaces42. In the DWBA, thin nanos- tructures at a substrate can be treated as a strong perturbation to the sample’s electron density and the electric ﬁeld in the out-of-plane direction. The sample electron density proﬁle in the z-direction is averaged over the in-plane directions to calculate perturbed incident and exit wavefronts. The scattering of a surface structure supported by a substrate can be summarized in Fig. 2a, the layered approach in the Nature Communications\| (2023) 14:5795 4 https://doi.org/10.1038/s41467-023-39984-3 Article . 3 \| Finite-element distorted-wave Born approximation (FE-DWBA) proach for coherent surface scattering. a The sketch shows the top and side ws of a highly heterogeneous sample and the corresponding FEA grid pattern of substrate-supported sample (not to scale). In the top view, the shading in yellow icates the X-ray footprint, while the gray-scale shading shows the lateral electron nsity variation in the surface pattern. In the side view, the shadings highlight the eral cells in the y-direction. b The four scattering contributions describe the full WBA terms in two adjacent lateral cells. The transmission and reﬂection coefﬁ- nts have also been indexed at each lateral interface. c CSSI pattern calculated h the FE-DWBA algorithm. The simulated scattering pattern reproduces the low t angle speckles and the intense interference fringes in the dashed boxes, which ect the nature of the multibeam or dynamical scattering mechanism, revealing pattern thickness. d Quantitative comparison between the FE-DWBA simulation he line scan at the center of the arc (equivalent to qx = 0) along the dashed black line from αf = 0 (horizon) to 0.3°, slightly above the silicon substrate critical angle (0.22°). The intensity oscillation, the signature of the dynamical scattering, below and near the critical angles of the substrate and the sample, matches well with the experimental data. Finite-element DWBA a The sketch shows the top and side views of a highly heterogeneous sample and the corresponding FEA grid pattern of the substrate-supported sample (not to scale). In the top view, the shading in yellow indicates the X-ray footprint, while the gray-scale shading shows the lateral electron density variation in the surface pattern. In the side view, the shadings highlight the lateral cells in the y-direction. b The four scattering contributions describe the full DWBA terms in two adjacent lateral cells. The transmission and reﬂection coefﬁ- cients have also been indexed at each lateral interface. c CSSI pattern calculated with the FE-DWBA algorithm. The simulated scattering pattern reproduces the low exit angle speckles and the intense interference fringes in the dashed boxes, which reﬂect the nature of the multibeam or dynamical scattering mechanism, revealing the pattern thickness. d Quantitative comparison between the FE-DWBA simulation —the line scan at the center of the arc (equivalent to qx = 0) along the dashed black approach for coherent surface scattering. a The sketch shows the top and side views of a highly heterogeneous sample and the corresponding FEA grid pattern of the substrate-supported sample (not to scale). In the top view, the shading in yellow indicates the X-ray footprint, while the gray-scale shading shows the lateral electron density variation in the surface pattern. In the side view, the shadings highlight the lateral cells in the y-direction. b The four scattering contributions describe the full DWBA terms in two adjacent lateral cells The transmission and reﬂection coefﬁ the length of the bar. The oscillation period corresponds to the bar length of 70.0 µm precisely. Therefore, this highlighted region in the single-view scattering image taken at an incident angle of 0.6° captures the 3D structural information of the sample. f 3D schematic of a more complex surface sample (shown not to scale), a petal-like pattern with a thickness of 55 nm. g Experimental data collected at 0.6° incident angle (right panel) and simulation by using the FE-DWBA method (left panel). The signature of the pattern thickness—the enhanced intensity modulations at the low exit angle is evident. The total scattering from the grid is then the modulus square of the scattering wave within a domain from the boundaries: dynamical scattering effect due to the in-plane inhomogeneity in the pattern (more in SI #3). Finite-element DWBA e Scattering intensity line scan along the dashed white line containing the information on the form factor of the thin-ﬁlm surface pattern along the length of the bar. The oscillation period corresponds to the bar length of 70.0 µm precisely. Therefore, this highlighted region in the single-view scattering image taken at an incident angle of 0.6° captures the 3D structural information of the sample. f 3D schematic of a more complex surface sample (shown not to scale), a petal-like pattern with a thickness of 55 nm. g Experimental data collected at 0.6° incident angle (right panel) and simulation by using the FE-DWBA method (left panel). The signature of the pattern thickness—the enhanced intensity modulations at the low exit angle is evident. line from αf = 0 (horizon) to 0.3°, slightly above the silicon substrate critical angle (0.22°). The intensity oscillation, the signature of the dynamical scattering, below and near the critical angles of the substrate and the sample, matches well with the experimental data. e Scattering intensity line scan along the dashed white line containing the information on the form factor of the thin-ﬁlm surface pattern along the length of the bar. The oscillation period corresponds to the bar length of 70.0 µm precisely. Therefore, this highlighted region in the single-view scattering image taken at an incident angle of 0.6° captures the 3D structural information of the sample. f 3D schematic of a more complex surface sample (shown not to scale), a petal-like pattern with a thickness of 55 nm. g Experimental data collected at 0.6° incident angle (right panel) and simulation by using the FE-DWBA method (left panel). The signature of the pattern thickness—the enhanced intensity modulations at the low exit angle is evident. Fig. 3 \| Finite-element distorted-wave Born approximation (FE-DWBA) Fig. 3 \| Finite-element distorted-wave Born approximation (FE-DWBA) h f h f h k h h h d line from αf = 0 (horizon) to 0.3°, slightly above the silicon substrate critical angle (0.22°). The intensity oscillation, the signature of the dynamical scattering, below and near the critical angles of the substrate and the sample, matches well with the experimental data. e Scattering intensity line scan along the dashed white line containing the information on the form factor of the thin-ﬁlm surface pattern along Fig. 3 \| Finite-element distorted-wave Born approximation (FE-DWBA) approach for coherent surface scattering. Nature Communications\| (2023) 14:5795 Finite-element DWBA Therefore, the interference and holography patterns, providing the 3D sample information from the single view, are in the dark ﬁeld, high contrast, and low noise, distinctly different from all previous Lloyd’s mirror holography themes. At αf = 0, the Fresnel reﬂectance is close to unity, and the phase shift is π, resulting in a node in the interference intensity similar to X-ray standing waves at X-ray mirror surfaces62,63. More speciﬁcally, when one point at a distance d above a mirror interferes with its mirrored point at −d, below the substrate surface, it behaves like a two-point source, separated by 2d, giving rise to scat- tering fringes with a period of 2π=ð2dÞ. The characteristic thickness 2d, directly determined by the interference period, is a dimension that doesn’t exist in the sample. This indicates that the near-horizon fringes are generated from a scattering mechanism that is very different from the kinematic scattering theory, which shows a dependence only on the sample thickness. Figure 4b shows the pattern thickness calculated from Lloyd’s mirror path difference is 57.2 nm. As αf increases, the Fresnel reﬂectivity drops as q4 z . Smeared by diffuse scattering from the surface roughness, the interference intensity due to Lloyd’s mirror effect decays and eventually is inundated in the background. Par- enthetically, we note that the original Lloyd’s mirror concept was deemed not practical for hard X-ray applications46,66,67. Now we demonstrate that it can be ideally suited for creating holography and coherent imaging at the surfaces as a high-resolution hard X- ray structural probe. The quantitative analysis of the dark-ﬁeld Lloyd’s- mirror-based interference and holography patterns is discussed next. The FE-DWBA method is applicable to reveal the dynamical scat- tering of more complex samples, e.g., the petal-like planar pattern presented in Fig. 3f. Figure 3g shows the side-by-side comparison of the corresponding simulated and experimental scattering pattern. The fringes along αf are even more prominent as the scattering data occupies more reciprocal space voxels due to the more complex 2D shape of the 3D sample. Again, the FE-DWBA simulation is well- validated by the experimental data. Because the FE-DWBA is based on layered cellular grids, it should be applicable to multilayer surface samples when a priori knowledge can be used to optimize the cell geometry and size in the direction normal to the surface. Finite-element DWBA In terms of the FE-DWBA computational aspect, the exit eigen- states, corresponding to each pixel on the detector, need to be com- puted for each of the more than 5 million cells, which can make it computationally intensive. To accelerate the algorithm, the vertical stacks that share the same electron density proﬁle are grouped. The eigenstates in each group only need to be computed once. In addition, we parallelize the computation of the scattering intensity for each exit state (Eq. 1) by using graphics processing units (GPUs). Without opti- mization, the total computation takes about 1 min on a single consumer-grade GPU of about 4352 cores (RTX 2080 Ti). To enable iterative reconstruction with FE-DWBA, the process can be accelerated I / X xp,yp X zp ψ f p∣ρp∣ψ i p D E + X xe,ye2boundaries X ze ψ f e1∣ρe∣ψi e2 D E 2 , ð1Þ ð1Þ where the ﬁrst term is the sum of all cells, which describes the dynamical scattering due to the wave distortion introduced by the layered surface structure, just like the conventional DWBA approach. The second term is the sum of cells on boundaries. e1 and e2 are cells downstream and upstream of cell e, which can vary in vertical electron density. These terms take care of the Nature Communications\| (2023) 14:5795 5 https://doi.org/10.1038/s41467-023-39984-3 Article signiﬁcantly by applying non-uniform FT to the computation of scat- tering (Eq. S22) and using multiple GPUs. the αf direction in the scattering pattern correspond to a dimension of twice the sample’s thickness. In SI #4 and Fig. S3, the scattering pat- terns taken at different incident angles (above the critical angle of the Si substrate) show that the period of the interference fringes is invar- iant for a given sample. With the in-plane inhomogeneities incorporated in the FE-DWBA simulation, this approach generates a high-ﬁdelity simulated scattering pattern. This is validated by the good quantitative agreement between simulation and experimental results, as shown in Fig. 3c (in the dashed- line boxes). The simulation not only resembles the overall scattering features but also produces matching positions and periods of fringes near the horizon. In addition, the simulation shows that the near- specular pattern is indeed dominated by kinematic scattering, reveal- ing the 2D structure in the x-y plane. Figure 3d, e show scans along the black and white dashed lines in Fig. 3c, respectively. Finite-element DWBA In the simulated pattern’s low exit angle region (αf < 0.3°), multibeam scattering/inter- ference fringes are prominent, matching the experimental data exceptionally well. The intensity oscillation along the αf direction in the scattering proﬁle, shown in Fig. 3d, indicates the surface pattern’s out- of-plane form factor, namely its thickness. The relationship between the oscillation period of the intensity proﬁle in Fig. 3d and the thickness of the sample can be signiﬁcant because the sample’s 3D structure can now be determined by a single scattering pattern at a single incident angle by using the FE-DWBA-based forward simulation. We note that the previous 3D reconstructions used the scattering data sets collected at multiple incident angles. An additional discussion of the out-of-plane dimension will follow in the next section. In Fig. 3e, it is visible that the fringe period along the 2θ-direction is sensitive to the pattern length along the X-ray beam direction (70 µm). The 3D structural determina- tion shows the powerfulness of the FE-DWBA method if the scattering data is measured and analyzed properly. Therefore, the single-shot 3D information can be used to monitor time-resolved changes of samples under stimuli with a high spatial and possibly temporal resolution. g p As illustrated in Fig. 4a, when a point at the 3D planar pattern surface or inside is illuminated by a coherent beam, the scattered beam becomes a secondary source. The sample scattering from the sec- ondary sourcehastwo major paths, one directly leading to the detector (Path A) and the other reﬂected by the substrate (Path B). The scattered wave along Path B experiences an additional phase shift, a function of the exit angle. These coherent waves from the two paths superpose and create interference patterns observed at the detector location. We emphasize here that in the direction along the sample/substrate hor- izon (<0.3°), the waves on both Paths A and B are different from the incident (primary) X-ray beam and its reﬂection from the substrate. With an incidence angle (0.6°) above the critical angle, the former is attenuated by the substrate and would not reach the detector. The latter, the reﬂection of the primary beam, reaches the detector at a reﬂection angle the same as the incident angle (0.6° in this case). The ﬁrst-principles holography simulation The ﬁrst-principles holography simulation While the FE-DWBA method produces high-ﬁdelity simulation results, like other numerical methods, the computation requires an excessive amount of computing resources. In many cases, the grid must be ﬁne enough to match the experimental conditions, and the electric ﬁeld in each cell must be computed. This may hinder its application in iterative image reconstruction algo- rithms, which involve multiple forward and backward calcula- tions. Close examination of the four terms in Eq. S12 reveals that the ﬁrst and the second term dominate if the incident angle αi is above and the exit angle αf is below the critical angles (i.e., the region where we observe the dynamical scattering fringes). The other two terms are relatively weak due to the scattering geo- metry. This enables the translation of the complex scattering process for samples on a surface into a much simpler approx- imation. The scattering is approximately the kinematically scat- tered wave of the sample mixed with its reﬂected wave from the substrate if the incident angle is above the system’s critical angles, in a simpliﬁed scheme without the presence of the cells Nature Communications\| (2023) 14:5795 Finite-element DWBA The advan- tage of the forward-calculation method is that it uses the full Parratt’s formalism31, which is well suited for computing dynamical scattering from surfaces and interfaces of complex planar 3D patterns such as multilayer and multicomponent structures in micro- and nanoelec- tronic circuits. However, the computation cost will be higher as fewer vertical stacks sharing the same electron density proﬁle can be grouped. Although hard X-rays are less sensitive to chemical compo- sition than soft X-rays and ultraviolet probes65, the sub-nm wavelength promises higher spatial resolution or sensitivity, as shown in what follows next. Another practical issue with this bilayer sample conﬁg- uration of 55-nm Au on top of the 5-nm Ti adhesion layer is that the simulation may only be sensitive to the nominal total thickness (60 nm), not the individual layer thicknesses. This is due to the grazing- incidence angle just slightly above the Au critical angle at which the relatively thick Au layer signiﬁcantly attenuates the X-rays and the Au/ Ti interface becomes less visible. Grazing-incident scattering holography a Schematic of surface holography near the substrate (not to scale). The scattered X-rays from the sample at low exit angles (close to or below the critical angles) can propagate towards the detector via the two pathways—the direct Path A and reﬂected Path B. They have a path length difference of approxi- mately 2dsinðαf Þ and an incident angle-dependent reﬂection phase shift. When αf is lower than or close to the substrate critical angle, the direct and reﬂected scattering intensities are comparable, creating a situation similar to Lloyd’s mirror and Young’s double-slit with an effective slit spacing of 2d and the additional variable phase shift due to the reﬂection. b The two-path interference period along the αf direction is invariable for incident angles (0.4, 0.6, and 0.8°) above the substrate critical angle of 0.22°. The fringes from experimental images correspond to 2d of 114.4 nm (see text for details). The error bars are estimated from the uncertainty Fig. 4 \| Grazing-incidence X-ray holography of the substrate-supported single- bar surface pattern. a Schematic of surface holography near the substrate (not to scale). The scattered X-rays from the sample at low exit angles (close to or below the critical angles) can propagate towards the detector via the two pathways—the direct Path A and reﬂected Path B. They have a path length difference of approxi- mately 2dsinðαf Þ and an incident angle-dependent reﬂection phase shift. When αf is lower than or close to the substrate critical angle, the direct and reﬂected scattering intensities are comparable, creating a situation similar to Lloyd’s mirror and (about 3 detector pixels) of the interference period along the αf-direction. c The holography-based ﬁrst-principles calculation generates a simulated pattern (left panel) that well matches the experimental data (right). d Line scans along the center of the arc (qx = 0) from αf = 0 (horizon) to 0.3° (black dashed line in panel c), showing the ﬁrst-principles Lloyd’s mirror calculation is validated by both the experimental data and full-ﬂedged FE-DWBA simulation at the low exit angles. e Line scan of the scattering proﬁle along the white dashed line in panel c, containing the information of the form factor of thin-ﬁlm surface pattern, more speciﬁcally, the length of the bar along the X-ray beam. The oscillation period indicates the bar length is 70.0 µm precisely. Grazing-incident scattering holography The ﬁrst-principles calculation indi- cates that the single-incident angle scattering pattern contains the precise sample structural information in 3D. (about 3 detector pixels) of the interference period along the αf-direction. c The holography-based ﬁrst-principles calculation generates a simulated pattern (left panel) that well matches the experimental data (right). d Line scans along the center of the arc (qx = 0) from αf = 0 (horizon) to 0.3° (black dashed line in panel c), showing the ﬁrst-principles Lloyd’s mirror calculation is validated by both the experimental data and full-ﬂedged FE-DWBA simulation at the low exit angles. e Line scan of the scattering proﬁle along the white dashed line in panel e Line scan of the scattering proﬁle along the white dashed line in panel c, containing the information of the form factor of thin-ﬁlm surface pattern, more speciﬁcally, the length of the bar along the X-ray beam. The oscillation period indicates the bar length is 70.0 µm precisely. The ﬁrst-principles calculation indi- cates that the single-incident angle scattering pattern contains the precise sample structural information in 3D. intensities are comparable, creating a situation similar to Lloyd’s mirror and Young’s double-slit with an effective slit spacing of 2d and the additional variable phase shift due to the reﬂection. b The two-path interference period along the αf direction is invariable for incident angles (0.4, 0.6, and 0.8°) above the substrate critical angle of 0.22°. The fringes from experimental images correspond to 2d of 114.4 nm (see text for details). The error bars are estimated from the uncertainty and layers, the interfering scattered waves in Eq. 1 and S12 can be: 2 require ﬁnite-element-based numerical computation, as detailed in SI #5. The simulated scattering with the simpliﬁed model again matches the experimental data very well, as shown in Fig. 4c. The line scans along the αf (Fig. 4d) and 2θ (Fig. 4e) directions also show an excellent match in both node and antinode positions, as well as oscillation periodicities related to the pattern thickness and the in-plane dimen- sions, respectively. The possibility of capturing all major scattering contributions validates our simpliﬁed computation method based on the two-beam holographic approach. I αi, αf , 2θ / Z ρ rð Þ 1 + R αf e2ikz αf ð Þz n o eiqrdr 2 ð2Þ ð2Þ where the ﬁrst term is the unperturbed scattering (Path A in Fig. Grazing-incident scattering holography The successful FE-DWBA simulations demonstrate that the physics responsible for the fringes is similar to the waveguide effects41. A detailed analysis of the fringes reveals that an important phenomenon —Lloyd’s mirror interferences, previously observed in other wave- length regimes, is now demonstrated in this hard X-ray grazing-inci- dence geometry near the critical angles. The periodic fringes seen in Nature Communications\| (2023) 14:5795 6 https://doi.org/10.1038/s41467-023-39984-3 Article Fig. 4 \| Grazing-incidence X-ray holography of the substrate-supported single- bar surface pattern. a Schematic of surface holography near the substrate (not to scale). The scattered X-rays from the sample at low exit angles (close to or below the critical angles) can propagate towards the detector via the two pathways—the direct Path A and reﬂected Path B. They have a path length difference of approxi- mately 2dsinðαf Þ and an incident angle-dependent reﬂection phase shift. When αf is lower than or close to the substrate critical angle, the direct and reﬂected scattering intensities are comparable, creating a situation similar to Lloyd’s mirror and Young’s double-slit with an effective slit spacing of 2d and the additional variable phase shift due to the reﬂection. b The two-path interference period along the αf direction is invariable for incident angles (0.4, 0.6, and 0.8°) above the substrate critical angle of 0.22°. The fringes from experimental images correspond to 2d of 114.4 nm (see text for details). The error bars are estimated from the uncertainty (about 3 detector pixels) of the interference period along the αf-direction. c The holography-based ﬁrst-principles calculation generates a simulated pattern (left panel) that well matches the experimental data (right). d Line scans along the center of the arc (qx = 0) from αf = 0 (horizon) to 0.3° (black dashed line in panel c), showing the ﬁrst-principles Lloyd’s mirror calculation is validated by both the experimental data and full-ﬂedged FE-DWBA simulation at the low exit angles. e Line scan of the scattering proﬁle along the white dashed line in panel c, containing the information of the form factor of thin-ﬁlm surface pattern, more speciﬁcally, the length of the bar along the X-ray beam. The oscillation period indicates the bar length is 70.0 µm precisely. The ﬁrst-principles calculation indi- cates that the single-incident angle scattering pattern contains the precise sample structural information in 3D. Fig. 4 \| Grazing-incidence X-ray holography of the substrate-supported single- bar surface pattern. Grazing-incident scattering holography 4a) and the second is the mirror image of the ﬁrst term but modiﬁed with a complex reﬂection coefﬁcient, Rðαf Þ, and the phase shift 2kzðαf Þz due to the mirroring effect by the total external reﬂection at the substrate surface (Path B, mirrored scattering, in Fig. 4a), in the condition of a higher incident angle (αi) and lower exit angle (αf). The distance between the two scattering sources atthe sample location is effectively 2z. With known substrate optical prosperities, the calculation of Rðαf Þ is relatively straightforward based on Parratt’s algorithm, which also predicts ∣Rðαf = 0Þ∣= 1 with a phase shift of π upon reﬂection in Path B. This constraint is shown in the holography images as the scattering intensity I(αi,αf = 0°,2θ) = 0, so that the intensity at the detector cor- responding to the direction of the sample/substrate horizon is always 0, an absolute constraint imposed onto the interfering waves, regardless of the incident angle and the sample structure. Therefore, within such angular ranges, the surface scattering is reduced to a two- beam holography mechanism, which can also be the preferred experimental condition for CSSI measurements. The simpliﬁed model demonstrates the true holographic nature of the multibeam scattering method that can be extended to using hard X-rays of tens-keV photon energy. where the ﬁrst term is the unperturbed scattering (Path A in Fig. 4a) and the second is the mirror image of the ﬁrst term but modiﬁed with a complex reﬂection coefﬁcient, Rðαf Þ, and the phase shift 2kzðαf Þz due to the mirroring effect by the total external reﬂection at the substrate surface (Path B, mirrored scattering, in Fig. 4a), in the condition of a higher incident angle (αi) and lower exit angle (αf). The distance between the two scattering sources atthe sample location is effectively 2z. With known substrate optical prosperities, the calculation of Rðαf Þ is relatively straightforward based on Parratt’s algorithm, which also predicts ∣Rðαf = 0Þ∣= 1 with a phase shift of π upon reﬂection in Path B. This constraint is shown in the holography images as the scattering intensity I(αi,αf = 0°,2θ) = 0, so that the intensity at the detector cor- responding to the direction of the sample/substrate horizon is always 0, an absolute constraint imposed onto the interfering waves, regardless of the incident angle and the sample structure. Nature Communications\| (2023) 14:5795 Grazing-incident scattering holography The top view highlights an unintended misalignment between the long axes of the bottom and top bars of about 0.009°, introduced during the preparation of the top bar. b The left half of the CSSI pattern of the stacked bar was taken at an incident angle of 0.6°. The dual- beam holographic scattering features are outlined by the rectangular box at low exit angles 0 <αf < 0.4°. At ﬁrst glance, the pattern in the box is similar to the scattering from the single-bar pattern, but it has many additional ﬁne features generated by the stacked bars. c Magniﬁed view of the experimental data in the marked in Fig. 5a (top view), and generated a scattering pattern with rich features, especially in the low exit angle and dynamical scattering region (Fig. 5b). The most notable ones, highlighted in Fig. 5c, include two primary arcs (labeled as the inner and outer arcs) and multiple secondary but eccentric arcs around the primary ones. The origin of the two-arc scattering pattern is discussed in SI #6 and Fig. S4. Using the two-beam holographic computation approach, we were able to model the stacked bars with seven parameters (i.e., dimensions of the two bars of identical length, the relative location of the top bar, and the misalignment angle), and simulate a scattering pattern that contains all the major features present in the experimental data, as shown in Fig. 5d. The simulation clearly shows that the double-arc results from the tiny angular misalignment of the top bar with respect to the bot- tom one. Although a perfect match of the entire speckle patterns between the experiment data and the simulation is not realistic, especially using a scattering model with signiﬁcant approximations, the level of the quantitative agreement is remarkable, as demonstrated as follows. The complexity of the scattering pattern/speckles, even at a single ﬁxed incident angle, is apparent, and all the global and ﬁne scattering signatures are contributed from all dimensions of the entire 3D sample pattern and the ﬁne features in 3D. 50 nm. Figure 5f, g show the simulations with ht = 10 and 30 nm, respectively, to highlight differences. The change of the large-period modulation along the outer arc is immediately apparent. The oscilla- tion period is susceptible to ht and varies dramatically over the range. In Fig. Grazing-incident scattering holography 5h, the scattering proﬁles were taken along the outer arc as a function of αf, showing the impact of the top-bar height unambigu- ously. In the meantime, the corresponding oscillations on the inner arc (Fig. 5i), more sensitive to the bottom-bar dimensions, speciﬁcally the thickness, remained invariant. More details are presented in SI #7 for the variation of the bottom-bar thickness hb. Therefore, the scattering intensity on the outer arc is mainly contributed from the top bar, while the inner arc is from the bottom bar. marked in Fig. 5a (top view), and generated a scattering pattern with rich features, especially in the low exit angle and dynamical scattering region (Fig. 5b). The most notable ones, highlighted in Fig. 5c, include two primary arcs (labeled as the inner and outer arcs) and multiple secondary but eccentric arcs around the primary ones. The origin of the two-arc scattering pattern is discussed in SI #6 and Fig. S4. Using the two-beam holographic computation approach, we were able to model the stacked bars with seven parameters (i.e., dimensions of the two bars of identical length, the relative location of the top bar, and the misalignment angle), and simulate a scattering pattern that contains all the major features present in the experimental data, as shown in Fig. 5d. The simulation clearly shows that the double-arc results from the tiny angular misalignment of the top bar with respect to the bot- tom one. Although a perfect match of the entire speckle patterns between the experiment data and the simulation is not realistic, especially using a scattering model with signiﬁcant approximations, the level of the quantitative agreement is remarkable, as demonstrated as follows. The complexity of the scattering pattern/speckles, even at a single ﬁxed incident angle, is apparent, and all the global and ﬁne scattering signatures are contributed from all dimensions of the entire 3D sample pattern and the ﬁne features in 3D. To understand the correlation between the intensity oscillation at the higher frequency and the sample dimension, we vary the width of the top bar, wt, (see Fig. 6a) from 2.5 to 4.0 µm, and two simulation results are shown in Fig. 6b, c. Note that the calculation of the scat- tering pattern in Fig. 5d uses the ground truth wt = 3.0 µm. In addition, we observe shorter-period (higher frequency) intensity oscillations superimposed on both inner and outer arcs along the 2θ direction. Grazing-incident scattering holography Therefore, within such angular ranges, the surface scattering is reduced to a two- beam holography mechanism, which can also be the preferred experimental condition for CSSI measurements. The simpliﬁed model demonstrates the true holographic nature of the multibeam scattering method that can be extended to using hard X-rays of tens-keV photon energy. To better demonstrate the effect of the holographic process that reveals the accurate 3D form factors of the surface patterns, we designed a sample based on two stacked bars. The bottom bar has dimensions of 300 µm (length) × 6.0 µm (width, wb) × 55 nm (thickness, hb). Again, only the simulation is sensitive to the combined thickness of the Au and Ti layers for the same practical reasons mentioned pre- viously. The top bar is also 300-µm long but 3.0-µm wide (wt) and 30- nm thick (ht). The true 3D sample is illustrated in Fig. 5a, including a 3D rendering, top view, and cross-section normal to the X-ray beam. The stacked bars create a non-uniform thickness and multiple interfaces in both y- and z-directions. In the sample fabrication process, the top bar was inadvertently misaligned by a minimal angle of γ = 0:009 (drawn not to scale). This angle was only revealed to be present by our ﬁrst- principles calculation below. To our pleasant surprise, the misaligned bars offer several unambiguous tell-tale features in the experiment and simulation to demonstrate the sensitivity of the holographic scattering imaging to the nm-scale features in this truely 3D sample. In the experiment, the X-ray beam impinged upon the left part of the sample with an incident angle of 0.6°, as the X-ray footprint is Based on the holography scheme, we developed a simulation using the simpliﬁed model in a ﬁrst-principles approach that does not 7 Nature Communications\| (2023) 14:5795 7 https://doi.org/10.1038/s41467-023-39984-3 Article Fig. 5 \| Grazing-incidence holography of a stacked-bar pattern to demonstrate the true 3D nature of surface scattering: dependence on the pattern thickness. a 3D schematics of the stacked-bar pattern deposited on a Si substrate with the X-ray incident direction indicated, top view, and side view along the X-ray direction with nominal (designed) dimensions (not to scale). The top view highlights an unintended misalignment between the long axes of the bottom and top bars of about 0.009°, introduced during the preparation of the top bar. Grazing-incident scattering holography At ﬁrst glance, the pattern in the box is similar to the scattering from the single-bar pattern, but it has many additional ﬁne features generated by the stacked bars. c Magniﬁed view of the experimental data in the dynamical multibeam scattering range of 2θ = (−0.7 – 0.4°) and αf = (0 – 0.4°). d First-principle simulation using the dual-beam holographic interference approximation. e Schematic of the stacked-bar sample pattern with ﬁxed bottom- bar dimensions and a top bar with a ﬁxed width of 3 µm but a varying height of ht. f, g Simulated holographic scattering patterns in the white box region for varying top-bar thicknesses ht of 10 and 30 nm, respectively. h Simulated scattering intensity proﬁles (line scans) along the outer arc as a function of αf from 0 to 0.4° for varying ht values 10, 30, and 50 nm. While the weaker and shorter-period oscillations remain similar over the full ht range, the large-period oscillations are sensitive to ht and vary dramatically. i Scattering proﬁles taken along the inner-ring arc showing that the proﬁles are insensitive to the top-bar height but sensitive to the bottom-bar thickness (see SI #7). dynamical multibeam scattering range of 2θ ( 0.7 0.4 ) and αf (0 0.4 ). d First-principle simulation using the dual-beam holographic interference approximation. e Schematic of the stacked-bar sample pattern with ﬁxed bottom- bar dimensions and a top bar with a ﬁxed width of 3 µm but a varying height of ht. f, g Simulated holographic scattering patterns in the white box region for varying top-bar thicknesses ht of 10 and 30 nm, respectively. h Simulated scattering intensity proﬁles (line scans) along the outer arc as a function of αf from 0 to 0.4° for varying ht values 10, 30, and 50 nm. While the weaker and shorter-period oscillations remain similar over the full ht range, the large-period oscillations are sensitive to ht and vary dramatically. i Scattering proﬁles taken along the inner-ring arc showing that the proﬁles are insensitive to the top-bar height but sensitive to the bottom-bar thickness (see SI #7). the true 3D nature of surface scattering: dependence on the pattern thickness. a 3D schematics of the stacked-bar pattern deposited on a Si substrate with the X-ray incident direction indicated, top view, and side view along the X-ray direction with nominal (designed) dimensions (not to scale). Grazing-incident scattering holography b The left half of the CSSI pattern of the stacked bar was taken at an incident angle of 0.6°. The dual- beam holographic scattering features are outlined by the rectangular box at low exit angles 0 <αf < 0.4°. At ﬁrst glance, the pattern in the box is similar to the scattering from the single-bar pattern, but it has many additional ﬁne features generated by the stacked bars. c Magniﬁed view of the experimental data in the dynamical multibeam scattering range of 2θ = (−0.7 – 0.4°) and αf = (0 – 0.4°). d First-principle simulation using the dual-beam holographic interference approximation. e Schematic of the stacked-bar sample pattern with ﬁxed bottom- bar dimensions and a top bar with a ﬁxed width of 3 µm but a varying height of ht. f, g Simulated holographic scattering patterns in the white box region for varying top-bar thicknesses ht of 10 and 30 nm, respectively. h Simulated scattering intensity proﬁles (line scans) along the outer arc as a function of αf from 0 to 0.4° for varying ht values 10, 30, and 50 nm. While the weaker and shorter-period oscillations remain similar over the full ht range, the large-period oscillations are sensitive to ht and vary dramatically. i Scattering proﬁles taken along the inner-ring arc showing that the proﬁles are insensitive to the top-bar height but sensitive to the bottom-bar thickness (see SI #7). Fig. 5 \| Grazing-incidence holography of a stacked-bar pattern to demonstrate the true 3D nature of surface scattering: dependence on the pattern thickness. Fig. 5 \| Grazing-incidence holography of a stacked-bar pattern to demonstrate the true 3D nature of surface scattering: dependence on the pattern thickness. a 3D schematics of the stacked-bar pattern deposited on a Si substrate with the X-ray incident direction indicated, top view, and side view along the X-ray direction with nominal (designed) dimensions (not to scale). The top view highlights an unintended misalignment between the long axes of the bottom and top bars of about 0.009°, introduced during the preparation of the top bar. b The left half of the CSSI pattern of the stacked bar was taken at an incident angle of 0.6°. The dual- beam holographic scattering features are outlined by the rectangular box at low exit angles 0 <αf < 0.4°. Nature Communications\| (2023) 14:5795 Grazing-incident scattering holography The ﬁner sensitivity value, better than 5-nm afforded by the detector data range, is from the over- sampling of the interference fringes. Therefore, we think that the probe sensitivity or resolution can be even better than 3 nm, espe- cially when higher coherent X-ray ﬂux, coupled with larger-format detectors, is used to generate the scattering images. bottom-bar width wb = 6 µm. The in-plane dimension change intro- duces a ﬁne but clear impact on the simulated pattern—on both inner and outer arcs, the signature of the interfering scattering off the ver- tical sides of the 3D structure at the different heights and horizontal surfaces of the top and bottom bars. The ﬁrst-principles two-beam interference calculation demon- strates quantitative agreement with the experimental data. Figure 6f shows the scattering intensity proﬁle, integrated over the 2θ-range of ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 0:62 α2 f q ± 0:03 (°) as a function of αf, showing the dynamical scattering in the low-exit angle region. Since the X-ray beam footprint is off-center near the edge of the sample, the interference oscillation of the integrated intensity proﬁle is dominated by the thickness of the larger bottom bar, 55 nm. The high-frequency inner-arc proﬁle con- ﬁrmed the width of the top bar of 3 µm by the excellent agreement between the simulation and the experiment (Fig. 6g). The ﬁrst- principles holographic simulation is sensitive to the angular displace- ment between the outer and inner arcs, shown in Fig. 6h. The simu- lation indicates that the top bar’s long axis has an angle of 0.009° with respect to the bottom one, counterclockwise in the top view. This slight angle originates from the misalignment during the preparation of the top bar, which was due to hardware limitations of the litho- graphy instrument. Surprisingly, it illustrates the extreme sensitivity of the surface holography method when probing elongated structures, such as micro- and nanoelectronics circuits and masks. With the sen- sitivity of the scattering patterns in the lower exit angle region to the width and height of the bars, every sample dimension can be obtained by ﬁtting the experimental data and the simulations, which agrees well with the designed parameters. In the out-of-the-plane or z-direction, the detector samples an αf range (or momentum transfer) much larger than the y-direction, which implies better sensitivity. Grazing-incident scattering holography They remain similar and independent of the ht and wt variation. In Fig. 6b, c, the effect of the top bar width is shown clearly in the 2D patterns. Quantitatively, in Fig. 6d, the integrated intensity proﬁles of the inner arc along αf as a function of 2θ contain many short-period high-contrast oscillation corresponding to the distance between the left edges of the top and bottom bars. The oscillations also appear in the proﬁle of the outer arc (Fig. 6e) but with a much-lessened contrast. As a result, the inner-arc proﬁles show high contrast in the fringes, whose periodicity is inversely proportional to (wb−wt)/2, as the To illustrate the holographic imaging concept and explain how the sample’s 3D dimensions impact the scattering patterns quantita- tively, we ﬁrst demonstrate explicitly that the sample information in the third dimension (i.e., normal to the stacking direction) is encoded in a single-view scattering pattern. For clarity, we use the top-bar height as the only variable, as shown schematically in Fig. 5e, and we performed simulations for three top-bar heights, ht, of 10, 30, and Nature Communications\| (2023) 14:5795 8 https://doi.org/10.1038/s41467-023-39984-3 Article Fig. 6 \| Grazing-incidence holography dependence on the stacked-bar pattern width and quantitative agreement between the data and holographic simula- tion. a Schematic of the stacked-bar sample pattern with ﬁxed bottom-bar dimensions and a top bar with a ﬁxed thickness of 30 nm but a varying width wt: 3D rendering and a cross-sectional view along the X-ray beam incidence. b, c Simulated holographic scattering patterns for wt = 2.5 and 4.0 µm. d Scattering proﬁle taken along the inner-ring arc as a function of 2θ from −0.60 to −0.45° showing the short- period oscillations are strong and the period is inversely proportional to the dis- tance between the side surfaces (marked by the two red arrows in panel a) in y-direction or (6 µm – wt)/2, as wt varies from 2.5 to 4 µm. e Simulated scattering intensity proﬁle along the outer arc as a function of 2θ as wt varies from 2.5 to 4.0 µm. While the larger-period intensity proﬁle remains unchanged over the wt range, the short-period modulation is observable but weak. f Scattering intensity proﬁle, integrated over a narrow range of 2θ as a function of αf shows the best agreement between the simulation and experiment data when the bottom-bar thickness is 55 nm. Grazing-incident scattering holography The simulation reveals that the top bar’s long axis has an unintended misalignment of 0.009° with respect to the bottom one, counterclockwise in the top view. tion. a Schematic of the stacked-bar sample pattern with ﬁxed bottom-bar dimensions and a top bar with a ﬁxed thickness of 30 nm but a varying width wt: 3D rendering and a cross-sectional view along the X-ray beam incidence. b, c Simulated holographic scattering patterns for wt = 2.5 and 4.0 µm. d Scattering proﬁle taken along the inner-ring arc as a function of 2θ from −0.60 to −0.45° showing the short- period oscillations are strong and the period is inversely proportional to the dis- tance between the side surfaces (marked by the two red arrows in panel a) in y-direction or (6 µm – wt)/2, as wt varies from 2.5 to 4 µm. e Simulated scattering intensity proﬁle along the outer arc as a function of 2θ as wt varies from 2.5 to simulation reveals wt = 3.0 µm. h The ﬁrst-principles holographic simulation shows the sensitivity of the angular displacement between the outer and inner arcs to the top and bottom bars’ axis rotational misalignment. The simulation reveals that the top bar’s long axis has an unintended misalignment of 0.009° with respect to the bottom one, counterclockwise in the top view. examples such as the top layer width (wt) and thickness (ht). In principle, the sensitivity (or spatial resolution) is determined by the scattering angles (or maximum momentum transfer in the respective directions). We estimated that in the 2θ direction, the value is limited by the detector sensor edge at 0.7°, which resulted in a resolution of ca. 5 nm. A direct demonstration is shown using numerical simula- tions with varying top bar width by a few nm around a hypothetic 5.000 µm. We chose this value different from the actual sample dimension (3 µm) to avoid reduced contrast in the interference fringes because of the detector pixel size effect in the experimental data, as shown in Fig. 6d. In Fig. 7a, we illustrated the line scans of the simulated scattering patterns with variations (Δwt) of 3-nm step size. While there are detectable differences in the speckle patterns, the varying speckle sizes (or interference periods) are immediately observable in the line-scan plots, Fig. 7a, showing the striking effects of 3-nm changes of the form factor. Grazing-incident scattering holography g With the sensitivity of the inner-arc scattering intensity proﬁle to the top-bar width wt, the best match between the experimental data and the simulation reveals wt = 3.0 µm. h The ﬁrst-principles holographic simulation shows the sensitivity of the angular displacement between the outer and inner arcs to the top and bottom bars’ axis rotational misalignment. The simulation reveals that the top bar’s long axis has an unintended misalignment of 0.009° with respect to the bottom one, counterclockwise in the top view. Fig. 6 \| Grazing-incidence holography dependence on the stacked-bar pattern width and quantitative agreement between the data and holographic simula- ti S h ti f th t k d b l tt ith ﬁ d b tt b Fig. 6 \| Grazing-incidence holography dependence on the stacked-bar pattern width and quantitative agreement between the data and holographic simula- tion. a Schematic of the stacked-bar sample pattern with ﬁxed bottom-bar dimensions and a top bar with a ﬁxed thickness of 30 nm but a varying width wt: 3D rendering and a cross-sectional view along the X-ray beam incidence. b, c Simulated holographic scattering patterns for wt = 2.5 and 4.0 µm. d Scattering proﬁle taken along the inner-ring arc as a function of 2θ from −0.60 to −0.45° showing the short- period oscillations are strong and the period is inversely proportional to the dis- tance between the side surfaces (marked by the two red arrows in panel a) in y-direction or (6 µm – wt)/2, as wt varies from 2.5 to 4 µm. e Simulated scattering intensity proﬁle along the outer arc as a function of 2θ as wt varies from 2.5 to 4.0 µm. While the larger-period intensity proﬁle remains unchanged over the wt range, the short-period modulation is observable but weak. f Scattering intensity proﬁle, integrated over a narrow range of 2θ as a function of αf shows the best agreement between the simulation and experiment data when the bottom-bar thickness is 55 nm. g With the sensitivity of the inner-arc scattering intensity proﬁle to the top-bar width wt, the best match between the experimental data and the simulation reveals wt = 3.0 µm. h The ﬁrst-principles holographic simulation shows the sensitivity of the angular displacement between the outer and inner arcs to the top and bottom bars’ axis rotational misalignment. Nature Communications\| (2023) 14:5795 Discussion a Line scans of the inner-arc intensity as a function of the in-plane scattering angle 2θ at various ﬁne deviations Δwt in top-bar width wt. b Line scans of the outer-arc intensity as a function of the off-plane scattering exit angle αf at various ﬁne deviations (Δht) in the top-bar thickness ht. The black dashed lines guide the eye and highlight the speckle phase shift. phase shift) as the reference beam. Thus, similar to dark-ﬁeld imaging, all interfering waves from the sample are well separated from the direct beam and its specular reﬂection. With the use of high electron- density substrates (such as noble metal coated silicon or glass), highly coherent ﬂux X-ray sources, and single-photon sensitive detectors, it’s expected that the surface hologram in the grazing-exit-angle direction will increase signiﬁcantly, unlocking new possibilities of surface char- acterizing technologies and application. The FE-DWBA simulation focused on one of the simplest sam- ples to illustrate the principle of the analysis, the tell-tale features of the coherent scattering patterns, and the sensitivity to the pattern’s 3D structures in a single incident-angle view. The simulation was also validated by the scattering images from a more complex pattern. Even though FE-DWBA treats the in-plane electric-ﬁeld boundary conditions with approximations, the simulation results are of high ﬁdelity with minimal computation load and are validated by the experiment data. We note that computational electromagnetics techniques, such as the ﬁnite-difference time-domain method68, can solve the distribution and propagation of the electromagnetic wave more rigorously. Still, they require enormous computational resources for X-rays with sub-nm wavelengths, even for simple pro- blems with low spatial resolution. The simulation methods and algorithm are limited to the forward calculation for illustrating multibeam coherent surface scattering and holography and have yet to be extended to reconstruct the real-space structure. As we observed, the information on the coherent scattering is abundant when the exit angle (af) is between 0 (sample horizon) and the nominal critical angles of the sample, which should not have existed in the kinematic approximation. We demonstrated in this work that this multibeam scattering region contains rich and crucial infor- mation about the sample form factors in all three dimensions. It will be our future work to evaluate whether the patterns in this ‘forbidden’ area have enough speckle oversampling rate for a full-ﬂedged recon- struction. Article https://doi.org/10.1038/s41467-023-39984-3 Fig. 7 \| Spatial sensitivity of the speckle size and phase of the holographic scattering on ﬁne changes in the critical dimensions of the stacked-bar sample. a Line scans of the inner-arc intensity as a function of the in-plane scattering angle 2θ at various ﬁne deviations Δwt in top-bar width wt. b Line scans of the outer-arc intensity as a function of the off-plane scattering exit angle αf at various ﬁne deviations (Δht) in the top-bar thickness ht. The black dashed lines guide the eye and highlight the speckle phase shift. Discussion In a most direct fashion, we demonstrated that coherent surface scattering at grazing incidence contains strong multiple scattering components, which encode the true 3D morphology of surface structures. More importantly, this 3D information can be extracted from a scattering pattern taken at a single incident angle, which does not require scanning the incident angle as required by the conven- tional CSSI analysis and should be proven suited for obtaining critical- dimension information in mesoscale surface patterns. To take advan- tage of this capability, we developed the FE-DWBA method that cap- tures the origin of multiple scattering physics and reproduces scattering patterns with high ﬁdelity. Furthermore, aided by the FE- DWBA simulation, we discovered that the multiple scattering resem- bles Lloyd’s mirror in the hard X-ray regime revealing the two-beam holography concept in the grazing-incidence condition. In the holo- graphy framework, a much-simpliﬁed ﬁrst-principles scattering simu- lation reduces the computational complexity even further while capturing the detailed 3D surface morphology qualitatively and quantitatively. The simpliﬁcation enables parametrizing the real-space object without the spatial resolution limit imposed by FE-DWBA and other ﬁnite-element-based scattering simulations, paving the way for applications such as 3D X-ray metrology of mesoscopic surface pat- terns with the improved spatiotemporal resolution or sensitivity of a few nm (in-plane and transverse to the X-ray beam) and even sub-nm in the out-of-plane direction. In addition, the multibeam scattering simulation and reconstruction have a signiﬁcant bearing on electron- beam and extreme-ultraviolet (EUV) imaging and scattering, where multiple scattering is a more commonly occurring phenomenon due to the strong interaction between the probes and matter. Fig. 7 \| Spatial sensitivity of the speckle size and phase of the holographic Fig. 7 \| Spatial sensitivity of the speckle size and phase of the holographic scattering on ﬁne changes in the critical dimensions of the stacked-bar sample Fig. 7 \| Spatial sensitivity of the speckle size and phase of the holographic scattering on ﬁne changes in the critical dimensions of the stacked-bar sample. a Line scans of the inner-arc intensity as a function of the in-plane scattering angle 2θ at various ﬁne deviations Δwt in top-bar width wt. b Line scans of the outer-arc intensity as a function of the off-plane scattering exit angle αf at various ﬁne deviations (Δht) in the top-bar thickness ht. The black dashed lines guide the eye and highlight the speckle phase shift. Grazing-incident scattering holography A similar simulation is performed with a variation (Δht) and ﬁner step of 1 nm around the top-bar thickness (ht) of the actual 30-nm value, shown in Fig. 7b. Again, the line scans demonstrate 1 nm, if not sub-nm, sensitivity in measuring the top-bar thickness. The simulation results of Δwt and Δht resemble an inter- ferometer when the shift of fringes is sensitive to small changes in optical paths. The sensitivity of this multiple scattering probe is further investigated as a function of the tell-tale features, again using Nature Communications\| (2023) 14:5795 9 Nature Communications\| (2023) 14:5795 Discussion In principle, with the forward-calculation algorithms such as FE-DWBA and the ﬁrst-principles analysis, reconstruction using the scattering images with a priori information should be possible in the multibeam scattering regime. However, the feasibility of such recon- struction on the complex 3D subject remains to be determined. An algorithm using the DWBA approach has been attempted but is yet to be validated experimentally in the case of a weak perturbation to the in-plane electric ﬁeld44. With strong sample-substrate multiple scat- tering, such as the system studied here, one of the solutions is to use the conventional kinematic reconstruction as a support to facilitate high-ﬁdelity dynamical reconstruction. Conversely, the CSSI recon- struction can also beneﬁt from the 3D morphological information captured in single-view holographic images. The simulation and experiments in this work are based on simple substrate-supported patterns with single or double layers of a single material. The FE-DWBA takes layered approaches in each cell, which resembles the X-ray reﬂectivity algorithm31 and is best suited for simulating multilayer samples of different compositions. However, the sensitivity to the chemical compositions has yet to be tested in the current simulation, which is the subject of future work. It is recognized that the chemical sensitivity of hard X-rays would be inferior to that in soft X-ray or EUV regimes65,69; anomalous scattering may improve the sensitivity if one of the constituent elements is tracked during struc- tural transformation. The concept of X-ray holography with Lloyd’s mirror setup was previously developed to use the primary and its reﬂectionbeams asthe reference waves, which requires the X-ray source to be coherent and divergent so that the reference beam can superpose with the reﬂected beam. The two conditions are difﬁcult to satisfy simultaneously, especially in the hard X-ray regime. In addition, the contrast and signal- to-noise ratio in such a bright-ﬁeld hologram is also limited because the reference beam is usually much stronger than the reﬂected beam. The surface holography technique developed in this work uses the sample scattering as the secondary source and its mirrored image (with well-characterized exit-angle-dependent intensity reduction and With the advent of novel fourth-generation X-ray sources with more coherent ﬂux, holographic imaging with dynamical surface scattering will immediately beneﬁt the research communities inter- ested in diffractive imaging, surface characterization, X-ray metrology, and X-ray reﬂective optics design. References 1. Holler, M. et al. High-resolution non-destructive three-dimensional imaging of integrated circuits. Nature 543, 402–406 (2017). 1. Holler, M. et al. High-resolution non-destructive three-dimensional imaging of integrated circuits. Nature 543, 402–406 (2017). 2. Jancar, J. et al. Current issues in research on structure–property relationships in polymer nanocomposites. Polymer 51, 3321–3343 (2010). Data availability The experimental and simulation data generated in this study have been deposited in the Figshare database [https://doi.org/10.6084/m9. ﬁgshare.23401400.v2]. One of the unique capabilities stemming from the dynamical or holographic scattering is the single-view 3D analysis of a surface pat- tern’s structure. This will prove useful for time-resolved in situ and operando measurements in realistic sample environments. We note that 3D structure determination from a single view has been long sought to beneﬁt broad ﬁelds of physical and life sciences72, which may now be enabled by the surface holographic imaging analysis devel- oped in this work. Discussion The 3D characterization capability Nature Communications\| (2023) 14:5795 10 https://doi.org/10.1038/s41467-023-39984-3 Article for collecting a coherent scattering pattern was between 1 and 64 s depending on the incident angle. associated with the surface coherent scattering method is set to answer several scientiﬁc questions more effectively than conventional coherent imaging techniques. For example, how metrology of 3D micro- and nanoscale electronics is performed nondestructively1, how thin ﬁlms and quantum dots grow at surfaces and interfaces3, how to advance nanopatterning using a combination of top-down and bottom-up techniques for the controlled fabrication of complex and multicomponent nanomaterials, how to control the morphology of thin-ﬁlm photovoltaics to optimize the efﬁciency of the devices70, and how physical and chemical processes and dynamics lead to hier- archical order in mesoscopic structures and the functionalities2,71. 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Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. © UChicago Argonne, LLC, Operator of Argonne National Labora- tory 2023 Competing interests Competing interests The authors declare no competing interests. 72. Raines, K. S. et al. Three-dimensional structure determination from a single view. Nature. 463, 214–217 (2010). Competing interests The authors declare no competing interests. Acknowledgements We acknowledge DESY (Hamburg, Germany), a member of the Helm- holtz Association HGF, for providing experimental facilities. Parts of this research were carried out at PETRA III, and we would like to thank Fabian Westermeier and Dmitry Dzhigaev for their assistance at the P10 beam- line. Beamtime was allocated for proposal I-20170155. We acknowledge the fruitful discussion with Sunil K. Sinha. We also thank Wei Jiang for our early discussion on the algorithm and Suresh Narayanan and Raymond Ziegler for their valuable assistance with the experiments at Sector 8 of the Advanced Photon Source (APS). We acknowledged that Jong Woo Kim and Pice Chen who contributed to the sample preparation and the data collection and Donald Walko who read and commented on part of the manuscript. Work was also performed at the APS Center for Nanoscale Materials of Argonne National Laboratory (ANL), both US Department of Energy (DOE) Ofﬁce of Science (SC) User Facilities. J.W., Z.J., M.C., M.W., and T.S. acknowledge the funding support provided by Laboratory Directed Research and Development (LDRD, 2017-073-N0) from ANL and by the US DOE, SC, Ofﬁce of Basic Energy Sciences (BES), under Contract No. DE-AC02-06CH11357. J.W., Z.J., and M.C. acknowl- edge partial funding support from the Accelerator and Detector Research Program of the US DOE, SC, BES under Contract No. DE-AC02- 06CH11357. Z.J. acknowledges partial support from the US DOE Early Career Research Award. Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41467-023-39984-3. Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41467-023-39984-3. Nature Communications\| (2023) 14:5795 Correspondence and requests for materials should be addressed to Miaoqi Chu or Jin Wang. Correspondence and requests for materials should be addressed to Miaoqi Chu or Jin Wang. Peer review information Nature Communications thanks the anon- ymous reviewers for their contribution to the peer review of this work. A peer review ﬁle is available. Methods Samples Shapiro, D. A. et al. Anultrahigh-resolution soft x-ray microscope for quantitative analysis of chemically heterogeneous nanomaterials. Sci. Adv. 6, eabc4904 (2020). 66. Giles, J. W. 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Author contributions J.W., M.C., and Z.J. conceptualized the experiment and the study. M.C., Z.J., T.S., M.S., and J.W. performed experiments. M.W. and M.C. pre- pared the samples. M.C., J.W., and Z.J. interpreted the data. M.C. (assisted by Z.J. and J.W.) developed the analysis method and algorithms and performed the simulations. M.C. prepared the ﬁrst draft. All authors contributed to revising the manuscript. J.W. and M.C. wrote the ﬁnal version of the manuscript. Nature Communications\| (2023) 14:5795 13
https://openalex.org/W3155511970	https://www.mdpi.com/1996-1073/14/8/2281/pdf	English	null	Bismuth-Graphene Nanohybrids: Synthesis, Reaction Mechanisms, and Photocatalytic Applications—A Review	Energies	2,021	cc-by	30,223	Citation: Usman, M.; Humayun, M.; Shah, S.S.; Ullah, H.; Tahir, A.A; Khan, A.; Ullah, H. Bismuth-Graphene Nanohybrids: Synthesis, Reaction Mechanisms, and Photocatalytic Applications—A Review. Energies 2021, 14, 2281. https://doi.org/ 10.3390/en14082281 Citation: Usman, M.; Humayun, M.; Shah, S.S.; Ullah, H.; Tahir, A.A; Khan, A.; Ullah, H. Bismuth-Graphene Nanohybrids: Synthesis, Reaction Mechanisms, and Photocatalytic Applications—A Review. Energies 2021, 14, 2281. https://doi.org/ 10.3390/en14082281 Keywords: bismuth/graphene; nanohybrids; photocatalysis; reaction mechanisms; energy; pollution energies energies Review Bismuth-Graphene Nanohybrids: Synthesis, Reaction Mechanisms, and Photocatalytic Applications—A Review n 1 , Muhammad Humayun 2 , Syed Shaheen Shah 1 , Habib Ullah 3 , Asif A Tahir 4 , d b b ll h 4 Muhammad Usman 1 , Muhammad Humayun 2 , Syed Shaheen Shah 1 , Habib Ullah 3 , Asif A Tahir 4 , Abbas Khan 5,* and Habib Ullah 4,* Muhammad Usman 1 , Muhammad Hum Abbas Khan 5,* and Habib Ullah 4,* 1 Center of Research Excellence in Nanotechnology, King Fahd University of Petroleum and Minerals (KFUPM), Dhahran 31261, Saudi Arabia; muhammadu@kfupm.edu.sa (M.U.); g201709190@kfupm.edu.sa (S.S.S.) 2 School of Optical and Electronic Information, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, China; 2017511018@hust.edu.cn 3 State Key Laboratory of Advanced Technology for Materials Synthesis and Processing, Wuhan University of Technology, Wuhan 430074, China; habib_aas86@outlook.com 4 Environment and Sustainability Institute, University of Exeter, Penryn TR10 9FE, Cornwall, UK; A.Tahir@exeter.ac.uk 5 Department of Chemistry, Abdul Wali Khan University, Mardan 23200, KP, Pakistan * Correspondence: abbas053@gmail.com (A.K.); hu203@exeter.ac.uk (H.U.) 5 Department of Chemistry, Abdul Wali Khan University, Mardan 23200, KP, Pakistan * Correspondence: abbas053@gmail.com (A.K.); hu203@exeter.ac.uk (H.U.) * Correspondence: abbas053@gmail.com (A.K.); hu203@exeter.ac.uk (H.U.) Abstract: Photocatalysis is a classical solution to energy conversion and environmental pollution control problems. In photocatalysis, the development and exploration of new visible light catalysts and their synthesis and modiﬁcation strategies are crucial. It is also essential to understand the mechanism of these reactions in the various reaction media. Recently, bismuth and graphene’s unique geometrical and electronic properties have attracted considerable attention in photocatalysis. This review summarizes bismuth-graphene nanohybrids’ synthetic processes with various design considerations, fundamental mechanisms of action, heterogeneous photocatalysis, beneﬁts, and challenges. Some key applications in energy conversion and environmental pollution control are discussed, such as CO2 reduction, water splitting, pollutant degradation, disinfection, and organic transformations. The detailed perspective of bismuth-graphene nanohybrids’ applications in various research ﬁelds presented herein should be of equal interest to academic and industrial scientists. 1. Introduction The increase in pollution due to urbanization and industrialization has become a signiﬁcant challenge for the sustainability of human society. The waste generated in different industries during crude oil storage, transportation, and reﬁnery has become a global problem [1,2]. The water and soil pollution caused by several pollutants’ discharge is a critical public health concern due to their toxicity. These pollutants can cause many health effects such as neurological toxicity, lung cancer, lethargy, fatigue, depression, headaches, nausea, dizziness, throat and eye irritation, and acute and chronic respiratory effects [3]. Toluene, benzene, xylene, ethyl benzene, and phenolic compounds some of the main compounds categorized as pollutants posing severe threats to our environment [4–6]. In the present situation, environmental pollution has increased several-fold due to the mismanagement of industrial waste. This can negatively affect the ecosystem and make lands unusable for agriculture and many other purposes [7]. Therefore, it is essential to remediate these toxic pollutants in our environment [8–10]. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). To eliminate organic pollutants from the environment, numerous technologies have recently been established for their degradation. Organic pollutants can be degraded by dif- ferent methods, such as physical, chemical, biological treatments and advanced oxidation techniques [9,11–15]. Organic pollutant photodegradation is an attractive “green” chemical https://www.mdpi.com/journal/energies Energies 2021, 14, 2281. https://doi.org/10.3390/en14082281 Energies 2021, 14, 2281 2 of 36 technology to control pollution, where photocatalysis is the most widely and potentially applied method used for demineralization and degradation of such pollutants [16,17]. technology to control pollution, where photocatalysis is the most widely and potentially applied method used for demineralization and degradation of such pollutants [16,17]. Various light sources have been applied for the excitation of heterogeneous cata- lysts [18], but the photodegradation approach is more economical if sunlight can be used compared to ultraviolet light [16,19,20]. The evolution of the term “photocatalysis” shows the development of certain fundamental concepts of photochemistry. The point where photochemistry became a discipline was when it became differentiated from thermal chem- istry. 1. Introduction Indeed, several researchers saw irradiation as one of the many methods available to catalyze a response that makes it quicker by, for example, heating or processing it with certain chemicals until the beginning of the 20th century [21]. Ciamician, the ﬁrst scientist to systematically understand the chemical effect of light, took great pains in ﬁnding out if he had “initiated heat” alone rather than “light” [22]. This was appropriately allotted the term “photochemical,” whilst the word “photocatalytic” applied to reactions caused by light, but with the same result as thermal reactions. Another step further was the identiﬁcation of electronically excited states, which became a general idea in 1914 and were part of Bodenstein’s photochemical reactions along with reactivity and thermodynamics. In an early stage, more distinction was made in the thermochemistry of the process itself. This allowed for photosynthesis to occur when part of photon energy in the products rose [22,23]. Around 43% of visible-light energy is solar, so visible-light catalysts are chosen in photoelectrocatalysis and photocatalysis processes. Until now, several semiconductive products have been utilized, including metal oxides (Ag2O, TiO2, Cu2O, ZnO, Fe2O3, Ta2O5), metal selenides (CdSe and MOSe2), metal phosphides (Ni2P), metal sulﬁdes (Bi2S3, ZnS, MoS2, and CdS), multi-structure oxides (Sr TiO3WO), metal halides and oxyhalides (AgBr, BiOBr) and metal-free materials (SiC, Si and g-C3N4), [24–28]. Those with a bandgap (Eg) greater than 3 eV, e.g., SrTiO3, TiO2, ZnO, KTaO3, ZnS, and SrTiO3, are called wide- bandgap photocatalysts, whereas catalysts with an Eg of less than 4 eV, e.g., Si, SiC, Ag2O, Bi2WO6, CdSe, InTaO4, Ag3VO4, CoO, Fe2O3, Cu2O, TaON, Ta3N5, CdS, Bi2S3, g-C3N4, and BiVO4, are photocatalysts that react to visible light [25,29]. p y g Heterogeneous catalysts play a vital role in environmental pollution control [30–32]. Powdered semiconductor photocatalysts are commonly used in various areas, such as car- bon reduction [33], selective organic transformations, environmental remediation [34], and water splitting [35]. There has been, in numerous applications, a growing interest in the use of semiconductors as photocatalysts. In 2015, around 5500 documents about photocatalytic applications were published, indicating that interest in heterogeneous photocatalysis was enormous and highly important in diverse research ﬁelds. This number has recently grown to over 13,000. A country-speciﬁc view of the increase in the number of publications on “photocatalytic degradation” is listed in Table 1. 1. Introduction No commercially accessible material can currently meet all application requirements, such as cost-effectiveness, stability, high visible-light quantum efﬁciency, and security [36]. For such tasks to be completed, a highly effective architecture and system for environmental remediation and energy supply are needed to examine new visible-light semiconductor materials. The development of nanomaterials has progressed from the synthesis of single- particles to multicomponent assemblies or hierarchical structures, where two or more pre-synthesized nanomaterials are coupled to obtain multifunctionality. Such multicompo- nent assemblies are termed nanohybrids. The development and use of these nanohybrids requires interdisciplinary knowledge from the energy and environmental sectors, including the applications reported in references [37–43]. There are previously published review articles on some types and uses of nanohybrids, including gold-graphene oxide nanohy- brids [39], organic/inorganic nanohybrids [44], polymer nanohybrids for oil recovery [45], nanohybrids of epoxy/polyamide with carbon nanotubes [46], protein-inorganic nanohy- brids [47], gold-based inorganic nanohybrids [48] and polymer-inorganic supramolecular nanohybrids [49]. Energies 2021, 14, 2281 3 of 36 Table 1. Country-wise publications growth on the photocatalytic degradation of organic pollutants. (Data acquired from SciFinder). S. No. Country No. of Publications 1 China 8838 2 India 1090 3 Iran 676 4 South Korea 384 5 United States of America 178 6 Japan 175 7 Malaysia 158 8 Saudi Arabia 103 9 Pakistan 84 10 Italy 77 11 Australia 73 12 Spain 72 13 Brazil 57 14 United Kingdom 48 Table 1. Country-wise publications growth on the photocatalytic degradation of organic pollutants. (Data acquired from SciFinder). Table 1. Country-wise publications growth on the photocatalytic degradation of organic pollutants. (Data acquired from SciFinder). Graphene is the basic structure of all other carbon allotropes. It is well noted that the potential applications of graphene its derivatives are mainly driven by progressive produc- tion of different graphene materials such as graphene oxide (GO), reduced graphene oxide (rGO), functionalized graphene oxide (fGO), and functionalized reduced graphene oxide (frGO) with speciﬁc attention to precise applications and this is expected to continue for at least a couple of decades as promising applications and requirements are disclosed [50,51]. Various literature reports on the synthesis, modiﬁcation and application of photocatalysts based on graphene for energy and environment solutions have already been published [52]. This review, therefore, summarizes and discusses r and their energy and environmental sector applications 2. Bismuth-Graphene Based Photocatalytic Materials 2.1. Bi2O3 and Bi2S3/Graphene Composites gy pp g phene is due to the vast available literature, as shown in Figure 1. Furthermore, most bis- muth-based photocatalysts are stable, reusable, photoactive, cheaper, and more environ- mentally friendly that other alternatives. Besides, due to some shortcomings of the pris- tine photocatalysts, such as charge carrier recombination, slow migration of charge carri- ers, and low visible light absorption [72,73], we discuss modification of graphene with bismuth species to produce improved photocatalysts for practical applications. The discussion above highlights the vital roles of graphene, bismuth, and nanohy- A signiﬁcant and the simplest bismuth compound is bismuth trioxide (Bi2O3). It can be used in various ceramics, fuel cells, and gas sensors [74,75]. It has also been used as a photocatalyst in organic pollutant decomposition and water splitting [76]. Bi2O3 is a visible-light-responding photocatalyst when acting as a semiconductor, and its bandgap ranges between 2.1 eV and 2.8 eV. Doping with noble materials and combination with other components have been used to increase graphene’s activity in photocatalytic (PC) form [77,78]. The discussion above highlights the vital roles of graphene, bismuth, and nanohy brids. Structural, chemical synthesis and mechanistic aspects of these nanohybrids are discussed, as are the suggested industrial applications of Bi-graphene. Recent literature on energy conversion, degradation of various pollutants, and the CO2 conversion process has been overviewed. Finally, the challenges associated with bismuth and graphene and possible solutions have been discussed. 2. Bismuth-Graphene Based Photocatalytic Materials 2.1. Bi2O3 and Bi2S3/Graphene Composites A significant and the simplest bismuth compound is bismuth trioxide (Bi2O3). It can be used in various ceramics, fuel cells, and gas sensors [74,75]. It has also been used as a photocatalyst in organic pollutant decomposition and water splitting [76]. Bi2O3 is a visi- ble-light-responding photocatalyst when acting as a semiconductor, and its bandgap ranges between 2.1 eV and 2.8 eV. Doping with noble materials and combination with other components have been used to increase graphene’s activity in photocatalytic (PC) form [77,78]. In recent times, the PC activity of some Bi-based semiconductors, e.g., BiVO4 [79], Bi2MoO6 [80,81], BiOX (X = Cl, Br, I), Bi2Sn2O7 [82], Bi2O3 [83], and BiSbO4 [84] in the deg- radation of pollutants has been described. 1. Introduction Finally, the chall solutions have been discussed nergies 2021, 14, x FOR PEER REVIEW overviewed. Finally, the challenges associated with bismuth and graphene and possible solutions have been discussed. Energies 2021, 14, x FOR PEER REVIEW 4 of 38 Figure 1. Annual numbers of published items in the last 10 years identified in SciFinder using the keywords: “Graphene- based photocatalysts” (a) and “Bismuth-based photocatalysts” (b). Figure 1. Annual numbers of published items in the last 10 years identiﬁed in SciFinder using the keywords: “Graphene-based photocatalysts” (a) and “Bismuth-based photocatalysts” (b). 1. Introduction Graphene, graphene and its derivatives [53,54], graphene in photocatalysis [55], graphene doping [56], graphene and graphene oxide sponge [57], nitrogen-doped graphene [58], structure of graphene and its disorders [59], strain engineering of graphene [60], me- chanics of graphene nanocomposites [61], chemical vapor deposition of graphene [62], functional modiﬁcation of graphene/graphene oxide [63], graphene-based ﬁbers [64], and graphene-based electrochemical micro-supercapacitors [65] are some of the subjects that have been reviewed. Considering the stability, reactivity, reusability, and light-responsive effect of bis- muth (Bi) it has been widely used as a photocatalyst. Several state-of-the-art review articles on topics including barium potassium bismuth oxide [66], bismuth-based compos- ite oxides [67], bismuth ferrite nanoparticles [68], bismuth vanadate-based materials [69], bismuth tungstate photocatalysts [70], and bismuth oxyhalides [71] have been published. Annual numbers of publications on graphene photocatalysts in the last ten years are shown in Figure 1a. Similarly, bismuth-containing compounds are signiﬁcant photocatalysts that react to visible light and fascinating research has been published in the ﬁeld of bismuth photocatalysis over the last ten years (Figure 1b). This review, therefore, summarizes and discusses recent Bi-graphene photocatalysts and their energy and environmental sector applications. The choice of bismuth with graphene is due to the vast available literature, as shown in Figure 1. Furthermore, most bismuth-based photocatalysts are stable, reusable, photoactive, cheaper, and more environ- mentally friendly that other alternatives. Besides, due to some shortcomings of the pristine photocatalysts, such as charge carrier recombination, slow migration of charge carriers, and low visible light absorption [72,73], we discuss modiﬁcation of graphene with bismuth species to produce improved photocatalysts for practical applications. The discussion above highlights the vital roles of graphene, bismuth, and nanohybrids. Structural, chemical synthesis and mechanistic aspects of these nanohybrids are discussed, as are the suggested industrial applications of Bi-graphene. Recent literature on energy conversion, degradation of various pollutants, and the CO2 conversion process has been Energies 2021, 14, 2281 4 of 36 overviewed. Finally, the challenges associated with bismuth and graphene and possible solutions have been discussed. nergies 2021, 14, x FOR PEER REVIEW 4 of 38 Figure 1. Annual numbers of published items in the last 10 years identified in SciFinder using the keywords: “Graphene- based photocatalysts” (a) and “Bismuth-based photocatalysts” (b). Figure 1. Annual numbers of published items in the last 10 years identiﬁed in SciFinder using the keywords: “Graphene-based photocatalysts” (a) and “Bismuth-based photocatalysts” (b). overviewed. This review, therefore, summarizes and discusses r and their energy and environmental sector applications 2. Bismuth-Graphene Based Photocatalytic Materials 2.1. Bi2O3 and Bi2S3/Graphene Composites Bismuth oxide was shown to be a strong can- In recent times, the PC activity of some Bi-based semiconductors, e.g., BiVO4 [79], Bi2MoO6 [80,81], BiOX (X = Cl, Br, I), Bi2Sn2O7 [82], Bi2O3 [83], and BiSbO4 [84] in the degradation of pollutants has been described. Bismuth oxide was shown to be a strong can- didate among the various Bi-based semiconductors because of its good PC and appropriate bandgap properties. Bi2O3’s PC activity is however restricted by quick recombination of the photogenerated carriers and by its susceptibility to photocorrosion. Because of the short distance between the conduction band (CB) of Bi2O3 and the valence band (VB), graphene can be designed for the sharing of Bi2O3 and graphene [85]. Under such condi- tions, electrons generated in the CB of Bi2O3 would quickly be coupled with graphene VB holes [86]. Therefore, the photogenerated electrons accumulated on the CB of graphene display strong reduction ability, and the photogenerated holes on the VB of Bi2O3, ex- hibit excellent oxidation ability [87,88]. The Z-Scheme PC activities are more effective than one component in terms of reduction and oxidation and advanced photocatalytic performance in the traditional photocatalysts [89,90]. Cui has reported a novel Z-scheme Bi2O3/graphene photocatalyst. Bi2S3 has a 1.7 eV bandgap and is a perfect photocatalytic material for light-harvesting due to its near-IR and visible light activation [91]. A number of Bi2S3 nanocrystal forms ranging from 1D nanorods and 2D nanosheets have been created with hot injection and standard non-oxidation techniques [92,93], while a solvothermal method produces 3D sea-urchin-like spheres [94]. p g didate among the various Bi-based semiconductors because of its good PC and appropri- ate bandgap properties. Bi2O3’s PC activity is however restricted by quick recombination of the photogenerated carriers and by its susceptibility to photocorrosion. Because of the Bismuth sulﬁde (Bi2S3) is a priviledged nontoxic inorganic semiconductor with excel- lent photocatalytic activity and chemical stability because of its good visible light response. It has been exploited and investigated mostly for optoelectronic applications. The photogen- Energies 2021, 14, 2281 5 of 36 erated holes and hydroxyl radicals (-OH) in the VB of Bi2S3 (1.62 eV) are mostly utilized in dye pollutant decomposition [92]. In combination with many other photocatalysts such as CdS [95], TiO2 [28,96], and Bi2WO6 [97], the recombination rate of electron-hole pairs could be lowered. An increase in visible light absorption enhances the photocatalytic activity. 2.2. Bi2MO6 (M = Cr, Mo, W)/Graphene Composites 2 6 , , p p Bi2MO6 (M = Mo, Cr, W) is considered the most common member of the Aurivillius family, Bi2An−1BnOn+3 (A = Sr, Ca, Ba, Bi, Pb, K, Na; B = Nb, Ti, Ta, Fe, W, Mo) is the general formula for Bi2MO6. The Bi2MO6 electronic structure is theoretically based on density functional theory (DFT) [98], while the Bi2MO6 crystal structure falls under orthorhombic space group Pca2(1). It was seen that both VB and CB of Bi2MO6 are composed of hybridized orbitals Bi6p, O2p, and Mnd (n = 3, 4, and 5) for Bi2CrO6, Bi2MoO6, and Bi2MO6, respectively [99]. Bi2MO6 compounds are suitable as visible-light-activated photocatalysts. Among all Bi2MO6 species Bi2CrO6 has a narrower bandgap, thus, it easily undergoes recombination of photogenerated holes and electrons and is thus not considered suitable as a photocatalyst and consequently few Bi2CrO6 studies are available in the ﬁeld of photocatalysis. For the preparation of Bi2MoO6 samples with a wider special surface area, smaller particles, and higher photocatalytic function, the solvothermal and hydrothermal methods are effective. Several Bi2MoO6 morphologies have been described, including ﬂoral hollow spheres (solvothermal process) and nanoplates (hydrothermal method). Moreover, microwave heating was applied to synthesize Bi2MoO6 samples with high photocatalytic activity in short periods [100,101]. Major applications of Bi2MO6 (and Bi2MoO6 and Bi2WO6) photocatalysts involve the removal of organic pollutants from polluted air and water. The key pollutants that have been tested in different studies include phenol [102], dyes [103], CHCl3 and CH3CHO in wastewater [104], and NO in air [105]. Microorganisms, e.g., E. coli, were also destroyed by the addition of Bi2WO6 [106] and Bi2MoO6 [107] under visible light irradiation. Current studies reveal the combined effect of plasmonic metals and graphene. The photocatalytic activity of semiconductors, e.g., TiO2 and ZnO, can be efﬁciently improved by increasing their photo-absorption ability and suppressing photogenerated electron-hole recombination. Compared to Bi2MoO6, Bi2MoO6-graphene binary composites have been developed and show improved photocatalytic performance. Graphene-based nanocompos- ites display desirable photocatalytic properties that their individual components do not have, therefore, improved Bi2MoO6 photocatalytic activity resulting from a combination of noble metals and graphene is expected. Bi et al. developed a rGO-Bi2MoO6/Au composite that displayed high catalytic activity for the photodegradation of rhodamine B [20]. Wang and Tian reported composites of GO-Bi2MoO6 and rGO-Bi2MoO6 [108,109]. These com- posites showed advanced phenol and rhodamine B degradation properties, respectively, compared to Bi2MoO6 alone [74]. This review, therefore, summarizes and discusses r and their energy and environmental sector applications 2. Bismuth-Graphene Based Photocatalytic Materials 2.1. Bi2O3 and Bi2S3/Graphene Composites A graphene/Bi2S3 nanocomposite with narrow bandwidth was recently synthesized. Compared with the individual components, the PC of this nanocomposite was much higher. Zhou et al. stated that the well-matched bandgap of graphene/Bi2S3 heterojunction could be tailored to increase the transfer and separation efﬁciency of photoinduced carriers and the visible light response. These graphene/Bi2S3 composites are effective photocatalysts for the photocatalytic degradation of environmental pollutants [74]. 2.3. BiVO4/Graphene Nanocomposites Bismuth vanadate (BiVO4) presents interesting physicochemical properties, including ionic conductivity and ferroelasticity. A theoretical bandgap of 2.047 eV was calculated by DFT for visible-light-driven photocatalysis [110]. Both O2 p- and V3 d-orbitals are included in the BiVO4 valence band. There are three forms of BiVO4, namely monoclinic fergusonite, tetragonal zircon, and tetragonal scheelite. Reversible monoclinic fergusonite and tetragonal scheelite phase transitions occur at 255 ◦C. A wide range of methods have been reported for BiVO4 preparation. Monoclinic BiVO4 is obtained by both high tempera- Energies 2021, 14, 2281 6 of 36 ture melting reactions and by solid-state reactions (SSR) [111]. Tetragonal BiVO4 has been synthesized at room temperature by a precipitation method [112]. The bandgap for the monoclinic form is 2.4 eV, while the bandgap for BiVO4 is 2.9 eV. This selective monoclinic BiVO4 preparation is advantageous for assembling effective photocatalysts with visible light shifts. There has been a report of an additional method for synthesizing monoclinic and tetragonal BiVO4 crystals in a simple water-based process [113]. A hydrothermal method has been used successfully in recent times for monoclinic BiVO4 preparation [114]. There are numerous advantages to this hydrothermal approach to selectively produce BiVO4 structures, i.e., mild experimental conditions, controllable conditions and simple experimental setups. Photocatalytic degradation under visible light is commonly used to decompose or- ganic pollutants (e.g., phenol and RhB) [115], and increased removal efﬁciency has been demonstrated [116]. BiVO4 was also used for the scission of water [117,118]. BiVO4 was shown to be an active photocatalyst for O2 evolution under visible light radiation since its conduction strip potential isn’t high enough to produce H2 by H2O reduction [119]. Booshehri et al., found BiVO4 to be a mild candidate for photocatalytic inactivation of bacteria in water under visible light irradiation [120]. For photocatalytic bactericidal activ- ity, surface redox reactions are essential for reactive species generation [121]. In addition, the interface for charge separation and transfer in hybrid catalysts is to be considered for two components [122]. The BiVO4/Ag/graphene photocatalyst showed improved activity for photocatalytic degradation of organic pollutants [123,124] or oxidation of nitrogen monoxide and water [125]. The probability of photocatalytic wastewater or disinfection of water by the Z-scheme BiVO4/graphene is however still unknown to the best of our knowledge. Moreover, at the molecular level the photocatalysis consistency is clearly not yet investigated [74]. 2.4. BiOX (X = F, Cl, Br, I)/Graphene Composites Bismuth oxyhalides’ (BioXoptical)’s properties can work as a photocatalyst. The structure of BiOX crystals is comprised of layer structure slabs [Bi2O2] which are inserted in two halogen atoms [126,127]. Biox contain X np (n = 2–5 for Cl, F, I, and Br respectively), O 2p, and Bi 6 p-orbitals both in the valence band (VB) and conduction band (CB). In theoretical terms, the bandgaps of BiOI, BiOF, BiOBr, and BiOCl are calculated to be 1.38 eV, 2.79 eV, 1.99 eV, or 2.34 eV, while experimentally, their bandgaps are estimated to be 1.77 eV, 3.64 eV [128], 2.64 eV, and 3.22 eV [129]. There are restrictions within the GGA method that cause these differences between the experimental and calculated bandgap results. However, both indicate the general decreasing tendency of the bandgaps as the atomic number increases. BIOF was used as a photocatalyst only under UV light, while BiOI was photocatalytically active both under near-IR and visible light. Because of their appropriate bandgaps, both BiOCl and BiOBr are therefore commonly tested. For BiOX synthesis with different morphologies, several methods can be effectively applied. In addition to direct precipitation techniques, the primary methods used to synthesize the BiOX with controlled nanostructures such as nanosheets, microsphere, and nanoﬁbers include hydrolysis, solvothermal and hydrothermal methods [130]. By adjusting the precursor pH, controlling hydrothermal treatment duration time and temperature, and by adding a template structure that can be selectively controlled, one can directly affect the photocatalytic performance. An extensive review of BiOX nanostructures was previously published [131]. p Signiﬁcant efforts have been carried out to design innovative photocatalysts [132,133]. Because of their excellent catalytic activity under visible light, the sequence of ternary bismuth oxyhalides (BiOX, X = Cl, Br, or I) has been commonly studied [134]. The charge separation and atomic polarization efﬁciency of the layered BiOX structures can be im- proved. BiOBr, with its crystalline PbFCl layer structure has been a big consideration among BiOX photocatalysts because of its excellent photocatalytic activity, appropriate bandgap, and high stability. The binary component and multi-component counterparts Energies 2021, 14, 2281 7 of 36 showed improved photocatalytic activity compared to single-component semiconductors. Multi-component synergies may overcome the single-component shortcomings, e.g., insuf- ﬁcient charge separation ability and wide-bandgap. Consequently, the BiOBr photocatalytic activity [135,136] with an indirect-transition bandgap (2.75 eV) may be efﬁciently enhanced by incorporating other materials. 2.4. BiOX (X = F, Cl, Br, I)/Graphene Composites Graphenes are currently used as a promising support platform for anchoring host NPs as well as acceptors for charge separation and superb electron transfer mediation with pecu- liar characteristics such as low density, high conductivity, and large surface areas [137–139]. The hydrothermal method has been used for the synthesis of Au/BiOBr/graphene com- posites [140,141]. A practical approach to shrink the bandgap, increase the catalytic activity and visible- light absorption was taken using black BiOCl material with the formation of oxygen vacan- cies. Although the black BiOCl is still subject to recombination of fast photocatalytic charge carriers, its photocatalytic activity is still not satisfactory. A simple and effective approach to resolve the above-related problems has been taken as the construction hetero-structures of BiOCls with the other appropriate photocatalysts. Thanks to their high electron mobility and a large surface area, the above issues could be well addressed by functional graphene- based semiconductor photocatalysts. A new BiOCl-Bi-Bi2O3/rGO heterojunction with oxygen vacancies has been developed, which provided a solid-solid, close-ﬁt interface and strong interaction between BiOCl, Bi, rGO, and Bi2O3. BiOCl-BI2O3/rGO hetero- junctions showed high photocatalytic performance due to the synergistic effect caused by effective charge separation among Bi2O3, BiOCl, rGO, and Bi-bridges. The BiOCl-Bi- Bi2O3/rGO heterojunction displayed high efﬁciency for photocatalytic degradation of 2-nitrophenol in industrial wastewater treatment. The signiﬁcant task is to demonstrate the superior long-term photostability of the BiOCl-Bi-Bi2O3/rGO heterojunctions. In ad- dition, a promising BiOCl-Bi-Bi-Bi2O3/rGO photocatalytic mechanism was proposed to describe primary phenomena taking place during the process, depending on multiple charge transfer channels [141]. 3. Synthesis of Bismuth/Graphene Nanohybrid Materials In composites based on bismuth graphene, the graphene acts as a substrate for im- mobilization and the other composites as a functional component. The robust conductive structure and wide graphene surfaces often facilitate the redox reaction, charge transfer, and the enforcement of the resulting composites’ mechanical strengths. The coupling of metal oxides with graphene will therefore enhance the efﬁciency for numerous energy conversion, storage, and catalytic reactions [166,167]. This section mainly focused on the recent progress to develop practical approaches to fabricate Bi- graphene nanocomposites. 2.5. BiPO4/Graphene Composites BiPO4 with high photocatalytic activity for organic pollutant degradation was fab- ricated for the ﬁrst time by a hydrothermal approach [142]. A faster hydrothermal way of synthesizing BiPO4 has also been reported [143]. The bandgap in BiPO4 prepared by hydrothermal methods is about 3.85 eV, higher than that of TiO2 (3.2 eV). BiPO4 nanocrys- tals synthesized with standard oxygen-free procedures have a bandwidth of around 4.6 eV. Only UV light can be used as a light source for large bandgap semiconductors. Although its bandgap is broader than that of TiO2, BiPO4 still has high photocatalytic degrada- tion kinetics. This is because the VB of BiPO4 is 3 eV, higher than that of TiO2, and it generates more oxidative holes in its VB compared to TiO2. Photocatalytic conversion of the gas-phase benzene into CO2 by BiPO4 has also been reported in addition to the degradation of the organic pollutant in an aqueous phase. A photocatalytic gas-phase transformation of benzene to CO2 was also reported during an aqueous phase organic pollutant degradation study [144,145]. BiPO4 photocatalysts still have several drawbacks as photocatalysts however, such as low photocatalytic activity, and comparatively rapid recombination of charge carriers, wide bandgaps, low adsorption ability, and large size, which would decrease the photocatalytic activity of BiPO4 and subsequently limit its industrial-scale applications [146,147]. Consequently, it is urgent to create and design photocatalytic materials based on BiPO4, with required and useful photocatalytic perfor- mance properties. To date, numerous efforts have been made to improve the photocatalytic activity of the BiPO4 photocatalyst by doping with non-metals or metals, surface hybridiza- tion, reducing the crystal size, forming heterostructures, or combinations of µ-structure materials [148,149]. BiPO4/rGO nanocomposites exposed the importance of graphene as the support of separating electron-hole pairs, which leads to a high photocurrent. Thus, the development of BiPO4/rGO hybrids is an efﬁcient way to improve the visible light catalytic Energies 2021, 14, 2281 8 of 36 8 of 36 performance of BiPO4. Extensive research has established a trend towards research in carbon-nanomaterials by doping with heteroatoms as they can adapt their fundamental properties successfully [150,151]. performance of BiPO4. Extensive research has established a trend towards research in carbon-nanomaterials by doping with heteroatoms as they can adapt their fundamental properties successfully [150,151]. 2.6. (BiO)2CO3/Graphene C omposites Bismuth subcarbonate is a known solid carbonate in the BI2O3-CO2-H2O system ((BiO)2CO3 or Bi2CO5) [152]. The bandgap of (BiO)2CO3 is 3.4 eV, so wavelengths under 365 nm can therefore stimulate the bandgap [153,154]. The CB of (BiO)2CO3 generally includes hybridized p-orbitals (O2 p and Bi 6p), while its VB consists of p-orbitals (O 2p, Bi 6p, and C 2p). A hydrothermal, template-free method has been used to efﬁciently synthesize (BiO)2CO3 with hollow microsphere orders whose structure is dependent on Ostwald’s growing properties. The compound showed photocatalytic activity for pollutant oxidation or disinfection of air and wastewater contamination [155,156]. Several articles have described p-n heterojunctions that exhibited enhanced photocatalytic activity [74,157]. p j p y y An innovative multi-component TiO2-Bi2O3/(BiO)2CO3-rGO nanocomposite has been synthesized and experimentally used for bisphenol A (BPA) photodegradation. The Bi2O3 was intended to be a visible light photosensitizer. The appropriate VB and CB’s positions TiO2 and (BiO)2CO3 were used as selective sinks for photogenerated holes and electrons, and rGO acted as a channel for charge carrier transport that extended the lifetime of the catalysts. BPA is an endocrine disruptive compound commonly used for the production of many common packaging materials [158]. These materials typically end up in waste dumps, leading to the slow leaching of BPA into water bodies. Accordingly, BPA has been chosen as the model for the photocatalytic activity of the designed photocatalysts based on environmental issues [158,159]. 2.7. M(BiO3)n/Graphene Composites Pentavalent bismuthates (M(BiO3)n (where n = 1, M = Li, Na, K, Ag; n = 2, M = Mg, Zn, Sr, Ba, and Pb) can be bought directly from commercial companies to synthesize additional Bi-based compounds, such as BiOX, as a Bi source [160]. The bandgaps of these compounds are MgBi2O6 1.61 eV, ZnBi2O6 1.53 eV, SrBi2O6 1.93 eV, SrBi2O6 1.93 eV, BaBi2O6 1.93 eV, PbBi2O6 1.92 eV [161], LiBiO3 1.8 eV, KBiO3 2.1 eV [162], NaBiO3 2.6 eV [163], and AgBiO3 2.5 eV, respectively [164]. The valency of Bi-based composites is +3 while the value of (BiO3)n is +5. The Bi3+ cation consists of two orbitals (10 d and 6 s). This indicates that the electronic structure of pentavalent bismuthates is different. Takei et al. tested nine bismuthates for degrading phenol and methylene blue [161]. High photo-catalytical activity under visible light irradiation was shown by NaBiO3, LiBiO3, BaBi2O6, and SrBi2O6. The d-electrons from Zn, Pb and Ag produce a large conduction range as well as consequently poor photocatalytic performance. Electronic systems greatly affect catalytic performance. Excellent visible-light photocatalytic activity recommends pentavalent bismuthates for different photocatalytic applications. M(BiO3)n could be used for efﬁcient visible light photocatalytic degradation of organic pollutants [161,165]. 3.1. Sol-Gel Method In this section, we focus on recent progress in the development of practical approaches for the fabrication of Bi-graphene nanocomposites [168–170]. The robust coupling offers Energies 2021, 14, 2281 9 of 36 many applications for hybrids, such as photocatalysis [171–173]. Anchoring and reactive areas for growth and the nucleation of NPs can be found in functional groups based on reduced graphene oxides (GO/rGO), which allow metal oxide nanostructures to be chemically attached to GO/RGO surfaces. pling offers many applications for hybrids, such as photocatalysis [171–173]. Anchoring and reactive areas for growth and the nucleation of NPs can be found in functional groups based on reduced graphene oxides (GO/rGO), which allow metal oxide nanostructures to be chemically attached to GO/RGO surfaces. A new sol gel-based electro-spinning process conﬁguration was adopted for the fabrication of TiO2/ZnO/Bi2O3 -Gr (TZB-Gr) composites photocatalyst. With this tech- nique, the rim effect was removed by rolling graphene into ‘spiral rolls’ implanted in TiO2/ZnO/Bi2O3 (TZB) nanoﬁbers, which allowed free electrons to move in the axis of nanoﬁbers on the graphene rolls unidirectional [174]. This new conﬁguration signiﬁ- cantly reduced the energy bandgap, enhanced the speciﬁc surface area, accelerated charge transport and delayed electron-hole pair recombination. In this unique conﬁguration, the electrons’ mobility and lifetime were enhanced [175]. The scheme of TZB-Gr nanoﬁbers is shown in Figure 2. A new sol gel-based electro-spinning process configuration was adopted for the fab- rication of TiO2/ZnO/Bi2O3 -Gr (TZB-Gr) composites photocatalyst. With this technique, the rim effect was removed by rolling graphene into ‘spiral rolls’ implanted in TiO2/ZnO/Bi2O3 (TZB) nanofibers, which allowed free electrons to move in the axis of nan- ofibers on the graphene rolls unidirectional [174]. This new configuration significantly reduced the energy bandgap, enhanced the specific surface area, accelerated charge transport and delayed electron-hole pair recombination. In this unique configuration, the electrons’ mobility and lifetime were enhanced [175]. The scheme of TZB-Gr nanofibers is shown in Figure 2. Figure 2. Schematic representation for TiO2 NPs deposition on graphene sheets; as-prepared TiO2/ZnO/Bi2O3-graphene (TZB-Gr) nanofibers. Adapted from [175]. Figure 2. Schematic representation for TiO2 NPs deposition on graphene sheets; as-prepared TiO2/ZnO/Bi2O3-graphene (TZB-Gr) nanoﬁbers. Adapted from [175]. Figure 2. Schematic representation for TiO2 NPs deposition on graphene sheets; as-prepared TiO2/ZnO/Bi2O3-graphene (TZB-Gr) nanofibers. Adapted from [175]. Figure 2. Schematic representation for TiO2 NPs deposition on graphene sheets; as-prepared TiO2/ZnO/Bi2O3-graphene (TZB-Gr) nanoﬁbers. Adapted from [175]. 3.2. Hydrothermal/Solvothermal Methods 3.2. Hydrothermal/Solvothermal Methods Hydrothermal/solvothermal methods are key tools for synthesizing inorganic nanocrys- tals that work at a high temperature in a limited volume under high pressure. With a one-pot hydrothermal/solvothermal approach, highly crystalline nanostructures can be prepared without post-synthetic calcination, and at the same time GO is reduced to rGO. The rational design of nanomaterials and fabrication with distinctive morphology has received a great deal of consideration because the material properties depend not only on the chemical phase and its composition, but also on its size and shape. The synthesis of nanomaterials with different sizes has inspired many researchers due to its potential applications and the size-dependent properties [176]. Consequently, numerous approaches have been devel- oped to make nanocrystals with controlled morphology. Among them, the hydrothermal method is considered to be effective because it is useful for controlling the size and shape of nanomaterials [177]. rGO/Bi2MoO6 nanocomposites are effectively synthesized via a simple hydrothermal process, with virtual uniformity and high-order direction. The rGO had also been added to the surface of Bi2MoO6. There is an extraordinary improvement in the photocatalytic activity for bacterial treatment over the Bi2MoO6–rGO nanocomposite compared to the pure Bi2MoO6. This enhancement is accredited to the high orientation of Bi2MoO6, which efﬁciently improved photogenerated electrons-holes pair’s separation. Energies 2021, 14, 2281 10 of 36 10 of 36 At the generation site, these electrons are quickly inserted into graphene, thus reducing charge recombination. Improved visible light catalytic wastewater treatment performance of Bi2MoO6–rGO nanocomposites can be accomplished [107]. The Bi2MoO6 microsphere surface contains different sizes of Ag3PO4 particles. The Bi2MoO6 and Ag3PO4 microspheres on both sides of the layer rGO are also well connected. The Ag3PO4/rGO/Bi2MoO6 structure can be established with a closed interface, which is beneﬁcial during the photocatalytic process to accelerate charge transfer. The appropriate porous structures and storage surface can offer substantial active surface sites to easily absorb more organic pollutants, which would favor an increase in the photocatalytic ac- tivity of the Ag3PO4/rGO/Bi2MoO6 composite [178]. Ag3PO4/rGO/Bi2MoO6 shows the broadest absorption edge and the highest absorption intensity in the visible light region. This suggests that this ternary composite can absorb a broad spectrum of visible light [179]. Figure 3 describes the synthesis process for Ag3PO4/rGO/Bi2MoO6 nanohybrid, a pho- tocatalytic mechanism for MB-degradation via Ag3PO4/rGO/Bi2MoO6 nanohybrid, and energy band structures of Ag3PO4 and Bi2MoO6. EW 11 of 38 Figure 3. 3.2. Hydrothermal/Solvothermal Methods (a) Synthesis process for Ag3PO4/RGO/Bi2MoO6 nanohybrid, (b) MB-photocatalytic mechanism via an Ag3PO4/RGO/Bi2MoO6 nanohybrid, and (c) energy band structures of Ag3PO4 and Bi2MoO6. Source: Adapted from [178]. Figure 3. (a) Synthesis process for Ag3PO4/RGO/Bi2MoO6 nanohybrid, (b) MB-photocatalytic mechanism via an Ag3PO4/RGO/Bi2MoO6 nanohybrid, and (c) energy band structures of Ag3PO4 and Bi2MoO6. Source: Adapted from [178]. Figure 3. (a) Synthesis process for Ag3PO4/RGO/Bi2MoO6 nanohybrid, (b) MB-photocatalytic mechanism via an Ag3PO4/RGO/Bi2MoO6 nanohybrid, and (c) energy band structures of Ag3PO4 and Bi2MoO6. Source: Adapted from [178]. Figure 3. (a) Synthesis process for Ag3PO4/RGO/Bi2MoO6 nanohybrid, (b) MB-photocatalytic mechanism via an Ag3PO4/RGO/Bi2MoO6 nanohybrid, and (c) energy band structures of Ag3PO4 and Bi2MoO6. Source: Adapted from [178]. The full GO reduction to graphene, the formation of BiPO4-nanorods, and appropri- ate mixing are carried out in a one-stage synthetic route using these two materials. As an essential agent for GO reduction, ethylene glycol (EG) plays an important role and does not require any additional agents. Besides, ethylene glycol is compatible with BiPO4 na- norod preparation. BiPO4-2% rGO is far more photocatalytic than pure BiPO4, and gra- phene for photodegradation of methyl orange under UV radiation is accredited to a wider For BiPO4 and graphene composite formation, two approaches are used. Because two-dimensional high-surface graphene platforms and exceptionally high conductivity can properly contact the target pollutants to provide plenty of reactive sites and efﬁciently accelerate the process of transferring photo-induced electrons from photocatalyst to reactant sites to suppress the photo-induced pair of electron-holes, graphene and nanocomposite integration with the appropriate graphene and BiPO4 may have desirable graphene and Energies 2021, 14, 2281 11 of 36 11 of 36 BiPO4 properties. This will signiﬁcantly improve the photocatalytic activity of the BiPO4 system. The two-step method of preparing the BiPO4/GO nanocomposites was ﬁrst used to synthesize oleylamine-coated BiPO4 and then assemble it onto a GO nanosheet at the water/toluene interface in the second step [180,181]. BiPO4 properties. This will signiﬁcantly improve the photocatalytic activity of the BiPO4 system. The two-step method of preparing the BiPO4/GO nanocomposites was ﬁrst used to synthesize oleylamine-coated BiPO4 and then assemble it onto a GO nanosheet at the water/toluene interface in the second step [180,181]. A two-step hydrothermal approach was used to synthesize BiPO4/rGO cuboids with low OH-related defects. 3.2. Hydrothermal/Solvothermal Methods Although nanocomposites are produced successfully with BiPO4-GO or BiPO4/rGO, the experiments still display a large number of inconveniences: (1) BiPO4/GO or BiPO4-rGO nanocomposite synthesis requires two or several steps that are tedious and time-consuming; (2) toxic organic solvents (toluene), hazardous reduc- ing agents (oleylamine) and other additives may cause many environmental protection problems and in the product post-treatment; (3) the weak interaction between graphene nanosheets and BiPO4 results from 2- or multi-step synthetic routes to BiPO4/rGO, so a simple, efﬁcient, and green approach has been used to synthesize nanocomposites. The full GO reduction to graphene, the formation of BiPO4-nanorods, and appropriate mixing are carried out in a one-stage synthetic route using these two materials. As an essential agent for GO reduction, ethylene glycol (EG) plays an important role and does not require any additional agents. Besides, ethylene glycol is compatible with BiPO4 nanorod preparation. BiPO4-2% rGO is far more photocatalytic than pure BiPO4, and graphene for photodegradation of methyl orange under UV radiation is accredited to a wider surface area, efﬁcient cargo transportation, the graphene introduction, and the close interfacial contact between graphene and BiPO4 have contributed to a much-increased adsorption and separation capacity [182,183]. BiPO4/rGO and BiPO4/GO composites synthesized, and simulated images are shown in Figure 4. W 12 of 38 Figure 4. Synthesis process for BiPO4/RGO and BiPO4/GO composites. Source: Adapted from Figure 4. Synthesis process for BiPO4/RGO and BiPO4/GO composites. Source: Adapted from [183]. Figure 4 Synthesis process for BiPO4/RGO and BiPO4/GO composites Source: Adapted from Figure 4. Synthesis process for BiPO4/RGO and BiPO4/GO composites. Source: Adapted from [183]. [183]. A BiPO4 NPs with MoS2/graphene-layered hybrid is manufactured via an easy hy- drothermal, microwave-assisted method, and the ternary BiPO4-MoS2/graphene photo- catalyst optimizes the activity of each component. This study demonstrates that the gra- phene and MoS2 nanoparticles as catalysts in the photocatalyst of BiPO4 can improve transport charges, eliminate the pair electron hole’s photogenerated recombination rate, and provide highly reactive locations for a photodegradation reaction This results in sig- A simple one-pot hydrothermal route was used to synthesize nanocomposites of biPO4/nitrogen-doped graphene hydrogel (BiPO4) to serve as a visible light-responsive material. The porous 3DNGH structure signiﬁcantly enhanced the photo-induced electron holes and the transfer and separation efﬁciency of BiPO4 visible illumination pairs. The BiPO4/3DNGH morphology has disclosed a cross-linked, porous structure, and 3DNGH nanorods are attached to the area. 3.2. Hydrothermal/Solvothermal Methods The 3DNGH surface was randomly dispersed with BiPO4 nanorods [181]. Energies 2021, 14, 2281 12 of 36 12 of 36 A BiPO4 NPs with MoS2/graphene-layered hybrid is manufactured via an easy hy- drothermal, microwave-assisted method, and the ternary BiPO4-MoS2/graphene pho- tocatalyst optimizes the activity of each component. This study demonstrates that the graphene and MoS2 nanoparticles as catalysts in the photocatalyst of BiPO4 can improve transport charges, eliminate the pair electron hole’s photogenerated recombination rate, and provide highly reactive locations for a photodegradation reaction. This results in signiﬁcantly improved photocatalytic activity for organic pollutant photodegradation by the attained BiPO4-MoS2/graphene photocatalyst. The GO, BiPO4, and MoS2 composite microstructure and morphology were characterized in the sense that GO has a layered stack- ing structure with some folds and wrinkles that can adsorb and photodegrade the color molecules on sufﬁciently large surfaces. The sample produced for MoS2 has an ultra-free nanosheet structure. In composites many BiPO4 NPs are dispersed compactly and homoge- nously on the surface of MoS2/graphene nanosheets. It is proposed to dispense, build, and attach the BiPO4 nanocrystals in MoS2/graphene by microwave-assisted techniques. There are distinct gate fringes on the BiPO4-MoS2/graphene composite. The gap from 0.328 nm to the monoclinic plane BiPO4 (200) corresponds very well, while the gap from 0.62 nm to the plane of (002) MOS2 can be assigned. The presence of close contact between MoS2/graphene nanosheets and BiPO4 NPs is predictable for building a necessary hetero structure [180]. BiPO4/rGO NCs were successfully synthesized by a simple solvothermal method. This composite possessed much advanced and best photocurrent performance. The as-prepared PEC sensor revealed a broader lower detection limit, linear range, and an excellent anti-interference capacity. In the formation of chlorpyrifos, the Bi-chlorpyrifos complex formation on BiPO4 NPs gave rise to an increase in steric hindrance. It thus stuck the BiPO4 NPs electron transfer toward the electrode surface, causing an observable fall in photocurrent [182]. rGO/Bi2MoO6 nanosheets were successfully synthesized using rGO/Bi2 (EG) pre- cursors using a two-stage solvothermal method. The introduction of graphene supports the recombination of electrons and holes generated by photogenerated rGO/Bi2MoO6 nanocomposite exhibits plate-on-plate enhanced Cr (VI) photoreduction structures with radiation from sunlight. With an ideal photocatalytic activity, the 2.5% rGO/Bi2MoO6 com- posite and a reduction of 94% to Cr(VI) at about 30 min, roughly twice that of pure Bi2MoO6. 3.2. Hydrothermal/Solvothermal Methods The rGO, which mainly functions as an electron collector and meaningfully promotes the photoinduced carrier separation, accommodates the improved photocatalytic efﬁcacy. Fur- thermore, rGO/Bi2MoO6 composites have excellent stability and can be recycled in an industrial process. The composite morphologies of 2.5% rGO/Bi2MoO6 are low-lying and non-regular plate-on-plate structures. This indicates that Bi2MoO6 nanoﬂocks are scattered to the surface of large graphene layers forming Bi2MoO6 nanoﬂocks and small ribs. Defects may cause wrinkles during the functioning of oxygen when GO was synthesized [184,185]. y g g yg y [ , ] A newer BWO/MG ternary heterojunction photocatalyst was designed with an im- proved load carrier separation using the two-step hydrothermal method through a pro- gressive load transfer route. MoS2 was used to improve the transition between graphene and BWO through the “stepping stone” approach. A positive synergetic effect between the graphene sheets and MoS2 is believed to occur. The cocatalyst components on photo- degradation can efﬁciently improve the interfacial charge transfer, suppress the recombi- nation of charges, and offer many photocatalytic reaction centers and active absorption sites [186]. The BWO/MG ternary hybrid facility is a visible and inexpensive environ- mental photocatalyst that expands the composite photocatalyst preparation range of MG hybrids and provides a prospective way to improve the performance of photocatalysts. The BWO catalyst has a microscopic structure and morphology with an average diameter of 3–4 microspheres. These microspheres consist of several hundred nanometers of lateral nanoplates. The BWO microspheres used the automatic spherical construction of nanosheet nanoplates. The SEM and TEM images of BWO and BWO/MG are shown in Figure 5. BWO nanosheets are not agglomerated during growth following MG modiﬁcations. The morphology of the BWO crystalline structures is controlled by the incorporation of MG, Energies 2021, 14, 2281 13 of 36 ction of hown in 13 of 36 ction of ho i which has increased photocatalytic performance in a larger speciﬁc area. The photogen- erated electrons should improve the photocatalytic efﬁciency and charging separation, a close relationship between BWO, graphene, and components achieved via hydrothermal processing [187]. tions. The morphology of the BWO crystalline structures is controlled by the incorporation of MG, which has increased photocatalytic performance in a larger specific area. The pho- togenerated electrons should improve the photocatalytic efficiency and charging separa- tion, a close relationship between BWO, graphene, and components achieved via hydro- thermal processing [187]. . Figure 5. 3.2. Hydrothermal/Solvothermal Methods SEM images of (A) BWO and (B) BWO/MG. (C) TEM image of BWO/MG and (D) HRTEM of BWO/MG. Adapted from [187]. Figure 5. SEM images of (A) BWO and (B) BWO/MG. (C) TEM image of BWO/MG and (D) HRTEM of BWO/MG. Adapted from [187]. Figure 5. SEM images of (A) BWO and (B) BWO/MG. (C) TEM image of BWO/MG and (D) HRTEM of BWO/MG. Adapted from [187]. Figure 5. SEM images of (A) BWO and (B) BWO/MG. (C) TEM image of BWO/MG and (D) HRTEM of BWO/MG. Adapted from [187]. Bi2WO6/rGO photocatalysts have been synthesized by an easy hydrothermal method and with 2 wt % rGO content display the highest photocatalyst performance. Enhanced photocatalytic activity for more efﬁcient cargo transport, maximum light absorption, and separation can be accredited to strong chemical bonds between rGO and Bi2WO6. In addition, Bi2WO6/rGO is highly stable and essential for applications in environmental protection applications [188,189]. 3.3. Self-Assembly Self-assembly is a useful and frequently favored method for assembling micro- and nano- substances into macroscopic systems [190–192]. It is used to produce functional materials such as composites, photonic crystals, and DNA structures. An innovative way of synthesizing ordered graphene-metal oxide hybrids via a surfactant-supported, ternary self-assembly process was established to achieve an interchangeable layer structure of ﬁnal composites [193]. The efﬁcient and easy electrostatic self-montage method is successfully used to produce BWO/rGO nanocomposites. BWO-nanocomposites RGO’s have been synthesized with hydrothermal reduction through electrostatic self-assembly processes. The uniform, electronically interacting, and close interface contact can be achieved with nanocomposites from the BWO/rGO. The adjacent interface contact stimulates the separa- tion of e/h+ pairs and extends the lifetime of the photo-induced charge carrier [194]. The charging balance and electronic interaction between rGO and BWO lead to VB change and change in conductive electricity and the valence band holes [195]. Nanocomposites of GO/BiPO4 were synthesized using an easy self-assembly two- phase method. The GO presence can substantially improve the visible light absorption of the load transfer facilitators, catalysts, and the pair of electron holes [196]. The GO/BiPO4 nanocomposites formation via a self-assembly method is shown in Figure 6. 14 of 36 / 14 of 36 / Energies 2021, 14, 2281 Figure 6. GO-BiPO4 nanocomposites formation via a self-assembly method. Adapted from [196]. Figure 6. GO-BiPO4 nanocomposites formation via a self-assembly method. Adapted from [196]. BiPO4 nanocomposites formation via a self assembly method. Adapted from [196]. An easy and fast approach to energy-generating chemical reactions is microwave ir- radiation. Graphene–metal oxide hybrids, for example, graphene-MnO2 have been syn- thesized using microwave irradiation [197] as has graphene–Co3O4 [198]. Direct electro- chemical deposition of inorganic crystals on graphene substrates is an intelligent ap- proach for thin film-based applications with no need for post-synthetic transfer of com- posite materials [199-209]. A summary of bismuth/graphene-based photocatalysts fabri- An easy and fast approach to energy-generating chemical reactions is microwave irradiation. Graphene–metal oxide hybrids, for example, graphene-MnO2 have been synthesized using microwave irradiation [197] as has graphene–Co3O4 [198]. Direct electro- chemical deposition of inorganic crystals on graphene substrates is an intelligent approach for thin ﬁlm-based applications with no need for post-synthetic transfer of composite materials [199–209]. A summary of bismuth/graphene-based photocatalysts fabrication methods, morphology, and applications is presented in Table 2. cation methods, morphology, and applications is presented in Table 2. 4. 4.1. Water Splitting To transform this ration of relevant p Hydrogen energy is considered as an ideal green energy source, and the product of hy- drogen combustion is H2O, so hydrogen, when used as fuel, it both solves the future fossil fuel crisis and shortage and lessens the environmental pollution from fossil fuel consump- tion [150,210,213–215]. In 1972, Fujishima et al. ﬁrst described the TiO2 photoelectrode water splitting phenomenon [216], and as a result, photocatalytic H2 production has gained much attention [217–220]. Hydrogen is one of the crucial pure fuels [221–223]. Hydrogen production using the appropriate photocatalyst and solar power is an important factor not just because it is an excellent way to supply large-scale renewable and clean hydrogen but also to prevent probable energy-storage problems. One of the more convenient methods in this respect is photocatalytic water splitting. To date, some nanocomposites based on graphene have been used for the photocatalytic cleavage of water [220,224]. To transform this technology into an industrial application, the development and exploration of relevant photocatalysts with outstanding performance are vital. In the past four decades, therefore, several semiconductors were tested as photocatalysts. Graphene is considered to have a great performance in this research ﬁeld [225,226]. In order to make a practical photocatalyst economically attainable, efforts have been made to improve the efﬁciency of the photocata- lysts. Amal’s group developed photocatalysts such as rGO/Ru/Sr, rGO/BiVO4, rGO/WO3, and TiO3, rGO/TiO2 [117,227,228]. In the case of the BiVO4/rGO composite, the evolution of the O2 and H2 on BiVO4/rGO was 0.21 mm and 0.75 mmol h−1, respectively, under visible light, while negligible gas production is detected in pure cells of BiVO4. This photo- catalytic water splitting has been accredited to the longer electron life of provoked BiVO4 electrons that promptly injected in rGO at the production site, leading to lower recombina- tion of charges (Figure 7). In recent times, an inspired Z-scheme photocatalysis system for dividing water under visible light radiation has been established. Photocatalytic systems for the artiﬁcial Z-scheme offer a blossoming approach for enhancing the performance of PH 2, by imitating the natural photosynthesis in typical green leaves [229]. p y g p p decades, therefore, several semiconductors were tested as photocatalysts. Graphene is considered to have a great performance in this research field [225,226]. In order to make a practical photocatalyst economically attainable, efforts have been made to improve the efficiency of the photocatalysts. 4. Applications of Bismuth/Graphene Nanohybrids drogen production using the appropriate photocatal factor not just because it is an excellent way to sup Bismuth-graphene-based composites have been used for the photodegradation of pollutants and also in many other domains, such as hydrogen production and photovoltaic cells linked to environmental preservation [210–212]. j y pp y g hydrogen but also to prevent probable energy-storage problems. One of the more conven- ient methods in this respect is photocatalytic water splitting. To date, some nanocompo- sites based on graphene have been used for the photocatalytic cleavage of water [220,224] 3.3. Self-Assembly Applications of Bismuth/Graphene Nanohybrids Bismuth-graphene-based composites have been used for the photodegradation of pollutants and also in many other domains, such as hydrogen production and photovol- taic cells linked to environmental preservation [210–212]. Table 2. Summary of bismuth/graphene-based photocatalyst fabrication methods, morphology, and applications. Photocatalyst Activities Morphology Method Refs. Bi2MoO6/Au/rGO RhB lattice fringes solvothermal and photochemical reduction [20] BiPO4/nitrogen-doped graphene hydrogel biomedical, food and environment analysis porous structure one-pot hydrothermal [181] BiPO4/GO MB sphere-like/rod two-phase self-assembly [196] BiPO4/MoS2/graphene RhB lattice fringes with wrinkles and folds one-pot microwave-assisted hydrothermal [180] Bi2MoO6/Pd-rGO phenol microspheres/ﬂake-like particles Solvothermal photoreduction method [200] BiOBr/Au/Graphene phenol ﬂower-like microstructure hydrothermal synthesis and reduction method [141] BiPO4/rGO Chlorpyrifos nanoparticles/nanosheets solvothermal method [182] TiO2-Bi2O3/(BiO)2 CO3-rGO bisphenol A nanoplates/nanosheet/nanorod hydrothermal procedure [159] TZB-Gr composite NO NPs/2D graphene sheets sol-gel based electrospinning process [175] black BiOCl-Bi-Bi2O3/rGO 2-nitrophenol (2NP) nanosheets sonication and mechanical stirring, in situ Fe reduction [141] BiPO4-graphene Methyl Orange MO wrinkles and folds one-step solvothermal [183] Bi2WO6-rGO NO microspheres/nanosheets hydrothermal method [188] Bi2MoO6/2D-rGO Cr(VI) reduction wrinkled nanoﬂakes hydrothermal method [184] BWO−RGO bisphenol A degradation uniform structure hydrothermal treatment [195] Bi2MoO6–RGO bacterial destruction highly oriented morphology hydrothermal process [107] Bi2MoO6/Ag3PO4/RGO MB microspheres/ﬂakes/irregular- sphere precipitation-solvothermal method [178] Bi/BiOBr/Graphene Degradation of RhB Nanosheets assemble into ﬂower-like microspheres One-step solvothermal [201] Bi-NPs/GO Remove ppb-level NO nanospheres Solution-based sonication [73] Bi-NPs/Graphene Disinfection and antibacterial activity towards Escherichia coli nanospheres Non-injection facile strategy [202] PbBiO2Br/GO CO2 conversion to CH4 nanolayers Hydrothermal synthesis [203] h-BiVO4/rGO BPA degradation and H2 evolution nanoplates embedded nanosheets Ultrasonication [204] BiVO4/rGO MB degradation nanoparticles Hydrothermal synthesis [205] BiFeO3/N-rGO RhB degradation nanoparticles Sol-gel method followed by hydrothermal synthesis [206] BONPs-NG/NGO Xylene removal nanoplates embedded nanosheets Carbon vapor deposition, stirring, and heating [207] Bi(PO4)/GO Ciproﬂoxacin degradation nanospheres embedded nanosheets Cross-linker polymerization [208] BiVO4/rGO Triethylamine (TEA) detection nanosheets wrapped with particles Hydrothermal synthesis [209] cation methods, morphology, and applications is presented in Table 2. smuth/graphene-based photocatalyst fabrication methods, morphology, and applications. Energies 2021, 14, 2281 15 of 36 hotoelec- tio ha 15 of 36 hotoelec ti h 4.1. Water Splitting To transform this ration of relevant p Amal’s group developed photocatalysts such as rGO/Ru/Sr, rGO/BiVO4, rGO/WO3, and TiO3, rGO/TiO2 [117,227,228]. In the case of the BiVO4/rGO composite, the evolution of the O2 and H2 on BiVO4/rGO was 0.21 mm and 0.75 mmol h−1, respectively, under visible light, while negligible gas production is detected in pure cells of BiVO4. This photocatalytic water splitting has been accredited to the longer electron life of provoked BiVO4 electrons that promptly injected in rGO at the production site, leading to lower recombination of charges (Figure 7). In recent times, an inspired Z- scheme photocatalysis system for dividing water under visible light radiation has been established. Photocatalytic systems for the artificial Z-scheme offer a blossoming ap- proach for enhancing the performance of PH 2, by imitating the natural photosynthesis in typical green leaves [229]. PVRO (PRGO/BiVO4, PRGO) and Ru/SrTiO3 photographic graphene oxide blends (PRGO/Ru/SrTiO3:Rh) can be synthesized in the presence of the photocatalytic reduction of GO on both BiVO4 and Ru/SrTiO3:Rh, in the presence of methanol as a hole scavenger. PRGO functions as a solid-state electron mediator in this system and transports electrons from the BiVO4 CB to vacancies in the Ru/SrTiO3:Rh impurity levels. In Ru/SrTiO3 elec- trons, the water is reduced H2 by a Ru cocatalyst, and the water is oxidized into O2 by holes from BiVO4, thus producing a full water decomposition cycle. The O2 and H2 time cycles have demonstrated that after the second cycle, this system is constant. This im- portant work provides a new entry to the use of g-C3N4 in the design of new and efficient water division systems [224]. Chong et al. [230] reported V2O5/rGO/BiVO4 heterojunction (Figure 8) as an efficient photo-electrochemical water division photoanode. . Figure 7. (A) SEM image of BiVO4/rGO; (B) visible light voltage–photocurrent functions of BiVO4, BiVO4/rGO, and TiO2 (under UV irradiation); (C) illustration of photocatalytic water splitting in photoelectrochemical cell based on BiVO4/rGO. Adapted from [224]. . Figure 7. (A) SEM image of BiVO4/rGO; (B) visible light voltage–photocurrent functions of BiVO4, BiVO4/rGO, and TiO2 (under UV irradiation); (C) illustration of photocatalytic water splitting in photoelectrochemical cell based on BiVO4/rGO. Adapted from [224]. PVRO (PRGO/BiVO4, PRGO) and Ru/SrTiO3 photographic graphene oxide blends (PRGO/Ru/SrTiO3:Rh) can be synthesized in the presence of the photocatalytic reduction of GO on both BiVO4 and Ru/SrTiO3:Rh, in the presence of methanol as a hole scavenger. 4.2. CO2 Reduction 4.2. CO2 Reduction Due to growing energy and environmental concerns, CO2 conversion into fuel is con- sidered a favorable approach [231–233]. Solar energy is mainly used for this due to its capacity to imitate the natural photosynthesis process to transform solar energy into chemical energy. The photocatalytic reduction of CO2 into valued fuels like formic acid, methane, and methanol is of particular importance [234–236]. In the last decades, this has received great attention and we have become acquainted with the enhanced release of the greenhouse gas CO2 into our atmosphere and the potential and real power supply short- age. The conversion of solar power into chemicals by photoelectrochemically or photo- catalytically reducing CO2, is also one of the most advantageous methods to solve envi- ronmental and energy problems simultaneously. CO2 molecules are chemically inert and therefore highly stable, with linear geometry and shell electronics [235]. The CO2 reduc- tion by photosensitive semiconductor catalysts yields highly sought products, e.g., formic acid, methane, formaldehyde, and methanol, etc. Several compounds, including metal complexes, can function as electrocatalysts for CO2 reduction [235,236]. Bismuth and gra- phene’s role is vital and has been studied widely in CO2 conversion to valued products. Bismuth is prominently used through electrochemical CO2 reduction reactions (ECRR), while there are several reports of photocatalysis by a bismuth-graphene nanohybrid cat- alyst. Sun el al. converted CO2 into formate using bismuth with bismuth oxides supported on graphene nanosheets (Bi/Bi2O3/NrGO). This hybrid electrocatalyst gives a high current density and low overpotential in ECRR due to the synergistic effect of bismuth and its oxides [237]. Similarly, a bismuth oxide-reduced graphene oxide quantum dots (rGO/BiO QDs) composite was synthesized, which provides excess photoelectrons and protons for CO2 reduction [238]. In another study, a nanoheterojunction electrocatalyst made of zinc phthalocyanine/graphene/BiVO4 showed higher performance than the BiVO4 nanocata- lysts due to the modulating presence of graphene [239]. Using defect engineering, oxygen vacancy-rich electrocatalysts were prepared by Yang et al. [240]. The electrocatalysts were prepared by a precipitation method from bismuth oxide and bismuth sulfide supported on reduced graphene oxide. This hybrid nanocatalyst facilitates CO and formate for- mation during ECRR at low overpotential with high stability during on-stream analysis. A l d bi h b id / h id l d d di d f h Due to growing energy and environmental concerns, CO2 conversion into fuel is considered a favorable approach [231–233]. 4.1. Water Splitting To transform this ration of relevant p PRGO functions as a solid-state electron mediator in this system and transports electrons from the BiVO4 CB to vacancies in the Ru/SrTiO3:Rh impurity levels. In Ru/SrTiO3 (PRGO/Ru/SrTiO3:Rh) can be synthesized in the presence of the photocatalytic reduction of GO on both BiVO4 and Ru/SrTiO3:Rh, in the presence of methanol as a hole scavenger. PRGO functions as a solid-state electron mediator in this system and transports electrons from the BiVO4 CB to vacancies in the Ru/SrTiO3:Rh impurity levels. In Ru/SrTiO3 Energies 2021, 14, 2281 16 of 36 electrons, the water is reduced H2 by a Ru cocatalyst, and the water is oxidized into O2 by holes from BiVO4, thus producing a full water decomposition cycle. The O2 and H2 time cycles have demonstrated that after the second cycle, this system is constant. This important work provides a new entry to the use of g-C3N4 in the design of new and efﬁcient water division systems [224]. Chong et al. [230] reported V2O5/rGO/BiVO4 heterojunction (Figure 8) as an efﬁcient photo-electrochemical water division photoanode. VIEW 18 of 38 of BiVO4/rGO; (B) visible light voltage–photocurrent functions of BiVO4, BiVO4/rGO, and TiO2 C) illustration of photocatalytic water splitting in photoelectrochemical cell based on BiVO4/rGO. . Figure 8. Photoelectrochemical water splitting system design and electron transfer mechanism schematics in V2O5/rGO/BiVO4 heterojunction photoanode. Adapted from [230]. Figure 8. Photoelectrochemical water splitting system design and electron transfer mechanism schematics in V2O5/ rGO/BiVO4 heterojunction photoanode. Adapted from [230]. Figure 8. Photoelectrochemical water splitting system design and electron transfer mechanism schematics in V2O5/rGO/BiVO4 heterojunction photoanode. Adapted from [230]. Figure 8. Photoelectrochemical water splitting system design and electron transfer mechanism schematics in V2O5/ rGO/BiVO4 heterojunction photoanode. Adapted from [230]. pp 4.3.1. NOx Conversion 4.3.1. NOx Conversion In addressing environmental problems associated with water and air pollutants, pho- tocatalytic processes in decomposition and inorganic compounds, along with the removal of dangerous gases, are of great importance [244–247]. The main pollutants caused by the combustion of industrial burners or fossil fuel in automotive engines are nitric oxide (NO) and nitric dioxide (NO2) [248]. Many catalytic processes for the transformation of nitrogen gases (e.g., NO and NO2) into nitrogen (N2), oxygen (O2), or nitrate (NO3−) have been es- tablished [249,250]. An ideal NOx conversion catalyst transforms NOx gases at lower- temperature [251]. TiO2 is one of the leading catalysts for the catalytic conversion of NOx gases into nitrous oxide (N2O) and N2 [252,253]. The majority of previous studies on the conversion of NOx gases have involved different lasers [254], spectroscopic (such as in- frared (IR), [255], and chemiluminescence (CL) [256] or electrochemical techniques [257] for the detection of NOx reaction products. The use of high-resolution MS for biomedical applications to detect NO [230] and indirectly semiconducting metal oxides [231] has been described. The main cause of water pollution is industrial wastewater discharge. Drinking polluted water for a long time poses potential health risks, and can also cause cancer, ter- atogenicity and mutagenicity. For this reason, it is very important to develop suitable techniques for the treatment of industrial wastewater to meet emission standards. Photo- catalysis is considered a sustainable and efficient water treatment technology. Old- pho- tocatalysts (such as ZnO and TiO2) with a wide bandgap are only active in the UV light region and their quick recombination of photo-generated holes and electrons leads to low quantum efficiencies that limit their application for wastewater treatment. The traditional inconveniences of these photocatalysts requires the development of new Bi-based semi- catalysts for real waste water treatment such as black BiOCl-Bi2O3/rGO nanocomposite In addressing environmental problems associated with water and air pollutants, photocatalytic processes in decomposition and inorganic compounds, along with the removal of dangerous gases, are of great importance [244–247]. The main pollutants caused by the combustion of industrial burners or fossil fuel in automotive engines are nitric oxide (NO) and nitric dioxide (NO2) [248]. Many catalytic processes for the transformation of nitrogen gases (e.g., NO and NO2) into nitrogen (N2), oxygen (O2), or nitrate (NO3−) have been established [249,250]. An ideal NOx conversion catalyst transforms NOx gases at lower-temperature [251]. 4.2. CO2 Reduction 4.2. CO2 Reduction Figure 9 presents an electron transfer mechanism and reducing adsorption and formate formation from CO2 molecules over the BiVO4 quantum dots/rGO composite [242,243]. IEW 19 of 38 bismuth graphene composites is needed in this field [241]. Figure 9 presents an electron transfer mechanism and reducing adsorption and formate formation from CO2 molecules over the BiVO4 quantum dots/rGO composite [242,243]. for the conversion of CO2 into methane under light [203]. The graphene-supported catalyst activity was much higher than without graphene, reﬂecting the importance of graphene in future environmental and energy conversion and storage applications. More research on bismuth graphene composites is needed in this ﬁeld [241]. Figure 9 presents an electron transfer mechanism and reducing adsorption and formate formation from CO2 molecules over the BiVO4 quantum dots/rGO composite [242,243]. IEW 19 of 38 bismuth graphene composites is needed in this field [241]. Figure 9 presents an electron transfer mechanism and reducing adsorption and formate formation from CO2 molecules over the BiVO4 quantum dots/rGO composite [242,243]. . Figure 9. (a) The electron transfer and reduction mechanism: a) adsorption of CO2 molecules, b) intermediate stabilization, and c) formate formation with desorption of formate in the electrolyte. (b) Schematic of charge transfer, separation, and the reaction of BiVO4 quantum dots/rGO composites for CO2 reduction. Adapted from [242,243]. Figure 9. (a) The electron transfer and reduction mechanism: a) adsorption of CO2 molecules, b) intermediate stabilization, and c) formate formation with desorption of formate in the electrolyte. (b) Schematic of charge transfer, separation, and the reaction of BiVO4 quantum dots/rGO composites for CO2 reduction. Adapted from [242,243]. . Figure 9. (a) The electron transfer and reduction mechanism: a) adsorption of CO2 molecules, b) intermediate stabilization, and c) formate formation with desorption of formate in the electrolyte. (b) Schematic of charge transfer, separation, and the reaction of BiVO4 quantum dots/rGO composites for CO2 reduction. Adapted from [242,243]. Figure 9. (a) The electron transfer and reduction mechanism: a) adsorption of CO2 molecules, b) intermediate stabilization, and c) formate formation with desorption of formate in the electrolyte. (b) Schematic of charge transfer, separation, and the reaction of BiVO4 quantum dots/rGO composites for CO2 reduction. Adapted from [242,243]. 4.2. CO2 Reduction 4.2. CO2 Reduction Solar energy is mainly used for this due to its capacity to imitate the natural photosynthesis process to transform solar energy into chemical energy. The photocatalytic reduction of CO2 into valued fuels like formic acid, methane, and methanol is of particular importance [234–236]. In the last decades, this has received great attention and we have become acquainted with the enhanced release of the greenhouse gas CO2 into our atmosphere and the potential and real power sup- ply shortage. The conversion of solar power into chemicals by photoelectrochemically or photocatalytically reducing CO2, is also one of the most advantageous methods to solve environmental and energy problems simultaneously. CO2 molecules are chemically inert and therefore highly stable, with linear geometry and shell electronics [235]. The CO2 reduction by photosensitive semiconductor catalysts yields highly sought products, e.g., formic acid, methane, formaldehyde, and methanol, etc. Several compounds, in- cluding metal complexes, can function as electrocatalysts for CO2 reduction [235,236]. Bismuth and graphene’s role is vital and has been studied widely in CO2 conversion to valued products. Bismuth is prominently used through electrochemical CO2 reduction reactions (ECRR), while there are several reports of photocatalysis by a bismuth-graphene nanohybrid catalyst. Sun el al. converted CO2 into formate using bismuth with bismuth oxides supported on graphene nanosheets (Bi/Bi2O3/NrGO). This hybrid electrocatalyst gives a high current density and low overpotential in ECRR due to the synergistic effect of bismuth and its oxides [237]. Similarly, a bismuth oxide-reduced graphene oxide quantum dots (rGO/BiO QDs) composite was synthesized, which provides excess photoelectrons and protons for CO2 reduction [238]. In another study, a nanoheterojunction electrocat- alyst made of zinc phthalocyanine/graphene/BiVO4 showed higher performance than the BiVO4 nanocatalysts due to the modulating presence of graphene [239]. Using defect engineering, oxygen vacancy-rich electrocatalysts were prepared by Yang et al. [240]. The electrocatalysts were prepared by a precipitation method from bismuth oxide and bismuth sulﬁde supported on reduced graphene oxide. This hybrid nanocatalyst facilitates CO and formate formation during ECRR at low overpotential with high stability during on-stream analysis. A lead bismuth oxobromide/graphene oxide catalyst was prepared and studied Energies 2021, 14, 2281 17 of 36 for the conversion of CO2 into methane under light [203]. The graphene-supported catalyst activity was much higher than without graphene, reﬂecting the importance of graphene in future environmental and energy conversion and storage applications. More research on bismuth graphene composites is needed in this ﬁeld [241]. pp 4.3.1. NOx Conversion 4.3.1. NOx Conversion Bismuth compounds have also been employed in combination with graphene to produce useful photocatalytic composites for NOx removal under visible light irradiation [271]. Zhihui et al. [272] prepared BiOBr-graphene nano- composites for efficient removal of NO via visible-light photocatalytic activity. The im- proved photocatalytic activity of the BiOBr-graphene nanocomposite was ascribed to the efficient charge separation, and enhanced transfer is due to robust chemical bonding be- tween graphene and BiOBr. Also, the N2-doped (BiO)2CO3/GO nanocomposites, reported by Chen et al., [273], play a pivotal role in higher photocatalytic performance for NOx removal under visible light irradiation. The rGO improved the electron-hole separation for pure Bi2WO6 and fully degrading RhB [274]. Ma et al. described an improved compo- site performance of rGO/Bi2WO6 photocatalytic in phenol and RhB degradations [275]. The selective photocatalytic 4-NP reduction on blank nanocomposites BWO, rGO and BWO/rGO after 30 min of irradiation is shown in Figure 10. . Figure 10. Photocatalytic selective reduction of 4-NP to 4-AP over blank BWO, RGO, and BWO/rGO nanocomposites after irradiation for 30 min. Source: Adapted from [195]. Figure 10. Photocatalytic selective reduction of 4-NP to 4-AP over blank BWO, RGO, and BWO/rGO nanocomposites after irradiation for 30 min. Source: Adapted from [195]. Figure 10. Photocatalytic selective reduction of 4-NP to 4-AP over blank BWO, RGO, and BWO/rGO nanocomposites after irradiation for 30 min. Source: Adapted from [195]. Figure 10. Photocatalytic selective reduction of 4-NP to 4-AP over blank BWO, RGO, and BWO/rGO nanocomposites after irradiation for 30 min. Source: Adapted from [195]. pp 4.3.1. NOx Conversion 4.3.1. NOx Conversion TiO2 is one of the leading catalysts for the catalytic conversion of NOx gases into nitrous oxide (N2O) and N2 [252,253]. The majority of previous studies on the conversion of NOx gases have involved different lasers [254], spectroscopic (such as infrared (IR), [255], and chemiluminescence (CL) [256] or electrochemical techniques [257] for the detection of NOx reaction products. The use of high-resolution MS for biomedical applications to detect NO [230] and indirectly semiconducting metal oxides [231] has been described. The main cause of water pollution is industrial wastewater discharge. Drinking polluted water for a long time poses potential health risks, and can also cause cancer, teratogenicity and mutagenicity. For this reason, it is very important to develop suitable techniques for the treatment of industrial wastewater to meet emission standards. Photocatalysis is considered a sustainable and efﬁcient water treatment technology. Old- photocatalysts (such as ZnO and TiO2) with a wide bandgap are only active in the UV light region and their quick recombination of photo-generated holes and electrons leads to low quantum efﬁciencies that limit their application for wastewater treatment. The traditional inconveniences of these photocatalysts requires the development of new Bi-based semi- catalysts for real waste water treatment such as black BiOCl-Bi2O3/rGO nanocomposite with high photocatalytic efﬁciency [141]. with high photocatalytic efficiency [141]. With economic growth, pollution, primarily air pollution, is becoming a serious con- cern and must be treated instantaneously. NOx plays an important role in acid rain for- mation, diseases and photochemistry. Therefore, the elimination of NOx is a hot topic in the area of environmental protection [258–261]. The photocatalytic oxidation of NO to NO2 is a good way to remove NO from flue gas, as NO2 can be removed simply by reacting i h h d b l N [262] Th h l i NO NO id i i With economic growth, pollution, primarily air pollution, is becoming a serious concern and must be treated instantaneously. NOx plays an important role in acid rain formation, diseases and photochemistry. Therefore, the elimination of NOx is a hot topic in the area of environmental protection [258–261]. The photocatalytic oxidation of NO to NO2 is a good way to remove NO from ﬂue gas, as NO2 can be removed simply by reacting with hydrocarbons to release N2 or water [262]. pp 4.3.1. NOx Conversion 4.3.1. NOx Conversion The photocatalytic NO-NO2 oxidation Energies 2021, 14, 2281 18 of 36 is observed as an essential reaction, and a great deal of effort has been made to develop appropriate NO-removal photocatalysts [263,264]. Bi2WO6 has attracted considerable attention as an Aurivillius oxide semiconductor with a 2.66 eV narrow bandgap. Bi2WO6 forms with different morphology can be synthesized by various approaches, like a cetyl- trimethylammonium bromide-assisted bottom-up route, hydrothermal processes and solid- state reactions [265–267]. It was used in several applications, including the decomposition of pollutants [266,267]. However, the photocatalytic activity fades due to fast recombination of photogenerated carriers in Bi2WO6, and its more practical applications are restricted. Graphene has been shown to successfully improve photocatalysts’ photoactivities through further separation of the electron-holes generated and helping photoinduced electrons to migrate and preventing the recombination of electron-holes and increasing the efﬁciency of quantization [268–270]. Bismuth compounds have also been employed in combination with graphene to produce useful photocatalytic composites for NOx removal under visible light irradiation [271]. Zhihui et al. [272] prepared BiOBr-graphene nanocomposites for efﬁcient removal of NO via visible-light photocatalytic activity. The improved photocatalytic activity of the BiOBr-graphene nanocomposite was ascribed to the efﬁcient charge separation, and enhanced transfer is due to robust chemical bonding between graphene and BiOBr. Also, the N2-doped (BiO)2CO3/GO nanocomposites, reported by Chen et al., [273], play a pivotal role in higher photocatalytic performance for NOx removal under visible light irradiation. The rGO improved the electron-hole separation for pure Bi2WO6 and fully degrading RhB [274]. Ma et al. described an improved composite performance of rGO/Bi2WO6 photocatalytic in phenol and RhB degradations [275]. The selective photocatalytic 4-NP reduction on blank nanocomposites BWO, rGO and BWO/rGO after 30 min of irradiation is shown in Figure 10. IEW 20 of 38 forms with different morphology can be synthesized by various approaches, like a cetyl- trimethylammonium bromide-assisted bottom-up route, hydrothermal processes and solid-state reactions [265–267]. It was used in several applications, including the decom- position of pollutants [266,267]. However, the photocatalytic activity fades due to fast re- combination of photogenerated carriers in Bi2WO6, and its more practical applications are restricted. Graphene has been shown to successfully improve photocatalysts’ photoactiv- ities through further separation of the electron-holes generated and helping photoinduced electrons to migrate and preventing the recombination of electron-holes and increasing the efficiency of quantization [268–270]. 4.3.2. Organic Degradation 4.3.2. Organic Degradation g g A dramatic surge in research in the visible light photocatalysis area was observed at the start of the 21st century, as evidenced by a promptly increasing number of publica- tions. Using visible light in combination with catalysts is effective for producing selective and efficient chemical transformations. Nature remarkably reveals the power of photo- synthesis by transforming CO2 and H2O into oxygen and carbohydrates, a process that is so far unequaled by any man made chemical procedure [276] A dramatic surge in research in the visible light photocatalysis area was observed at the start of the 21st century, as evidenced by a promptly increasing number of publications. Using visible light in combination with catalysts is effective for producing selective and efﬁcient chemical transformations. Nature remarkably reveals the power of photosynthesis by transforming CO2 and H2O into oxygen and carbohydrates, a process that is so far unequaled by any man-made chemical procedure [276]. so far unequaled by any man-made chemical procedure [276]. The use in organic synthesis of solar energy as a motiving power is now beginning. Key solar energy components include UV (λ = 200–400 nm), visible light (λ = 400–800 nm), and infrared light (λ > 800 nm), accounting for almost 5%, 43%, and 52%, respectively. UV energy can directly trigger certain organic molecules to provide highly reactive interme- diates, resulting in poor product selectivity. Furthermore, for the vast majority of organic reactions the infrared wavelength with relatively low energy does not meet the energy The use in organic synthesis of solar energy as a motiving power is now beginning. Key solar energy components include UV (λ = 200–400 nm), visible light (λ = 400–800 nm), and infrared light (λ > 800 nm), accounting for almost 5%, 43%, and 52%, respectively. UV energy can directly trigger certain organic molecules to provide highly reactive intermedi- ates, resulting in poor product selectivity. Furthermore, for the vast majority of organic reactions, the infrared wavelength with relatively low energy does not meet the energy Energies 2021, 14, 2281 19 of 36 demand. In comparison, UV and visible light are abundant, but the reactant molecules can usually not directly adsorb them to drive reactions. Therefore, it will be important for visible photocatalysts to work as bridging media for energy transfer between the sub- strate and visible light. 4.3.2. Organic Degradation 4.3.2. Organic Degradation These photocatalysts may be assigned to ﬁve different groups: plasmonic-metal NPs, homogenous photocatalysts, opposite heterogeneous semiconductor photocatalysts, other new photoelectric materials, and organic dyes. Various semiconduc- tors show different widths and positions of the string so that there are different reduction and oxidation potential for the electrons and hole pairs created in situ. When the carriers (holes and electrons) travel to the catalyst surface, which lowers photo-catalytic efﬁciency, electron and hole pair recombination occurs frequently. Many approaches have been de- veloped to improve the separation efﬁciency of electron-hole pairs, such as supporting a photocatalyst on graphene with a big surface or using a valuable metal materials such as Pt so photogenerated charge transfer could be accelerated. In organic reactions, H2O is considered an ideal solvent. However, the problem is that, under photocatalytic conditions, the semiconductor VB hole can oxidize H2O into a highly active OH radical form, making the reaction system complicated. Bi2WO6 photocat- alyst VB’s inherent reduction potential is +1.77 V vs. Ag/AgCl, which is negative to the H2O/ANOH. H2O as a solvent is possible when Bi2WO6 is used as a catalyst. Recently, a selective oxidation of benzyl alcohols into aldehydes has been effectively developed with a Bi2WO6/H2O/air system [277]. Although the different synthetic applications of visible light photocatalysis are awe-inspiring, there is still scope for improvement. In several instances, the reaction times for many conversions are fairly long. In order to make photocatalytic changes faster and more energy-efﬁcient, the quantum efﬁciency must be extremely enhanced. A better mechanistic consideration could beneﬁt the rational design of new transformations and the expectation of the substrate scope. The reachable potential should be stretched for the exchange of chemically reduced single-electron or stoichiometric oxidizing reagents by photocatalytic reactions. There is no examination of the various photocatalytic energies of transformations, and chemists have just begun to produce organic conversions that are promising with additional light energies. Finally, we must ﬁnd out how this can be extended to ions and carbenes and how the common visible light’s common photocatalytic reactions continue through the radical intermediates. There are plenty of opportunities for future development in photocatalysis. We should have followed Ciamician’s initial ideas for sustainable and innovative organic syntheses using visible light much earlier [278]. g g In comparison with applications such as organic contaminant degradation, heteroge- neous semi-conducting photocatalysis addresses more complex problems. 4.3.2. Organic Degradation 4.3.2. Organic Degradation In particular, rationally designed nanophotocatalyst nanomaterials have tremen- dous potential here to produce robust and adequate reactive species using solar light (the most plentiful, and accessible renewable energy source on Earth). The bactericidal activity of photocatalysts sextends to all reactive species formed during the photocatalytic process. In addition, visible light corresponds to the strongest solar irradiance range. A photocata- lyst, which can efficiently absorb the visible light to produce reactive species, is a condi- tion for achieving fast photocatalytic disinfection [283]. Jamshaid et al. [284] synthesized a BiOCl/GO composite and utilized it under visible light, full solar light, and UV photo- catalytic degradation of diclofenac sodium (DCF) (Figure 12). As an efﬁcient, non-toxic, and stable method, photocatalytic disinfection was shown to be superior to traditional methods for water disinfection, including UV irradiation, ozonation and chlorination, since they form carcinogenic disinfection by-products, and are causes of global warming due to the formation of chemical-intensive or energy-intensive products. Highly successful and innovative wastewater disinfection approaches need to be implemented and maintained, that are less dependent on fossil fuels and chemicals [282]. In particular, rationally designed nanophotocatalyst nanomaterials have tremendous po- tential here to produce robust and adequate reactive species using solar light (the most plentiful, and accessible renewable energy source on Earth). The bactericidal activity of photocatalysts sextends to all reactive species formed during the photocatalytic process. In addition, visible light corresponds to the strongest solar irradiance range. A photo- catalyst, which can efﬁciently absorb the visible light to produce reactive species, is a condition for achieving fast photocatalytic disinfection [283]. Jamshaid et al. [284] synthe- sized a BiOCl/GO composite and utilized it under visible light, full solar light, and UV photocatalytic degradation of diclofenac sodium (DCF) (Figure 12). catalytic degradation of diclofenac sodium (DCF) (Figure 12). As a photocatalyst, BiVO4/rGO nanocomposite exhibits efficient catalytic activity to- wards organic dye degradation [285,286]. The photodegradation results showed that the BiVO4-rGO nanocomposite catalyst could effectively degrade organic dyes in a variety of wastewaters. Similarly, a one-step hydrothermally synthesized Bi-TiO2/graphene nano- composite is considered an efficient photocatalyst for remarkable organic pollutant deg- radation under visible light irradiation [287]. The Z-scheme photocatalyst systems pro- vided a promising approach of simultaneously removing heavy metals and organic pol- lutants. Acong et al. [288] reported an all-solid-state Z-scheme system containing BiOI/Bi2S3/rGO composites for simultaneous removal of aqueous Cr(VI) and phenol [288]. 4.3.2. Organic Degradation 4.3.2. Organic Degradation The photoin- duced charging transfers resulting from semiconductor interfaces with holes or electrons used as reducers and oxidizers, respectively, are the basis of all types of photocatalytic applications. In photocatalytic selective organic synthesis, the critical problem is how to reg- ulate the method of interfacial charge transfer to ensure only the selective transformation of speciﬁc functional groups in organic substrata while the remaining molecular struc- ture remains intact [279]. Because the VB holes photogenerated as a stable photocatalyst (e.g., WO3, TiO2, and ZnO) have strong oxidation power, VB holes tend to oxidize non- selectively and degrade whole molecules, respectively. For RhB degradation, BiOCl/rGO is considered an effective photocatalyst [280]. The mechanism is schematically shown in Figure 11. At present, there are several technical difﬁculties and knowledge gaps in the organic synthesis research ﬁeld. The photocatalytic method is heterogeneous. It is expected that individual photocatalysts will offer enhanced selectiveness for selective reactions, sim- ilar to organic degradation processes. It is estimated that for individual organic synthesis reaction cases, each photocatalyst must be optimized as selectivity control depends on the molecular structure and the particular organic substrate characteristics as well as on the photocatalyst [281]. 20 of 36 22 of 38 20 of 36 22 of 38 Energies 2021, 14, 2281 Energies 2021, 14, x FO Figure 11. Illustration of the RhB degradation mechanism via BiOCl/rGO photocatalysts by (A).O2− radicals and (B) single oxygen under the white LED irradiation. Adapted from [280]. Figure 11. Illustration of the RhB degradation mechanism via BiOCl/rGO photocatalysts by (A) .O2−radicals and (B) single oxygen under the white LED irradiation. Adapted from [280]. Figure 11. Illustration of the RhB degradation mechanism via BiOCl/rGO photocatalysts by (A).O2− radicals and (B) single oxygen under the white LED irradiation. Adapted from [280]. Figure 11. Illustration of the RhB degradation mechanism via BiOCl/rGO photocatalysts by (A) .O2−radicals and (B) single oxygen under the white LED irradiation. Adapted from [280]. As an efficient, non-toxic, and stable method, photocatalytic disinfection was shown to be superior to traditional methods for water disinfection, including UV irradiation, ozo- nation and chlorination, since they form carcinogenic disinfection by-products, and are causes of global warming due to the formation of chemical-intensive or energy-intensive products. Highly successful and innovative wastewater disinfection approaches need to be implemented and maintained, that are less dependent on fossil fuels and chemicals [282]. 4.3.2. Organic Degradation 4.3.2. Organic Degradation A series of bismuth-graphene nanocomposite systems were summarized by Yu-Hsun et al. [289] for adequate catalytic activity and stability, acting as visible-light-driven photo- catalysts in efficient organic pollutant degradation p y g g As a photocatalyst, BiVO4/rGO nanocomposite exhibits efﬁcient catalytic activity towards organic dye degradation [285,286]. The photodegradation results showed that the BiVO4-rGO nanocomposite catalyst could effectively degrade organic dyes in a vari- ety of wastewaters. Similarly, a one-step hydrothermally synthesized Bi-TiO2/graphene nanocomposite is considered an efﬁcient photocatalyst for remarkable organic pollutant degradation under visible light irradiation [287]. The Z-scheme photocatalyst systems provided a promising approach of simultaneously removing heavy metals and organic pollutants. Acong et al. [288] reported an all-solid-state Z-scheme system containing BiOI/Bi2S3/rGO composites for simultaneous removal of aqueous Cr(VI) and phenol [288]. A series of bismuth-graphene nanocomposite systems were summarized by Yu-Hsun et al. [289] for adequate catalytic activity and stability, acting as visible-light-driven photo- catalysts in efﬁcient organic pollutant degradation. 21 of 36 23 of 38 23 of 38 Energies 2021, 14, 2281 Energies 2021, 14, x FOR Energies 2021, 14, x FOR . Figure 12. (a) Schematic representation for DCF photocatalytic degradation mechanism onto Bi- OCl/GO composite, (b) Effect of different scavengers on DCF degradation, and (c) plot for regen- eration of spent BiOCl/GO composite. Copied from [284]. Figure 12. (a) Schematic representation for DCF photocatalytic degradation mechanism onto BiOCl/GO composite, (b) Effect of different scavengers on DCF degradation, and (c) plot for regener- ation of spent BiOCl/GO composite. Copied from [284]. . Figure 12. (a) Schematic representation for DCF photocatalytic degradation mechanism onto Bi- OCl/GO composite, (b) Effect of different scavengers on DCF degradation, and (c) plot for regen- eration of spent BiOCl/GO composite. Copied from [284]. . . Figure 12. (a) Schematic representation for DCF photocatalytic degradation mechanism onto Bi- OCl/GO composite, (b) Effect of different scavengers on DCF degradation, and (c) plot for regen- eration of spent BiOCl/GO composite Copied from [284] Figure 12. (a) Schematic representation for DCF photocatalytic degradation mechanism onto BiOCl/GO composite, (b) Effect of different scavengers on DCF degradation, and (c) plot for regener- ation of spent BiOCl/GO composite. Copied from [284]. Figure 12. (a) Schematic representation for DCF photocatalytic degradation mechanism onto Bi- OCl/GO composite, (b) Effect of different scavengers on DCF degradation, and (c) plot for regen- eration of spent BiOCl/GO composite. Copied from [284]. 4 3 3 G S i 4.3.3. Gas Sensing 4.3.3. Gas Sensing 4.3.3. Gas Sensing The exploitation and design of photoelectrochemical (PEC) sensors with innovative nanomaterials are of great significance to attain the goal of inexpensive and sensitive de- tection. Therefore, BiPO4/rGO nanocomposite, a novel PEC sensor platform, can offer a delicate approach in chlorpyrifos detection and the resulting BiPO4/rGO nanocomposite ll l f h E l d l The exploitation and design of photoelectrochemical (PEC) sensors with innovative nanomaterials are of great signiﬁcance to attain the goal of inexpensive and sensitive detection. Therefore, BiPO4/rGO nanocomposite, a novel PEC sensor platform, can offer a delicate approach in chlorpyrifos detection and the resulting BiPO4/rGO nanocomposite is a potentially active catalyst for the PEC-related applications (Figure 13) [182]. The exploitation and design of photoelectrochemical (PEC) sensors with innovative nanomaterials are of great significance to attain the goal of inexpensive and sensitive de- tection. Therefore, BiPO4/rGO nanocomposite, a novel PEC sensor platform, can offer a delicate approach in chlorpyrifos detection and the resulting BiPO4/rGO nanocomposite is a potentially active catalyst for the PEC-related applications (Figure 13) [182]. Figure 13. The PEC sensor illustration for chlorpyrifos. Adapted from ref [182]. Figure 13. The PEC sensor illustration for chlorpyrifos. Adapted from ref [182]. Figure 13. The PEC sensor illustration for chlorpyrifos. Adapted from ref [182]. Figure 13. The PEC sensor illustration for chlorpyrifos. Adapted from ref [182]. Figure 13. The PEC sensor illustration for chlorpyrifos. Adapted from ref [182]. Figure 13. The PEC sensor illustration for chlorpyrifos. Adapted from ref [182]. Figure 13. The PEC sensor illustration for chlorpyrifos. Adapted from ref [182]. Figure 13. The PEC sensor illustration for chlorpyrifos. Adapted from ref [182]. Figure 13. The PEC sensor illustration for chlorpyrifos. Adapted from ref [182]. Energies 2021, 14, 2281 Energies 2021, 14, x FOR 22 of 36 24 of 38 22 of 36 24 of 38 In the modern nanotechnology ﬁeld, considerable attention has been given to an architecture-controlled combination of nanomaterials because of their astonishing chemical and physical properties and promising applications in different ﬁelds, e.g., optics, electron- ics, catalysis, and so on [290,291]. Similarly, using innovative conﬁgurations with implanted graphene for a broad surface, long electron life can be supported by other photonic de- vices such as solar cells and non-photonic devices, like lithium batteries and biochemical sensors. Low band-gap energy, reduced recombination rate, and fast charge transit e.g., spiral rolls-implanted graphene in the TiO2/ZnO/Bi2O3 (TZB) nanoﬁber [175,292]. 4 3 3 G S i 4.3.3. Gas Sensing 4.3.3. Gas Sensing The BiPO4/3DNGH and BiVO4/rGO provide a new platform for speciﬁc biomedical, food, and environmental detection applications [181,293,294]. TEA and H2S are highly toxic gases that can pollute the atmosphere and damage the human respiratory system. Consequently, it is important to be able to easily detect low levels of TEA and H2S in our everyday lives. Shouli et al. [209] developed a pine dendritic BiVO4/rGO hybrid heterojunction, which improves not only BiVO4 response and speeds up the response time but also has good selectivity and stability to 10 ppm TEA at 180 ◦C operating temperature. The formation of heterojunction and the integration of rGO are responsible for the change. Ketkaeo et.al. [295] investigated Bi2WO6 nanoparticles loaded with rGO nanosheets for H2S gas sensing applications. The developed sensor exhibited high H2S selectivity against numer- ous volatile organic compounds and some other environmental gases. The H2S sensing mechanism via Bi2WO6/rGO composite is illustrated in Figure 14. In the modern nanotechnology field, considerable attention has been given to an ar- chitecture-controlled combination of nanomaterials because of their astonishing chemical and physical properties and promising applications in different fields, e.g., optics, elec- tronics, catalysis, and so on [290,291]. Similarly, using innovative configurations with im- planted graphene for a broad surface, long electron life can be supported by other pho- tonic devices such as solar cells and non-photonic devices, like lithium batteries and bio- chemical sensors. Low band-gap energy, reduced recombination rate, and fast charge transit e.g., spiral rolls-implanted graphene in the TiO2/ZnO/Bi2O3 (TZB) nanofiber [175,292]. The BiPO4/3DNGH and BiVO4/rGO provide a new platform for specific biomed- ical, food, and environmental detection applications [181,293,294]. TEA and H2S are highly toxic gases that can pollute the atmosphere and damage the human respiratory system. Consequently, it is important to be able to easily detect low levels of TEA and H2S in our everyday lives. Shouli et al. [209] developed a pine dendritic BiVO4/rGO hybrid heterojunction, which improves not only BiVO4 response and speeds up the response time but also has good selectivity and stability to 10 ppm TEA at 180 °C operating temperature. The formation of heterojunction and the integration of rGO are responsible for the change. Ketkaeo et.al. [295] investigated Bi2WO6 nanoparticles loaded with rGO nanosheets for H2S gas sensing applications. The developed sensor exhibited high H2S selectivity against numerous volatile organic compounds and some other environmental gases. 5. Drawbacks/Challenges Related to Bismuth and Graphene Although, there has been diversiﬁed study on bismuth and graphene nanohybrids for large-scale applications of such photocatalysts, there remains several drawbacks/challenges such as the site of attachment of dopant, the overall efﬁcient doping mechanism, assess- ment of integration, photocatalyst degradation, and visible light absorption that remain to be unraveled. The improved Hummers process has been commonly used to synthesize graphene, which is the most recent and best method. However, despite the low experimental complex- ity, the experimental procedures to complete the graphene fabrication are time-consuming. As a result, the substitution or elimination of such chemicals must be studied further to reduce fabrication times and produce a better fabrication processes. Furthermore, the amount of chemicals used in the fabrication process or replacing them with less expen- sive alternatives could make the whole process more cost-effective and applicable to real-world applications. Challenges also remain in the exploration of graphene-based nanohybrids for high performance practical applications. High-quality graphene nanohybrids with tailored functionalization, tunable structures, and optimized properties need to be fabricated in a more simple, effective, and economical approach. In graphene functionalization, attention must be paid to the control distribution, amount, and afﬁnity to graphene nanosheets and the dispersibility and functionality of nanohybrids. p y y y Graphene sheets tend to form aggregates in solution due to hydrogen bonding or strong van der Waals force interactions in polar solvents. Chemical functionaliza- tion [291,296] and electrostatic stabilization [297] are used to avoid this aggregation. Graphene reduction using simple methods facilitates graphene applications to synthe- size composite materials in cost-effective, scalable approaches with low cost of produc- tion [167,298]. GOs may be synthesized using the Hummers and Offeman method and then by sonication exfoliated using strong graphite chemical oxidation. Most studies have concentrated on Bi3+-containing compounds, like Bi2O3 BiOX (X = Cl, Br, I), BiPO4, BiVO4, BiFeO3Bi4Ti3O12, Bi2WO6, Bi2O2CO3, Bi12TiO20, Bi0.5K0.5TiO3, and Bi3TiNbO9. Among them, a majority of the compounds possess a plate-like appearance and layered structures. Visible light can excite Bi5+-containing compounds, e.g., KBiO3, LiBiO3, and NaBiO3. Hy- bridized O 2p and Bi 6s2 orbitals can inﬂuence the valence bands in Bi(III) compounds (VBs). Therefore, the Bi compounds’ band gap is usually less than 3.0 eV and can easily be excited by visible light. 4 3 3 G S i 4.3.3. Gas Sensing 4.3.3. Gas Sensing The H2S sensing mechanism via Bi2WO6/rGO composite is illustrated in Figure 14. . Figure 14. H2S sensing approaches via (a) Bi2WO6 nanoparticles, (b) moderate loaded rGO over Bi2WO6 nanoparticles, and (c) high loaded rGO over Bi2WO6 nanoparticles. Adapted from [295]. Figure 14. H2S sensing approaches via (a) Bi2WO6 nanoparticles, (b) moderate loaded rGO over Bi2WO6 nanoparticles, and (c) high loaded rGO over Bi2WO6 nanoparticles. Adapted from [295]. . Figure 14. H2S sensing approaches via (a) Bi2WO6 nanoparticles, (b) moderate loaded rGO over Bi2WO6 nanoparticles, and (c) high loaded rGO over Bi2WO6 nanoparticles. Adapted from [295]. Figure 14. H2S sensing approaches via (a) Bi2WO6 nanoparticles, (b) moderate loaded rGO over Bi2WO6 nanoparticles, and (c) high loaded rGO over Bi2WO6 nanoparticles. Adapted from [295]. Figure 14. H2S sensing approaches via (a) Bi2WO6 nanoparticles, (b) moderate loaded rGO over Bi2WO6 nanoparticles, and (c) high loaded rGO over Bi2WO6 nanoparticles. Adapted from [295]. Figure 14. H2S sensing approaches via (a) Bi2WO6 nanoparticles, (b) moderate loaded rGO over Bi2WO6 nanoparticles, and (c) high loaded rGO over Bi2WO6 nanoparticles. Adapted from [295]. Energies 2021, 14, 2281 23 of 36 23 of 36 6. Summary and Outlook Future developments would be part of the present start of this new century. Bi/graphene- based semiconductors’ fascinating physiochemical features have attracted researchers’ atten- tion and signiﬁcantly motivated research, especially on visible-light photocatalytic activities. This review has discussed the most frequently studied bismuth/graphene photocatalysts. In addition, key challenges, including the broad bandwidth, high photogeneration carrier recombination rates, and low-capacity reduction in the conduction band, are outlined. The work reported has supported recommending achievable approaches to overcome these challenges. Though photocatalysts based on bismuth/graphene can considerably lessen the inconvenience, further efforts are still necessary to achieve signiﬁcant advancements. To date, these prepared bismuth/graphene materials’ major applications are to purify polluted air and destroy pollutants in wastewater. Applying the formation of Z-scheme structures or modifying energy bands, improves the photocatalytic H2 production. The work on these advanced nanocomposites should extend to other major areas, such as photocatalytic improvements, photocatalytic organic synthesis, and the recovery of heavy metals. The practical uses of photocatalysts using bismuth/graphene are seldom described. Integrating the application in different directions and many other areas with other suitable techniques, such as biotechnology, membrane technology, and electrochemistry, can lead to rapid advancements. Although many nanocomposites with bismuth/graphene have been reported to be active using visible and high photocatalytic light, the use of these advanced materials is still in the early stages of commercialization. Photocatalysts can selectively degrade pollutants. g p Nanomaterial photocatalysis, especially nanophotocatalysts, exhibits huge potential because solar light can produce powerful and abundant reactive species. The visible light range is intended to achieve maximum photocatalytic decontamination and fast output rates. A photocatalyst capable of efﬁciently absorbing visible light is a prerequisite for pro- ducing reactive species. Two well-investigated visible-light-driven photocatalysts among various semiconductors are bismuth (Bi) and graphene. Due to their chemical stability, bulk availability, they have great potential for water disinfection applications and environ- mental friendliness. A bismuth/graphene hybrid effectively suppressed e−and h+ pair recombination, promoted the interfacial electron transfer, and enhanced the photocatalytic process of reactive species generation. While this review is incomplete in the context of photocatalytic pollutant breakdown of bismuth/graphene nanocomposites, important aspects have been addressed concerning fundamental applications and principles. Author Contributions: Conceptualization, M.U., M.H., A.K. and H.U. (Habib Ullah 4); validation, M.H., A.K., A.AT., S.S.S., H.U. (Habib Ullah 3), M.U., S.S.S. and H.U. (Habib Ullah 4); writing— original draft preparation, M.U.; writing—review and editing, M.H., A.K. and H.U. 5. Drawbacks/Challenges Related to Bismuth and Graphene However, the photocatalytic performance of bulk Bi-based semiconductors is not as high as the performance of photocatalysts from nano Bi-sources, like photogenerated holes and electrons have not been used and used efﬁciently. The photocatalysts in bulk are smaller in area and have less light absorption than photocatalysts in the nanoscale range. A variety of attempts to improve bulk semiconductors have been made to achieve the ideal photocatalytic activity. In addition, changes in components, e.g., doping, alteration of stoichiometry, and preparation of solid solutions, are current methods used to change the Bi-based semiconductor band structures. Therefore for Bi photocatalysts, a suitable component change is promising [25]. It has been studied that the Bi6 s-orbital decreases the bandgap while increasing photogenerated charge carriers’ mobility [299]. While a majority of the Bi-based compounds have about a 3.0 eV bandgap. Bi-based compounds, including Bi2O3, Bi2MO6 (M = W, Mo, and Cr), BiVO4, BiOX (X = I, Br and Cl), BiPO4, pentavalent bismuthate and (BiO)2CO3, were tested as a large number of photocatalytic compounds. In environmental protection applications, Bi-based semicon- ductors have been used for the oxidation of gaseous pollutants, such as NO [105], organic dye degradation in wastewater [300], and CO2 photoreduction [94]. During various studies, photocatalytic water division for generating O2 and H2 was reported [301]. A fﬁi id d h f i i b h l ’ An efﬁcient strategy considered a new approach for improving bare photocatalysts’ catalytic performance is by combining a new Z-scheme structure with the appropriate band position. The Z-scheme design can retain a high redox capacity to forgive both semiconductors, except for e-h pairs’ recombination. Thanks to the band structure’s Energies 2021, 14, 2281 24 of 36 adaption, environmental ease, and low cost, graphene was reported as another component by modifying Bi to change a Z-scheme system and doping [302]. adaption, environmental ease, and low cost, graphene was reported as another component by modifying Bi to change a Z-scheme system and doping [302]. Conﬂicts of Interest: The authors declare no conﬂict of interest. Acknowledgments: We are thankful to the Engineering and Physical Science Research Council, UK (EPSRC under the research grant no. EP/V049046/1 and EP/T025875/, for ﬁnancial support. M. U also acknowledges the support from Saudi Aramco Chair Programme (ORCP2390). References Sci. 2014, 26, 59–65. [CrossRef] 8. Hu, G.; Li, J.; Zeng, G. Recent development in the treatment of oily sludge from petroleum industry: A review. J. Hazard. Mater. 2013, 261, 470–490. [CrossRef] 7. Ali, S.M.; Pervaiz, A.; Afzal, B.; Hamid, N.; Yasmin, A. 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https://openalex.org/W4200147273	https://avianres.biomedcentral.com/track/pdf/10.1186/s40657-021-00309-x	English	null	Organising a juvenile ratio monitoring programme for 10 key waterbird species in the Yangtze River floodplain: analysis and proposals	Avian research	2,021	cc-by	11,661	Abstract Background: In the face of continued degradation and loss of wetlands in the Yangtze River floodplain (YRF), there is an urgent need to monitor the abundance and distribution of wintering waterbirds. To understand fully observed annual changes, we need to monitor demographic rates to understand factors affecting global population size. Annual reproduction success contributes to dynamic changes in population size and age structure, so an assessment of the juvenile ratio (i.e. first winter birds as a proportion of total number aged) of overwintering waterbirds can be an important indicator of the reproductive success in the preceding breeding season. Methods: During 2016–2019, we sampled juvenile ratios among 10 key waterbird species from the wetlands in the YRF. Based on these data, we here attempt to establish a simple, efficient, focused and reliable juvenile ratio monitor- ing scheme, to assess consistently and accurately relative annual breeding success and its contribution to the age structure among these waterbird species. Results: We compared juvenile ratio data collected throughout the winter and found that the optimal time for undertaking these samples was in the early stages of arrival for migratory waterbirds reaching their wintering area (early to mid-December). We recommend counting consistently at key points (i.e. those where > 1% biogeographi- cal flyway population were counted) at sites of major flyway importance (Poyang Lake, East Dongting Lake, Shengjin Lake, Caizi Lake, Longgan Lake and Chen Lake). Based on this, the error rate of the programme (155 planned points, the count of 10 waterbird species is 826–8955) is less than 5%. Conclusions: We established a juvenile ratio monitoring programme for 10 key waterbird species in the wetlands of the YRF, and discuss the feasibility and necessity of implementing such a future programme, and how to use these data in our monitoring and understanding of the population dynamics of these waterbird populations. Keywords: Juvenile ratio, Monitoring scheme, The Yangtze River floodplain, Statistical independence Organising a juvenile ratio monitoring programme for 10 key waterbird species in the Yangtze River floodplain: analysis and proposals Yuxi Wang1, Iderbat Damba2,3,4, Qingshan Zhao2, Yanbo Xie5, Xueqing Deng2,3, Rdi Ga6, Guanhua Liu7, Zhiwen Xu7, Yue Li8, Dali Gao9, Wenbin Xu10, Guoxun Chen11 and Lei Cao2,3* Wang et al. Avian Research (2021) 12:72 https://doi.org/10.1186/s40657-021-00309-x Wang et al. Avian Research (2021) 12:72 https://doi.org/10.1186/s40657-021-00309-x Avian Research Avian Research Open Access Backgroundh The Yangtze River stretches for 1893 km, and drains an area of about 800,000 km2 (Ma 2014). The monsoon pre- cipitation is concentrated mainly in summer, so water levels are highest in late summer but fall through winter (Nakayama and Shankman 2013). These periodic water Correspondence: leicao@rcees.ac.cn Correspondence: leicao@rcees.ac.cn 2 State Key Laboratory of Urban and Regional Ecology, Research Center for Eco Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China Full list of author information is available at the end of the article © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 13 Wang et al. Avian Research (2021) 12:72 Wang et al. Avian Research (2021) 12:72 Wang et al. Avian Research (2021) 12:72 no overlap in migration routes, staging areas and Arc- tic breeding grounds between the Bewick’s Swans (Cyg- nus columbianus bewickii) that use Japan to winter and those individuals wintering in China (Fang et al. 2020). Similarly, Greater White-fronted Geese (Anser albifrons) wintering in Japan and Korea almost never overlap with individuals wintering in China during migration and on their Arctic breeding areas (Deng et al. 2020). This gives us some confidence in regarding these wintering groups as discrete entities (“biogeographical populations”) in the sense that inter-annual differences in abundance are most affected by the balance between new recruits (first year birds) and the death of birds between successive winters, because their discreteness implies minimal immigration and emigration to and from these biogeographical popu- lations. Backgroundh Insight into factors affecting the size of such pop- ulations requires an understanding of whether the causes of population decline is a reduction in annual reproduc- tive success (Bromley and Rothe 2003) or annual survival (or a combination of both). Most large-bodied waterbirds are relatively long-lived and therefore sensitive to even small changes despite their relatively high annual survival rate (Johnson et al. 1992). Hence, an understanding of annual reproductive success can provide an insight into the causes for between-year fluctuations in winter abun- dance (for example, predator abundance cycles on Arctic breeding grounds; Nolet et al. 2013). level changes have created many seasonal wetlands, especially among the vast natural wetlands in the YRF, attracting 1,000,000 waterbirds to overwinter here from breeding areas further north (Barter et al. 2004, 2006). However, increasing water abstraction, pollution, aqua- culture, overfishing, river traffic and other anthropo- genic phenomena have caused habitat degradation and loss throughout the YRF, resulting in loss of biodiver- sity (Chen et al. 2017). As upper trophic level consum- ers, waterbirds are good indicators of many aspects of the ecosystems they live in. Hence, we need to monitor the distribution and abundance of waterbirds in the YRF, and also to assess the quality of their environment. To do this effectively, we need to understand their population dynamics that are driving their overall annual changes in abundance, which requires an understanding of their breeding success (annual gains to the overall popula- tion), balanced against mortality (as a measure of annual losses). Waterbird abundance at a given wintering site may decrease because there has been a drop in the overall total population size since last winter, or because they have moved to better foraging site elsewhere (a fact which may indicate local habitat loss/degradation). It is therefore essential to obtain comprehensive simultane- ous counts of waterbirds from throughout the flyway winter quarters to determine their abundance and dis- tribution. In recent years, coordinated counts of winter- ing waterbirds throughout the YRF have brought China into line with a longer tradition of counts from Korea and Japan, which share several waterbird species in the Far East Asian flyway (Rees and Fox 2020). Backgroundh The analysis of these counts is the subject of a series of publications published as a special issue of the journal Wildfowl (Rees and Fox 2020), and their annual compilation enables us to determine changes in numbers at any given site in the context of changes elsewhere in the flyway. Plumage characteristics of most waterbirds enable us to distinguish between birds hatched in the preceding sum- mer and those that are older (Baldassarre 2014). This pro- vides an opportunity to distinguish, at very least, between first calendar year birds and older individuals in the autumn after reproduction in the northern hemisphere (Howes et al. 2019). As a result, there is a long tradition of assessing the relative reproductive success of popula- tions by determining the juvenile ratios of waterbirds on their winter quarters. Such determinations are unable to determine the true reproductive output of a population, since many birds will have died or been lost from the population on the breeding areas or during migration. However, such juvenile ratios provide an assessment of the proportions of young that have returned to the winter quarters following the summer on the breeding quarters. l While shifts in wintering distribution can give us insights into the effects of winter site quality on local abundance, population declines (which are likely mani- fest at all sites equally) requires explanation at the so- called “flyway” level. The concept of a waterbird flyway is a concept based on observed biological phenomena. Capture-mark-recapture studies throughout the world show waterbirds generally are highly site faithful to their breeding, staging and wintering sites (Schock et al. 2018). Recently, international collaboration has achieved extensive tagging of waterbirds throughout Far East Asia to follow their annual migrations (Rees and Fox 2020). This detailed information on movements of individuals throughout their annual cycle has enabled us to obtain a greater understanding of flyway structure. In this way, for example, it has been possible to show that there is Most North American goose populations are moni- tored annually (U.S. Fish and Wildlife Service 2019). This is achieved within population-specific management plans for most identifiable goose populations, relying on monitoring data to support their conservation (Hestbeck et al. 1990; Cooch et al. 2001; Bromley and Rothe 2003). Hence, knowledge of juvenile ratios contributes greatly to our understanding of the causes of change in waterbird abundance (Johnson et al. 2018). Backgroundh Our knowledge of Far East Asian waterbird populations is very different from Wang et al. Avian Research (2021) 12:72 Page 3 of 13 Wang et al. Avian Research (2021) 12:72 those in North America and Europe, but thanks to inter- national cooperation throughout the flyway, this situation is improving rapidly. As outlined above, we are develop- ing networks in China to annually census the numbers of overwintering species in the YRF, which are beginning to accumulate data over more than a decade. Our next challenge is to develop demographic monitoring systems to help us better understand trends in distribution and abundance, and central to this, we consider, is the compi- lation of juvenile ratio data from overwintering waterbird species in the YRF, which has rarely been reported to the present. about how to best design our sampling programme for deployment throughout the Yangtze. This is especially important when considering the goals of the programme in relation to our understanding of the drivers of the population dynamics of each species and its age structure (Prevett and MacInnes 1980; Cowardin and Blohm 1992; Spaans et al. 1993; Alisauskas and Lindberg 2002). g Advances in our knowledge from the recent telemetry and count monitoring programmes have enabled us to define the flyway populations of waterbirds wintering in the Yangtze and assess their annual wintering distribu- tion and abundance. On this basis, we considered it an essential next step to investigate the possibilities and potential problems associated with developing a sam- pling programme to monitor the reproductive output of a series of 10 key species for which we have accumu- lated wintering distribution and abundance. We selected Bewick’s Swan, Swan Goose (Anser cygnoides), Lesser White-fronted Goose (Anser erythropus), Greater White- fronted Goose, Greylag Goose (Anser anser), Bean Goose (Anser fabalis), Siberian Crane (Grus leucogeranus), Hooded Crane (Grus monachal), White-naped Crane (Grus vipio) and Common Crane (Grus grus). As a base- line, and to aid our programme design, we gathered juve- nile ratio data on all these species throughout the YRF from 2016 to 2019, using the methods of Barter et al. (2004) to distinguish the species and to ensure a balanced consistent data-gathering regime in the field. In this paper, we analyse juvenile ratio data from these 4 years to attempt to understand the distribution and age structure of the 10 waterbird species wintering in these wetlands. Backgroundh Our aim here is to establish a simple, efficient and reli- able juvenile ratio monitoring scheme for the future. The development of such a long-term monitoring scheme will enable a deeper understanding of the population dynam- ics of these species and provide the future scientific basis for the more effective management of these species. p Before we embark on considerations about how to design a sampling programme for juvenile ratio assess- ment for deployment throughout the Yangtze, it is important to consider the experiences of others, which show that juvenile ratio samples vary in time and space (e.g. Ward et al. 2018). Telemetry has shown that non- breeding or failed breeding geese moult and initiate autumn migration in Greater Snow Geese (Anser caer- ulescens atlantica) earlier than those successful breeders with broods (Reed et al. 2003), so samples undertaken too early (for example very in early winter, before the arrival of families) will underestimate juvenile ratios. Similar patterns have been confirmed for midcontinent Greater White-fronted Geese (Anser albifrons frontalis) at a northern autumn staging area (Schock et al. 2018). Waterbirds breeding at high latitudes also tend to arrive at wintering areas later than those that bred at low lati- tudes (Ely and Takekawa 1996; Ely et al. 2013). Again, early sampling may underestimate to contribution to overall population juvenile ratios of these elements of the population. Regular within-season changes in juvenile ratios are therefore something to be aware of (Lambeck 1990a), but for many goose species, moult also affects our ability to accurately assign individuals to age class. For instance, many grey geese of the genus Anser moult feathers in autumn and winter, which make juveniles more difficult to separate from adults with certainty from November onwards. The feeding profitability of habi- tats may also affect juvenile ratios. Large family groups among goose flocks are behaviourally dominant over small families, pairs and individuals (Boyd 1953) and so large families tend to feed in optimal foraging situations, which may segregate families and non-breeding birds between different habitats (Lambeck 1990b). This also explains the difference between the underrepresentation of first-winter Brent Geese (Branta bernicla bernicla) in samples taken from the leading edge of dense flocks and the underrepresentation of adults in the centres (Lambeck 1990a). Hence, it is important to consider an optimal timing and design sampling procedures. These considerations must be taken into account when thinking Timing of migration In order to minimise between-observer bias, all age determination work was led by one observer (Iderbat Damba) with considerable experience of waterbird plum- ages and age characteristics, supported by other observ- ers with less experience. Field team compositions are listed in Additional file 1: Table S1. During each field survey, experienced observers used telescopes to detect individuals of all 10 species from a fixed viewing location and the total number of each species and the number of juveniles were counted (Gregory et al. 2004). We used results from our ongoing telemetry tracking studies of the different species to judge the timing of migration to establish the duration of time when these 10 species are present in the YRF. This was important to judge when all of the birds have returned to their ulti- mate wintering grounds after completion of autumn migration. Full details of the duration of 8 species (Great White-fronted Goose, Swan Goose, Greylag Goose, Bean Goose, Lesser White-fronted Goose, Bewick’s Swan, White-naped Crane, Siberian Crane) on the wintering grounds are shown in Additional file 1: Table S3 (Bat- bayar et al. 2013; Huang et al. 2018; Deng et al. 2019; Li et al. 2019, 2020a, b; Ao et al. 2020; Yang et al. 2020; Yi Data collection Juvenile ratio data were sampled from 10 waterbird species (Swan Goose, Lesser White-fronted Goose, Siberian Crane, Hooded Crane, White-naped Crane, Greater White-fronted Goose, Greylag Goose, Bean Goose, Bewick’s Swan and Common Crane) from 2016 to 2019. Sampling points are identified as key points (i.e. points supporting more than 1% of the flyway populations of one or more of the 10 species, N = 258) and other, non-key points (N = 243) Goose, Siberian Crane, Hooded Crane and White-naped Crane) and 5 non-endangered species (Greater White- fronted Goose, Greylag Goose, Bean Goose, Bewick’s Swan and Common Crane). Their wintering numbers in the YRF represents about 45% of the total number of waterbirds in the region (Barter et al. 2006). The morpho- logical differences between the adults and the juveniles are shown in Additional file 1: Table S2. from designated observation points. In this case, we will replace the old points with new ones, so as to obtain sim- ilar coverage of the lake. On the other hand, based on the new information about previously unknown wintering areas of birds revealed by tracked individuals and sugges- tions of local reserve managers, we also added a few new points in some lakes in some years to obtain better data. Data collection Data were collected twice a year from 2016 to 2019. The field surveys were conducted at 17 lakes in the YRF, an area that supports the highest density of shallow lakes in China (Qin et al. 2002). Detailed sampling sites and points are listed in Additional file 1: Table S1 (Point Dis- tribution Map; see Fig. 1). During the course of the 4 years of the juvenile ratio survey, we tried our best to ensure the consistent use of the same observation points at all of the lakes. How- ever, some were rendered inaccessible due to construc- tion work, changes in water level, the physical loss of some lakes and wetland habitats and other factors, all of which resulted in our inability to survey consistently Wang et al. Avian Research (2021) 12:72 Page 4 of 13 Wang et al. Avian Research Fig. 1 Distribution map showing the positions of all the 501 sampling points used to gather juvenile ratio data of wintering waterbirds in middle and lower reaches of Yangtze River from 2016 to 2019. Juvenile ratio data were sampled from 10 waterbird species (Swan Goose, Lesser White-fronted Goose, Siberian Crane, Hooded Crane, White-naped Crane, Greater White-fronted Goose, Greylag Goose, Bean Goose, Bewick’s Swan and Common Crane) from 2016 to 2019. Sampling points are identified as key points (i.e. points supporting more than 1% of the flyway populations of one or more of the 10 species, N = 258) and other, non-key points (N = 243) Fig. 1 Distribution map showing the positions of all the 501 sampling points used to gather juvenile ratio data of wintering waterbirds in middle and lower reaches of Yangtze River from 2016 to 2019. Juvenile ratio data were sampled from 10 waterbird species (Swan Goose, Lesser White-fronted Goose, Siberian Crane, Hooded Crane, White-naped Crane, Greater White-fronted Goose, Greylag Goose, Bean Goose, Bewick’s Swan and Common Crane) from 2016 to 2019. Sampling points are identified as key points (i.e. points supporting more than 1% of the flyway populations of one or more of the 10 species, N = 258) and other, non-key points (N = 243) Fig. 1 Distribution map showing the positions of all the 501 sampling points used to gather juvenile ratio data of wintering waterbirds in middle and lower reaches of Yangtze River from 2016 to 2019. Data analysis h In the model, we approxi- mate the field observation survey to the sampling, and assume that the number of young birds drawn in the sampling process is related to the juvenile ratio in the population as a whole, given that in the observation process, there are only two possibilities, of assigning birds as adult or young. In theory, when our obser- vation times reach infinity, the P value of observing young birds is equal to the juvenile ratio of the spe- cies throughout the year. After the actual N counts, the mathematical expectation of this observation is NP, and the variance of this is σ2 = NP (1 − P). Mean- while, due to errors in the observation itself, we believe that the number of young birds within the interval N (P − 0.01) to N (P + 0.01) is consistent with the real sit- uation after N experiments, where 0.01 is the maximum allowable error value we assume, although the accept- able error value in the actual results is greater than our assumed value. After N observations, because N is generally large and can satisfy the conditions of NP > 5 and N (1 − P) > 5, we can use the Gaussian distribution to estimate the result of this binomial distribution and select E – σ < X < E + σ. Therefore: To distinguish between sampling units, throughout this paper, we make the three definitions. The first is a “key site” as a lake where more than 1% biogeographical fly- way population was counted, the second is a “key point” as a point where more than 1% biogeographical flyway population was counted and the last is a “site of major fly- way importance” which is a lake that supports than 5% of total numbers of the defined population in that year. The 1% biogeographical flyway population of 10 waterbird species is shown in Additional file 1: Table S4 (Wetlands International 2021).h The juvenile ratio sample results from the 10 spe- cies of waterbirds from the 4 years are presented in the form of a mean and standard deviation. We denote the number of individuals of a given species sampled for age class as N, the number of juveniles as n, and the YRF sampled juvenile ratio of this species as P = n/N. Data analysis h For each species, the overall annual juvenile ratio for each year was derived from the combined juvenile ratio deter- minations at all of the different lakes from which samples were taken in a given season (the sum of the juveniles/ the sum of the juveniles and adults). For those lakes for which only one survey was conducted in a given year, we took that juvenile ratio for these lakes as representative of that year. If two surveys were undertaken using the same observation points in the same year, we selected the larger sample of the two as representative of that lake in that year.h If we define the number of key points in the sites of major importance as O (O ≤ M), the error rate of key points in the sites of major importance can be calcu- lated as \|(O 1 ni/ O 1 Ni)−P\| P × 100% . The error rate reflects the difference between the theoretical value and the YRF sampled value when only the key points in sites of major importance are considered.i If we define the result of the previous investiga- tion period as P11 and the result from the subsequent period as P12, then the error rate can be calculated as \|(P11−P12)\| P × 100% . In all statistical analyses, we used the comparisons of the mean ± SD of the 4-year results to measure the changes between the annual results. The programme aimed to derive extensive juvenile ratio data from all of the numerically most important dis- crete lakes for a given species during the 4-year investi- gation, lakes that also had the added advantage of being geographically dispersed. For this reason, we make the assumption that the juvenile ratio structure derived for each species in each of the 4 years reflects the true under- lying juvenile ratios in the populations of these species on their wintering areas in those years. This assumption remains to be tested, but given the large sample sizes and their distribution from extensive sampling among large and small flocks from geographically dispersed lakes, we consider this a reasonable assumption to make. g We constructed a mathematical model to calculate the theoretical size of the sample population needed to derive a robust estimate of YRF sampled juvenile ratio in the population as a whole. Study species Our focal 10 large-bodied waterbird species included 5 endangered species (Swan Goose, Lesser White-fronted Wang et al. Avian Research (2021) 12:72 Page 5 of 13 et al. in press). There are no representative satellite track- ing results of the other 2 waterbird species considered here (Hooded Crane, Common Crane), but their autumn migration arrival phenology is similar to that of the other 8 waterbird species. the percentage of data contributed by a given lake of a given species in that year as k 1 Ni N × 100% , to illustrate the importance of the sites of major flyway importance, then the number of all sites of major flyway importance isM = K . We defined the error rate of the juve- nile ratio results of the survey in sites of major flyway importance as \|(M 1 ni/ M 1 Ni)−P\| P × 100% , and the error rate indicates the difference between the subsample value and the YRF sampled value for sites of major fly- way importance.i Juvenile ratio of 10 waterbird species in 2016–2019h Juvenile ratio of 10 waterbird species in 2016 2019 The overall annual juvenile ratios for each of the 10 spe- cies are shown in Table 1 and Additional file 1: Table S5. Across all species, 2017 was the year with lowest juve- nile ratio, 2016 and the following two years were higher than 2017 (Table 1). The average juvenile ratio of 10 spe- cies was 0.169 ± 0.031, among which the juvenile ratios of Hooded Crane, White-naped Crane, Common Crane, Greylag Goose and Bewick’s Swan were higher than the average, while the juvenile ratios of Siberian Crane, Swan Goose, Greater White-fronted Goose, Lesser White- fronted Goose and Bean Goose were lower than the average. In order to measure independence, we still need to assume that the investigation process is independ- ent, which means for each observation point, we need to obtain the difference value between the theoreti- cal value and the actual value. By analysing the differ- ence value, we will be able to obtain the independence of each species and correct N according to these val- ues. This requires the introduction of three extra vari- ables as follows: P, the overall YRF sampled (actual) of encountering a young of a give species in a given year (i.e. the overall YRF sampled juvenile ratio of the spe- cies in that year); Ni, the total number of birds counted at the point i of the species survey in that year and Pi, the probability of occurrence of juveniles at point i of the species survey in the that year (i.e. the point sam- pled juvenile ratio at point i of the species in the that year). Data analysis h At a given point i, we define the number of juveniles as ni, the total sample aged is Ni, and the number of points covered within a lake is K. When considering only sites of major flyway importance, we calculated h (1) E −σ = N(P −0.01) (2) E + σ = N(P + 0.01) E −σ = N(P −0.01) (1) (2) Wang et al. Avian Research (2021) 12:72 Page 6 of 13 Wang et al. Avian Research (2021) 12:72 Wang et al. Avian Research (1) and (2) can be reformulated as: (1) and (2) can be reformulated as: 5. Weight: defined as √Ni/ √Ni , where Ni refers to the number of birds counted at this point i; 6. Time2_Weight: defined as Weight × Time2; 7. sum_Weight: defined as (Weight × Time); 8. sqrt_sum_Weight: defined as (Weight × Time2). (3) NP − NP(1 −P) = N(P −0.01) (4) NP + NP(1 −P) = N(P + −0.01) (3) (4) Squaring (3) and (4) before reformulation gives: Among these, Time2_Weight is used to measure the independence of survey points, while sqrt_sum_Weight is used to measure the independence of each year. In the revision process, since the value of (Weight) in each species survey is 1, the theoretical population number in the actual survey needs to be revised to Time2 × N, but since we are conducting a long-term survey (Year > 8 years), we believe that the theoretical population number in a single year survey can be revised to Time2×N Year . (5) NP(1 −P) = 0.0001 × N 2 (5) (5) N = P(1 −P) 0.0001 N = P(1 −P) 0.0001 N = P(1 −P) 0.0001 Therefore, in order to ensure the accuracy of the investigation, N = P(1−P) 0.0001h 0.0001 This analysis assumes complete independence of observations in the investigation. However, in the real process, we cannot guarantee the complete independ- ence of field observations, so it is necessary to design a scheme to measure the independence level of different species in field observation so that we can adjust the theoretical investigation value of N. Optimising the timing of the juvenile ratio survey As well as optimising the geographical extent of the field survey to maximise efficiency, it is important to deter- mine the optimal time for undertaking the juvenile ratio surveys because of migration and turnover in the popula- tions of waterbirds being sampled. During 2016 to 2019, two surveys were conducted in different survey periods within the same year and comparing these generated an overall error rate of 38.0% ± 40.6% (Mean ± SD, for full details see Additional file 1: Table S9) for the same spe- cies at the same lakes. This shows that differences in juve- nile ratio results between different survey periods at the same lake are very large, so we cannot arbitrarily select a specific sampling period for monitoring juvenile ratios. Compared with the surveys reported here, we would recommend reducing the number of lakes covered by future surveys, without loss of information. Based on the relative proportion of the 10 waterbird species that each lake held, we identified 6 lakes that meet the site of major flyway importance for the 10 waterbird species, namely Poyang Lake, East Dongting Lake, Shengjin Lake, Caizi Lake, Longgan Lake and Chen Lake (Additional file 1: Table S6). The distribution of waterbirds between the sites of major flyway importance was related to the habi- tat and feeding selectivity of different species. Of these, Poyang Lake played a vital role in providing the overwin- tering habitat of 9 waterbird species (45.9–100%), exclud- ing the Lesser White-fronted Goose which was almost totally confined to East Dongting Lake (> 98%). i For this reason, we used data from satellite tracking studies of individuals from these wintering waterbird populations to provide the basis for the most effective timing for our monitoring programme. The telemetry results were shown in Additional file 1: Table S3. In sum- mary, all of the eight species of waterbirds that we are concerned about here have completed their migration by early December. Given that the plumage characteristics of first winter birds change during the course of the win- ter as they moult (making it more difficult to reliably dif- ferentiate first winter from older birds), we recommend that observers start to undertake the juvenile ratio sam- pling in early to mid-December. Juvenile ratio of 10 waterbird species in 2016–2019h We also introduce eight new parameters: Table 1 The overall annual juvenile ratio (expressed as proportion of first winter birds out of all birds aged) among 10 large-bodied waterbird species sampled in the Yangtze River Floodplain from 2016 to 2019 The values in bold represent the lowest juvenile ratio from all 4 years for each species, which for all species fell in 2017 Species 2016 2017 2018 2019 Mean ± SD Common Crane 0.187 0.135 0.184 0.241 0.187 ± 0.037 Siberian Crane 0.178 0.039 0.146 0.173 0.134 ± 0.056 Hooded Crane 0.257 0.169 0.231 0.252 0.227 ± 0.035 White-naped Crane 0.146 0.125 0.188 0.253 0.178 ± 0.049 Swan Goose 0.133 0.096 0.108 0.175 0.128 ± 0.030 Lesser White-fronted Goose 0.344 0.060 0.090 0.113 0.152 ± 0.112 Greater White-fronted Goose 0.164 0.122 0.138 0.187 0.153 ± 0.025 Greylag Goose 0.259 0.104 0.165 0.222 0.188 ± 0.059 Bean Goose 0.184 0.104 0.153 0.137 0.145 ± 0.029 Bewick’s Swan 0.241 0.125 0.198 0.251 0.204 ± 0.050 1. Expectation: defined as P(1−P) Ni , where P is the overall YRF sampled juvenile ratio of the species in that year, and Ni refers to the number of birds counted at this point i. It represents the theoretical error; 2. Error: defined as (Pi − P), where P is the YRF sampled juvenile ratio of the species in that year, Pi refers to the juvenile ratio results at point i of the species in that year. It represents true error; 3. Time: defined as Error/Expectation;i 3. Time: defined as Error/Expectation;i i 4. sqrt_Total: defined as √Ni , where Ni refers to the number of birds counted at this point i; The values in bold represent the lowest juvenile ratio from all 4 years for each species, which for all species fell in 2017 Page 7 of 13 Wang et al. Avian Research (2021) 12:72 Wang et al. Avian Research (2021) 12:72 Wang et al. Avian Research The basis for a Yangtze monitoring programme for the juvenile ratio of 10 waterbird species Rationalising cover of sites of major flyway importance Based on the survey experience and results in 2016– 2019, we have developed proposals for a monitoring programme to sample the juvenile ratio of 10 waterbird species in the future. points surveyed in the original work, a reduction in the extent of coverage of almost 50%. Optimising the timing of the juvenile ratio survey i When the samples derived from only the sites of major flyway importance in the monitoring programme, the overall error rate was under 5%, which shows that our recommended plan for only sampling at sites of major fly- way importance (N = 6 of the most important lakes) can robustly reflect the overall juvenile ratio results of each of the years from 17 lakes (Additional file 1: Table S7). On this basis, we only need to survey one third of the area covered in the full surveys to generate robust estimates of juvenile ratios for all 10 waterbird species. Testing for statistical independence f Our monitoring programme for juvenile ratio relies on field samples, while the effectiveness of the field investi- gation relies on its ability to reflect the underlying species population statistics and the effectiveness of identifying juveniles from adults. It is important to ensure the sta- tistical independence of the sampling process and how this may affect the design of our juvenile ratio monitoring programme. In order to understand statistical independ- ence, we have calculated the corresponding eight param- eter results (see Additional file 1: Table S10). Rationalising cover of key observation pointsf The map shows proposed sampling points for the future juvenile ratio field survey (green), all of which were located on key lakes (a Poyang Lake, N = 108; b East Dongting Lake, N = 13; c Longgan Lake, N = 6; d Shengjin Lake, N = 15; e Caizi Lake, N = 6; f Chen Lake, N = 7). N in each case is the number of planned points. Remaining points (shown in white) that were surveyed in 2016–2019 are not proposed to be sampled in the future unless circumstances change species of waterbird (all Anatidae species, sqrt_sum_ Weight = 4.817), Swan Goose, Lesser White-fronted Goose, Greylag Goose, Bean Goose, Greater White- fronted goose and Bewick’s Swan have weak independ- ence (see Additional file 1: Table S11 for a complete breakdown of species by year). species of waterbird (all Anatidae species, sqrt_sum_ Weight = 4.817), Swan Goose, Lesser White-fronted Goose, Greylag Goose, Bean Goose, Greater White- fronted goose and Bewick’s Swan have weak independ- ence (see Additional file 1: Table S11 for a complete breakdown of species by year). Rationalising cover of key observation pointsf To further reduce sampling effort, we looked at possibili- ties to reduce the number of points necessary to generate robust juvenile ratio estimates. j We extracted all the observation points that meet the definition of key points from the sites of major flyway importance, and statistically re-analysed (see the distri- bution of observation points shown in Fig. 2). During the 4-year investigation period, the error rate of key points is shown in Additional file 1: Table S8. The overall error rate was about 5%, which shows that our recommended plan for only sampling at key points of sites of major fly- way importance (N = 258 of the most important points) can robustly reflect the overall juvenile ratio based on the results of all the years from 501 points (Additional file 1: Table S8). On this basis, we can generate robust estimates from only 258 observation points out of the original 501 i Time2_Weight is used to measure the independence of counts from a given point. When the counting pro- cess is a completely independent statistical process, sqrt_sum_Weight will be 1 in the overall YRF survey. So, the closer the sqrt_sum_Weight is to 1 for a given spe- cies, the stronger its statistical independence. Based on our results, the four species of crane (family Gruidae, sqrt_sum_Weight = 1.607), Siberian Crane, Hooded Crane, White-naped Crane and Common Crane showed the strongest independence. However, the other six Wang et al. Avian Research (2021) 12:72 Wang et al. Avian Research Page 8 of 13 Fig. 2 Sites of major flyway importance for the 10 large-bodied waterbirds in the Yangtze River Floodplain. The map shows proposed sampling points for the future juvenile ratio field survey (green), all of which were located on key lakes (a Poyang Lake, N = 108; b East Dongting Lake, N = 13; c Longgan Lake, N = 6; d Shengjin Lake, N = 15; e Caizi Lake, N = 6; f Chen Lake, N = 7). N in each case is the number of planned points. Remaining points (shown in white) that were surveyed in 2016–2019 are not proposed to be sampled in the future unless circumstances change Fig. 2 Sites of major flyway importance for the 10 large-bodied waterbirds in the Yangtze River Floodplain. Establishing optimal species‑specific sample size for the juvenile ratio surveys Assuming the statistical independence of the sampling process, we can calculate the theoretically optimal sample size for each species in each year to generate a robust juvenile ratio estimate (see “Methods” section for details), the results of which are shown in Table 2. The sqrt_sum_Weight values for the crane species Wang et al. Avian Research (2021) 12:72 Page 9 of 13 Page 9 of 13 Table 2 The optimal future survey sample sizes calculated based on analysis of the degree of statistical independence achieved for each of 10 large-bodied waterbird species in each year from 2016 to 2019 By then applying the overall sqrt_sum_Weight of each species (see “Methods” section, values derived from Additional file 1: Table S11), we can generate a value for the optimal future survey sample size to generate robust species juvenile ratios. IH means the independence hypothesis Species 2016 2017 2018 2019 2016–2019 Age ratio Survey sample (IH) Age ratio Survey sample (IH) Age ratio Survey sample (IH) Age ratio Survey sample (IH) Optimal future survey sample Swan Goose 0.133 1153 0.096 868 0.108 963 0.175 1444 2761 Lesser White-fronted Goose 0.344 2257 0.060 564 0.090 819 0.113 1002 826 Siberian Crane 0.178 1463 0.039 375 0.146 1247 0.173 1431 1129 Hooded Crane 0.257 1909 0.169 1404 0.231 1776 0.252 1885 1744 White-naped Crane 0.243 1839 0.125 1094 0.188 1527 0.253 1890 1588 Greater White-fronted Goose 0.164 1371 0.122 1071 0.138 1190 0.187 1520 6364 Greylag Goose 0.259 1919 0.104 900 0.170 1411 0.220 1716 1312 Bean Goose 0.180 1476 0.104 900 0.150 1275 0.140 1204 8955 Bewick’s Swan 0.240 1824 0.125 1056 0.200 1600 0.250 1875 5844 Common Crane 0.190 1539 0.135 1204 0.180 1476 0.240 1824 1511 Table 2 The optimal future survey sample sizes calculated based on analysis of the degree of statistical independence achieved for each of 10 large-bodied waterbird species in each year from 2016 to 2019 By then applying the overall sqrt_sum_Weight of each species (see “Methods” section, values derived from Additional file 1: Table S11), we can generate a value for the optimal future survey sample size to generate robust species juvenile ratios. IH means the independence hypothesis By then applying the overall sqrt_sum_Weight of each species (see “Methods” section, values derived from Additional fil optimal future survey sample size to generate robust species juvenile ratios. Establishing optimal species‑specific sample size for the juvenile ratio surveys IH means the independence hypothesis varied between 0.7 and 2.7, with many reasonably close to 1, suggesting that these species attained a reason- able level of statistical independence, which suggest the recommended sample sizes were likely to be sufficient for the purposes of the survey. However, for the Anati- dae species, sqrt_sum_Weight values varied between 1.1 and 13.3 (mean 4.817) which showed the level of statistical independence was not so good, necessitat- ing the correction of the optimal theoretical sampling size using the sqrt_sum_Weight of each species. These optimal future survey sample sizes are shown for each species in Table 2, where the required samples for Bean Goose (8955) and Great White-fronted Goose (6364) can be seen to be more inflated than for the other spe- cies. In future, we need to check to ensure the recom- mended sample sizes for these species are maintained at a sufficient level in order to maintain the statistical independence of the sampling procedure. The monitoring programme for juvenile ratio of 10 waterbird species in the YRF 3 The distribution map of the 155 proposed points for the future sampling of juvenile ratios among large-bodied waterbirds in the middle and lower reaches of the Yangtze River based on the results from analysis of data from the monitoring scheme. The proposed monitoring scheme will conduct juvenile age ratios among the 10 large-bodied waterbirds from early December to mid-December. The 155 planned points are confined to six sites of major flyway importance (Poyang Lake, East Dongting Lake, Shengjin Lake, Longgan Lake, Caizi Lake, Chen Lake) Fig. 3 The distribution map of the 155 proposed points for the future sampling of juvenile ratios among large-bodied waterbirds in the middle and lower reaches of the Yangtze River based on the results from analysis of data from the monitoring scheme. The proposed monitoring scheme will conduct juvenile age ratios among the 10 large-bodied waterbirds from early December to mid-December. The 155 planned points are confined to six sites of major flyway importance (Poyang Lake, East Dongting Lake, Shengjin Lake, Longgan Lake, Caizi Lake, Chen Lake) 10 waterbird species over 4 winters. In this analysis, we show that it is feasible to sample in a statistically robust method, the juvenile ratios of 10 large-bodied waterbird species using a fraction of the effort expended in the orig- inal surveys. We show that we can reduce the time spent surveying and the number of observation points by a half (reducing the overall workload by two-thirds) without reduction in the statistical power of the survey results. observational range of a telescope (2 km). Based on these new recommendations, the predicted juvenile ratio of the investigation of sites of major flyway importance, key points and planned points (Fig. 3) and the theoretical juvenile ratio of the theoretical underlying populations does not differ substantially from the data generated from the entire survey (Additional file 1: Table S12). Compared with the effort required to achieve the level of survey coverage in 2016–2019, the new programme saves about 50% of the investigation time, reduces the work- load in the investigation area by 66.7%, and reduces the numbers of observation points to be visited by 48.3%. The monitoring programme for juvenile ratio of 10 waterbird species in the YRF p y In the subsampling design for the future composition of the monitoring scheme, we will focus on coverage of the six most important lakes (Poyang, East Dongting, Shengjin, Longgan, Caizi and Chen Lakes), because these lakes support all the key observation points based on the last four winter surveys for the 10 waterbird species examined here. We would especially target Poyang Lake, which we have shown is extremely important for nine of the wintering waterbird species considered here, for intensive sampling of juvenile ratios. This is consistent with other studies that show many of the species consid- ered here are increasingly concentrated within the YRF at Poyang Lake, making it of increasing significance for those species (Cao et al. 2008; Cao and Fox 2009). The results of this study point to sampling at other large lakes, The monitoring programme for juvenile ratio of 10 waterbird species in the YRF Based on the above results, we obtained the 258 key points from the sites of major flyway importance from 2016 to 2019. For the purpose of future surveys (Table 3), we were able to reduce the 258 key points to 155 planned points for the 10 waterbird species based on the typical Table 3 Final recommendations for the juvenile ratio sampling programme for 10 species of large-bodied waterbird species in the Yangtze River floodplain, by investigating their respective planned points based on the monitoring scheme See “Methods” section for explanation for generating Error Rates PYH Poyang Lake, DDTH East Dongting Lake, SJH Shengjin Lake, LGH Longgan Lake, CZH Caizi Lake, CH Chen Lake Species Recommended timing of the surveys Recommended sites of major flyway importance for each species to be visited Recommended number of planned observation points for each species Error rate (%) Swan Goose 12/01–12/15 PYH, SJH 26 0.82 Lesser White-fronted Goose 12/01–12/15 DDTH 7 4.17 Siberian Crane 12/01–12/15 PYH 19 1.58 Hooded Crane 12/01–12/15 PYH, SJH, CZH 19 1.20 White-naped Crane 12/01–12/15 PYH 21 0.50 Greater White-fronted Goose 12/01–12/15 PYH, SJH, DDTH 52 4.92 Greylag Goose 12/01–12/15 PYH, LGH, CH 15 1.77 Bean Goose 12/01–12/15 PYH, SJH, DDTH, CZH 59 1.24 Bewick’s Swan 12/01–12/15 PYH, LGH 31 2.39 Common Crane 12/01–12/15 PYH 42 3.73 Total 12/01–12/15 PYH, DDTH, SJH, LGH, CZH, CH 155 2.23 ndations for the juvenile ratio sampling programme for 10 species of large-bodied waterbird species in the by investigating their respective planned points based on the monitoring scheme Table 3 Final recommendations for the juvenile ratio sampling programme for 10 species of large-bodied Yangtze River floodplain, by investigating their respective planned points based on the monitoring scheme Wang et al. Avian Research (2021) 12:72 Wang et al. Avian Research Page 10 of 13 Fig. 3 The distribution map of the 155 proposed points for the future sampling of juvenile ratios among large-bodied waterbirds in the middle and lower reaches of the Yangtze River based on the results from analysis of data from the monitoring scheme. The proposed monitoring scheme will conduct juvenile age ratios among the 10 large-bodied waterbirds from early December to mid-December. The 155 planned points are confined to six sites of major flyway importance (Poyang Lake, East Dongting Lake, Shengjin Lake, Longgan Lake, Caizi Lake, Chen Lake) Fig. Discussion A major contribution of the study to the effective man- agement of this massive and globally important resource is to monitor the annual reproductive success of the waterbirds that use the Yangtze wetlands as winter quar- ters. This study has provided a statistically defensible means to organise a more concise, efficient, less time- consuming, reliable sampling scheme for future research- ers and managers for the first time based on the survey data and experience from sampling the juvenile ratios of Wang et al. Avian Research (2021) 12:72 Wang et al. Avian Research (2021) 12:72 Page 11 of 13 Crane habitat in the Yellow River Delta has not been visited on the ground or protected (Yang et al. 2020). If birds from the same biogeographical population are win- tering outside the YRF, these should be incorporated into the programme if it is to be able to deliver robust juvenile ratio estimates for the entire population. Therefore, we strongly recommend that satellite tracking of these over- wintering waterbird populations be extended as much as possible to improve on our current understanding of their wintering abundance and distribution to ensure the representativeness of our juvenile ratio sampling. By combining satellite tracking and monitoring pro- grammes, we are confident that we can better monitor the distribution and age structure of these 10 waterbird species in the YRF, and support their effective conserva- tion through better protection measures (de Boer et al. 2011; Pavón-Jordán et al. 2020). such as East Dongting Lake, likely because their size accommodates birds that aggregate in large numbers, but also because larger lakes support a greater diversity of habitat types. This latter feature is important because the species considered here all differ in their diet and habitat use. For example, the Swan Goose forages for submerged tubers (An et al. 2018, 2019), Bean Goose grazes the above ground parts of Carex, as well as consuming grain seeds and plant buds (Yang et al. 2016), while the fam- ily Gruidae can prey on fish, crustaceans, invertebrates and other animals (BirdLife International 2017). This also means that any sampling regime needs to account for the differential distribution of the 10 migra- tory waterbird species within and between the lakes, for which our approach has taken account. In the future, climate, hydrological and wetland land-use change, as well as human activity, will affect habitat distribution and quality in the YRF. Discussion This means that the habitat upon which waterbirds depend will change with time, which will bring challenges to the sustainability of our monitor- ing programme. While using the experience from earlier surveys to inform how best to undertake juvenile ratios in the future is inevitably based on a few recent years of experience, we also consider this approach does make the design future-proofed to some degree. Our recom- mendations for a future monitoring programme focus on the sites which are currently of major flyway importance, based on the current distribution and abundance of the 10 species. In order to take account of any changes in waterbird utilization of wetlands in the future, we would advocate maintaining our recommended programme, while reviewing our approach regularly based on the results from satellite tracking. In the face of habitat destruction and degradation, the waterbird juvenile ratio survey programme must be able to adapt sequentially to changes in the distribution and abundance of the 10 spe- cies within the YRF (Ydenberg and Prins 1981; Cromsigt et al. 2009; Heuermann et al. 2011). Should conditions change so much that birds start to redistribute outside of the YRF, the programme will need to address the change at larger spatial scales. This means that the programme need to communicate and cooperate with neighbor- ing management units (such as Japan, South Korea and Russia, etc.) as well as in the YRF in monitoring, protec- tion and data. At the same time, it will be essential to strengthen international cooperation in monitoring and protection in order to maintain and enhance waterbird and wetland protection in the future. Conclusion Retrospective data analysis of the juvenile ratio data from 10 species of waterbirds sampled in 2016–2019 enabled us to see the degree to which a reduced set of lakes and samples from observation points could generate esti- mates that still reflected the overall juvenile ratios in the entire data set. Using this approach, we were able to recommend how to sample juvenile ratios in the future at much reduced effort without loss of statistical power. Because the 10 species of waterbirds are highly concen- trated at relatively few sites of major flyway importance on their wintering areas, large samples (reflecting rela- tively high proportions of the entire wintering popula- tions) could be sampled in this way. This was especially the case at Poyang Lake in Jiangxi Province, which has become of disproportionate importance in the YRF for many waterbird species. In addition, the results from the surveys suggested that the YRF sampled juvenile ratios of Swan Goose (VU), Lesser White-fronted Goose (VU) and Siberian Crane (CR) were lower than the average for all 10 species. This is especially alarming for the two goose species, because historical juvenile ratios and those from elsewhere in the range are thought to be higher and because their numbers are decreasing more than other species. This confirms the need for greater attention to be paid by researchers to understand factors affecting reproductive success and by protection and management personnel for the habitat and management needs of the species while on the winter quarters. The online version contains supplementary material available at https://doi. org/10.1186/s40657-021-00309-x. Supplementary Information We see the programme we have initiated here as focus- ing on the YRF, but for a truly holistic species biogeo- graphical flyway approach, we also need to extend the geographical scope outlined here. For instance, previous satellite tracking studies showed that potential Siberian The online version contains supplementary material available at https://doi. org/10.1186/s40657-021-00309-x. The online version contains supplementary material available at https://doi. org/10.1186/s40657-021-00309-x. Wang et al. Avian Research (2021) 12:72 Page 12 of 13 Page 12 of 13 Wang et al. Avian Research National Nature Reserve, the Longgan Lake National Nature Reserve and the East Dongting Lake National Nature Reserve. Additional file 1: Table S1. The fieldwork that generated the data analysed here came from 17 lakes and 501 points in the YRF from 2016 to 2019. Table S2. Photographic information relating to the 10 waterbird species involved in the juvenile ratio survey of Yangtze River, illustrating the salient plumage differences and other features that are characteristic of adult and young birds of all species (Brazil 2009). Table S3. Autumn and spring migration schedules of eight large waterbird species in China based on published tracking studies. Table S4. The 1% biogeographi- cal flyway population levels for each of the 10 large-bodied waterbirds wintering in the Yangtze River Floodplain analysed here. Table S5. The sample sizes and juvenile ratio from each of the 10 large waterbird species surveyed in each year from 2016 to 2019 (see “Methods” – Data analysis). Table S6. The percentage of each large waterbird species counted in a given year at six sites of major flyway importance in the Yangtze River Floodplain. Table S7. The error rate between predicted juvenile ratio and YRF sampled juvenile ratio of 10 large-bodied waterbirds in Yangtze River Floodplain from 2016 to 2019. Table S8. The error rate between predicted juvenile ratio and Yangtze River Floodplain sampled juvenile ratio of 10 large-bodied waterbird species in sites of major flyway importance from 2016 to 2019. Table S9. The error rate between sampled juvenile ratio in November and sampled juvenile ratio in December of 10 large-bodied waterbirds in the same lake from 2016 to 2019. Table S10. The theoreti- cal error, real error and T2_Weight results of 10 large-bodied waterbird species in the Yangtze River Floodplain based on statistical independence test analysis. Table S11. The sqrt_sum_Weight results of 10 large-bodied waterbird in YRF from 2016 to 2019 based on statistical independence test analysis. Declarations Cao L, Fox AD. Birds and people both depend on China’s wetlands. Nature. 2009;460:173. Funding Batbayar N, Takekawa JY, Newman SH, Prosser DJ, Natsagdorj T, Xiao XM. Migration strategies of Swan Geese Anser cygnoides from northeast Mongolia. Wildfowl. 2013;61:90–109. Our study was supported by the National Natural Science Foundation of China (Grant Nos. 31870369, 31970433) and China Biodiversity Observation Networks (Sino BON). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Innovative Research Group Project of the National Natural Science Foundation of China (CN), No. 31670424. Batbayar N, Takekawa JY, Newman SH, Prosser DJ, Natsagdorj T, Xiao XM. Migration strategies of Swan Geese Anser cygnoides from northeast Mongolia. Wildfowl. 2013;61:90–109. BirdLife International. BirdLife International and handbook of the birds of the world (2017) Bird species distribution maps of the world. 2017. BirdLife International. BirdLife International and handbook of the birds of the world (2017) Bird species distribution maps of the world. 2017. Boyd H. On encounters between wild White-fronted Geese in winter flocks 1. Behaviour. 1953;5:85–128. Brazil M. Birds of East Asia: China, Taiwan, Korea, Japan, and Russia. New York: A&C Black; 2009. Availability of data and materials The datasets used in the present study are available from the corresponding author on reasonable request. Bromley RGH, Rothe TC. Conservation assessment for the dusky Canada goose (Branta canadensis occidentalis Baird). US Department of Agriculture, For- est Service, Pacific Northwest Research Station; 2003. Acknowledgements We are very grateful to the Professor Anthony David Fox of Department of Bioscience, Aarhus University, Denmark, who provided so much help with the structure and writing of the paper. We would sincerely like to thank all the volunteers, like Yuqing Han, Fujia Dong, Xiaoping Lu, Wenwen Niu, Taoping Wu and Itgelt, for their contributions to our field surveys. We would also like to thank the staff members of the Poyang Lake National Nature Reserve, the Nanji Wetland National Nature Reserve, Shengjin Lake National Nature Reserve, the Longgan Lake National Nature Reserve and the East Dongting Lake National Nature Reserve for their cooperation. Finally, thanks to all mem- bers of the investigation team responsible for the surveys of overwintering waterbirds in the Yangtze River. An A, Zhang Y, Cao L, Jia Q, Wang X. A potential distribution map of wintering Swan Goose (Anser cygnoides) in the middle and lower Yangtze River floodplain, China. Avian Res. 2018;9:1–10. An A, Cao L, Jia Q, Wang X, Zhu Q, Zhang JJ, et al. Changing abundance and distribution of the wintering Swan Goose Anser cygnoides in the middle and lower Yangtze River floodplain: an investigation combining a field survey with satellite telemetry. Sustainability. 2019;11:1398. Ao PR, Wang X, Solovyeva D, Meng FJ, Ikeuchi T, Shimada T, et al. Rapid decline of the geographically restricted and globally threatened Eastern Palearc- tic Lesser White-fronted Goose Anser erythropus. Wildfowl. 2020;9:206–43. Baldassarre G. Ducks, geese, and swans of North America. New York: JHU Press; 2014. References Alisauskas RT, Lindberg MS. Effects of neckbands on survival and fidelity of white-fronted and Canada geese captured as non-breeding adults. J Appl Stat. 2002;29:521–37. Consent for publication Not applicable. Consent for publication Not applicable. Authors’ contributions Barter M, Chen L, Cao L, Lei G. Waterbird survey of the middle and lower Yang- tze River floodplain in late January and early February 2004. Beijing: China Forestry Pulishing House; 2004. LC, YW and YX conceived the ideas and designed methodology; ID, XD and RG collected the data; YW analyzed the data. YW led the writing of the manu- script, with contributions from LC. All authors contributed critically to the drafts. All authors read and approved the final manuscript. Barter M, Lei G, Cao L. Waterbird survey of the middle and lower Yangtze River floodplain (February 2005). Beijing: World Wildlife Fund-China & China Forestry Publishing House; 2006. Author details 1 School of Life Science, University of Science and Technology of China, Hefei 230026, China. 2 State Key Laboratory of Urban and Regional Ecology, Research Center for Eco Environmental Sciences, Chinese Academy of Sci- ences, Beijing 100085, China. 3 University of Chinese Academy of Sciences, Beijing 100049, China. 4 Institute of Biology, Mongolian Academy of Sciences, Ulaanbaatar 13330, Mongolia. 5 Department of Modern Physics, University of Science and Technology of China, Hefei 230026, China. 6 College of Life Sci- ence and Technology, Inner Mongolia Normal University, Huhehaote 010000, China. 7 Jiangxi Poyang Lake National Reserve Authority, Nan Chang 330038, China. 8 Authority of Jiangxi Duchang Provincial Natural Reserve, Duch- ang 332600, China. 9 Authority of East Dongting Lake National Nature Reserve, Yueyang 414000, China. 10 Anhui Shengjin Lake National Reserve Author- ity, Dongzhi 247000, China. 11 Authority of Longgang Lake National Nature Reserve, Huanggang 435500, China. Received: 26 February 2021 Accepted: 29 November 2021 test analysis. Table S12. Cross-comparison table showing juvenile ratios generated (j/N, where j is the number of juveniles in sample N) from each annual sample (sample size N) for each of the 10 large-bodied waterbird species in each year, 2016 to 2019. Supplementary Information Table S12. Cross-comparison table showing juvenile ratios generated (j/N, where j is the number of juveniles in sample N) from each annual sample (sample size N) for each of the 10 large-bodied waterbird species in each year, 2016 to 2019. Ethics approval and consent to participate Cao L, Barter M, Lei G, Yang Q. Anatidae in the Yangtze floodplain in winter 2004 and 2005. Casarca. 2008;11:146–60. 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Breeding population inventories and measures of recruitment. In: Batt BDJ, Afton AD, Anderson MG, Ankney CD, Johnson DH, Kadlec JA, Krapu GL, editors. Ecology and management of breeding waterfowl. Minneapolis: University of Minnesota Press; 1992. p. 423–45.f Reed ET, Bêty J, Mainguy J, Gauthier G, Giroux JF. Molt migration in relation to breeding success in greater snow geese. Arctic. 2003;56:76–81. H, Kadlec JA, Krapu GL, editors. Ecology and management of breeding aterfowl. Minneapolis: University of Minnesota Press; 1992. p. 423–45. p gy g aterfowl. Minneapolis: University of Minnesota Press; 1992. p. 423–4 Cromsigt JPGM, Prins HHT, Olff H. Habitat heterogeneity as a driver of ungulate diversity and distribution patterns: interaction of body mass and diges- tive strategy. Divers Distrib. 2009;15:513–22. Rees EC, Fox AD. Flyways, population trends and conservation challenges for the Anatidae in Far East Asia. Wildfowl. 2020;6:1–319. the Anatidae in Far East Asia. Wildfowl. 2020;6:1–319. Schock WG, Fischer JB, Ely CR, Stehn RA, Welker JM, Causey D. Variation in age ratio of midcontinent greater White-fronted Geese during fall migration. J Fish Wildl Manage. 2018;9:340–7. de Boer WF, Cao L, Barter M, Wang X, Sun MM, Oeveren H, et al. Comparing the community composition of European and Eastern Chinese waterbirds and the influence of human factors on the China waterbird community. Ambio. 2011;40:68–77. Spaans B, Stock M, Joseph AS, Bergmann HH, Ebbinge BS. Ethics approval and consent to participate Breeding biology of dark-bellied brent geese Branta b. bernicla in Taimyr in 1990 in the absence of arctic foxes and under favourable weather conditions. Polar Res. 1993;12:117–30. Deng XQ, Zhao QS, Fang L, Xu ZG, Wang X, He HR, et al. Spring migration duration exceeds that of autumn migration in Far East Asian Greater White-fronted Geese (Anser albifrons). Avian Res. 2019;10:1–11. U.S. Fish and Wildlife Service. Waterfowl population status, 2019. U.S. Depart- ment of the Interior, Washington, D.C. 2019. https://www.fws.gov/migra torybirds/pdf/surveys-and-data/Population. Deng XQ, Zhao QS, Solovyeva D, Lee H, Harmey IB, Xu ZG, et al. Contrasting trends in two East Asian populations of the Greater White-fronted Goose Anser albifrons. Wildfowl. 2020;6:181–205. Ward DH, Amundson CL, Stehn RA, Dau CP. Long-term trends in fall juvenile ratios of black brant. J Wildlife Manage. 2018;82:362–73. Ward DH, Amundson CL, Stehn RA, Dau CP. Long-term trends in fall juvenile ratios of black brant. J Wildlife Manage. 2018;82:362–73. Wetlands International. Waterbird population estimates. 2021. wpe.wetlands. org. Accessed 13 Feb 2021. Ely CR, Takekawa JY. Geographic variation in migratory behavior of Greater White-Fronted Geese (Anser albifrons). Auk. 1996;113:889–901. Wetlands International. Waterbird population estimates. 2021. wpe.wetlands. org. Accessed 13 Feb 2021. Ely CR, Nieman DJ, Alisauskas RT, Schmutz JA, Hines JE. Geographic variation in migration chronology and winter distribution of midcontinent greater white-fronted geese. J Wildlife Manage. 2013;77:1182–91. Yang YZ, Zhan AB, Cao L, Meng FJ, Xu WB. Selection of a marker gene to construct a reference library for wetland plants, and the application of metabarcoding to analyse the diet of wintering herbivorous waterbirds. Peer J. 2016;4:e2345. Fang L, Zhang JJ, Zhao QS, Solovyeva D, Vangeluwe D, Rozenfeld SB, et al. Two distinct flyways with different population trends of Bewick’s Swan Cygnus columbianus bewickii in East Asia. Wildfowl. 2020;6:13–42. Yang XL, Jiang HX, Zou CL, Wang Y, Lin BQ, Li LS. Changes in migration pat- tern and important stopover sites of the eastern population of Siberian Cranes. For Sci. 2020;56:123–33 (In Chinese). Gregory RD, Gibbons DW, Donald PF. Bird census and survey techniques. Bird Ecol Conserv. 2004;17:56. Cranes. For Sci. 2020;56:123–33 (In Chinese). Ydenberg RC, Prins HHT. 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Results from a Far East Asia telemetry study. Ibis. 2019;162:548–55. • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? 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Choose BMC and benefit from: • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: y y Li XH, Wang X, Fang L, Batbayar N, Natsagdorj T, Davaasuren B, et al. Annual migratory patterns of Far East Greylag Geese (Anser anser rubrirostris) revealed by GPS tracking. Integr Zool. 2020;15:213–23. Ma JH. Establishment of water resources support and guarantee for Yangtze River economic zone. Yangtze River. 2014;45:1–6 (In Chinese). Nakayama T, Shankman D. Impact of the Three-Gorges Dam and water transfer project on Changjiang floods. Global Planet Change. 2013;100:38–50. Li XH, Wang X, Fang L, Batbayar N, Natsagdorj T, Davaasuren B, et al. Annual migratory patterns of Far East Greylag Geese (Anser anser rubrirostris) revealed by GPS tracking. Integr Zool. 2020;15:213–23. g y p y g ( ) revealed by GPS tracking. Integr Zool. 2020;15:213–23. Ma JH. Establishment of water resources support and guarantee for Yangtze River economic zone. Yangtze River. Ethics approval and consent to participate 2014;45:1–6 (In Chinese). Nakayama T, Shankman D. Impact of the Three-Gorges Dam and water transfer project on Changjiang floods. Global Planet Change. 2013;100:38–50. Ma JH. Establishment of water resources support and guarantee for Yangtze River economic zone. Yangtze River. 2014;45:1–6 (In Chinese). Nakayama T, Shankman D. Impact of the Three-Gorges Dam and water transfer project on Changjiang floods. Global Planet Change. 2013;100:38–50. Nolet BA, Bauer S, Feige N, Kokorev YI, Popov BS, Ebbinge BS. Faltering lem- ming cycles reduce productivity and population size of a migratory Arctic goose species. J Anim Ecol. 2013;82:804–13. Pavón-Jordán D, Abdou W, Azafzaf H, Balaž M, Bino T, Borg JJ, et al. Positive impacts of important bird and biodiversity areas on wintering waterbirds under changing temperatures throughout Europe and North Africa. Biol Conserv. 2020;246:108549. Pavón-Jordán D, Abdou W, Azafzaf H, Balaž M, Bino T, Borg JJ, et al. Positive impacts of important bird and biodiversity areas on wintering waterbirds under changing temperatures throughout Europe and North Africa. Biol Conserv. 2020;246:108549.
https://openalex.org/W4378376576	https://www.nature.com/articles/s44172-023-00077-0.pdf	English	null	Aerial and aquatic biological and bioinspired flow control strategies	Communications engineering	2,023	cc-by	15,557	Aerial and aquatic biological and bioinspired ﬂow control strategies Ahmed K. Othman1, Diaa A. Zekry1,2, Valeria Saro-Cortes 1,2, Kyung Jun “Paul” Lee1,2 & Aimy A. Wissa 1✉ Flow control is the attempt to favorably modify a ﬂow ﬁeld’s characteristics compared to how the ﬂow would have developed naturally along the surface. Natural ﬂyers and swimmers exploit ﬂow control to maintain maneuverability and efﬁciency under different ﬂight and environmental conditions. Here, we review ﬂow control strategies in birds, insects, and aquatic animals, as well as the engineered systems inspired by them. We focus mainly on passive and local ﬂow control devices which have utility for application in small uncrewed aerial and aquatic vehicles (sUAVs) with beneﬁts such as simplicity and reduced power consumption. We also identify research gaps related to the physics of the biological ﬂow control and opportunities for device development and implementation on engineered vehicles. N N atural ﬂyers and swimmers operate under various conditions. The same animal, for example, a ﬂyer, can repeatedly take off, hover, glide, ﬂap, and perch in different environmental conditions, such as during gusts and thermals or closer to the ground or the water surface. This agility and maneuverability are possible because of the ability of these biological systems to alter the ﬂow around their lifting and thrusting surfaces, using what is referred to as ﬂow control. Flow control is deﬁned as attempting to favorably modify a ﬂow ﬁeld’s characteristics compared to how the ﬂow would have developed naturally along the surface1,2. Flow control mechanisims include postponing boundary layer separation, delaying or advancing laminar-turbulent transition, and altering vortical structures and dynamics (Fig. 1). Such mechanisms often lead to drag reduction, lift enhancements, or noise suppression and can be implemented through ﬂow control devices such as ﬂaps, slats, or synthetic jets, to mention a few. Multiple classiﬁcations exist for ﬂow control devices; one of the most common ways is to classify ﬂow control techniques as active or passive based on their energy expenditure. Passive ﬂow control requires no actuation nor energy expenditure, making it less complex and more affordable; however, it cannot always provide enough control authority or adaptability, especially in complex applications3,4. On the other hand, active ﬂow control devices require actuation and power to affect the ﬂow5. Another classiﬁcation is related to the area affected by the ﬂow control device, namely local versus global ﬂow control (Fig. 1). COMMUNICATIONS ENGINEERING \| (2023)2:30 \| https://doi.org/10.1038/s44172-023-00077-0 \| www.nature.com/commseng 1 Department of Mechanical and Aerospace Engineering, Princeton University, Princeton, NJ 08544, USA. 2These au Valeria Saro-Cortes, Kyung Jun “Paul” Lee. ✉email: awissa@princeton.edu COMMUNICATIONS ENGINEERING \| https://doi.org/10.1038/s44172-023-00077-0 COMMUNICATIONS ENGINEERING \| https://doi.org/10.1038/s44172-023-00077-0 Fig. 1 Flow control mechanisms and classiﬁcations and their implementations as ﬂow control devices. The superscripts indicate the ﬂow control mechanism implemented by each ﬂow control device. The ﬁgure classiﬁes ﬂow control mechanisms into 1- separation control [Reproduced from ref. 160, The National Aeronautics and Space Administration (NASA)]160, 2- transition control [Reproduced from ref. 161, The National Aeronautics and Space Administration (NASA)]161, 3- vortex tailoring [Reproduced from ref. 162, The National Aeronautics and Space Administration (NASA)]162. Fig. 1 Flow control mechanisms and classiﬁcations and their implementations as ﬂow control devices. The superscripts indicate the ﬂow control mechanism implemented by each ﬂow control device. The ﬁgure classiﬁes ﬂow control mechanisms into 1- separation control [Reproduced from ref. 160, The National Aeronautics and Space Administration (NASA)]160, 2- transition control [Reproduced from ref. 161, The National Aeronautics and Space Administration (NASA)]161, 3- vortex tailoring [Reproduced from ref. 162, The National Aeronautics and Space Administration (NASA)]162. to well-deﬁned ﬂow control mechanisms and classiﬁcations (Table 1). The article mainly focuses on passive and local ﬂow control devices because they can be easily integrated into small engineered vehicles compared to active and global ﬂow control devices. Finally, the article highlights some of the gaps in the literature and the exciting opportunities for bioinspired ﬂow control research. bioinspired ﬂow control techniques help in two crucial ways. First, these techniques provide new insight into biological loco- motion and uncover the physics that enable these organisms to be adaptable and efﬁcient across multiple ﬂight conditions. Second, bioinspired ﬂow control techniques are more suitable for small- scale uncrewed vehicles than traditional approaches developed for large-scale and high-speed operations. Due to the limited volume and payload capabilities of small-scale vehicles, traditional ﬂow control devices’ size, complexity, and weight penalties are prohibitive1,6,7,10,11. Scaling down conventional ﬂow control devices may also not be suitable for the mission demands of small-scale vehicles. Traditional ﬂow control techniques are most effective over a limited range of angles of attack and operational conditions. In contrast, the mission demands and applications of small uncrewed aerial or aquatic vehicles (sUAVs) often require them to operate over a wide range of angles of attack. Thus, biological ﬂow control techniques offer a suitable solution to augment the performance of sUAVs because of the similarities between the operational conditions of these vehicles and the biological organisms. Aerial and aquatic biological and bioinspired ﬂow control strategies Local ﬂow control devices introduce localized changes to the ﬂow structures at a particular area of the lifting surface. In contrast, global ﬂow control devices usually affect the ﬂow over the entire wing or lifting surface. Examples of local ﬂow control devices include synthetic jet actuators or roughness strips, while wing sweep and camber morphing systems would be considered global ﬂow control devices. 3 4 6 7 There have been extensive studies and reviews on ﬂow control techniques and devices3,4,6,7. Most of these studies focus on engineered ﬂow control devices, large-scale aircraft, and fully developed turbulent conditions (Reynolds number (Re) ≥106)8. More recently, studies have focused on bioinspired and biological ﬂow control techniques9. Understanding biological and MMUNICATIONS ENGINEERING \| (2023)2:30 \| https://doi.org/10.1038/s44172-023-00077-0 \| www.nature.com/commseng 1 REVIEW ARTICLE COMMUNICATIONS ENGINEERING \| (2023)2:30 \| https://doi.org/10.1038/s44172-023-00077-0 \| www.nature.com/commseng Avian ﬂow control d h Bird wings have multiple sets of feathers that serve numerous functions, from thermal insulation to improved ﬂight perfor- mance and sound damping13–15. Aerodynamically, aside from generating the necessary aerodynamic forces for ﬂight, feathers also serve as ﬂow control devices15–18. A detailed description of bird wing geometry, internal and external structures, and feathers’ form and function can be found in15. This section reviews four feather systems or feather features for ﬂow control, namely the covert feathers, the alula, the emargination of the primary feathers that create slots towards the wingtips, and leading edge serrations that have been mainly observed in owl feathers. g g This article reviews biological ﬂow control techniques and devices implemented by birds, insects, and aquatic animals and the engineered systems inspired by them. The article expands on recent review articles focused on biological ﬂow control9,12 by including the biological ﬂow control mechanisms and the bioinspired devices inspired by them, highlighting key research gaps and opportunities for sUAVs. The article is divided into three main sections: ﬂow control in avian and avian-scale ﬂiers, ﬂow control in insect and insect-scale ﬂiers, and ﬂow control in marine animals and bioinspired aquatic systems. For each section, the article categorizes the ﬂow control devices according Covert feathers. The covert feathers are a set of contour feathers that exist on the upper and lower surfaces of birds’ wings and tails. Wing coverts consist of multiple overlapping rows or layers, where the row near the leading edge is named the lesser coverts, followed by the median coverts and the greater coverts (Fig. 2a-i)19. Understanding the role of the coverts in avian ﬂight as a ﬂow control device was crucial for the development of similar bio-inspired ﬂow control devices16. In a study done by Carruthers et al.17, they examined the coverts deployment of a steppe eagle (Aquila nipalensis) during different ﬂapping and perching 2 REVIEW ARTICLE COMMUNICATIONS ENGINEERING \| https://doi.org/10.1038/s44172-023-00077-0 Summary of selected biological and bioinspired ﬂow control devices studies. ations under the study type are as follows: E is an experimental study, F is a free-ﬂight experiment, and S refers to numerical simulations. The icons in the ﬂow control mechanis cons used in Fig. 1. Summary of selected biological and bioinspired ﬂow control devices studies. ions under the study type are as follows: E is an experimental study, F is a free-ﬂight experiment, and S refers to numerical simulations. Avian ﬂow control d h also observed that the underwing coverts deploy at take-off, landing, and ﬂapping perching sequences. The underwing coverts, highlighted by the orange circle in Fig. 2, were noticed to deploy all together as one unit, which makes them analogous to Kruger ﬂaps. Kruger ﬂaps pro- trude from the leading edge of engineered wings to augment and extend the lift curve slope20. They are typically used during take- off and landing, which is also when the underwing coverts were noticed to deploy. On the other hand, the greater upperwing coverts (Fig. 2a-i) were noticed to deploy during the downstroke of ﬂapping ﬂight and in response to gust. Moreover, secondary upperwing coverts were noticed to deploy during both gliding and ﬂapping perching sequences. The study suggests that the upperwing and underwing coverts appear to be used for ﬂow control to enhance unsteady maneuvers, and may also provide sensory feedback to the bird17. sequences both indoors and outdoors using a high-speed digital video camera placed on the upper wing. In their study, the cov- erts’ response was classiﬁed as a passive aeroelastic response, since the bird muscles can only apply force at the base of those feathers, while during the experiments, the deployment of the coverts was initiated from the feather tip17. The upperwing and underwing coverts were noticed to deploy during high-angle of attack maneuvers, such as perching, take-off, landing, and gust maneuvers. Carruthers et al. also observed that the underwing coverts deploy at take-off, landing, and ﬂapping perching sequences. The underwing coverts, highlighted by the orange circle in Fig. 2, were noticed to deploy all together as one unit, which makes them analogous to Kruger ﬂaps. Kruger ﬂaps pro- trude from the leading edge of engineered wings to augment and extend the lift curve slope20. They are typically used during take- off and landing, which is also when the underwing coverts were noticed to deploy. On the other hand, the greater upperwing coverts (Fig. 2a-i) were noticed to deploy during the downstroke of ﬂapping ﬂight and in response to gust. Moreover, secondary upperwing coverts were noticed to deploy during both gliding and ﬂapping perching sequences. The study suggests that the upperwing and underwing coverts appear to be used for ﬂow control to enhance unsteady maneuvers, and may also provide sensory feedback to the bird17. Avian ﬂow control d h b-ii An alula-inspired device applied to an engineered wing is also effective at delaying separation compared to a wing without an alula, as indicated by Particle Image Velocimetry (PIV) results43,45. [adapted with permission from ref. 45] c Flow visualization of vortex structures around the slotted wing tip of a jackdaw (i and iii) shows the effect of slots on the vortex structures at the wingtips [adapted with permission from ref. 54] with similar studies performed on engineered wings showing similar vortex structures (ii and iv) [adapted with permission from ref. 65]. Fig. 2 Selected avian and avian-scale ﬂow control devices inspired by feathers. Selected feather systems shown here are the coverts (a), the alula (b), and the slotted wingtips (c). a–i Coverts on the upper surface of a heron wing where the initials PC, SLC, SMC, and SGC stand for Primary Coverts, Secondary Lesser Coverts, Secondary Median Coverts, and Secondary Greater Coverts, respectively. [Source: Pixabay] a-ii Numerical vortex contours show the streamlines superimposed with the vorticity contours. Blue and yellow contours represent clockwise and counterclockwise vorticity, respectively. The ﬁgure shows that the covert-inspired ﬂaps can block reverse ﬂow and enhance lift29. [Adapted with permission from ref. 29] b-i The alula in birds generates tip vortices, which delay separation and boundary layer reversal42. [Reproduced from ref. 42]. b-ii An alula-inspired device applied to an engineered wing is also effective at delaying separation compared to a wing without an alula, as indicated by Particle Image Velocimetry (PIV) results43,45. [adapted with permission from ref. 45] c Flow visualization of vortex structures around the slotted wing tip of a jackdaw (i and iii) shows the effect of slots on the vortex structures at the wingtips [adapted with permission from ref. 54] with similar studies performed on engineered wings showing similar vortex structures (ii and iv) [adapted with permission from ref. 65]. sequences both indoors and outdoors using a high-speed digital video camera placed on the upper wing. In their study, the cov- erts’ response was classiﬁed as a passive aeroelastic response, since the bird muscles can only apply force at the base of those feathers, while during the experiments, the deployment of the coverts was initiated from the feather tip17. The upperwing and underwing coverts were noticed to deploy during high-angle of attack maneuvers, such as perching, take-off, landing, and gust maneuvers. Carruthers et al. Avian ﬂow control d h The icons in the ﬂow control mechanis ns used in Fig 1 Table 1 Summary of selected biological and bioinspired ﬂow control devices studies. COMMUNICATIONS ENGINEERING \| (2023)2:30 \| https://doi.org/10.1038/s44172-023-00077-0 \| www.nature.com/commseng 3 COMMUNICATIONS ENGINEERING \| https://doi.org/10.1038/s44172-023-00077-0 REVIEW ARTICLE Fig. 2 Selected avian and avian-scale ﬂow control devices inspired by feathers. Selected feather systems shown here are the coverts (a), the alula (b), and the slotted wingtips (c). a–i Coverts on the upper surface of a heron wing where the initials PC, SLC, SMC, and SGC stand for Primary Coverts, Secondary Lesser Coverts, Secondary Median Coverts, and Secondary Greater Coverts, respectively. [Source: Pixabay] a-ii Numerical vortex contours show the streamlines superimposed with the vorticity contours. Blue and yellow contours represent clockwise and counterclockwise vorticity, respectively. The ﬁgure shows that the covert-inspired ﬂaps can block reverse ﬂow and enhance lift29. [Adapted with permission from ref. 29] b-i The alula in birds generates tip vortices, which delay separation and boundary layer reversal42. [Reproduced from ref. 42]. b-ii An alula-inspired device applied to an engineered wing is also effective at delaying separation compared to a wing without an alula, as indicated by Particle Image Velocimetry (PIV) results43,45. [adapted with permission from ref. 45] c Flow visualization of vortex structures around the slotted wing tip of a jackdaw (i and iii) shows the effect of slots on the vortex structures at the wingtips [adapted with permission from ref. 54] with similar studies performed on engineered wings showing similar vortex structures (ii and iv) [adapted with permission from ref. 65]. Fig. 2 Selected avian and avian-scale ﬂow control devices inspired by feathers. Selected feather systems shown here are the coverts (a), the alula (b), and the slotted wingtips (c). a–i Coverts on the upper surface of a heron wing where the initials PC, SLC, SMC, and SGC stand for Primary Coverts, Secondary Lesser Coverts, Secondary Median Coverts, and Secondary Greater Coverts, respectively. [Source: Pixabay] a-ii Numerical vortex contours show the streamlines superimposed with the vorticity contours. Blue and yellow contours represent clockwise and counterclockwise vorticity, respectively. The ﬁgure shows that the covert-inspired ﬂaps can block reverse ﬂow and enhance lift29. [Adapted with permission from ref. 29] b-i The alula in birds generates tip vortices, which delay separation and boundary layer reversal42. [Reproduced from ref. 42]. Avian ﬂow control d h As for underwing covert-inspired devices studies, an experimental investigation by Wang et al. compared the aerodynamic effects of ﬂaps, made from real feathers, on the upper and lower sides of a NACA 0012 airfoil at Re = 30, 000. The ﬂap mounted on the suction (upper) side was found to have a positive impact at post-stall angles. On the other hand, when mounted on the pressure (lower) side, the ﬂaps show aerodynamic beneﬁts at small angles of attack (α = −4∘to 8∘). Using PIV measurements, the authors attributed the lift beneﬁts of the pressure side ﬂaps to the increase in leading-edge vorticity and the larger dead ﬂow region in the vicinity of the ﬂapped airfoil pressure surface, leading to generation of high pressure and greater downward ﬂow momentum beneath the pressure surface for the ﬂapped case24. In addition to wind tunnel experiments and numerical simulations, Meyer et al.31 performed free-ﬂight experiments to test the effectiveness of the covert-inspired ﬂaps during actual ﬂight. Freely moving ﬂaps were mounted on top of a STEMME S10 motor glider at Re = 106. It was found that the ﬂap improved maximum lift by about 10% at post-stall angles of attack, which matches the results of both wind tunnel experiments and simulations. Pilot comments indicated that changes to ﬂight behavior were moderate when the ﬂaps were mounted at the inner part of the wing and that keeping ﬂight speed near stall values was easier with the movable ﬂaps attached. On the other hand, the same study examined the use of covert-inspired ﬂaps on swept and tapered wings, and it was found that in these cases, ﬂow separation is dominated by 3D secondary ﬂows, which makes these ﬂaps less effective. Moreover, the ﬂaps were found not to be compatible with vortex generators upstream of the ﬂaps; this is because the vortex generators generate wrapped free shear layers with which the ﬂaps cannot interact in a meaningful way. Furthermore, under attached ﬂow conditions (small angles of attack), the freely moving ﬂaps get slightly raised, which results in a slight drag increase and a lift detriment due to the small separation regime at the end of the ﬂap. f h d d Avian alula. Another feather system investigated for its role in ﬂow control is the alula. The alula is a collection of feathers on the hand-wing of most birds15,37. Avian ﬂow control d h birds covert feathers are limited to the front half of the wing, engineering studies experimented with covert-inspired ﬂaps at different locations ranging from the leading edge of the wing to the trailing edge. Moreover, the implementation of covert- inspired devices in engineering studies ranges from real feathers and hair-like ﬂaps to rigid plastic and metal ﬂaps with various geometries21–24. Furthermore, ﬂaps with various mobility have also been studied, starting from simple static ﬂaps at a ﬁxed angle, to freely moving ﬂaps, to torsionally-hinged ﬂaps with adjustable stiffness25–30. Despite the different structural and mobility forms of the covert-inspired ﬂaps, most studies conﬁrmed that upperwing coverts act as a lift enhancement ﬂow control device at post-stall conditions. p Most of the engineering studies have focused on upperwing covert-inspired devices. Duan and Wissa25 showed that rigid static covert-inspired ﬂaps placed between 40% and 80% of the chord from the leading edge can improve post-stall lift up to 23% at a Re ~ (105). Several studies 26,27,29,31 also examined the effects of freely moving upperwing ﬂaps at Re ~ (105−106). Bechert et al.27,28, and Meyer et al.31 examined the effects of a freely moving ﬂap on two different airfoils using both wind tunnel testing and numerical simulations and observed lift improvement of more than 10% and stall delay in both studies. They attributed the lift enhancement of the trailing edge coverts to the ﬂap blocking the reverse ﬂow and adverse pressure from advancing towards the leading edge of the airfoil in what they called a “pressure dam” effect. Coverts-enabled lift enhancement was also evident at a wide range of laminar low Reynolds number studies, Re ~ (103−104) both numerically and experimentally30,32,33. Nair y Due to their simplicity and aerodynamic advantages, the coverts were adapted on engineering wings in multiple forms to obtain a passive yet adaptive high-lift ﬂow control device for stall mitigation (Fig. 2a-ii). Engineering studies have expanded the parameter space beyond what is in nature; for example, while in COMMUNICATIONS ENGINEERING \| (2023)2:30 \| https://doi.org/10.1038/s44172-023-00077-0 \| www.nature.com/commseng 4 4 REVIEW ARTICLE COMMUNICATIONS ENGINEERING \| https://doi.org/10.1038/s44172-023-00077-0 control during high angle of attack maneuvers to enhance post- stall lift. They improve lift by blocking the reverse separated ﬂow from propagating upstream of the wing. Avian ﬂow control d h We classiﬁed the coverts ﬂow control mechanism as separation control across all Reynolds numbers regimes (Table 1) However, despite many studies on covert-inspired ﬂow control devices, there are still gaps that warrant further investigation. For example, there is a need for more studies focused on the interaction between upperwing and underwing coverts as a function of the ﬂow conditions and the ﬂaps’ properties. Furthermore, the interaction of covert-inspired ﬂaps with traditional ﬂow and ﬂight control techniques such as ﬂaps, ailerons, and slats has not been studied. Finally, covert- inspired devices have yet to be implemented on small-scale UAVs during ﬂight to evaluate their beneﬁts and downsides critically and assess how these ﬂaps change the wing’s stability parameters and the aircraft’s propulsive efﬁciency. and Goza33 and Othman et al.29 analyzed the ﬂuid-structure interaction of an upperwing torsionally hinged covert-inspired ﬂap at two different Re regimes (Re = 1000 and Re = 200, 000) to understand the effect of different ﬂap structural properties (i.e., ﬂap inertia and hinge stiffness) on the vortex structure and shedding frequency. Their study shows that low-inertia ﬂaps were more effective at improving lift than high-inertia ﬂaps. They also found that there are several similarities between both Reynolds numbers. For example, the covert-inspired ﬂaps do not alter the vortex shedding frequency of the original airfoil at both high and low Re ﬂows, with some unsteadiness for the high Re regimes due to turbulent transition29. Thus, despite the difference in the ﬂow physics between laminar [Re ¼ Oð103Þ] to transitional [Re ¼ Oð104 105Þ] to turbulent ﬂows [Re ¼ Oð106Þ], the effectiveness of the covert-inspired ﬂaps in improving lift across Reynolds numbers regimes suggests their suitability as a ﬂow control device for different ﬂight regimes and different UAV conﬁgurations24,32–34. g Fewer studies have considered the role of multiple upperwing covert-inspired ﬂaps and underwing devices. Duan and Wissa studied the effect of multiple ﬂaps arranged chordwise on a 2D airfoil at a Re ~ (105), and they found that the ﬂaps have an additive effect on lift post-stall where the lift improvements increased with the number of ﬂaps35. Nair et al.36, also studied the effect of multiple ﬂaps numerically at a much lower Re ~ (103), and found that at this low Re deploying a single ﬂap near the trailing edge alone results in the greatest lift improve- ment compared to the multiple ﬂap cases. COMMUNICATIONS ENGINEERING \| (2023)2:30 \| https://doi.org/10.1038/s44172-023-00077-0 \| www.nature.com/commseng Avian ﬂow control d h Structurally, the alula is a skeletal bone digit named the alular digit. It is attached to the carpome- tacarpus bone on the wing15. The alula consists of 2–3 feathers at the leading edge of most birds’ wings15,37. On average, the alula size varies between one-ﬁfth to one-tenth of the total span of a bird’s wing15,37,38. Birds with high wing loading or that frequently maneuver at high angles of attack are the birds with the most pronounced alula according to the weight, span, and alula mea- surements reported in reference13. Savile et al. also studied the evolution of the avian wing with a focus on the aerodynamic performance of different birds. They observed that birds mainly used their alulas during low-speed ﬂight control for landing, takeoff, and perching maneuvers. The connection between the alular digit and the carpometacarpus allows complex motion of the alula, which facilitates the performance of such maneuvers. The alula moves both in-plane and out of the plane of the wing, meaning it can abduct, adduct, pronate, and supinate15,37. The alula can be found in insects and birds. We focus on birds’ alula in this section and insects’ alula in a later section. Fewer studies have considered the role of multiple upperwing covert-inspired ﬂaps and underwing devices. Duan and Wissa studied the effect of multiple ﬂaps arranged chordwise on a 2D airfoil at a Re ~ (105), and they found that the ﬂaps have an additive effect on lift post-stall where the lift improvements increased with the number of ﬂaps35. Nair et al.36, also studied the effect of multiple ﬂaps numerically at a much lower Re ~ (103), and found that at this low Re deploying a single ﬂap near the trailing edge alone results in the greatest lift improve- ment compared to the multiple ﬂap cases. As for underwing covert-inspired devices studies, an experimental investigation by Wang et al. compared the aerodynamic effects of ﬂaps, made from real feathers, on the upper and lower sides of a NACA 0012 airfoil at Re = 30, 000. The ﬂap mounted on the suction (upper) side was found to have a positive impact at post-stall angles. On the other hand, when mounted on the pressure (lower) side, the ﬂaps show aerodynamic beneﬁts at small angles of attack (α = −4∘to 8∘). Avian ﬂow control d h Using PIV measurements, the authors attributed the lift beneﬁts of the pressure side ﬂaps to the increase in leading-edge vorticity and the larger dead ﬂow region in the vicinity of the ﬂapped airfoil pressure surface, leading to generation of high pressure and greater downward ﬂow momentum beneath the pressure surface for the ﬂapped case24. dd d l d l Aerodynamically, the alula is a post-stall lift enhancement device studied in the literature through numerical simulations, wind tunnel testing, and ﬂight observation of birds. Savile et al13. proposed that the mechanism by which the alula works is such that it acts as a leading-edge slotted ﬂap that deploys passively by large birds or small active birds. The slotting accelerates the ﬂow between the wing and the ﬂap, which mixes and energizes the ﬂow past the ﬂap, thus delaying separation and increasing maneuverability. Multiple studies hypothesized that the deploy- ment of the alula is passive37,39,40. However, Carruthers et al.41 observed the alula deployment through video recording and divided it into two stages: a) passive peeling in response to changes in the ﬂow conditions, and b) active protraction of the alula through a 45∘sweep forward to the leading edge41. Austin et al.39 went further and proposed an additional mechanism for the alula. They proposed that the alula suppresses the vortex bursting over the upper surface by generating vortical structures that maintain the lift over the hand wing. Lee et al.42 studied the alula based on ﬂight observations and wind tunnel experiments. Using digital particle image velocimetry, Lee supported the hypothesis in ref. 39 and showed that the alula increases lift at high angles of attack (AoA) by working as a vortex generator (Fig. 2b-i). momentum beneath the pressure surface for the ﬂapped case . In addition to wind tunnel experiments and numerical simulations, Meyer et al.31 performed free-ﬂight experiments to test the effectiveness of the covert-inspired ﬂaps during actual ﬂight. Freely moving ﬂaps were mounted on top of a STEMME S10 motor glider at Re = 106. It was found that the ﬂap improved maximum lift by about 10% at post-stall angles of attack, which matches the results of both wind tunnel experiments and simulations. Pilot comments indicated that changes to ﬂight behavior were moderate when the ﬂaps were mounted at the inner part of the wing and that keeping ﬂight speed near stall values was easier with the movable ﬂaps attached. REVIEW ARTICLE During ﬂight, such slots cause the wingtip feathers to separate or spread horizontally and vertically13,18. Liu et al. present a detailed review of the aerodynamic effects of these wingtip slots on biological and engineered wings52. However, we will summarize here some of the key results and the enabling physics of these ﬂow control devices. Wind tunnel experiments of birds’ wings with the slotted wingtips have detailed the aerodynamic beneﬁts of slotted wingtips18,53–56. Many of these studies noted that the slotted wingtips reduce the lift-induced drag of the wing. The drag reduction caused by slotted wingtips can be attributed to the shape of the wingtips during ﬂight under aerodynamic loading. In ﬂight, due to aerodynamic loading, the tips of the slotted wingtips bend upwards and separate vertically, while the broad root of the slotted wingtips overlap, limiting root twist and bending. The bending and separation at the tip of the slotted wingtips form a non-planar slotted wingtip conﬁguration18,53,54. The non-planar wing can generate less induced drag and similar lift compared to a planar wing, that is straight from tip to tip, at the same speed18,53. For example, Tucker et al. show that the unclipped wingtips, which form a non-planar conﬁguration, have 70% to 90% of the drag of the clipped wingtips, which form a planar conﬁguration53. Hence, slotted wingtips can improve aerodynamic efﬁciency, deﬁned as the ratio of lift to drag, by generating a similar lift with less drag than a wing without slots. Linehan and Ito studied the alula experimentally in a steady ﬂow at Reynold’s number 75,000 and 100,000–120,000, respec- tively. Linehan studied the effects of the different parameters by modeling the wing and alula as ﬂat plates. Ito et al. added complexity to the geometry using an S1223 airfoil and a bird- inspired alula proﬁle. However, both studies lack the addition of ﬂapping or aeroelastic effects. A study by Bao49 added the ﬂapping complexity by studying a sinusoidal ﬂapping input numerically on an alula similar in geometry and ﬂow conditions to Ito. The numerical solver used Unsteady Reynold’s-Averaged Navier-Stokes with a κ–ω turbulence model. Results support that the alula works through slat and vortex interaction effects. Further, it shows that the slot effect dominates at the start of the upstroke. However, the vortex effect dominates in the midpart of the upstroke, and both play a role in the downstroke. REVIEW ARTICLE The authors also report the effect of different design parameters on the alula trailing edge vortex and the alula streamwise vortex, affecting the lift response49. Finally, Zekry et al.50 used a design of experiment approach with linear regression models to produce response surfaces for lift and drag forces as a function of the ﬂight conditions and the alula parameters, which can be later used in control laws for ﬂight control systems. Flow visualization was also used to understand the effect of the wingtip slots on the ﬂow ﬁeld and tip vortices around birds’ wings. KleinHeerenbrink et al. used particle image velocimetry to measure the airﬂow around the slotted wingtip of a jackdaw (Corvus monedula) in a wind tunnel during gliding and ﬂapping ﬂight. Their results conﬁrm that the separated primary feathers produce individual wingtip vortices, spreading the overall wingtip vorticity in the horizontal and vertical planes, which was previously associated with improved efﬁciency54. March et al. also used ﬂow visualization around the wingtips of a Great Horned Owl (Bubo virginianus). Their results show smooth streamlines near the slotted wingtips55. Thus, these ﬂow visualization studies suggest that the slotted wingtips spread the vorticity behind the wings, decompose the upwash from the tip vortex, and reduce the induced drag (Fig. 2c-i,iii)54,55. Researchers continued to study the physics of the slotted wingtips through the application and study of engineered wingtips/wingtip sails. Wingtips or wingtip sails are small devices attached to the tip of engineered wings, inspired by the slotted wingtips, to enhance the aerodynamic performance57–59. Some of the wingtip parameters explored in the literature include the number of wingtips, the dihedral, twist, incidence angles of individual wingtips, and the gap between the wingtips. Results show that using multiple wingtips with various dihedral angles reduces the overall tip vortex by splitting it into smaller vortices and reducing the effective downwash at the wing (Fig. 2c-ii,iv). These effects often resulted in increased aerodynamic efﬁciency and delayed stall without incurring considerable lift penalties52,57–67. Negative incidence angles have increased the aerodynamic efﬁciency by re-orienting the lift vector forward and counteracting the drag62,63,68. Finally, the increase in the gap distance between the wingtips was shown to weaken the tip- vortex suppression52,65, suggesting the importance of tuning the gap size to enable vorticity spreading and wingtip vortex suppression. REVIEW ARTICLE REVIEW ARTICLE COMMUNICATIONS ENGINEERING \| https://doi.org/10.1038/s44172-023-00077-0 ﬂow control devices on sUAVs (Fig. 2b-ii)). Mandadzhiev et al. mounted an alula-inspired device on an S1223 airfoil (i.e., a 2D experiment)43. Their study shows that the alula-inspired device enhanced lift at post-stall angles of attack and that the lift enhancements are a function of the device’s deployment parameters. Ito44,45 expanded on Mandadzhiev’s study43 by mounting the alula-inspired device on a ﬁnite wing rather than an airfoil (i.e., a 3D experiment). The study showed that the lift enhancements for the 3D experiment (37%) were more pronounced compared to the 2D experiment (8%). The additional lift enhancement was attributed to the hypothesis that the alula- inspired device works not only as a slotted ﬂap but also that it generates a tip vortex that acts as a boundary layer fence preventing the propagation of stall outboard44. Thus, Ito’s44,45 results show that the alula is a 3D post-stall lift enhancement device, supporting biological observation in refs. 39,37,46. Linehan et al.47,48 used PIV to study the vortical structures created by the alula-inspired device and their interaction with the wing vortices. The authors proposed that the alula works through leading-edge vortex roll-up and tilting. More speciﬁcally, the alula causes a spanwise ﬂow which energizes the shear layer and directs the ﬂow towards the wingtip vortex, enhancing post-stall lift production47,48. In further work, Linehan et al.38 studied the scale effect and location of the alula, showing that a lift- maximizing position also corresponds to the actual evolutionary location of alula in birds. Slotted wingtips. Another local and passive feather ﬂow control system in birds is the wingtip slots. In some species, the ﬁve to six primary feathers at the tip of the wings emarginate, or narrow down, forming slots or gaps near the wingtip. During ﬂight, such slots cause the wingtip feathers to separate or spread horizontally and vertically13,18. Liu et al. present a detailed review of the aerodynamic effects of these wingtip slots on biological and engineered wings52. However, we will summarize here some of the key results and the enabling physics of these ﬂow control devices. Slotted wingtips. Another local and passive feather ﬂow control system in birds is the wingtip slots. In some species, the ﬁve to six primary feathers at the tip of the wings emarginate, or narrow down, forming slots or gaps near the wingtip. COMMUNICATIONS ENGINEERING \| (2023)2:30 \| https://doi.org/10.1038/s44172-023-00077-0 \| www.nature.com/commseng Avian ﬂow control d h On the other hand, the same study examined the use of covert-inspired ﬂaps on swept and tapered wings, and it was found that in these cases, ﬂow separation is dominated by 3D secondary ﬂows, which makes these ﬂaps less effective. Moreover, the ﬂaps were found not to be compatible with vortex generators upstream of the ﬂaps; this is because the vortex generators generate wrapped free shear layers with which the ﬂaps cannot interact in a meaningful way. Furthermore, under attached ﬂow conditions (small angles of attack), the freely moving ﬂaps get slightly raised, which results in a slight drag increase and a lift detriment due to the small separation regime at the end of the ﬂap. In more recent years, a transition towards using engineering wings with alula-inspired devices occurred in order to system- atically study the design parameters of the alula and their effect on aerodynamic performance, as well as to implement them as g In summary, covert feathers and covert-inspired systems can be considered high-lift devices that can be used for separation 5 Insect-scale ﬂow control Leading edge serrations (LES), deﬁned as comb-like hooks structures that exist on the outer vanes of the feathers at the leading edge of the wing, and trailing edge fringes, deﬁned as unconnected barbs at the ends in feathers, have been studied extensively for noise reduction and silent ﬂight, especially in owls14,70–76. However, given the focus of this article on ﬂow control, rather than noise suppression, we will highlight the few studies related to the role of leading edge serrations on aero- dynamic tailoring. Alongside their noise-canceling capabilities, LES can be considered ﬂow control devices for their role in improving aerodynamic efﬁciency and tailoring laminar to turbulent transition70,72,77. In 1971, Kroeger et al.74 used ﬂow visualization on two prepared owl wings and found that LES act as co-rotating vortex generators, which stabilize the ﬂow over the upper surface of the owl’s wing and prevent laminar separation. As a result, these vortices are able to drive higher momentum ﬂow towards the wall surface, which can also delay stall, similar to vortex generators. These ﬁndings inspired engineering studies using similar serrations designs on traditional airfoils and rotor blades to test their functionality as a passive ﬂow control device. In the post-stall regime, multiple studies show that the LES can effectively improve the aerodynamic performance of the stalled airfoil by increasing lift, reducing drag, and reducing the ﬂuctuation of aerodynamic forces78–80. Wenzen et al. and Rao and Hiu81,82 studied the Reynolds number dependency of LES and found that they are effective as post-stall ﬂow control devices for Re ≥40, 000. Muthuramalingam et al.83 studied the LES effect on 3D backward swept wings and found that the serrations have a ﬂow-turning effect that counteracts the outboard cross-span ﬂow that typically appears for swept-back wings. This effect can attenuate cross-ﬂow instabilities and delay laminar to turbulent transition. g While the aerodynamic properties of several insect species (e.g., dragonﬂies, damselﬂies, hoverﬂies, butterﬂies, locusts, fruit ﬂies, blowﬂies, and beetles) have been investigated, most studies have focused on the corrugated wings of dragonﬂies since they exhibit higher aerodynamic efﬁciency (lift to drag ratio) compared to those of other insects89–92,94–97,99–105. Results from the aero- dynamic force measurements show that the corrugated wings of dragonﬂies generally have higher aerodynamic efﬁciency (lift-to- drag ratio) compared to a ﬂat plate and streamlined airfoils100–102. Insect-scale ﬂow control Insect wings generate aerodynamic forces to accelerate or to stay aloft in the air. Unlike ﬂying vertebrates, like birds and bats, which use limbs and muscles to control their wing shape, the muscles of insects do not extend beyond the body. Hence, the shape of the insect wing is controlled passively as its ﬂexible wing constantly deforms in response to in-ﬂight forces84–86. g p Despite the numerous studies on the aerodynamic effects of wingtip slots, an optimal conﬁguration across all vehicles or systems does not exist since the aerodynamic beneﬁts of using engineered wingtips depend on the in-ﬂight conditions such as the Reynolds number and angle of attack. There is also a need for an adaptive slotted wingtip system62,67,68. An adaptive system is needed to adjust the wingtip dihedral angle, incidence angle, and gap space as a function of the ﬂight conditions to yield the desired aerodynamic effects. Additionally, most of the research on engineered wingtips was conducted in wind tunnels and through numerical simulations, rather than ﬂight tests on actual ﬂight vehicles. Hence, more studies on the applications of engineered wingtips are required to assess the in-ﬂight aerodynamic performance for future research59,69. Wing corrugations. One of the main features of insect wings is corrugation. Wing corrugations provide aerodynamic beneﬁts by controlling airﬂow, and structural beneﬁts by withstanding aerodynamic loads during ﬂight87,88. This review mainly focuses on the aerodynamic role of wing corrugations. y g g There have been several morphological studies focused on characterizing the corrugations of insect wings using 3D recon- struction of cross-sectional 2D imaging (photogrammetry)89–95 and 3D scanning (micro-CT) techniques96 (Fig. 3-i). Morphological studies on the wings of dragonﬂies, damselﬂies, hoverﬂies, butterﬂies, locusts, fruit ﬂies, blowﬂies, and beetles show that the wings of the insects are corrugated and their wing cross sections at different spanwise locations display varying corrugation patterns89–97. Following the morphological studies, there have been a few wind tunnel experiments conducted on the actual insects to study their wing’s aerodynamics. Experiments on dragonﬂies show an increase in maximum lift coefﬁcient and maximum lift-to-drag ratio compared to a ﬂat plate (Fig. 3-ii)93,98. However, studies on actual insect wings are limited, given their scale and measurement limitations. Thus, most studies on the aerodynamic effects of corrugations are performed either numerically or experimentally on engineered wing sections with corrugation proﬁles measured from morphological studies. Leading edge serrations. REVIEW ARTICLE COMMUNICATIONS ENGINEERING \| https://doi.org/10.1038/s44172-023-00077-0 because they mainly spread the wingtip vortices (Table 1). Such a ﬂow control mechanism was also implemented for ﬂight control. The induced drag effect of the wingtips was shown to provide yaw stability when implemented asymmetrically on the left and right wings56. Some researchers have also applied wingtip slots/sails to aircraft during ﬂight52,59. In general, ﬂight testing results conﬁrmed the wind tunnel results, indicating that slotted wingtips or wingtip sails can be used to improve aerodynamic efﬁciency by reducing drag compared to a planar or a conventional wingtip. acoustic wind tunnels, which differs from real scenarios in which turbulence may have a notable impact. Finally, the interaction between LES and other traditional or bio-inspired ﬂow control techniques has not been fully studied, so there is a lack of understanding of how LES would alter the ﬂight envelope or stability parameters of an actual ﬂying vehicle. COMMUNICATIONS ENGINEERING \| (2023)2:30 \| https://doi.org/10.1038/s44172-023-00077-0 \| www.nature.com/commseng REVIEW ARTICLE In summary, the alula is a very effective high-lift device as it can maintain ﬂow attachment over the wing at high AoA and low- speed ﬂight. It accelerates the ﬂow between the wing and the alula lower surface, energizing the ﬂow and delaying separation. The alula also generates a vortex at its tip that acts as a boundary layer fence to prevent the propagation of stall outboard of the wing. Thus, we classiﬁed the ﬂow control mechanisms for the alula and alula-inspired devices as separation control and vortex tailoring (Table 1). Furthermore, in addition to ﬂow control, alula-inspired devices can be used for ﬂight control on sUAVs if deployed asymmetrically on the left and right wings, as they can produce lift and drag differentials resulting in rolling and yawing moments51. Despite all these advantages, alula literature is still limited and the structure is yet to be implemented on ﬂight vehicles. Alula- inspired devices should also be tested in ﬂight on vehicles across various ranges of speeds and scales. Future work should also focus on characterizing the alula’s dynamics and aeroelasticity during gliding and ﬂapping ﬂights, since no papers discuss these issues. Thus, in both biological and engineering studies, the ﬂow control mechanism for the slotted wing tips is vortex tailoring 6 REVIEW ARTICLE Insect-scale ﬂow control 3 Summary of biological and bioinspired studies on the role of wing corrugations in insects. There have been several (top) biological and (bottom) bioinspired studies on insect wings especially focusing on the role of corrugations. (i) Wing corrugations in the hind (left) and fore (right) wings of dragonﬂies vary along the chord and span [adapted with permission from ref. 93]. (ii) Drag polar diagrams from wind tunnel force measurements of fore (left) and hind (right) wings of a dragonﬂy at different Reynolds numbers showing high L/D ratio across the range of Reynolds number tested [adapted with permission from ref. 98]. The effect of insect wing corrugations has also been studied on engineered wing sections (iii and iv). (iii) Corrugations trap vortices in the peaks and valleys of the wing proﬁle. Trapped vortices energize the boundary layer and mitigate ﬂow separation. (iv) Compared to a ﬂat plate proﬁle (right), the corrugated proﬁle (left) produces 32% higher L/D [adapted with permission from ref. 103]. ﬂexibility, and deformation are needed to understand the aerodynamic effects of corrugated wings better. corrugations are ﬁlled in with recirculating vortices, the wing maintains a laminar boundary layer, and the shear drag of the system is reduced. Shear drag is a major contributor to the total drag at a lower Reynolds number, as viscous effects are more dominant. At Reynolds number of 10,000, the shear drag produced by the corrugated airfoil constitutes 24% of the total drag, in contrast to 65% for a smooth airfoil or a ﬂat plate103. In summary, the thin and lightweight corrugated wings of insects act like a proﬁled cross-section for aerodynamic beneﬁts with the advantages of low mass, reduced overall drag, and delayed separation. Insect alula. Another local ﬂow control device that has been investigated for insects is the alula. The alula in insects is a hinged ﬂap found at the base of the wings of some insects. The alula is actuated via a component at the wing hinge. During ﬂight, the alula can either be ﬂipped or ﬂat113. In their study, Walker et al. studied the wing beat cycle of hoverﬂies with the alula in the ﬂipped or ﬂat states. The alula state was associated with different wing kinematics. The study concluded that the alula state might indicate the ﬂight mode, and that when the alula is ﬂipped, the wings produce less aerodynamic forces113. Insect-scale ﬂow control For instance, a corrugated proﬁle based on dragonﬂies wing generates 32% and 5% higher lift to drag ratio compared to a ﬂat plate and the streamlined airfoil, respectively at α = 5∘and Re = 10,000103. Studies show that the aerodynamic enhancements are due to the increase in lift92,100,102 and drag reduction101. Flow visualization experiments, such as PIV, and numerical simulations have also been used to analyze the ﬂow ﬁelds around corrugated wings. The results from the experiments and simulations on the corrugated wing sections of dragonﬂies, hoverﬂies, butterﬂies, and locusts show that the vortices generated by corrugated wings play an important role in delaying ﬂow separation89–91,94,101–106. Results show vortices trapped in between the peaks and valleys of the corrugated wings (Fig. 3- iii, iv). These vortices induce high-velocity ﬂow, energizing the boundary layer, and mitigating ﬂow separation94,101,102,104. In addition, the vortices between the peaks and valleys alter the effective wing proﬁle making it more airfoil-like. As the Leading-edge serrations implement various ﬂow control mechanisms, including transition and separation control, as well as vortex tailoring (Table 1). However, despite their potential as ﬂow control devices, there is still no full understanding of the exact physical mechanism by which LES mitigate stall and tailor transition, preventing implementation on existing UAVs. More- over, most studies on LES have been conducted in low-turbulence 7 7 REVIEW ARTICLE COMMUNICATIONS ENGINEERING \| https://doi.org/10.1038/s44172-023-00077-0 Fig. 3 Summary of biological and bioinspired studies on the role of wing corrugations in insects. There have been several (top) biological and (bottom) bioinspired studies on insect wings especially focusing on the role of corrugations. (i) Wing corrugations in the hind (left) and fore (right) wings of dragonﬂies vary along the chord and span [adapted with permission from ref. 93]. (ii) Drag polar diagrams from wind tunnel force measurements of fore (left) and hind (right) wings of a dragonﬂy at different Reynolds numbers showing high L/D ratio across the range of Reynolds number tested [adapted with permission from ref. 98]. The effect of insect wing corrugations has also been studied on engineered wing sections (iii and iv). (iii) Corrugations trap vortices in the peaks and valleys of the wing proﬁle. Trapped vortices energize the boundary layer and mitigate ﬂow separation. (iv) Compared to a ﬂat plate proﬁle (right), the corrugated proﬁle (left) produces 32% higher L/D [adapted with permission from ref. 103]. Fig. COMMUNICATIONS ENGINEERING \| (2023)2:30 \| https://doi.org/10.1038/s44172-023-00077-0 \| www.nature.com/commseng Insect-scale ﬂow control a Surface Secretions157 [source: Pixabay]- A 2D simulation of a ﬁsh represented by a deforming ﬂapping foil (top view) in ﬂuids of varying viscosity. From left to right, the columns show data from a shear-thinning ﬂuid, a Newtonian ﬂuid, and a shear-thickening ﬂuid. The shear-thinning ﬂuid represents a simpliﬁed consistency of ﬁsh slime. (I) The wake of the foil in the shear-thinning ﬂuid indicates thrust rather than drag, which is seen in the other two ﬂuids. The colors indicate vorticity, where blue indicates a non-dimensional value of −5 and red indicates 5. (II) Plotted are contours for the second invariant of rate of the strain tensor, where blue and red indicate non-dimensional values of to 400, respectively. The boundary layer is seen to be thinner for the shear-thinning ﬂuid, suggesting reduced drag156. b Dorsal125 and Anal126 Fins, [source: Pixabay]- PIV visualizations of lateral momentum transfer jets induced by dorsal and anal ﬁns of a rainbow trout (top view). Shown is the wake of the dorsal ﬁn (left edge) as it approaches the caudal ﬁn (right edge). This wake, which is the incident ﬂow seen by the caudal ﬁn, contains alternating vortices which are markedly different from the freestream. Jets are also visible in the wake, directed laterally for stability124. c Tubercles133 [source: Pixabay]- Comparison of lift coefﬁcient, CL, drag coefﬁcient, CD, and lift-to-drag ratio, L/D, results from steady wind tunnel experiments for a whale ﬂipper model with tubercles (open circles) and one without tubercles (closed circles). Stall is delayed by ~5° in the model with tubercles. At higher angles of attack, AoA, lift is higher, drag is lower, and thus L/D is higher for the ﬁn with tubercles132. d Denticles146 [reproduced from ref 146, 2019, Biomimetics]146 Time resolved-DPIV results for ﬂow over a plate without shark skin (I) and one with shark skin (II). The shark skin covered plate causes transition to occur sooner, reattachment to occur more rapidly, and induces a smaller laminar separation bubble141. control from a bioinspired perspective while building on the previous review with more recent studies. control from a bioinspired perspective while building on the previous review with more recent studies. mostly ﬂat plate wings, with most focusing on designing actuation mechanisms at the root of the wing. Insect-scale ﬂow control Therefore, future research on the application of insect wings’ corrugations, ﬂexibility, and deformations on insect-scale robots can be used to understand insect ﬂight better and enhance current robotic wings with the aerodynamic and structural beneﬁts highlighted by the previous research115,116. Fins. Fish ﬁns have a variety of uses, such as sensing, locomotion, and ﬂow control118, but when it comes to ﬂow control, the median ﬁns play the most signiﬁcant role in many ﬁsh119. The median ﬁns in ﬁsh include the dorsal and anal ﬁns, which are centered along the mid-lines of the dorsal and ventral surfaces of many ﬁsh120,121. The wakes of these ﬁns determine the nature of the incident incoming ﬂow seen by the caudal ﬁn, a median ﬁn primarily used for thrust generation. Depending on the ﬁsh species and the swimming speed, the dorsal ﬁn may undulate and generate its own vortical wake with an average velocity higher than that of the free-stream122. This wake then becomes the incoming ﬂow seen by the caudal ﬁn and is hypothesized to increase the momentum shed in the caudal wake123 (Fig. 4b)124–126. PIV visualizations of the dorsal ﬁn wake also show lateral jets that are suggested to be used for stability124,127. While studies have mostly been performed on dorsal ﬁns, anal Insect-scale ﬂow control a Surface Secretions157 [source: Pixabay]- A 2D simulation of a ﬁsh represented by a deforming ﬂapping foil (top view) in ﬂuids of varying viscosity. From left to right, the columns show data from a shear-thinning ﬂuid, a Newtonian ﬂuid, and a shear-thickening ﬂuid. The shear-thinning ﬂuid represents a simpliﬁed consistency of ﬁsh slime. (I) The wake of the foil in the shear-thinning ﬂuid indicates thrust rather than drag, which is seen in the other two ﬂuids. The colors indicate vorticity, where blue indicates a non-dimensional value of −5 and red indicates 5. (II) Plotted are contours for the second invariant of rate of the strain tensor, where blue and red indicate non-dimensional values of to 400, respectively. The boundary layer is seen to be thinner for the shear-thinning ﬂuid, suggesting reduced drag156. b Dorsal125 and Anal126 Fins, [source: Pixabay]- PIV visualizations of lateral momentum transfer jets induced by dorsal and anal ﬁns of a rainbow trout (top view). Shown is the wake of the dorsal ﬁn (left edge) as it approaches the caudal ﬁn (right edge). This wake, which is the incident ﬂow seen by the caudal ﬁn, contains alternating vortices which are markedly different from the freestream. Jets are also visible in the wake, directed laterally for stability124. c Tubercles133 [source: Pixabay]- Comparison of lift coefﬁcient, CL, drag coefﬁcient, CD, and lift-to-drag ratio, L/D, results from steady wind tunnel experiments for a whale ﬂipper model with tubercles (open circles) and one without tubercles (closed circles). Stall is delayed by ~5° in the model with tubercles. At higher angles of attack, AoA, lift is higher, drag is lower, and thus L/D is higher for the ﬁn with tubercles132. d Denticles146 [reproduced from ref 146, 2019, Biomimetics]146 Time resolved-DPIV results for ﬂow over a plate without shark skin (I) and one with shark skin (II). The shark skin covered plate causes transition to occur sooner, reattachment to occur more rapidly, and induces a smaller laminar separation bubble141. Fig 4 A summary of major ﬂow control strategies found in swimming organisms A model swimmer is presented in the center as a generic ﬁsh163 Fig. 4 A summary of major ﬂow control strategies found in swimming organisms. A model swimmer is presented in the center as a generic ﬁsh163 [source: Pixabay], with speciﬁc examples of aquatic ﬂow control devices indicated by the inserted graphics144,164. COMMUNICATIONS ENGINEERING \| (2023)2:30 \| https://doi.org/10.1038/s44172-023-00077-0 \| www.nature.com/commseng Insect-scale ﬂow control In a more recent study, numerical simulations were used to investigate the role of the alula on a simpliﬁed rectangular wing. Their results show that the wings with the alula require less aerodynamic power than those without the alula. The authors also show that the phasing between the wing and the alula matters. The wing with alula ﬂapping in phase produces the largest lift, but the efﬁciency is the lowest. The alula also provides a stabilizing effect on the leading edge vortex for the wings when the alula ﬂaps 45∘ahead or in phase114. p In contrast to studies that support the aerodynamic role of the corrugates, a few studies disagree with the aerodynamic beneﬁts of corrugations89,91,94,105,107. Experimental and numerical studies found that structural corrugation does not signiﬁcantly impact aerodynamic performance89,94,105,107. The corrugated and ﬂat plates produced similar aerodynamic forces, and some even show that the corrugations increased drag at the peak gliding ratios. Flow simulations have demonstrated that corrugated wings produce high-pressure drag compared to ﬂat plates due to the increase in the thickness of the viscous region91,105,107. Studies thus far have focused on the static aerodynamic effects of corrugations. However, insects mainly use ﬂapping ﬂight for locomotion. Hence, there is a need for experiments and simulations focused on the role of corrugations during ﬂapping to implement such proﬁles on insect-scale robots. Moreover, corrugated insect wings undergo deformation due to their ﬂexibility during ﬂapping ﬂight, and limited studies have investigated the combined effects of the aerodynamic, ﬂow control effects, ﬂexibility, and deformation of ﬂapping corrugated wings108–112. Therefore, studies incorporating wing kinematics, Table 1 highlights some studies on insect ﬂow control devices. The ﬂow control mechanism for the wing corrugations combines all three mechanisms identiﬁed in this article, while the alula’s mechanism can be classiﬁed as mainly vortex tailoring. Compared to avian and avian-inspired ﬂow control devices, insects’ passive and local ﬂow control devices are less studied, except for the wing corrugations. There have been few implementations of insect-inspired ﬂow control devices on engineered vehicles. Current ﬂying insect-scale robots have 8 REVIEW ARTICLE COMMUNICATIONS ENGINEERING \| https://doi.org/10.1038/s44172-023-00077-0 Fig. 4 A summary of major ﬂow control strategies found in swimming organisms. A model swimmer is presented in the center as a generic ﬁsh163 [source: Pixabay], with speciﬁc examples of aquatic ﬂow control devices indicated by the inserted graphics144,164. COMMUNICATIONS ENGINEERING \| https://doi.org/10.1038/s44172-023-00077-0 COMMUNICATIONS ENGINEERING \| https://doi.org/10.1038/s44172-023-00077-0 ﬁns have been shown to behave nearly symmetrically to the dorsal ﬁns in some species and thus have similar impacts on the caudal ﬁn incident ﬂow. These ﬁns often work together such that the rolling torques generated by one stabilize those generated by the other127. A notable robotic implementation of dorsal and anal ﬁns for thrust improvement is seen in ref. 128, where model dorsal and anal ﬁns are mounted to a robotic ﬁsh. Linear acceleration of the mechanical model was improved by up to 32.5% when the ﬁns were added. Another example is a snake-like swimming robot whose swimming speed is observed to change depending on parameters including ﬁn spacing and the amplitudes and angular velocities of the oscillatory swimming waveform129. Several other robotic dorsal/anal ﬁn applications exist but are focused mainly on their role in stability120,130, rather than ﬂow control. case study, the mucus layer in puffer ﬁsh is observed to decom- pose as velocity increases. However, drag is still reduced due to the newly uncovered tips of their spines that texture the surface of the ﬁsh. Thus, the mechanism changes from reducing skin- friction drag via a slip layer to reducing pressure drag via the textured surface153. The mucus layer as an engineering ﬂow control strategy is problematic due to the gradual degradation of the layer in the presence of an external ﬂow156. To combat this issue, Lee et al. presented a slippery lubricant-infused surface, “LIS,” which was shown to be robust when exposed to steady shear from the surrounding ﬂuid while reducing friction drag by 18%150. Again, it would be interesting to see how this ﬂuid withstands shear in accelerating external ﬂows similar to the ones experienced by ﬁsh. Other ﬂow control strategies that are not discussed in detail in the present work include several studies on dolphins, which are known to be highly efﬁcient swimmers. Wainwright et al.158 demonstrated that ridges on dolphin skin may improve sprinting performance, but the data is not conclusive. Another recent study on the color of dolphin skin strongly suggests that darker colors on the dorsal surfaces of swimmers can reduce skin friction in turbulent conditions due to thermal effects159. Surface structures. In whale ﬁns, “tubercles” are the structures of interest for ﬂow control. Tubercles are lumps located on the leading edge of their pectoral ﬁns131. Outlook Thi i This article highlighted several passive and local biological and bioinspired ﬂow control devices for birds, insects, and aquatic animals. The number of species and animals that inspire these ﬂow control devices shows the efﬁciency of these devices in favorably altering the ﬂow across scales and locomotion regimes. Even though ﬂow control devices in nature have different morphologies and manifestations, they all modulate the ﬂow through three common mechanisms: separation control, transi- tion control, and vortex tailoring. The studies highlighted in this article also showcase the diverse research methods involving these devices, ranging from experimental studies using wind tunnels and free-ﬂight tests to numerical simulations and analytical models. Another widely studied surface feature for aquatic ﬂow control, primarily known to reduce drag, is shark scales, also known as “denticles”140. Each denticle has a ridge known as a “riblet” that is aligned with the body axis of the shark, creating a rough texture on the shark skin141. Riblets have been observed to decrease turbulent skin friction drag in steady ﬂow independently of whether they are on denticles or a smooth surface142. Denticles are also known to bristle, a movement that can be described as varying their pitch angles relative to the shark surface. The bristling is hypothesized to reduce pressure drag by inhibiting backﬂow in the boundary layer143. For an accelerating foil, a denticle-like texture was found to have delayed boundary layer separation compared to a foil with no texture144,145 (Fig. 4d)141,146. Wen et al.147 presented 3D-printed shark denticles that can be easily modiﬁed and applied to surfaces. Others have studied aspects of biomimetic denticles, such as swimming performance and boundary layer separation on an accelerating body148,149. Still, there is a lack of research on denticles directly applied to swimming robots. The article also identiﬁed research gaps for each ﬂow control device. However, some gaps are related to most of the ﬂow control devices discussed in this article. For example, few studies have considered the ﬂuid-structure interaction between these devices and the unsteady aerodynamic ﬂow surrounding them. Some studies have also limited this interaction by assuming the device is rigid rather than ﬂexible or deformable under aero- dynamic or hydrodynamic forces. Furthermore, the lack of con- sistency across studies in terms of ﬂow regimes, Reynolds numbers, and the adaptation of the bioinspired ﬂow control devices make it difﬁcult to perform quantitative comparisons between the studies. COMMUNICATIONS ENGINEERING \| https://doi.org/10.1038/s44172-023-00077-0 These lumps have been shown to help keep the ﬂow attached at higher angles of attack, thus delaying stall and improving the lift to drag ratio at these angles of attack (Fig. 4c)132133. Tubercles improve the maneu- verability of whale ﬁns, which perform less complex 3D kine- matics compared to ray-ﬁnned ﬁshes, by giving them a greater range of mobility119. It is important to note that whales swim within a higher Reynolds number regime (around 106) than many ﬁsh do (as low as 103)132. Experimental134 and numerical135 studies on ﬁns with tubercles in low Reynolds number regimes of approximately Re < 300,000 found that tubercles degraded rather than improved the hydrodynamic performance of the foils. Tubercles have been investigated on several airfoils showing various aerodynamic and hydrodynamic beneﬁts. These studies are summarized in refs. 136–139. Most of these studies have been conducted in steady ﬂow conditions. Thus, there is a dearth of studies performed on the inﬂuence of tubercles when the ﬂow is unsteady. Such studies would greatly beneﬁt the bio-inspired swimming community, as unsteady ﬂow is a critical component of biologically inspired swimming. Table 1 summarizes some studies on aquatic ﬂow control devices. The ﬂow control mechanisms of such devices vary from separation control (e.g., tubercules), to vortex tailoring (e.g., ﬁns) and transition control (e.g., denticles), and some of these devices implement more than one ﬂow control mechanism. Most of these aquatic ﬂow control devices have not been implemented on unmanned aquatic vehicles or ﬁsh-inspired robots, creating research opportunities towards improving the performance of the bioinspired systems. COMMUNICATIONS ENGINEERING \| (2023)2:30 \| https://doi.org/10.1038/s44172-023-00077-0 \| www.nature.com/commseng Aquatic bio-inspired ﬂow control l l f Flow control from aquatic creatures can be attributed to devices such as 1) ﬁns, 2) secretions, and 3) surface textures. This is summarized in Fig. 4, which details some of the most studied aquatic ﬂow control devices, including dorsal/anal ﬁns on ﬁshes, mucus secretions on ﬁsh bodies, denticles on shark skin, and tubercles on whale ﬂippers. A few other, less studied ﬂow control devices are mentioned brieﬂy at the end of this section. Fish and Lauder performed an in-depth review of ﬂow control in swim- ming animals in ref. 117, and much work has been done since then. This review provides a broad overview of aquatic ﬂow 9 REVIEW ARTICLE References 1. Gad-el Hak, M., Pollard, A. & Bonnet, J.-P. Flow Control Fundamentals and Practices. Lecture Notes in Physics Monographs, 1st edn, Vol. 3. 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Ke, W., Hashem, I., Zhang, W. & Zhu, B. Inﬂuence of leading-edge tubercles on the aerodynamic performance of a horizontal-axis wind turbine: a numerical study. Energy 239, 122186 (2022). 13 COMMUNICATIONS ENGINEERING \| (2023)2:30 \| https://doi.org/10.1038/s44172-023-00077-0 \| www.nature.com/commseng Competing interests Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. 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https://openalex.org/W4386086367	https://www.researchsquare.com/article/rs-1972401/latest.pdf	English	null	An investigation on the Monkeypox virus dynamics in human and rodent populations for a deterministic mathematical model	Research Square (Research Square)	2,023	cc-by	11,884	An Investigation on the Monkeypox Virus Dynamics in Human and Rodent Populations for a Deterministic Mathematical Model Leonce Leandry (  leonceleandry@gmail.com ) Jordan University College https://orcid.org/0000-0002-4816-5424 Eunice Mureithi University of Dar es Salaa, ˚Corresponding author Email address: leonceleandry@gmail.com ( ) Abstract A mathematical deterministic model for the dynamics of Monkeypox disease is developed. Monkeypox is a viral zoonotic disease that can be transmitted to humans, through contact with infected rodents. The model captures both the human and rodent populations and incorporates control strategies such as vaccines and quarantine for the human population. The model is analysed for local and global stability of the equilibrium solutions. In addition, numerical simulations of the model equations and sensitivity analysis of the parameters are carried out. The solutions obtained show that an increase in vaccination and quarantine measures could reduce the number of reproductions and ultimately eradicate the virus. Keywords: Monkeypox; Vaccination, Quarantine; Mathematical modelling; Reproduction number; Sensitivity analysis Keywords: Monkeypox; Vaccination, Quarantine; Mathematical modelling; Reproduction number; Sensitivity analysis An Investigation on the Monkeypox Virus Dynamics in Human and Rodent Populations for a Deterministic Mathematical Model An Investigation on the Monkeypox Virus Dynamics in Human and Rodent Populations for a Deterministic Mathematical Model Leonce Leandry2,˚1, Eunice Mureithi1 1. Department of Mathematics, College of Natural and Applied Sciences, University of Dar-es-Salaam, Tanzania Preprint submitted to Informatics in Medicine Unlocked Research Article Posted Date: August 24th, 2023 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Version of Record: A version of this preprint was published at Informatics in Medicine Unlocked on August 25th, 2023. See the published version at https://doi.org/10.1016/j.imu.2023.101325. Preprint submitted to Informatics in Medicine Unlocked August 7, 2023 Leonce Leandry2,˚1, Eunice Mureithi1 1. Department of Mathematics, College of Natural and Applied Sciences, University of Dar-es-Salaam, Tanzania 2. Department of Economics, Mathematics and statistics, Jordan University College, Morogoro, Tanzania 1,˚ Email address: leonceleandry@gmail.com ( ) ˚Corresponding author Email address: leonceleandry@gmail.com ( ) 2. Department of Economics, Mathematics and statistics, Jordan University College, Morogoro, Tanzania 1,˚ ˚Corresponding author E l dd 1. Introduction Monkeypox is a viral disease that aﬀects rodents (e.g. rats) and is trans- mitted to humans through contact with the infected rodents. Monkeypox was Preprint submitted to Informatics in Medicine Unlocked August 7, 2023 ﬁrst discovered in 1958 when two outbreaks of a pox-like disease occurred in colonies of monkeys kept for research; hence the name ‘monkeypox’. The ﬁrst human case of monkeypox was recorded in 1970 in the Democratic Republic of Congo, during a period of intensiﬁed eﬀorts to eliminate smallpox. Since then, monkeypox has been reported in humans in other central and western African countries [1]. The most recent outbreak of the monkeypox virus was in May 2022, and the ﬁrst case was conﬁrmed on May 6th, 2022, by an individual with a travel history to Nigeria. According to the Centres for Disease Control and Prevention (CDC), there were 87,314 conﬁrmed infected cases by February 2023, in more than 112 countries, since the outbreak. The outbreak is ongoing, and aﬀected countries include the United Kingdom and some countries in Europe, North and South America, Asia, Australia and Africa. Due to the unexpected and large geographical spread of the disease, it is likely that the actual number of cases will be underestimated [2, 3]. This is in line with the Global Health Report which reported 98% of the cases conﬁrmed were male of an average age of 41. On 23 July 2022, the Director General of the World Health Organisation (WHO) declared the outbreak of the disease and called upon the public health under emergency [4, 3]. According to WHO[4] monkeypox virus is a type of or- thopox virus in the same family of viruses that causes smallpox. The symptoms of monkeypox are similar, though less severe and less contagious, than those of smallpox. In a study done by Mackenzie [5], the processes of zoonotic viral emergence, the intricacies of host virus interactions, and the distinct role of biological tran- sitions and modifying factors are well documented. Additionally, the study showed that the process of emergence is conceptualised as having two transition stages typical of disease emergence: contact of humans with the infectious agent and cross-species transmission of the agent. 1. Introduction The report on the response to the monkeypox outbreak of the Nigeria Centre for Disease Control [6] highlighted that the primary route of transmission of the disease to humans was contact with infected animals or their bodily ﬂuids or 2 the eating of inadequately cooked meat from infected animals. In the book titled “Learning from Experience: The Public Health Response to West Nile Virus, SARS, monkeypox, and Hepatitis A Outbreaks in the United States” which was written by Stoto[7], there is a need to prepare resources in the health sector to accommodate people infected with emerging outbreaks such as the monkeypox virus. Additionally, there is a need to understand the dynamics and take precautionary measures well before the disease becomes out of control. There is no licenced vaccine available speciﬁcally for the monkeypox virus. However, some experimental vaccines have been tested on animals and humans [8]. One example is the modiﬁed Vaccinia Ankara (MVA) vaccine, which has been shown to be eﬀective in preventing monkeypox in non-human primates. A phase 1 clinical trial of the MVA vaccine was also conducted in humans and found to be safe and well-tolerated [9]. Presently, there are no clear treatments available for monkeypox infection, though vaccination against smallpox has been shown to be successful by 85 percent in the prevention of monkeypox. Therefore, vaccination remains the best way to reduce the incidence and spread of the virus, as shown in the studies by Shaban & Moﬁ[10] and Rwezaura [11] in other virally spread diseases. A study by Martcheva[12] has shown that in order to typically contain the disease, quarantine must be made compulsory. Quarantine is applied to any individual who has come into contact with an infectious individual. Infectious diseases are currently posing a huge challenge not only to individuals but also to the global community, demanding the development of mathematical modelling approaches to improve our understanding of their dynamics. This is particularly relevant in the context of diseases such as Monkeypox. Recent literature has made valuable contributions to this domain, with notable work by Naik et al. [13] on the approximate solution of nonlinear fractional-order HIV models using the homotopy analysis method, Naik et al. [14] on the complex dynamics of a seasonally forced discrete-time SIR epidemic model, Farman et al. [15] on the numerical treatment of a nonlinear dynamical hepatitis B model using an evolutionary approach, and Ahmad et al. 1. Introduction [16] on 3 the modelling and numerical investigation of fractional-order bovine babesiosis disease. These works have provided valuable insights into the application of mathematical modelling to understanding and combating infectious diseases. In recent years, several studies have been conducted, including the work of Peter et al.[17]; Usman & Isa Adamu[18]; Lasisi et al [19]; Emeka et al.[20]; Al- Shomrani et al[21]; Marwa et al [22] and Stoto et al.[7], among others. However, more studies are needed to understand the dynamics and control of monkeypox. According to the WHO [4], the monkeypox virus remains a signiﬁcant threat to the world [23]. Therefore, the current study makes a signiﬁcant contribution to mitigating the spread of the virus as it investigates the dynamics of the deterministic model of the monkeypox virus with quarantine and vaccination interventions, which have not been extensively explored before. 2. Material and Methods 2.1. Mathematical Model Formulation 2.1. Mathematical Model Formulation 2.1. Mathematical Model Formulation A mathematical model is formulated with two sub-populations, namely, the human sub-population denoted by NH and the rodent (the non-human primates) sub-population denoted by NR. The model assumptions are as follows: • The human sub-population has six compartments, that is, the susceptible humans, SH , exposed EH, the quarantined, QH, infectious IH, recovered RH, and vaccinated individuals VH. The rodent subpopulation has three compartments, namely susceptible SR, exposed rodents ER, and infectious rodents RH. • Immigrants contribute to an increase in susceptible humans SH at a rate of πH. This will later be taken as πH “ 2.7 per every 1000 human pop- ulation as shown by [24] or as by the International Migration Report of 2020 (see more details in the link here https://www.un.org/en/desa/ international-migration-report-2020-highlights). 4 SN Variable Description 1 SH Susceptible human population 2 SR Susceptible rodent population 3 EH Number of exposed human individuals 4 ER Number of exposed rodent 5 IH Number of infectious human 6 IR Number of infectious rodent 7 RH Number of recovered human beings 8 VH Number of vaccinated human beings 9 QH Number of quarantined human beings Table 1: Variable descriptions SN Variable Description • Only susceptible and recovered individuals can be vaccinated, at rates of ϑ1 and ϑ2, respectively. The vaccine used is that for smallpox, which is not eﬃcient 100% for the monkeypox virus [25]. Thus, vaccinated individuals return to the susceptible class at a rate ω. • Only susceptible and recovered individuals can be vaccinated, at rates of ϑ1 and ϑ2, respectively. The vaccine used is that for smallpox, which is not eﬃcient 100% for the monkeypox virus [25]. Thus, vaccinated individuals return to the susceptible class at a rate ω. The contact rate between rodents and susceptible humans is indicated by ξ1, while that between humans and humans is indicated by ξ2 and the proportion of undetected cases from the quarantined population after diagnosis is ψ. A proportion ppq of immigrants are vaccinated, while the rest 1 ´ p are not and therefore belong to the susceptible class. From the exposed class, some individuals transition to either the infected class at the rate σ1 or the quarantine class at the rate σ2. Likewise, the pro- gression from quarantined class to infected class is ζ and while the recovery rate from infection is λ. Humans die naturally at rate µH or due to monkeypox at rate δH. 2.1. Mathematical Model Formulation The recruitment rate for the rodent sub-population is denoted as πR, while the rodent-to-rodent contact rate is ξ3. The progression rate of rodents from exposed to infectious is denoted as σ3. Rodents die naturally at the rate µR or due to disease at the rate of δR. 2.2. Schematic Diagram The schematic diagram for monkeypox dynamics, based on the assumptions above, is shown in Figure 1. The solid arrows represent the transition of the disease from one compartment to another, while the dotted arrows represent the 5 interactions between the rodent subpopulation and the human subpopulation. The direction of transmission indicated by the dotted arrow is from rodents to humans, meaning the disease is from the rodent population to humans. Figure 1: The Schematic diagram of Monkeypox virus transition dynamics Figure 1: The Schematic diagram of Monkeypox virus transition dynamics 6 6 2.3. Model equations (1) $ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ dSH dt “ πHp1 ´ pq ´ pξ1IR ` ξ2IHq SH NH ´ pµH ` ϑ1qSH ` ωVH ` ψQH dEH dt “ pξ1IR ` ξ2IHq SH NH ´ pσ1 ` σ2 ` µHqEH dIH dt “ σ1EH ` ζQH ´ pµH ` δH ` λqIH dQH dt “ σ2EH ´ pψ ` ζ ` µH ` δHqQH $ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ & ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ % dSH dt “ πHp1 ´ pq ´ pξ1IR ` ξ2IHq SH NH ´ pµH ` ϑ1qSH ` ωVH ` ψQH dEH dt “ pξ1IR ` ξ2IHq SH NH ´ pσ1 ` σ2 ` µHqEH dIH dt “ σ1EH ` ζQH ´ pµH ` δH ` λqIH dQH dt “ σ2EH ´ pψ ` ζ ` µH ` δHqQH dRH dt “ λIH ´ pµH ` ϑ2qRH dVH dt “ πHp ` ϑ1SH ` ϑ2RH ´ pµH ` ωqVH dSR dt “ πR ´ ξ3IR SR NR ´ µRSR dER dt “ ξ3IR SR NR ´ pµR ` σ3qER dIR dt “ σ3ER ´ pµR ` δRqIR. (1) (1) dVH dt “ πHp ` ϑ1SH ` ϑ2RH ´ pµH ` ωqVH 2.4. Values for each parameter used for simulation 2.4. Values for each parameter used for simulation 2.3. Model equations 2.3. Model equations From the schematic diagram in Figure 1 we obtain the following diﬀerential model equations $ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ & ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ % dSH dt “ πHp1 ´ pq ´ pξ1IR ` ξ2IHq SH NH ´ pµH ` ϑ1qSH ` ωVH ` ψQH dEH dt “ pξ1IR ` ξ2IHq SH NH ´ pσ1 ` σ2 ` µHqEH dIH dt “ σ1EH ` ζQH ´ pµH ` δH ` λqIH dQH dt “ σ2EH ´ pψ ` ζ ` µH ` δHqQH dRH dt “ λIH ´ pµH ` ϑ2qRH dVH dt “ πHp ` ϑ1SH ` ϑ2RH ´ pµH ` ωqVH dSR dt “ πR ´ ξ3IR SR NR ´ µRSR dER dt “ ξ3IR SR NR ´ pµR ` σ3qER dIR dt “ σ3ER ´ pµR ` δRqIR. Table 2: Parameter Values used in Numerical simulation Table 2: Parameter Values used in Numerical simulation 2.4. Values for each parameter used for simulation From the model equations 1, it is clearly seen that dSHptq dt ě ´pξ1IRptq ` ξ2IHptqq SHptq NHptq ´ pµH ` ϑ1qSHptq (2) (2) Solving (2) we have, Solving (2) we have, SHptq ě SHp0q exp " ´ ż t 0 ppξ1IRpηq ` ξ2IHpηqq SHpηq NHpηq ` pµH ` ϑ1qSHpηqqdη * (3) (3) Before the onset of disease, IR “ EH “ IH “ 0 and after the onset of disease when IR ą 0, EH ą 0, IH ą 0 as t Ñ 8 in equation 3 we have; lim tÑ8 SHptq ą 0 (4) (4) Similarly, following the same procedure for the other equations of 1 gives pSH, IH, EH, VH, QH, RH, SR, ER, IRq P R6 `ˆR3 ` of the model 1 are non-negative for all time t ą 0. For details of the proof one can read the work of [12, 17]. Similarly, following the same procedure for the other equations of 1 gives pSH, IH, EH, VH, QH, RH, SR, ER, IRq P R6 `ˆR3 ` of the model 1 are non-negative for all time t ą 0. For details of the proof one can read the work of [12, 17]. 2.6. Steady States of the Model 2.6. Steady States of the Model 2.4. Values for each parameter used for simulation The following Table 2 presents the parameter descriptions and values that have been used for the simulation: 7 Parameter Description Value Unit Source πH Human Recruitment rate 1,160,000 year´1 [Estimated] πR Rodents Recruitment rate 200000 year´1 [Estimated] ξ1 Force of infection from Rodents to Human 0.0025 year´1 [26] ξ2 Force of infection from Human to Human 0.000063 year´1 [26] ξ3 Force of infection from Rodents to Rodents 0.0027 year´1 [26] σ1 rate of transition from exposed human to infected human 0.2 year´1 [17] σ2 Transition rate from exposed to isolated cases 2.0 year´1 [17] σ3 transition from exposed rodents to infected rodents 3.0 year´1 [Assumed] ψ fraction of those isolated that is not infected 2.0 year´1 [17] ζ Progression from isolated to in- fected class 0.52 year´1 [17] λ Humans recovery rate 0.83 year´1 [27] µH Natural death rate of human 1.5 year´1 [26] µR Natural death rate of rodents 0.002 year´1 [26] δR Disease induced death rate for rodents 0.5 year´1 [17] δH Death rate due to disease for hu- mans 0.2 year´1 [28] ϑ1 Vaccination rate of humans from susceptible class 0.1 year´1 [18] ϑ2 Vaccination rate of humans from Recovered class 0.01 year´1 [Assumed] ω Rate loss of Vaccination eﬀec- tiveness 0.003 year´1 [Assumed] p fraction of vaccinated immi- grants 0.0004 year´1 [Assumed] 8 2.5. Model Analysis 2.5. Model Analysis 2.5.1. Invariant Domain 2.5.1. Invariant Domain Theorem 2.1. Let the initial conditions of the monkeypox disease dynamical system 1 be given as SHp0q ě 0, IHp0q ě 0, EHp0q ě 0, VHp0q ě 0, QHp0q ě 0, RHp0q ě 0, SRp0q ě 0, ERp0q ě 0, IRp0q ě 0 Then, the solutions Ω“ pSH, IH, EH, VH, QH, RH, SR, ER, IRq P R9 ` of the model 1 are non-negative for all time t ą 0 Theorem 2.1. Let the initial conditions of the monkeypox disease dynamical system 1 be given as SHp0q ě 0, IHp0q ě 0, EHp0q ě 0, VHp0q ě 0, QHp0q ě 0, RHp0q ě 0, SRp0q ě 0, ERp0q ě 0, IRp0q ě 0 Then, the solutions Ω“ pSH, IH, EH, VH, QH, RH, SR, ER, IRq P R9 ` of the model 1 are non-negative for all time t ą 0 Proof of Theorem 2.1. 2.6.1. Monkeypox Free Equilibrium state (MFE), ε0 2.6.1. Monkeypox Free Equilibrium state (MFE), ε0 An equilibrium solution for the system of equations 1 is obtained by setting the derivatives equal to zero and solving the algebraic equations. One of the equilibrium solutions obtaining from solving the system of al- gebraic equations represents no disease, E˚ H “ Q˚ H “ R˚ H “ E˚ R “ I˚ R “ 0, S˚ H “ πHp1 ´ pqpµH ` ωq ` πHpω pµH ` ϑ1qpµH ` ωq ` ωϑ1 , V ˚ H “ ϑ1S˚ H ` pπH µHpµH ` ϑ1q and S˚ R “ πR µR . We deﬁne this equilibrium solution as Monkeypox-free-equilibrium (MFE) and denote it using ε0. ε0 “ ˜ πHp1 ´ pqpµH ` ωq ` πHpω pµH ` ϑ1qpµH ` ωq ` ωϑ1 , 0, 0, 0, 0, ϑ1S˚ H ` pπH µHpµH ` ϑ1q, πR µR , 0, 0 ¸ (5) (5) Where, Where, S˚ H “ πHp1 ´ pqpµH ` ωq ` πHpω pµH ` ϑ1qpµH ` ωq ` ωϑ1 . 2.6.2. Monkeypox virus Endemic equilibrium (MEE) 2.6.2. Monkeypox virus Endemic equilibrium (MEE) 2.6. Steady States of the Model In this section the model system is analyzed qualitatively by determining the equilibrium points, ﬁnding out their corresponding stability (local and global for disease free equilibrium and endemic equilibrium) and subsequently interpreting the sensitivity of the model parameters. 9 9 2.6.1. Monkeypox Free Equilibrium state (MFE), ε0 2.6.2. Monkeypox virus Endemic equilibrium (MEE) 2.6.2. Monkeypox virus Endemic equilibrium (MEE) A second equilibrium solution obtaining from solving the system of algebraic equations represents the case when IH, IR ‰ 0. This solution is termed as the 10 endemic equilibrium solution and is given as S˚ H “ πHp1 ´ pq ` ωV ˚ H ` ψQ˚ H ξ1I˚ R ` ξ2I˚ H ` µH ` ν1 , E˚ H “ ξ1I˚ R ` ξ2I˚ HS˚ H σ1 ` σ2 ` µH , I˚ H “ σ1E˚ H ` ζQ˚ H µH ` δH ` λ , Q˚ H “ σ2E˚ H ψ ` ζ ` µH ` δH , R˚ H “ λI˚ H µH ` ν2 , V ˚ H “ pπHν1S˚ H ` ν2R˚ H µH ` ω , S˚ R “ pµR ` σ3qpµR ` δRq ξ3σ3 , E˚ R “ pπRσ3ξ3q pµR ` σ3q2pµR ` δRqp pπRσ3ξ3q pµR ` σ3qpµR ` δRq ´ µRq, I˚ R “ πRσ3 pµR ` σ3qpµR ` δRq ´ µR ξ3 . (6) E˚ H “ ξ1I˚ R ` ξ2I˚ HS˚ H σ1 ` σ2 ` µH , I˚ H “ σ1E˚ H ` ζQ˚ H µH ` δH ` λ , Q˚ H “ σ2E˚ H ψ ` ζ ` µH ` δH , R˚ H “ λI˚ H µH ` ν2 , V ˚ H “ pπHν1S˚ H ` ν2R˚ H µH ` ω , S˚ R “ pµR ` σ3qpµR ` δRq ξ3σ3 , E˚ R “ pπRσ3ξ3q pµR ` σ3q2pµR ` δRqp pπRσ3ξ3q pµR ` σ3qpµR ` δRq ´ µRq, I˚ R “ πRσ3 pµR ` σ3qpµR ` δRq ´ µR ξ3 . QH “ ψ ` ζ ` µH ` δH , R˚ H “ λI˚ H µH ` ν2 , V ˚ H “ pπHν1S˚ H ` ν2R˚ H µH ` ω , S˚ R “ pµR ` σ3qpµR ` δRq ξ3σ3 , E˚ R “ pπRσ3ξ3q pµR ` σ3q2pµR ` δRqp pπRσ3ξ3q pµR ` σ3qpµR ` δRq ´ µRq, I˚ R “ πRσ3 pµR ` σ3qpµR ` δRq ´ µR ξ3 . (6) E˚ R “ pπRσ3ξ3q pµR ` σ3q2pµR ` δRqp pπRσ3ξ3q pµR ` σ3qpµR ` δRq ´ µRq, I˚ R “ πRσ3 pµR ` σ3qpµR ` δRq ´ µR ξ3 . Endemic equilibrium occurs when the infection (monkeypox virus) persists in the population. 2.6.3. Computing eﬀective reproduction number pRvq 2.6.3. Computing eﬀective reproduction number pRvq The next-generation approach is used (see Martcheva [12] and Al-Shomrani[21]) as a techniques for computing eﬀective reproduction number (Rv). The idea for the next-generation approach rests on the observation that Rv is characterized by regarding the infection transmission as producing oﬀspring in an epidemio- logical sense that is, giving birth to a new infected individual [19]. The approach used here is that developed by Van den Driessche and Watmough whereby, we divided the compartments into two broad categories: infected compartments and non-infected (healthy) compartments [29]. The compartments for which 11 individuals are infected but not infectious (such as Quarantined and Exposed individuals), are also among the infected compartments. Clearly, in this model the disease free compartments are SH, RH, VH and SR while infectious com- partments are EH, IH, QH, ER and IR. 2.6.3. Computing eﬀective reproduction number pRvq Then by Next generation approach, the compartments of infected groups is taken then split and the model can be written as; dX dt “ FpXq ´ V pXq That is, That is, dX dt “ $ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ & ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ % pξ1IR ` ξ2IHq SH NH ´ pσ1 ` σ2 ` µHqEH σ1EH ` ζQH ´ pµH ` δH ` λqIH σ2EH ´ pψ ` ζ ` µH ` δHqQH ξ3IR SR NR ´ pµR ` σ3qER σ3ER ´ pµR ` δRqIR (7) (7) Then after splitting the right hand side in the infected compartments gives the following Fi “ $ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ & ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ % pξ1IR ` ξ2IHq SH NH 0 0 ξ3IR SR NR 0 (8) (8) 12 and and Vi “ $ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ & ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ % pσ1 ` σ2 ` µHqEH ´σ1EH ´ ζQH ` pµH ` δH ` λqIH ´σ2EH ` pψ ` ζ ` µH ` δHqQH pµR ` σ3qER ´σ3ER ` pµR ` δRqIR (9) (9) Now, the partial derivatives with respect to the dependent variables EH, IH, ER, IR in equation 8 and 9 are taken and then upon substituting the MFE values we get Now, the partial derivatives with respect to the dependent variables EH, IH, ER, IR in equation 8 and 9 are taken and then upon substituting the MFE values we get F “ ¨ ˚ ˚ ˚ ˚ ˚ ˚ ˚ ˝ 0 ξ2 πHp1 ´ pqpµH ` wq ` πHpω NHrpµH ` ν1qpµH ` ωq ` ων1s 0 0 ξ1 πHp1 ´ pqpµH ` wq ` πHpω NHrpµH ` ν1qpµH ` ωq ` ων1s 0 0 0 0 0 0 0 0 0 ξ3πR NRµR 0 0 0 0 0 ˛ ‹‹‹‹‹‹‹‚ (10) F “ ¨ ˚ ˚ ˚ ˚ ˚ ˚ ˚ ˝ 0 ξ2 πHp1 ´ pqpµH ` wq ` πHpω NHrpµH ` ν1qpµH ` ωq ` ων1s 0 0 ξ1 πHp1 ´ pqpµH ` wq ` πHpω NHrpµH ` ν1qpµH ` ωq ` ων1s 0 0 0 0 0 0 0 0 0 ξ3πR NRµR 0 0 0 0 0 ˛ ‹‹‹‹‹‹‹‚ (10) V “ ¨ ˚ ˚ ˚ ˚ ˚ ˚ ˚ ˚ ˚ ˝ σ1 ` σ2 ` µH 0 0 0 0 ´σ1 µH ` δH ` λ ´ζ 0 0 ´σ2 0 ψ ` ζ ` µH ` δH 0 0 0 0 0 µR ` σ3 0 0 0 0 ´σ3 µR ` δR ˛ ‹‹‹‹‹‹‹‹‹‚ . 2.6.3. Computing eﬀective reproduction number pRvq (11) F “ ¨ ˚ ˚ ˚ ˚ ˚ ˚ ˚ ˝ 0 ξ2 πHp1 ´ pqpµH ` wq ` πHpω NHrpµH ` ν1qpµH ` ωq ` ων1s 0 0 ξ1 πHp1 ´ pqpµH ` wq ` πHpω NHrpµH ` ν1qpµH ` ωq ` ων1s 0 0 0 0 0 0 0 0 0 ξ3πR NRµR 0 0 0 0 0 ˛ ‹‹‹‹‹‹‹‚ (10) V “ ¨ ˚ ˚ ˚ ˚ ˚ ˚ ˚ ˚ ˚ ˝ σ1 ` σ2 ` µH 0 0 0 0 ´σ1 µH ` δH ` λ ´ζ 0 0 ´σ2 0 ψ ` ζ ` µH ` δH 0 0 0 0 0 µR ` σ3 0 0 0 0 ´σ3 µR ` δR ˛ ‹‹‹‹‹‹‹‹‹‚ . 2.6.3. Computing eﬀective reproduction number pRvq (11) ( ) We need to ﬁnd FV ´1: 13 For simplicity we can let; $ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ & ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ % v11 “ σ1 ` σ2 ` µH v22 “ µH ` δH ` λ v33 “ ψ ` ζ ` µH ` δH v44 “ µR ` σ3 v55 “ µR ` δR $ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ & ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ % v11 “ σ1 ` σ2 ` µH v22 “ µH ` δH ` λ v33 “ ψ ` ζ ` µH ` δH v44 “ µR ` σ3 v55 “ µR ` δR $ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ & ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ % v11 “ σ1 ` σ2 ` µH v22 “ µH ` δH ` λ v33 “ ψ ` ζ ` µH ` δH v44 “ µR ` σ3 v55 “ µR ` δR Then the matrix V in equation 11 becomes, V “ ¨ ˚ ˚ ˚ ˚ ˚ ˚ ˚ ˚ ˚ ˝ v11 0 0 0 0 ´σ1 v22 ´ζ 0 0 ´σ2 0 v33 0 0 0 0 0 v44 0 0 0 0 σ3 v55 ˛ ‹‹‹‹‹‹‹‹‹‚ (12) (12) Computing the determinant we get, \|V \| “ v11v22v33v44v55 The inverse of V is given by: The inverse of V is given by: V ´1 “ 1 \|V \| ¨ ˚ ˚ ˚ ˚ ˚ ˚ ˚ ˚ ˝ v22v33v44v55 0 0 0 0 σ1v33v44v55 v11v33v44v55 0 0 0 0 0 v11v22v44v55 0 0 0 0 0 v11v22v33v55 0 0 0 0 σ3v11v22v33 v11v22v33v44 ˛ ‹‹‹‹‹‹‹‹‚ (13) V ´1 “ 1 \|V \| ¨ ˚ ˚ ˚ ˚ ˚ ˚ ˚ ˚ ˝ v22v33v44v55 0 0 0 0 σ1v33v44v55 v11v33v44v55 0 0 0 0 0 v11v22v44v55 0 0 0 0 0 v11v22v33v55 0 0 0 0 σ3v11v22v33 v11v22v33v44 ˛ ‹‹‹‹‹‹‹‹‚ (13) 14 Then, FV ´1 “ 1 \|V \| ¨ ˚ ˚ ˚ ˚ ˚ ˚ ˚ ˚ ˝ σ1v33v44v55ξ2k v11v33v44v55ξ2k 0 σ3v11v22v33ξ1k v11v22v33v44ξ1k 0 0 0 0 0 0 0 0 0 0 0 0 0 σ3v11v22v33ξ3l v11v22v33v44ξ3l 0 0 0 0 0 ˛ ‹‹‹‹‹‹‹‹‚ (14) where, k “ πHp1 ´ pqpµH ` wq ` πHpω NHrpµH ` ν1qpµH ` ωq ` ων1s and l “ πR NRµR FV ´1 “ 1 \|V \| ¨ ˚ ˚ ˚ ˚ ˚ ˚ ˚ ˚ ˝ σ1v33v44v55ξ2k v11v33v44v55ξ2k 0 σ3v11v22v33ξ1k v11v22v33v44ξ1k 0 0 0 0 0 0 0 0 0 0 0 0 0 σ3v11v22v33ξ3l v11v22v33v44ξ3l 0 0 0 0 0 ˛ ‹‹‹‹‹‹‹‹‚ (14) where, k “ πHp1 ´ pqpµH ` wq ` πHpω NHrpµH ` ν1qpµH ` ωq ` ων1s and l “ πR NRµR The basic reproduction number is deﬁned as the largest eigenvalue (spectral radius) of the next generation matrix. 2.6.3. Computing eﬀective reproduction number pRvq Deﬁnition 2.1. The spectral radius of a matrix A is deﬁned as the maximum of the absolute values of the eigenvalues of A:ρpAq “ supt\|λ\| : λ P σpAqu, where σpAq denotes the eigenvalues of A. Consequently, it is easily shown that the eigenvalues of A are: $ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ & ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ % λ1 “ σ1ξ2k v11v22 λ2 “ 0 λ3 “ 0 λ4 “ σ3ξ3l v44v55 λ5 “ 0 (15) (15) The Basic reproduction number is the largest eigenvalue of the next generation matrix, from Equation 15. The Eﬀective reproduction number is Rv “ max " σ1ξ2k v11v22 , σ3ξ3l v44v55 * . (16) (16) 15 That is, That is, Rv “ max $ ’ ’ ’ ’ & ’ ’ ’ ’ % σ1ξ2 pσ1 ` σ2 ` µHµH ` δH ` λq πHp1 ´ pqpµH ` wq ` πHpω pNHrpµH ` ϑ1qpµH ` ωq ` ωϑ1sq , σ3ξ3 pµR ` σ3µR ` δRq πR pNRµRq , / / / / . / / / / - . (17) (17) Since λ1 is clearly on dynamics related to human population and λ4 is for Rodent population, then RH “ σ1ξ2 pσ1 ` σ2 ` µHqpµH ` δH ` λq. πHp1 ´ pqpµH ` wq ` πHpω pNHrpµH ` ϑ1qpµH ` ωq ` ωϑ1sq RR “ σ3ξ3 pµR ` σ3µR ` δRq πR pNRµRq (18) (18) In the absence of vaccine and quarantine, that is ϑ1 “ p “ ω “ 0 “ ϑ2 “ σ2, then we have the basic reproduction number as R0 “ max " σ1ξ2 pσ1 ` µHqpµH ` δH ` λq πHµH pNHµHq, σ3ξ3 pµR ` σ3µR ` δRq πR pNRµRq * . (19) (19) Therefore, since we are interested in reducing the transmission of the virus to the human population then the analysis done here considers all the possibilities of the basic reproduction number for the two populations such as the case when RH ą RR, RH ă RR and RH “ RR. Throughout this study the analysis has been done with the main objective of investigation on how to reduce the transmission to the human population when RH ă 1and RR ă 1. 2.6.3. Computing eﬀective reproduction number pRvq which is always negative which implies that the increase in quarantine for sus- pected cases will reduce the reproduction number. The same applies to the impacts of Vaccination, when analyzing we see that, BRv Bϑ1 “ ´ σ1ξ2 pσ1 ` σ2 ` µHqpµH ` δH ` λq pπHp1 ´ pqpµH ` ωq ` πHpωqpµH ` 2ωq pNHrpµH ` ϑ1qpµH ` ωq ` ωϑ1sq2 (23) which also is always negative, which implies that increasing the Vaccination could also reduce the reproduction number. BRv Bϑ1 “ ´ σ1ξ2 pσ1 ` σ2 ` µHqpµH ` δH ` λq pπHp1 ´ pqpµH ` ωq ` πHpωqpµH ` 2ωq pNHrpµH ` ϑ1qpµH ` ωq ` ωϑ1sq2 (23) (23) which also is always negative, which implies that increasing the Vaccination could also reduce the reproduction number. 2.6.3. Computing eﬀective reproduction number pRvq Therefore, since we are interested in reducing the transmission of the virus to the human population then the analysis done here considers all the possibilities of the basic reproduction number for the two populations such as the case when RH ą RR, RH ă RR and RH “ RR. Throughout this study the analysis has been done with the main objective of investigation on how to reduce the transmission to the human population when RH ă 1and RR ă 1. 2.6.4. Analysis of the basic eﬀective reproduction number pRvq Theorem 2.2. The possibility that the monkeypox virus could be eradicated from the community is when Rv ă 1 which implies that RH ă 1 and RR ă 1 and the derivatives with respect to the transmission rate ξ2 and ξ3 should always be positive to reduce the transmission rate, also the derivative with respect to quarantine, σ2 and vaccination, ϑ1 rate should always be negative to reduce the reproduction number [30] Proof of Theorem 2.2. Now taking the partial derivatives of Rv with respect to ξ2 and ξ3 we have: 16 BRv Bξ2 “ σ1 pσ1 ` σ2 ` µHqpµH ` δH ` λq πHp1 ´ pqpµH ` ωq ` πHpω pNHrpµH ` ϑ1qpµH ` ωq ` ωϑ1sq (20) For ξ3 BRv Bξ3 “ σ3 pµR ` σ3µR ` δRq πR pNRµRq (21) BRv Bξ2 “ σ1 pσ1 ` σ2 ` µHqpµH ` δH ` λq πHp1 ´ pqpµH ` ωq ` πHpω pNHrpµH ` ϑ1qpµH ` ωq ` ωϑ1sq (20) (20) For ξ3 For ξ3 Rv ξ3 “ σ3 pµR ` σ3µR ` δRq πR pNRµRq (21) (21) which are always positive and this implies that the transmission could reduce the reproduction number linearly. which are always positive and this implies that the transmission could reduce the reproduction number linearly. Moreover, this could be accomplished by isolation or quarantine the exposed cases in human population. That is, taking the partial derivative of R with respect to σ2 and ϑ1 we have, BRv Bσ2 “ ´ σ1ξ2 pσ1 ` σ2 ` µHq2pµH ` δH ` λq πHp1 ´ pqpµH ` ωq ` πHpω pNHrpµH ` ϑ1qpµH ` ωq ` ωϑ1sq (22) (22) which is always negative which implies that the increase in quarantine for sus- pected cases will reduce the reproduction number. 2.7. Global stability of the Monkey-pox virus Free Equilibrium We used the approach as that done by Mumbu[31] where he used the Lya- punov function, in detail it has also been shown by Martcheva [12] and Li [32] to determine the global stability of the monkeypox virus free equilibrium one needs to prove that the Lyapunov function L is asymptotically stable. Theorem 2.3. If a function Lpxq is globally positive deﬁnite and radially un- bounded, and its time derivative is globally negative, L1pxq ă 0 for all x ‰ x˚ then the equilibrium x is globally stable. 17 17 Proof of Theorem 2.3. For the proof details see in [12, 31] p f [ , ] Suppose RH ą 1 and RR ą 1, in which the endemic equilibrium point exists, then to prove the global stability, the Lyapunov function L is deﬁne and derive as follows: The approach used here is by letting the non-negative constants, Ci for i “ 1, 2, . . . , 6 and multiply to all the compartments which give zero values at the MFE. 2.7. Global stability of the Monkey-pox virus Free Equilibrium L “ pSH ´ S˚ H ´ S˚ H ln SH S˚ H q ` C1EH ` C2IH ` C3QH ` C4RH` `pVH ´ V ˚ H ´ V ˚ H ln VH V ˚ H q ` pSR ´ S˚ R ´ S˚ R ln SR S˚ R q ` C5ER ` C6IR (24) (24) Then diﬀerentiating with respect to t and supposing, SH ď S˚ H, VH ď V ˚ H and SR ď S˚ R then the equation reduces to Then diﬀerentiating with respect to t and supposing, SH ď S˚ H, VH ď V ˚ H and SR ď S˚ R then the equation reduces to dL dt “ C1 dEH dt ` C2 dIH dt ` C3 dQH dt ` C4 dRH dt ` C5 dER dt ` C6 dIH dt (25) (25) Substituting the values from Equation1 we get, Substituting the values from Equation1 we get, dL dt “ C1ppξ1IR ` ξ2IHq SH NH ´ pσ1 ` σ2 ` µHqEHq ` C2pσ1EH ` ζQH ´ pµH ` δH ` λqIHq` dL dt “ C1ppξ1IR ` ξ2IHq SH NH ´ pσ1 ` σ2 ` µHqEHq ` C2pσ1EH ` ζQH ´ pµH ` δH ` λqIHq` 3pσ2EH ´ pφ ` ζ ` µH ` δHqQHq ` C4pλIH ´ pµH ` ϑ2qRHq` `C5pξ3IR SR NR ´ pµR ` σ3qERq ` C6pσ3ER ´ pµR ` δRqIRq (26) `C3pσ2EH ´ pφ ` ζ ` µH ` δHqQHq ` C4pλIH ´ pµH ` ϑ2qRHq` (26) (26) `C5pξ3IR SR NR ´ pµR ` σ3qERq ` C6pσ3ER ´ pµR ` δRqIRq Rearranging terms with trivial solution in MFE by collecting like terms we get, dL dt ď pC1ξ1 S˚ H NH ` C5ξ3 S˚ R NR ´ C6pµR ` δRqqIR ` pC1ξ2 S˚ H NH ´ C2pµH ` δH ` λq ` C4λqIH `pC1p´pσ1 ` σ2 ` µHqq ` C2σ1 ` C3σ2qEH ` pC3p´pψ ` ζ ` µH ` δHqq ` C2ζqQH` (27) Rearranging terms with trivial solution in MFE by collecting like terms we get, dL dt ď pC1ξ1 S˚ H NH ` C5ξ3 S˚ R NR ´ C6pµR ` δRqqIR ` pC1ξ2 S˚ H NH ´ C2pµH ` δH ` λq ` C4λqIH `pC1p´pσ1 ` σ2 ` µHqq ` C2σ1 ` C3σ2qEH ` pC3p´pψ ` ζ ` µH ` δHqq ` C2ζqQH` (27) `pC1p´pσ1 ` σ2 ` µHqq ` C2σ1 ` C3σ2qEH ` pC3p´pψ ` ζ ` µH ` δHqq ` C2ζqQH` (27) `p´C4pµH ` ϑ2qRHq ` C5p´pµR ` σ3q ` C6σ3qER `p´C4pµH ` ϑ2qRHq ` C5p´pµR ` σ3q ` C6σ3qER 18 18 Then equating each term to zero and substituting the values of S˚ H and S˚ R, as we are looking for the points in the system state space where the function L is at an equilibrium, we obtain the following $ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ & ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ % C1ξ1 S˚ H NH ` C5ξ3 S˚ R NR ´ C6pµR ` δRq “ 0 C1ξ2 S˚ H NH ´ C2pµH ` δH ` λq ` C4λ “ 0 ´C1pσ1 ` σ2 ` µHq ` C2σ1 ` C3σ2 “ 0 ´C3pψ ` ζ ` µH ` δHq ` C2ζ “ 0 ´C4pµH ` ϑ2q “ 0 ´C5pµR ` σ3q ` C6σ3 “ 0 (28) ´C1pσ1 ` σ2 ` µHq ` C2σ1 ` C3σ2 “ 0 (28) ´C3pψ ` ζ ` µH ` δHq ` C2ζ “ 0 ´C4pµH ` ϑ2q “ 0 Solving for the constants, we have C4 “ 0 and C3 “ 0 substituting them back we have Solving for the constants, we have C4 “ 0 and C3 “ 0 substituting them back we have $ ’ ’ ’ ’ & ’ ’ ’ ’ % C1ξ2 S˚ H NH ´ C2pµH ` δH ` λq “ 0 ´C1pσ1 ` σ2 ` µHq ` C2σ1 “ 0 (29) (29) Making C2 the subject we get, Making C2 the subject we get, Making C2 the subject we get, Making C2 the subject we get, C2 “ C1 σ1 ` σ2 ` µH σ1 (30) C2 “ C1 σ1 ` σ2 ` µH σ1 (30) Similarly for C6 C6 “ C5 σ3 ` µR σ3 (31) (30) Similarly for C6 C6 “ C5 σ3 ` µR σ3 (31) Similarly for C6 σ3 ` µR Similarly for C6 C6 “ C5 σ3 ` µR σ3 C6 “ C5 σ3 ` µR σ3 (31) (31) σ3 19 Substituting back to Equation 27 and Simplifying we get, Substituting back to Equation 27 and Simplifying we get, dL dt ď C˚ 1 p σ1ξ2 pσ1 ` σ2 ` µHqpµH ` δH ` λq πHp1 ´ pqpµH ` wq ` πHpω pNHrpµH ` ϑ1qpµH ` ωq ` ωϑ1sq ´ 1q `C˚ 5 p σ3ξ3 pµR ` σ3µR ` δRq πR pNRµRq ´ 1q (32) dL dt ď C˚ 1 p σ1ξ2 pσ1 ` σ2 ` µHqpµH ` δH ` λq πHp1 ´ pqpµH ` wq ` πHpω pNHrpµH ` ϑ1qpµH ` ωq ` ωϑ1sq ´ 1q (32) `C˚ 5 p σ3ξ3 pµR ` σ3µR ` δRq πR pNRµRq ´ 1q (32) That is, dL dt ď C˚ 1 pRH ´ 1q ` C˚ 5 pRR ´ 1q (33) (33) Clearly, dL dt ă 0 if and only if when RH ă 1 and RR ă 1 , which proves the theorem, therefore the MFE is globally stable. 2.7. Global stability of the Monkey-pox virus Free Equilibrium Clearly, dL dt ă 0 if and only if when RH ă 1 and RR ă 1 , which proves the theorem, therefore the MFE is globally stable. 2.7.1. Global stability of the Monkeypox Endemic Equilibrium (MEE) Theorem 2.4. Endemic equilibrium is asymptotically stable when RH ą 1 (for Human) and RR ą 1 (for rodents) and unstable when RR ă 1 and RR ă 1 [33] 2.7.1. Global stability of the Monkeypox Endemic Equilibrium (MEE) Theorem 2.4. Endemic equilibrium is asymptotically stable when RH ą 1 (for Human) and RR ą 1 (for rodents) and unstable when RR ă 1 and RR ă 1 [33] Proof of Theorem 2.4. For this study we used the approach as that of Masan- dawa et.al [34], with Logarithmic Lyapunov function as p “ 9ÿ i“1 Cipxi´x˚ i q lnpxiq Where, Ci is a positive constant xi is population of the compartment i and x˚ i is the equilibrium level value. 2.7.1. Global stability of the Monkeypox Endemic Equilibrium (MEE) Theorem 2.4. Endemic equilibrium is asymptotically stable when RH ą 1 (for Human) and RR ą 1 (for rodents) and unstable when RR ă 1 and RR ă 1 [33] ppSH, EH, IH, QH, RH, VH, SR, ER, IRq “ C1ppSH ´ S˚ Hq ln SHq` `C2ppEH ´ E˚ Hq ln EHq ` C3ppIH ´ I˚ Hq ln IHq ` C4ppQH ´ Q˚ Hq ln QHq` `C5ppRH ´ R˚ Hq ln RHq ` C6ppVH ´ V ˚ Hq ln VHq ` C7ppSR ´ S˚ Rq ln SRq` `C8ppER ´ E˚ Rq ln ERq ` C9ppIR ´ I˚ Rq ln IRq (34) (34) 20 Now taking the derivative of p with respect to time and simplifying we have, dp dt “ C1ppSH ´ S˚ Hq SH dSH dt q ` C2ppEH ´ S˚ Hq EH dEH dt q ` C3ppIH ´ I˚ Hq IH dIH dt q` `C4p pQH´Q˚ Hq QH dQH dt q ` C5ppRH ´ R˚ Hq RH dRH dt q ` C6ppVH ´ V ˚ Hq VH dVH dt q` `C7p pSR´S˚ Rq SR dSR dt q ` C8ppER ´ E˚ Rq ER dER dt q ` C9ppIR ´ I˚ Rq IR dIR dt q (35) (35) Then substitute Equation 1 into Equation 35 we have, Then substitute Equation 1 into Equation 35 we have, dp dt “ C1ppSH ´ S˚ Hq SH πHp1 ´ pq ´ pξ1IR ` ξ2IHq SH NH ´ pµH ` ϑ1qSH` `ωVH ` ψQHq ` C2p pEH´S˚ Hq EH ξ1IR ` ξ2IHq SH NH ´ pσ1 ` σ2 ` µHqEHq` `C3p pIH´I˚ Hq IH σ1EH ` ζQH ´ pµH ` δH ` λqIHq` `C4p pQH´Q˚ Hq QH σ2EH ´ pψ ` ζ ` µH ` δHqQHq` `C5p pRH´R˚ Hq RH λIH ´ pµH ` ϑ2qRHq` `C6p pVH´V ˚ H q VH πHp ` ϑ1SH ` ϑ2RH ´ pµH ` ωqVHq` `C7p pSR´S˚ Rq SR πR ´ ξ3IR SR NR ´ µRSRq` `C8p pER´E˚ Rq ER ξ3IR SR NR ´ pµR ` σ3qERq` `C9p pIR´I˚ Rq IR σ3ER ´ pµR ` δRqIRq (36) dp dt “ C1ppSH ´ S˚ Hq SH πHp1 ´ pq ´ pξ1IR ` ξ2IHq SH NH ´ pµH ` ϑ1qSH` `ωVH ` ψQHq ` C2p pEH´S˚ Hq EH ξ1IR ` ξ2IHq SH NH ´ pσ1 ` σ2 ` µHqEHq` `C3p pIH´I˚ Hq IH σ1EH ` ζQH ´ pµH ` δH ` λqIHq` `C4p pQH´Q˚ Hq QH σ2EH ´ pψ ` ζ ` µH ` δHqQHq` pR R˚ q `C5p pRH´R˚ Hq RH λIH ´ pµH ` ϑ2qRHq` (36) (36) `C6p pVH´V ˚ H q VH πHp ` ϑ1SH ` ϑ2RH ´ pµH ` ωqVHq` `C7p pSR´S˚ Rq SR πR ´ ξ3IR SR NR ´ µRSRq` `C8p pER´E˚ Rq ER ξ3IR SR NR ´ pµR ` σ3qERq` `C9p pIR´I˚ Rq IR σ3ER ´ pµR ` δRqIRq As done by Martcheva [12] one of the classical ﬁrst steps here is to replace πH 21 and πR with its equal from the equilibria equations, that is, and πR with its equal from the equilibria equations, that is, πH “ 1 p1 ´ pqppξ1I˚ R ` ξ2I˚ Hq S˚ H NH ` pµH ` ϑ1qS˚ H ´ ωV ˚ H ´ ψQ˚ Hq πR “ ξ3I˚ R S˚ R NR ´ µRS˚ R (37) πH “ 1 p1 ´ pqppξ1I˚ R ` ξ2I˚ Hq S˚ H NH ` pµH ` ϑ1qS˚ H ´ ωV ˚ H ´ ψQ˚ Hq πR “ ξ3I˚ R S˚ R NR ´ µRS˚ R (37) (37) Then ppµH ` σ1qS˚ H ´ ψS˚ Hq ´ ppµH ` σ1qSH ´ ψSHqcan be combined with the ﬁrst term in the product to yield a negative term. 2.7.1. Global stability of the Monkeypox Endemic Equilibrium (MEE) Theorem 2.4. Endemic equilibrium is asymptotically stable when RH ą 1 (for Human) and RR ą 1 (for rodents) and unstable when RR ă 1 and RR ă 1 [33] We multiply out all other products: We now have to apply the Krasovkii-LaSalle theorem. We consider the set where the Lyapunov function is equal to zero, that is L “ tx P ℜn\|V 1pxq “ 0u (38) (38) Clearly from equation 36 the V 1pxq “ 0 if and only if SH ´ S˚ H “ 0, EH “ E˚ H, IH “ I˚ H, QH “ Q˚ H, RH “ R˚ H, VH “ V ˚ H, SR “ S˚ R, ER “ E˚ Rand IR “ I˚ R , other terms are negative when pEH, IH, QH, RH, VH, SR, ER, IRq ą 0. Therefore, the MEE is globally asymptotically stable when RH ą 1 and RR ą 1 this results concludes and proves the theorem. 3.1. Sensitivity analysis The goal of sensitivity analysis is to qualitatively decide which parameters are most inﬂuential in the model output. Sensitivity analysis can be performed on a dynamical system or on static quantities such as the reproduction number or equilibria prevalence [11, 35]. We compute the sensitivity indices of the basic reproductive number R0 to the parameters in model. These indices allow us to see how important each parameter is to disease transmission [36]. The normalized forward sensitivity index of R that depends diﬀerentiability on the parameter p is deﬁned as γR p “ BR Bp ˆ p R , where γR p is the sensitivity index of R 22 with respect to parameter p is positive if R is increasing with respect to p and is negative if R is decreasing with respect to p [10, 18, 34, 37, 35]. 3.1. Sensitivity analysis Using the eﬀective reproduction number Rv we can take partial derivative with respect to each parameter and compute sensitivity, for human and rodent reproduction numbers populations, RH and RR, respectively and then was ﬁlled in the Table 3 Figure 2: The Sensitivity Indices Figure 2: The Sensitivity Indices Figure 2: The Sensitivity Indices 23 Table 3: Sensitivity expression and index for each parameter SN Parameter Expression of the Sensitivity parameter Sensitivity Value 1 πH 1 1 2 ξ2 ξ2σ1πHp1 ´ pqpµH ` ωq σ1ξ2πHp1 ´ pqpµH ` ωq ` πHpω 0.00005999976 3 σ1 1 ´ σ1 σ1 ` σ2 ` µH 0.945945945 4 σ2 ´ σ2 σ1 ` σ2 ` µH ´0.54054054 5 λ ´ λ pµH ` δH ` λq ´0.328063241 6 µH µH pµH ` ωq ` πHpω ` p1 ` 1 pµH ` δH ` λqq ˆ µH pσ1ξ3πHp1 ´ pqpµH ` ωq ` πHpωq 1.622222519 7 δH ´ δH pµH ` δH ` λq ´0.0790514 8 ϑ1 ´ 2ω ` µH pµH ` ϑ1qpµH ` ωq ` ωϑ1 ´0.62616939 9 ω ξ2σ1πHp1 ´ pqpµH ` ωq σ1ξ2πHp1 ´ pqpµH ` ωq ` πHpω ´ ωpµH ` 2ϑ1q pσ1 ` σ2 ` µHqpµH ` δH ` γqppµH ` ϑ1qpµH ` ωq ` ωϑ1q 0.064051357 10 πR 1 1 11 ξ3 1 1 12 σ3 1 ´ σ3 pµR ` σ3q 0.000666222 13 µR ´ µ2 Rp3µR ` 2δR ` 2σ3q pµR ` σ3qpµR ` δRqµR ´0.009297918 14 δR ´ δR µR ` δR ´0.996015936 24 Table 3: Sensitivity expression and index for each parameter ´ 2ω ` µH pµH ` ϑ1qpµH ` ωq ` ωϑ1 24 The results from the sensitivity analysis clearly indicate that increasing the values of parameters πH, ξ2, ω, and σ1 will lead to an increase in the spread of the monkeypox virus in the human population (for details see Figure 2). Conversely, increasing the values of parameters σ2, λ, µH, δH, and ϑ1 will contribute to a decrease in the spread of the virus among humans. In the case of the rodent population, increasing the values of parameters πR, ξ3, and σ3 will result in an increased infection rate of the monkeypox virus among rodents, while increasing the parameters δR and µR will lead to the elimination of the virus in the rodent population. 3.1. Sensitivity analysis The most sensitive parameters in the analysis are the vaccination rate of susceptible individuals, the progression rate from exposed to infectious individ- uals, and the progression rate from exposed to individuals in quarantine, as well as the death rate of the rodents. 3.2. Model simulation The proposed model was simulated and analysed using MATLAB R2018a with the parameter values stated in Table 2. The following initial population densities for people and rodents were considered for the analysis: NH “ rSH, EH, IH, QH, RH, VHs “ r4000000, 2000000, 100000, 500000, 200000, 400000s NH “ rSH, EH, IH, QH, RH, VHs “ r4000000, 2000000, 100000, 500000, 200000, 400000s NR “ rSR, ER, IRs “ r1000000, 500000, 300000s respectively which was simulated for a time span of one year to the developed model. The results of reproduction numbers Rv “ 5.374900247 while RH “ 2.4768 ˆ 10´7 and RR “ 5.374900247 Clearly, the obtained reproduction num- bers for the data simulated indicate that the main contribution to transmission comes from rodents, as their transmission can result in more than ﬁve (5) new secondary infections. This clearly demonstrates that the primary mode of virus transmission is from rodents to humans, while the transmission from human to human is minimal, with a rate less than one. Con- sequently, by eﬀectively controlling the transmission among the rodent population, the virus can be eradicated in the human population. This observation is well depicted in Figure 5, Figure 4 and Figure 7, where the formulated model shows that the disease will eventually die out in the human population while persisting in the rodent population. Furthermore, the graphs illustrating the population dynamics in both humans (Figure 3) and rodents (Figure 4) provide additional support for this fact, showcasing the proportional relationship between the populations. To gain a better understanding of the dynamics, the initial conditions were adjusted to a smaller population size. The model was further analyzed by modifying the initial conditions 25 Figure 3: Human population Proportions evolution over time Figure 3: Human population Proportions evolution over time 26 Figure 4: Rodent evolution against time for the proposed mathematical model Figure 4: Rodent evolution against time for the proposed mathematical model Figure 4: Rodent evolution against time for the proposed mathematical model 27 Figure 5: Human and rodent population evolution against time for the proposed model Figure 5: Human and rodent population evolution against time for the proposed model Figure 5: Human and rodent population evolution against time for the proposed model 28 as follows: For the human population, we set SH “ 40, EH “ 3, IH “ 2, QH “ 0, RH “ 0, and VH “ 0. 3.2. Model simulation Similarly, for the rodent population, we set SR “ 10, ER “ 2, and IR “ 1. These adjustments allow for a more detailed exploration of the system’s behavior and its response to diﬀerent initial conditions. The details are explained in Figure 6 and Figure 7. Figure 6: Evolution of the human population over time in the proposed model: It is evident that implementing the suggested model leads to a signiﬁcant reduction in the number of infected and exposed individuals within the human population. At the same time, it eﬀectively increases the number of individuals who have recovered from the infection and enhances the size of the susceptible population, contributing to overall population health and resilience. The model highlights the potential beneﬁts of the proposed interventions in controlling the spread of the disease and mitigating its impact on the human population. Figure 6: Evolution of the human population over time in the proposed model: It is evident that implementing the suggested model leads to a signiﬁcant reduction in the number of infected and exposed individuals within the human population. At the same time, it eﬀectively increases the number of individuals who have recovered from the infection and enhances the size of the susceptible population, contributing to overall population health and resilience. The model highlights the potential beneﬁts of the proposed interventions in controlling the spread of the disease and mitigating its impact on the human population. 3.3. Eﬀects of Quarantine and Vaccination on the Transmission of Monkeypox Virus 3.3. Eﬀects of Quarantine and Vaccination on the Transmission of Monkeypox Virus This is well depicted in Figure 8, Figure 9, Figure 10 Figure 11 and Figure 12 with detailed explanation in their caption. From Figure 9, it is evident that increasing the vaccination rate and implementing both quarantine measures and vaccination (Figure 8) have a signiﬁcant impact on reducing the number of infectious individuals in the human population. Additionally, controlling the spread of the virus among rodents is also crucial, as demonstrated in Figure 10. By decreasing the force of infection from rodents to humans, the number of infectious individuals in the human population is eﬀectively reduced. This outcome is clearly depicted in Figure 12, where the reduction in the progression rate from exposed rodents to infectious rodents successfully reduces the infectious human population. 29 Figure 7: Evolution of the rodent population over time in the proposed model: In the ab- sence of interventions, the dynamics demonstrate a persistent infected population within the rodent population. This highlights the need to address the human-to-rodent contact to eﬀec- tively eliminate the spread of monkeypox. We recommend implementing measures to reduce human-rodent interactions as a crucial step in controlling the disease and safeguarding public health. By minimizing contact, we can mitigate the transmission of monkeypox from rodents to humans and contribute to the overall prevention and eradication eﬀorts. Figure 7: Evolution of the rodent population over time in the proposed model: In the ab- sence of interventions, the dynamics demonstrate a persistent infected population within the rodent population. This highlights the need to address the human-to-rodent contact to eﬀec- tively eliminate the spread of monkeypox. We recommend implementing measures to reduce human-rodent interactions as a crucial step in controlling the disease and safeguarding public health. By minimizing contact, we can mitigate the transmission of monkeypox from rodents to humans and contribute to the overall prevention and eradication eﬀorts. 30 Figure 8: The human infected population increases when no intervention is taken but reduces signiﬁcantly when both quarantine and vaccination measures are implemented. This observa- tion highlights the eﬀectiveness of these interventions in controlling the spread of monkeypox. Without any intervention, the disease can rapidly propagate within the human population, leading to a higher number of infected individuals. However, with the implementation of quarantine measures and vaccination, virus transmission is signiﬁcantly curtailed, resulting in a notable reduction in the population of infected humans. 3.3. Eﬀects of Quarantine and Vaccination on the Transmission of Monkeypox Virus This underscores the importance of proactive measures, such as quarantine and vaccination, in mitigating the impact of mon- keypox and protecting public health. Figure 8: The human infected population increases when no intervention is taken but reduces signiﬁcantly when both quarantine and vaccination measures are implemented. This observa- tion highlights the eﬀectiveness of these interventions in controlling the spread of monkeypox. Without any intervention, the disease can rapidly propagate within the human population, leading to a higher number of infected individuals. However, with the implementation of quarantine measures and vaccination, virus transmission is signiﬁcantly curtailed, resulting in a notable reduction in the population of infected humans. This underscores the importance of proactive measures, such as quarantine and vaccination, in mitigating the impact of mon- keypox and protecting public health. 31 Figure 9: Infected human dynamics under varying vaccination rates: Figure 9 demonstrates the relationship between the vaccination rate and the infected human population. As the vaccination rate increases, there is a noticeable reduction in the number of infected humans. This ﬁnding highlights the signiﬁcance of a higher vaccination rate in mitigating the spread and impact of monkeypox among the human population. Figure 9: Infected human dynamics under varying vaccination rates: Figure 9 demonstrates the relationship between the vaccination rate and the infected human population. As the vaccination rate increases, there is a noticeable reduction in the number of infected humans. This ﬁnding highlights the signiﬁcance of a higher vaccination rate in mitigating the spread and impact of monkeypox among the human population. 32 Figure 10: Infected human dynamics under the variation of force of infection from rodents, which shows that an increase in the force of infection will lead to an increase in the population of infected humans. This observation highlights the crucial role of rodents as a source of transmission of monkeypox to humans. As the force of infection from rodents increases, the likelihood of transmission to humans also increases, resulting in a larger population of infected individuals. This emphasizes the importance of eﬀective control measures targeting rodents, such as improved rodent control practices and habitat modiﬁcation, to mitigate the risk of monkeypox transmission and reduce the burden of human infections. Figure 10: Infected human dynamics under the variation of force of infection from rodents, which shows that an increase in the force of infection will lead to an increase in the population of infected humans. 3.3. Eﬀects of Quarantine and Vaccination on the Transmission of Monkeypox Virus This observation highlights the crucial role of rodents as a source of transmission of monkeypox to humans. As the force of infection from rodents increases, the likelihood of transmission to humans also increases, resulting in a larger population of infected individuals. This emphasizes the importance of eﬀective control measures targeting rodents, such as improved rodent control practices and habitat modiﬁcation, to mitigate the risk of monkeypox transmission and reduce the burden of human infections. 33 Figure 11: Dynamics of infected rodents with varying progression rates from the exposed to infectious population: The plot illustrates a direct proportional relationship between the increase in the progression rate and the number of infected humans. As the progression rate from the exposed to infectious population in rodents increases, it leads to a corresponding increase in the number of infected humans. This ﬁnding emphasizes the importance of under- standing and managing the transmission dynamics within the rodent population to eﬀectively control and prevent the spread of monkeypox to humans. Figure 11: Dynamics of infected rodents with varying progression rates from the exposed to infectious population: The plot illustrates a direct proportional relationship between the increase in the progression rate and the number of infected humans. As the progression rate from the exposed to infectious population in rodents increases, it leads to a corresponding increase in the number of infected humans. This ﬁnding emphasizes the importance of under- standing and managing the transmission dynamics within the rodent population to eﬀectively control and prevent the spread of monkeypox to humans. Figure 11: Dynamics of infected rodents with varying progression rates from the exposed to infectious population: The plot illustrates a direct proportional relationship between the increase in the progression rate and the number of infected humans. As the progression rate from the exposed to infectious population in rodents increases, it leads to a corresponding increase in the number of infected humans. This ﬁnding emphasizes the importance of under- standing and managing the transmission dynamics within the rodent population to eﬀectively control and prevent the spread of monkeypox to humans. 34 Figure 12: The contour plots demonstrates the contribution of the proposed interventions. Starting from the top left, it shows that increasing the rate of quarantine and reducing the human-to-human contact rate help to reduce the reproduction number. The same principle applies to the vaccine in the top right and bottom sections. They exhibit the same property. 3.3. Eﬀects of Quarantine and Vaccination on the Transmission of Monkeypox Virus Figure 12: The contour plots demonstrates the contribution of the proposed interventions. Starting from the top left, it shows that increasing the rate of quarantine and reducing the human-to-human contact rate help to reduce the reproduction number. The same principle applies to the vaccine in the top right and bottom sections. They exhibit the same property. 35 4. Conclusions In our study, we formulated a deterministic dynamic model for monkeypox virus trans- mission in which we calculated the DFE. The reproduction number is Rv “ 5.3749, with RH “ 2.4768 ˆ 10´7 for for humans and RR “ 5.3749 for rodents, for the simulated results, which shows that the main contribution of infection by this virus is from the rodent popu- lation. The global stability of the model at MFE and MEE was analysed, which revealed it to be stable. The sensitivity analysis was done for the reproduction numbers of both human and rodent populations. The most sensitive parameters that should be taken into account were the rate of vaccination of susceptibles, the progression rate from exposed to infectious and exposed to quarantine, and the mortality rate of the rodent population. Generally, the study shows that for the proposed mathematical model, increasing vaccination, quarantine, and avoiding contact with infected rodents will eradicate the spread of the virus; therefore, we propose this model be used to explore the dynamics of the monkeypox virus outbreak. In summary, this study contributes to understanding the dynamics of monkeypox virus transmission and provides information for eﬀective control measures. The proposed determin- istic dynamic model, with its calculated reproduction numbers and stability analysis, oﬀers valuable guidance for targeted interventions. The sensitivity analysis identiﬁes key parameters for control, while the proposed numerical scheme enhances the model’s predictive capacity. Overall, this research has practical implications for outbreak management and can inform public health interventions to eradicate the spread of the monkeypox virus. Acknowledgments The authors express their gratitude for the support received from the NORHED II project and the Department of Mathematics at the University of Dar es Salaam in Tanzania, as well as the University of Bergen in Norway. These institutions provided essential facilities that contributed to the successful completion of this research. Additionally, the authors acknowledge the anonymous reviewers for their valuable comments that greatly contributed to the improvement of this work. Ethical Approval Not applicable Funding This work has been supported by the Mathematics for Sustainable Development (MATH4SDG) project at the University of Dar es Salaam -Tanzania funded through the NORHED II pro- gramme under the Norwegian Agency for Development Cooperation (NORAD, project no. 68105). Availability of data and materials The numerical data used to support the ﬁndings of this study have been taken from previously published articles and are cited in Table 2 of this article. Also, others are available online in https://www.cdc.gov/poxvirus/mpox/response/2022/world-map.html and https: //ourworldindata.org/monkeypox. Competing interests The authors have no conﬂict of interest. 36 Authors’ contributions • Leonce Leandry: Writing original draft, Conceptualization, Methodology, Formal analysis, Software and Review. • Leonce Leandry: Writing original draft, Conceptualization, Methodology, Formal analysis, Software and Review. • Eunice Mureithi: Validation, Writing review, editing and Supervision. • Eunice Mureithi: Validation, Writing review, editing and Supervision. References [1] P. v. Magnus, E. K. Andersen, K. B. Petersen, A. 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https://openalex.org/W4313485352	https://www.researchsquare.com/article/rs-2426437/latest.pdf	English	null	Environmental-related technologies and carbon neutrality challenges in emerging economies? A case study of China	Research Square (Research Square)	2,023	cc-by	10,886	Highlights 1. This study introduces the key factors of carbon neutrality (CN) in China. 1. This study introduces the key factors of carbon neutrality (CN) in China. 2. FR and environmental taxes significantly contribute to carbon neutrality. 3. GDP and international cooperation do not work perfectly for carbon neutrali 4. Environmental-related technologies are against CN’s theme in China. 5. Policy suggestions are given to attain the zero-emissions. 2. FR and environmental taxes significantly contribute to carbon neutrality. 3. GDP and international cooperation do not work perfectly for carbon neu 4. Environmental-related technologies are against CN’s theme in China. 5. Policy suggestions are given to attain the zero-emissions. Research Article Keywords: Carbon Neutrality, International cooperation, Environmental technology, Fertility rate, China Posted Date: January 4th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-2426437/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/29 Page 1/29 Abstract This study provides new evidence for an unclear link between international cooperation (IC) and environmental-related technologies (ERT) with carbon neutrality (CN) in China. This research contributes to environmental economics literature by opening a new door for economic growth, fertility rate, environmental taxes, IC, and ERT from 1990 to 2020. Considering the sustainability initiatives taken by China, it is crucial to examine the mentioned factors' role in carbon neutrality. Therefore, we employed advanced techniques to investigate the study’s objectives. Estimated results show the harmful impact of economic progress, IC, and ERT on CN. Moreover, fertility rate and environmental taxes help out in emissions reduction. In addition, this study uses Breitung and Candelon causality test and supports the uni-directional association between economic progress to emissions and emissions to fertility rate in China. However, this study also proposes imperative policies to achieve the carbon neutrality target. JEL Codes: O14, J13, O44, H23. JEL Codes: O14, J13, O44, H23. 1- Introduction Nowadays, economic progress and urbanization are directly associated with the industrial sector. Such an association increases the development of other sectors with an increase in population. The world’s population is expected to grow by approximately 8 billion to 10 billion by 2050. This trend will require more than 80% energy and 70% food to maintain the same living standard. Since the last three decades, there has been immense exploitation of natural resources by human kinds. This phenomenon has occurred in exchange for massive deforestation and utilization of petroleum resources to meet excess demand for necessities. Thus, these factors are being introduced as a crux of global warming and climate change. Upto 2050, 50% of global emissions would increase by a share of carbon emissions via the energy sector. If this trend continues over time, it may exceed the limit and cause irreversible climate change. Therefore, it is the best era to fight against global warming. However, the globe has taken several initiatives for environmental sustainability, and the Paris agreement is one of them. Moreover, all economies have agreed to keep global warming less than 2 0C to achieve carbon neutrality (CN). But global temperature is increasing over time, and economies are trying to achieve zero emissions. The CN target will not only reduce the emissions level but also cause clean air. It is necessary to work together as CN has become a severe global challenge. However, more than 120 countries have committed Page 2/29 Page 2/29 to keeping their country temperature at 2 0C and will try to include it in carbon-neutral economies (K. Hu, Raghutla, Chittedi, Zhang, & Koondhar, 2021). Similarly, Suriname and Bhutan have become carbon neutral in the last decade and have contributed negatively. The remaining economies set their targets to rise as carbon-neutral economies in a different era, such as Uruguay by 2030, Finland by 2035, and the UK, France, Germany, South Africa, and South Korea by 2050. However, China has set its target to achieve zero carbon emissions by 2060. China has become the largest carbon emitter globally, and upto 2020 it has contributed 32% in total global emissions. Such a scenario makes it difficult for China to achieve CN targets. Therefore, China’s president and climate experts are trying to reach its peak by 2030 and commit to carbon neutrality by 2060. Table 1: Summary of the Past Studies 1- Introduction Therefore, to tackle this issue, international cooperation is necessary to fight against climate variation. In the 14th five-year development plan, carbon peaking and neutrality is also considered in the construction and other human activities. From this initiative, it is clear that china is extremely serious about zero emissions and takes it as an urgent strategy. Due to immense attention towards zero emissions, china has significantly declined by 48% to 18% in the last decade. However, in the existing literature, numerous case studies tried to convey a clear message about CN, but they have reached a single point. For instance, (Can, 2021) demonstrated that technical innovation, finance, and collaboration might cause emission reduction to zero. Besides, there is an urgent need to re- check the innovative determinants of carbon neutrality. Likewise, Table 1 presents the existing studies relevant to carbon neutrality. Table 1: Summary of the Past Studies Table 1: Summary of the Past Studies Page 3/29 Author Region Technique Findings. CN (Abbasi, Hussain, Redulescu, & Ozturk, 2021) UK D-ARDL EU, IVA, NRD CO2 Yes (K. Hu et al., 2021) India JC RE CO2 Yes (C. Yang, Hao, & Irfan, 2021) China MC EU CO2 Yes (H. Zhang, 2021) BRICS FE PT CO2 Yes (Shao, Zhong, Li, & Altuntaş, 2021) The USA DOLS, FMOLS ERR&D CO2 Yes (Tan et al., 2021) China ARDL EU & FD CO2 Yes (Nair, Arvin, Pradhan, & Bahmani, 2021) 67 developing countries FMOLS IQ CO2 Yes (J. Hu, Wu, Zhao, & Wang, 2021) Beijing, China Sub-energy increment EU CO2 Yes (Cheng et al., 2021) China FMOLS, DOLS & CCR EE & ECI CO2 Yes (H. Yang, Shahzadi, & Hussain, 2021) The USA DOLS GG CO2 Yes (H.-S. Li, Geng, Shinwari, Yangjie, & Rjoub, 2021) Top exporting countries CS-ARDL, AMG Exports & RE CO2 Yes (Shan, Genç, Kamran, & Dinca, 2021) Turkey Bootstrapping ARDL GTI & RE CO2 Yes (I. Khan et al., 2022) Pakistan NARDL TR & FDI CO2 Yes (Xu, Dong, Xu, & Bhattarai, 2022) China PCA Doesn’t reach carbon peak Yes (A. A. Khan et al., 2022) G7 CS-ARDL and AMG IQ & RE CO2 Yes (Hossain et al., 2022) India D-ARDL MVA CO2 Yes (Saqib, 2022) 63 emerging and developed economies AMG and CCE-MG RE and FD CFP Yes (X. 1- Introduction Sun, Zhang, Ahmad, & Xue, 2022) 36 Chinese cities DKSE URB CO2 Yes (Murshed, Mahmood, Ahmad, Rehman, & Alam, 2022) Argentina ARDL TI CO2 Yes CN Author CN Page 4/29 (Y. Sun, Guan, Cao, & Bao, 2022) China DID GF CO2 Yes (Kurramovich et al., 2022) Japan ARDL REG CO2 Yes (B. Li & Haneklaus, 2022) China ARDL RE CO2 Yes (Ahmed, Ahmad, & Ahmed, 2022) Pakistan OLS IND & GDP CO2 Yes (Wang, Raza, Adebayo, Yi, & Shah, 2023) China QR NE, BIO & HY EFP Yes Similarly, in the existing literature, as summarized in Table, numerous case studies have tried to investigate the possibilities regarding carbon neutrality. However, past studies did not consider the critical determinants of zero emissions, such as international cooperation, environment-related technologies, environmental taxes, and fertility rate across the globe. Therefore, the present study tries to incorporate these factors and may introduce a new chapter for investigation. In order to fill up the past flaws, this research will contribute in the following ways. Firstly, the impact of gross domestic product (GDP) on carbon emissions has been justified by various case studies, but these studies have not proposed a systematic way to reduce carbon emissions. Therefore, it is important to use this factor in this study and try to convey a clear message why China cannot meet the green environment targets set by the UN. In the current era, economies care for their progress at the cost of environmental pollution, and China is not free from this condition. Due to such reasons, it is crucial to investigate whether GDP harms or protects environmental quality. In order to fill up this gap, the current study tries to explore the impact of GDP on carbon emissions in China. Secondly, China settled one child policy in 1980 and ended it in 2016, but it has long run effect on China’s economy. Since the last three decades, china has had a low fertility rate; therefore, it is essential to add this factor to the environment-growth model and investigate its impact on environmental sustainability. Due to the low fertility rate, the older population has increased; thus, examining its effects on environmental situation could be informative. Thirdly, numerous case studies have focused on the linkages of GDP, FR and CO2 emissions, while ignoring environment-related technologies (ERT). 1- Introduction On behalf of such schemes and their outcomes, a door to rethink zero emission is still open. As mentioned in prior stages, the overall focus of existing studies has been on economic progress, energy use, trade sector, etc.; however, there has not found any clear message for ERT across the globe and especially for China’s economy. Undoubtedly, numerous key Page 5/29 Page 5/29 factors play a distinctive role in zero emissions due to geo, social and political scenarios. Therefore, this empirical study tries to focus on ERT, and its inclusion may help-out to achieve CN. Because various sectors are directly associated with technologies, green technologies may possibly reduce the share of emissions from each sector and ultimately cause zero-emissions. Hence, it is necessary to include ERT in the emission-growth model. Fourthly, environmental-related taxes (Tax) also perform well in maintaining environmental quality (Elkins & Baker, 2001). Numerous researchers commonly believe that taxes are extremely important for emissions reduction (Hammar & Sjöström, 2011). There has been an inverse association between CO2 emissions and environmental taxes (Sundar, Mishra, & Naresh, 2016). However, it is impossible to reduce emissions to a certain level only with carbon taxes; hence, environmental taxes and technologies are core factors in dealing with environmental concerns. Therefore, the present study tries to answer whether environmental taxes significantly contribute to CN. Fifthly, international cooperation is considered another core factor that may help reduce environmental pollution across the border. However, the nexus of international cooperation (IC) and the environment is a new research approach (Scott, 2008). Similarly, the World Polity theory also demonstrates that social, political, and cultural aspects of international cooperation can explain the policymakers’ behavior in the development process. Furthermore, this theory also discusses strengthening institutions to deal with issues concerning human beings. Therefore, international cooperation may help reduce the environmental pressure and cause emissions reduction. Neglecting the ecological system may not be a good choice; therefore, emerging economies must cooperate with other nations and put zero emissions in development strategies. Thus, this study investigates this theme and suggests suitable policies for further cooperation with other nations. Finally, this study employs a series of empirical estimators to investigate more robust results. This analysis is based on five different estimators: Dynamic ARDL, ARDL, fully modified ordinary least square (FMOLS), dynamic ordinary least square (DOLS), and canonical correlation regression (CCR). 1- Introduction We are using these econometric techniques to deal with problems in time series data. However, the variables data collected from different sources and the obtained outcomes may suggest some imperative policies to attain the zero-emissions level. The rest of the paper is organized as follows; section 2 consists of data and methods, section 3 consists of results and discussion, and section 4 consists of conclusion and policy recommendations. 2.1. Variables Selection Since the last two decades, environmental taxes (TAX) have been considered one of the most suitable ecological legislation to improve environmental sustainability. Since 1979 China has focused on environmental legislation (Mu, Bu, & Xue, 2014). By having this initiative, China has introduced a pollution discharge fee to reduce carbon emissions. Upto 2017 this legislation has remained one of the top environmental policies, but it has faced several challenges (Khan, Sisi, & Siqun, 2019). Besides such hurdles, China is committed to working for zero emissions up to 2060. In the recent decade, another eco- Page 6/29 friendly development plan has been introduced by China’s leadership, and its motto was to minimize emissions from the cement and steel industry. Since 2014, the emissions level has stagnated; therefore, eco-friendly initiatives or legislation will cause peak emissions by 2030 (Its & Least, 2017). Furthermore, economic growth is being considered another determent for carbon neutrality. Therefore, there may be a change that GDP can influence the environmental quality via two effects, i.e., scale and technique effects. There may be a possibility that the scale effect becomes dominant and cause of CO2 emissions; otherwise, in case of technique effect it may cause CN by reducing emissions. However, ERT is significantly playing a crucial role in emissions reduction. It may be possible that ERT, such as energy efficiency, eco-friendly protective innovations etc., cause of decline in emissions and may further improve environmental sustainability. Undoubtedly, economies across the border are helping each other resolve environmental issues. In order to bring world temperature by approximately 2 0C, it needs time to cooperate and try to solve un-friendly problems. Lastly, the fertility rate is a determinant of environmental degradation and is directly associated with environmental deterioration. Therefore, environmental variation may cause household status and indirectly affect the fertility rate (Adsera & Menendez, 2011). Moreover, due to limited budget constraints, the cost and affordability of children may feel impossible, which would cause fewer human activities and reduce emissions (Sellers & Gray, 2019). However, unexpected human behavior, such as conflicts between spouses and employment migration, is directly associated with the fertility rate, and as a result, it decreases emissions (Axinn, Ghimire, & Smith- Greenaway, 2017; Hidrobo & Fernald, 2013). Moreover, the data sources, units, and variables notions are provided in Table 2. 2.1. Variables Selection Table 2: Data Description Variable Unit Source Expected sign CN Carbon emissions (Kt) WDI N/A GDP Gross Domestic Product (US $) WDI +VE FR Fertility rate (Children per woman) WDI -VE ET Environmental Related Technologies (% of all technologies) Knoema +VE IC International Cooperation (index) QGISD +VE Tax Environmental-related tax (% of GDP) OECD -VE Note: QGISD: Quality of Government Institute Standard Dataset Table 2: Data Description Note: QGISD: Quality of Government Institute Standard Dataset However, some initial tests, such as descriptive statistics and pairwise correlation tests, can investigate the data's reliability. Table 3 presents the outcomes of descriptive statistics of selected variables, and there was no considerable difference between mean and median values. This supports the concept that there is no outlier in the given data. Page 7/29 Table 3: Descriptive Statistics of Variables Table 3: Descriptive Statistics of Variables Page 7/29 LCO2 LGDP LFR LET IC LTAX Mean 4.757 6.850 0.226 0.891 0.701 -0.125 Median 4.747 6.852 0.212 0.912 0.757 -0.064 Maximum 4.982 7.372 0.363 1.003 0.936 0.224 Minimum 4.466 6.218 0.202 0.564 0.238 -0.642 Std. Dev. 0.153 0.360 0.037 0.093 0.185 0.237 Skewness -0.401 -0.134 2.469 -1.559 -0.974 -0.715 Kurtosis 2.141 1.752 8.373 6.054 2.969 2.714 Jarque-Bera 1.782 2.102 68.792 24.627 4.905 2.751 Probability 0.000 0.049 0.000 0.000 0.046 0.002 Similarly, Table 4 shows the outcomes regarding pairwise correlation tests. The given results show that FR and environmental technologies are inversely associated with explained variables. In light of estimated outcomes, there is no problem with multicollinearity. In order to verify this statement, the current study also runs the VIF test. LCO2 LGDP LFR LET IC LTAX Mean 4.757 6.850 0.226 0.891 0.701 -0.125 Median 4.747 6.852 0.212 0.912 0.757 -0.064 Maximum 4.982 7.372 0.363 1.003 0.936 0.224 Minimum 4.466 6.218 0.202 0.564 0.238 -0.642 Std. Dev. 0.153 0.360 0.037 0.093 0.185 0.237 Skewness -0.401 -0.134 2.469 -1.559 -0.974 -0.715 Kurtosis 2.141 1.752 8.373 6.054 2.969 2.714 Jarque-Bera 1.782 2.102 68.792 24.627 4.905 2.751 Probability 0.000 0.049 0.000 0.000 0.046 0.002 Similarly, Table 4 shows the outcomes regarding pairwise correlation tests. The given results show that FR and environmental technologies are inversely associated with explained variables. In light of estimated outcomes, there is no problem with multicollinearity. In order to verify this statement, the current study also runs the VIF test. Similarly, Table 4 shows the outcomes regarding pairwise correlation tests. 2.1. Variables Selection The given results show that FR and environmental technologies are inversely associated with explained variables. In light of estimated outcomes, there is no problem with multicollinearity. In order to verify this statement, the current study also runs the VIF test. Table 4: Pairwise Correlation test LCO2 LGDP LFR LET IC LTAX LCO2 1.000 LGDP 0.6776* 1.000 0.000 LFR -0.5582* -0.4734** 1.000 0.001 0.007 LET -0.0862* -0.0012** 0.4917* 1.000 0.005 0.042 0.005 IC 0.5736* 0.6617* -0.7439* -0.2480* 1.000 0.000 0.000 0.000 0.005 LTAX 0.7048* 0.6744* -0.7548* -0.4373** 0.6876* 1.000 0.000 0.000 0.000 0.013 0.000 Note: * and ** show the significance level at 1% and 5%, respectively. Note: * and ** show the significance level at 1% and 5%, respectively. Table 5 shows the outcomes of the VIF test and accepts the alternative hypothesis: there is no multicollinearity in the given data. However, Figure 1 shows the box-plot for each selected variable. Table 5: VIF Test Table 5: VIF Test Table 5: VIF Test VIF 1/VIF LGDP 5.75 0.1740 LFR 3.33 0.3000 LET 1.69 0.5905 IC 4.54 0.2202 LTAX 6.51 0.1535 Mean VIF 4.37 2.2. Model Construction: 2.2. Model Construction: The general form of the selected variables can be written in a given function, The general form of the selected variables can be written in a given function, CNt = f(β0, GDPβ1, FR β2, ET β3, IC β4, Tax β5, µ) (1) (1) In the given function (Eq. 1), CN, GDP, FR, ET, IC, and Tax are denoted with carbon neutrality, gross domestic product, fertility rate, environmental-related technologies, international cooperation, and environmental taxes. Whereas t is the time series data and µ is the white noise error term. However, the transform model can be written as follows, LCNt = β0 + β1 LGDPt + β2 LFRt + β3 LETt + β4 ICt + β5 LTaxt + µ (2) LCNt = β0 + β1 LGDPt + β2 LFRt + β3 LETt + β4 ICt + β5 LTaxt + µ (2) (2) In equation 2, LCN, LGDP, LFR, LET, and LTax are denoted with the natural log of carbon neutrality, gross domestic products, fertility rate, environment-related technologies, and environmental taxes (Abbas et al., 2020; X. Li, Abbas, Dongling, Baig, & Zhang, 2022). Similarly, IC indicates with the international cooperation index; there, we do not have its natural log. However, this study proposes some key hypotheses concerning association of explained and explanatory variables. Firstly, this study supposes that gross domestic product (GDP) significantly contributes to carbon emissions and causes of hurdle for CN (Jiakui, Abbas, Najam, Liu, & Abbas, 2023; Zafar, Shi, Yang, Abbas, & Chen, 2022). This connection would happen due to its scale effect on Chin’s economy, and its slope will be (β > 0). Similarly, the fertility rate may be suitable for selected environmental model and may cause a decline in other non-productive expenditure to childcares expenditures; therefore, its slope will be by (β < 0). Moreover, ERT may be suitable for the emerging economy’s environment (Talib Hussain, Abbas, Li, Aman, & Ali, 2017; Y. Li, Al- Sulaiti, Dongling, Abbas, & Al-Sulaiti, 2022; X. Zhang et al., 2022; Zhuang et al., 2022). Due to the massive usage of dirty inputs by firms in the production process there may be a chance of rising emissions; thus, the current research supposes that it would cause environmental pollution (β > 0). 2.2. Model Construction: Likewise, international In equation 2, LCN, LGDP, LFR, LET, and LTax are denoted with the natural log of carbon neutrality, gross domestic products, fertility rate, environment-related technologies, and environmental taxes (Abbas et al., 2020; X. Li, Abbas, Dongling, Baig, & Zhang, 2022). Similarly, IC indicates with the international cooperation index; there, we do not have its natural log. However, this study proposes some key hypotheses concerning association of explained and explanatory variables. Firstly, this study supposes that gross domestic product (GDP) significantly contributes to carbon emissions and causes of hurdle for CN (Jiakui, Abbas, Najam, Liu, & Abbas, 2023; Zafar, Shi, Yang, Abbas, & Chen, 2022). This connection would happen due to its scale effect on Chin’s economy, and its slope will be (β > 0). Similarly, the fertility rate may be suitable for selected environmental model and may cause a decline in other non-productive expenditure to childcares expenditures; therefore, its slope will be by (β < 0). Moreover, ERT may be suitable for the emerging economy’s environment (Talib Hussain, Abbas, Li, Aman, & Ali, 2017; Y. Li, Al- Sulaiti, Dongling, Abbas, & Al-Sulaiti, 2022; X. Zhang et al., 2022; Zhuang et al., 2022). Due to the massive usage of dirty inputs by firms in the production process there may be a chance of rising emissions; thus, the current research supposes that it would cause environmental pollution (β > 0). Likewise, international Page 9/29 Page 9/29 cooperation is a key solution to fighting ecological concerns. Since the last three decades, the globe has tried to settle sustainability targets, but they don’t help each other for it because every economy has the ambition to compete with the world via economic progress at any cost of environmental damage. Consequently, this study supposes that IC may contribute to emissions, and its slope will be positive (β > 0). Finally, environmental taxes (Tax) would be a key solution for environmental sustainability and it supposes that this factor would contribute (β < 0) to carbon neutrality. Consequently, this study supposes that IC may contribute to emissions, and its slope will be positive (β > 0). Finally, environmental taxes (Tax) would be a key solution for environmental sustainability and it supposes that this factor would contribute (β < 0) to carbon neutrality. Similarly, Figure 2 presents the overall flow of the estimation strategy. 2.2. Model Construction: However, the study begins with the Augmented Dickey-Fuller (ADF) and Phillips-Perron (PP) unit root test to identify whether the data is integrated. Both mentioned UR tests unable to elaborate on structural breaks in the data; to identify this issue, this study also employed the Zivot unit root test. Furthermore, to check out the long-run relationship among selected variables, the current study uses the ARDL bound test, Bayer-Hanck test, and fisher cointegrations for the time series data. Such interesting outcomes make it necessary to use an advanced econometric technique for robust results (Abbasi, Abbas, & Tufail, 2021; Abbasi, Adedoyin, Abbas, & Hussain, 2021; Raza Abbasi et al., 2021). Therefore, this study adopts the novel estimation strategy proposed by (Jordan & Philips, 2018). This estimation technique aims to resolve all problems faced by the old ARDL model (Iorember, Iormom, Jato, & Abbas, 2022). It has various advantages over old ARDL, such as estimating, plotting forecasts (Jordan & Philips, 2018). However, the general form of the dynamic ARDL can be presented as, LCNt = β0 LCO2, t-2 + β1 LGDPt + β2 LGDPt-2 + β3 LFRt + β4 LFRt-2 + β5 LETt + β6 LETt-2 + β7 ICt + β8 ICt-2 + β9 LTaxt + β10 LTaxt-2 + ϵt (3) (3) However, for the robustness, this study also employs the FMOLS, DOLS, and ARDL tests. Beside, to check out the causal association, we employ the BC-Spectral Granger causality test by (Breitung & Candelon, 2006). However, for the robustness, this study also employs the FMOLS, DOLS, and ARDL tests. Beside, to check out the causal association, we employ the BC-Spectral Granger causality test by (Breitung & Candelon, 2006). 3- Results And Discussion Note: The critical values are significant at the 1% level. 3- Results And Discussion Table 6 consists of data integration results by ADF and PP unit root tests. According to the given outcomes, all selected variables are integrated at 1st difference. Similarly, both tests cannot check out the structural break in data; therefore, we employ the Zivot unit root test. Table 6: ADF and PP unit root tests Page 10/29 Variable ADF unit root PP unit root Level 1st difference Level 1st difference LCO2 -1.0547 -4.4186* -1.0442 -4.4248* LGDP -2.4364 -5.6305* -2.9184 -4.7702* LFR -0.7922 -11.080* -14348 -3.3729** LET -2.7144 -7.1560* -2.6556 -7.2639* IC -2.0877 -4.9622* -2.0025 -4.1108* LTax -2.5264 -4.4091* -2.3776 -4.5285* ** h h i ifi l l 1% d 5% i l Note: * and ** show the significance level at 1% and 5%, respectively. Similarly, Table 7 presents the Zivot unit root test's outcome and finds the different structural breaks at intercept and trend for each variable. Similarly, Table 7 presents the Zivot unit root test's outcome and finds the different structural breaks at intercept and trend for each variable. Table 7: Zivot Unit root test Intercept Trend Variable Value Break Value Break LCO2 -4.2806* 2014 -4.1968* 2011 LET -3.9962* 1996 -4.2734* 2000 LFR -6.9031* 2012 -7.1928* 1999 LGDP -1.5345** 2005 -4.0048* 2015 IC -2.7435* 2014 -6.8128* 2013 LTax -3.1465* 1997 -3.9425* 2006 Note: * and ** show the significance level at 1% and 5%, respectively. Note: * and ** show the significance level at 1% and 5%, respectively. Note: * and ** show the significance level at 1% and 5%, respectively. Table 8 shows the results of long-run cointegration among selected variables. The upper part of Table 8 shows the ARDL bound test outcomes, while the lower part shows the cointegration results by the Bayer- Hanck cointegration test. Estimated outcomes reject the null hypothesis of no cointegration among variables and accept the Alternative hypothesis that long-term cointegration exists. Table 8: Cointegration Tests Table 8: Cointegration Tests Page 11/29 Upper Part Test Statistic Value F-statistic 6.4986 Critical Value Bounds Significance Level Lower Bound Upper Bound 10% 2.26 3.35 5% 2.62 3.79 1% 3.41 4.68 Lower Part EG-JOH EG-JOH-BOBDM Cointegration 13.856* 47.3549* Yes Upper Part Test Statistic Value F-statistic 6.4986 Critical Value Bounds Significance Level Lower Bound Upper Bound 10% 2.26 3.35 5% 2.62 3.79 1% 3.41 4.68 Lower Part EG-JOH EG-JOH-BOBDM Cointegration 13.856* 47.3549* Yes Note: The critical values are significant at the 1% level. 3.1. Model Stability tests However, to move forward for long-term association among selected variables via D-ARDL, FMOLS, DOLS, CCR, and ARDL estimators, here it may be fascinating if this study provides a piece of the evidence whether the selected model is stable or not. To answer this, we employ various stability tests, and all of them verify our selected model is stable. Table 9: Model Stability Tests There may be a possibility that if the fertility rate increased, that would cause more expenditure on child care and less on other energy-intensive activities. Consequently, a rise in fertility rate would cause emissions reduction and support the carbon neutrality concept. Secondly, an increase in the fertility rate would cause tightness in budget constraint and insist to use on productive work. Nowadays, parents prioritize investing more and more in a child’s education. Accordingly, the cost of education may cause of reduction in expenditures on other activities that bring eco-friendly consumption patterns. Thus, from this outcome, it is clear that an increase in fertility rate would be a supportive instrument for CN. Therefore, to achieve CN, it is necessary to increase the population in China. This outcome is in line with the study of China (L. Yang, Hu, & Wei, 2023). that would cause more expenditure on child care and less on other energy-intensive activities. Consequently, a rise in fertility rate would cause emissions reduction and support the carbon neutrality concept. Secondly, an increase in the fertility rate would cause tightness in budget constraint and insist to use on productive work. Nowadays, parents prioritize investing more and more in a child’s education. Accordingly, the cost of education may cause of reduction in expenditures on other activities that bring eco-friendly consumption patterns. Thus, from this outcome, it is clear that an increase in fertility rate would be a supportive instrument for CN. Therefore, to achieve CN, it is necessary to increase the population in China. This outcome is in line with the study of China (L. Yang, Hu, & Wei, 2023). However, environmental-related technology (ERT) is positively associated with CO2 emissions. It refers to a 1% rise in this factor would cause to increase in environmental pollution by 0.026% in China. In simple words, this factor does not play a supportive role in mitigating environmental stress. This outcome can be supported with some authentic evidence. As discussed in early paragraphs, at this stage of economic progress, China tries to export maximum output via the trade sector while the environmental-related technologies are not perfectly mature to secure EQ. Therefore, ERT does not help enough to reduce traditional energy use and causes environmental deterioration at this stage of China's progress. Thus, the key point is that ERT is directly associated with clean and green energy adoption, which may secure the environment in the future. Table 9: Model Stability Tests In order to investigate the long-run impact among the selected variables, this study employs a series of econometric tests, and D-ARDL is one of them. Estimated outcomes show the positive association of the gross domestic product with explained variable. It implies that a 1% change in this factor would cause to increase in environmental pollution by 0.048% in China. There are several logics behind the scene. Firstly, economic growth may affect the environmental quality (EQ) by scale and technique effect. If the scale effect becomes dominant on the technique effect, it could cause ED and vice versa. At this stage in China, the Scale effect is prevalent in the China economy and generates long-run missions. Secondly, there may be another logic that, at this stage, it causes industrial development at the cost of massive emissions. Undoubtedly, an immense amount of energy is being utilized in industrial activities, raising CO2 emissions. However, emerging economies mostly use natural resources in gigantic amounts for their industrial and economic purposes (Abbas et al., 2020; Al Halbusi, Al-Sulaiti, Abbas, & Al-Sulaiti, 2022; Asad, Abbas, Irfan, & Raza, 2017; Azadi et al., 2021; Farzadfar et al., 2022; Ge et al., 2022). In this scenario, they focus on infrastructure development based on energy-intensive activities and, as a result, cause emissions (Geng et al., 2022; Touseef Hussain et al., 2021). In concluding remarks, it may be noteworthy that china's economic model is resource-intensive, and many other emerging economies are following this. However, this outcome is in line with the study of the Iranian economy (Shabani & Shahnazi, 2019). Similarly, the fertility rate is another influential factor for carbon neutrality. The outcome shows the inverse association of FR with CO2 emissions. It infers that a 1% rise in this factor would cause a decline in emissions by -1.375%. Some facts can explain the inverse association. Firstly, the fertility rate and household resources are directly associated. There may be a possibility that if the fertility rate increased, that would cause more expenditure on child care and less on other energy-intensive activities. Similarly, the fertility rate is another influential factor for carbon neutrality. The outcome shows the inverse association of FR with CO2 emissions. It infers that a 1% rise in this factor would cause a decline in emissions by -1.375%. Some facts can explain the inverse association. Firstly, the fertility rate and household resources are directly associated. Table 9: Model Stability Tests Table 9: Model Stability Tests Table 9: Model Stability Tests y Test Value. Prob. Value. Ramsey RESET Test 1.9054 0.068 BG-LM Test 2.2168 0.699 Breusch-Pagan-Godfrey 2.0567 0.105 Glejser Test 2.1441 0.093 ARCH Test 1.0927 0.287 White Test 1. 6210 0.099 over, CUSUM and CUSUM square also validate by the graph that the selected Moreover, CUSUM and CUSUM square also validate by the graph that the selected model is stable (Figure 3). Similarly, Figure 4 shows the stability model by recursive graphs for the selected Moreover, CUSUM and CUSUM square also validate by the graph that the selected model is stable (Figure 3). Similarly, Figure 4 shows the stability model by recursive graphs for the selected 3.2. Long run Results Dynamic ARDL estimator Page 12/29 In order to investigate the long-run impact among the selected variables, this study employs a series of econometric tests, and D-ARDL is one of them. Estimated outcomes show the positive association of the gross domestic product with explained variable. It implies that a 1% change in this factor would cause to increase in environmental pollution by 0.048% in China. There are several logics behind the scene. Firstly, economic growth may affect the environmental quality (EQ) by scale and technique effect. If the scale effect becomes dominant on the technique effect, it could cause ED and vice versa. At this stage in China, the Scale effect is prevalent in the China economy and generates long-run missions. Secondly, there may be another logic that, at this stage, it causes industrial development at the cost of massive emissions. Undoubtedly, an immense amount of energy is being utilized in industrial activities, raising CO2 emissions. However, emerging economies mostly use natural resources in gigantic amounts for their industrial and economic purposes (Abbas et al., 2020; Al Halbusi, Al-Sulaiti, Abbas, & Al-Sulaiti, 2022; Asad, Abbas, Irfan, & Raza, 2017; Azadi et al., 2021; Farzadfar et al., 2022; Ge et al., 2022). In this scenario, they focus on infrastructure development based on energy-intensive activities and, as a result, cause emissions (Geng et al., 2022; Touseef Hussain et al., 2021). In concluding remarks, it may be noteworthy that china's economic model is resource-intensive, and many other emerging economies are following this. However, this outcome is in line with the study of the Iranian economy (Shabani & Shahnazi, 2019). Table 9: Model Stability Tests By having this response of ERT toward carbon neutrality, the policymakers may Page 13/29 Page 13/29 not refer to it as a security instrument for CN in China's emerging economy. Therefore, higher authorities and analysts should rethink their environmental policies and try to achieve a carbon peak by 2030. This outcome is in line with the study of China (Gu, Zhao, Yan, Wang, & Li, 2019). International cooperation (IC) performs well across the border to limit environmental issues, mainly in the COVID-19 challenges (Q. Liu, Qu, Wang, Abbas, & Mubeen, 2021; Su et al., 2022; Yu, Abbas, Draghici, Negulescu, & Ain, 2022). Therefore, the present study also tries to add this exciting factor as a key instrument for carbon neutrality. Unfortunately, the given coefficient value shows an increasing trend in emissions by 0.038% due to a unit change in IC. From this outcome, it is clear to the world that IC is not at the forefront policy agenda. There are different logics for why IC has no diminishing effect on CO2 emissions. Firstly, there has been an unprecedented rise in societies, and their human & economic activities are directly linked with weather or climate change. Secondly, domestic and across-the-border countries cannot wait for a long time to see benefits in the future. However, they tried to invest more and more in development activities and believed climate change would automatically adjust over time. Thirdly, some economies want a deep dose of climate change up to a certain point; Russia has both i lt l d f t t th t li t iti I d t i t t f l ti h International cooperation (IC) performs well across the border to limit environmental issues, mainly in the COVID-19 challenges (Q. Liu, Qu, Wang, Abbas, & Mubeen, 2021; Su et al., 2022; Yu, Abbas, Draghici, Negulescu, & Ain, 2022). Therefore, the present study also tries to add this exciting factor as a key instrument for carbon neutrality. Unfortunately, the given coefficient value shows an increasing trend in emissions by 0.038% due to a unit change in IC. From this outcome, it is clear to the world that IC is not at the forefront policy agenda. There are different logics for why IC has no diminishing effect on CO2 emissions. Table 9: Model Stability Tests Firstly, there has been an unprecedented rise in societies, and their human & economic activities are directly linked with weather or climate change. Secondly, domestic and across-the-border countries cannot wait for a long time to see benefits in the future. However, they tried to invest more and more in development activities and believed climate change would automatically adjust over time. Thirdly, some economies want a deep dose of climate change up to a certain point; Russia has both agricultural and forest sectors that are climate sensitive. In order to remain constant for a long-time such economy wants warm weather. Fourthly, economies across the globe do not have an equal interest in fighting climate change. These are the four reasons international cooperation worldwide, especially for emerging economies like China, is not working well. Therefore, all economies must pay equal attention to the fight against climate change. This result is against (M. Liu & Lo, 2021) and in line with (Paroussos et al., 2019). Finally, environmental taxes (Tax) are another factor for the carbon neutrality concept. This factor has a surprising association with explained variable, which implies that a 1% rise in this factor would cause a decline in emissions by 0.051%. For instance, an inverse association may help achieve long-term CN. It is a common phenomenon that a tax increase would decrease environmental pollution. In order to minimize the negative externalities, firms use advanced or eco-friendly technologies for their business activities. H i l d l i h k di i f ’ i SDG Finally, environmental taxes (Tax) are another factor for the carbon neutrality concept. This factor has a surprising association with explained variable, which implies that a 1% rise in this factor would cause a decline in emissions by 0.051%. For instance, an inverse association may help achieve long-term CN. It is a common phenomenon that a tax increase would decrease environmental pollution. In order to minimize the negative externalities, firms use advanced or eco-friendly technologies for their business activities. However, strict rules and regulations are the key directions for government’s commitment to SDGs. Therefore, such revenue must allocate to research & development activities or eco-friendly technologies and inputs to attain the CN. The obtained result also verifies the outcomes of (P. Li, Lin, Du, Feng, & Zuo, 2021; Z. Li & Zhao, 2017; Xiao, Niu, Guo, & Xu, 2015). Table 9: Model Stability Tests Therefore, such revenue must allocate to research & development activities or eco-friendly technologies and inputs to attain the CN. The obtained result also verifies the outcomes of (P. Li, Lin, Du, Feng, & Zuo, 2021; Z. Li & Zhao, 2017; Xiao, Niu, Guo, & Xu, 2015). Table 10: Results of D-ARDL Estimator Page 14/29 Variable Coefficient Std. Error T-Value Prob. Value CO2 t-1 -0.6916** 0.2521 -2.74 0.013 LGDP 0.0484* 0.1530 0.32 0.005 LFR -1.3752* 0.7076 -1.94 0.001 LET 0.0260** 0.0661 0.39 0.025 IC 0.0381* 0.1629 0.23 0.000 LTax -0.0511** 0.0418 -1.22 0.043 Cons. 0.8696* 0.5817 1.49 0.002 R2 0.973 Adjusted R2 0.965 F-Statistics 111.17 (0.000) Note: * and ** shows the significance level at 1% and 5%, respectively. Note: * and ** shows the significance level at 1% and 5%, respectively. s the real variation between the explained and explanatory variables of the Similarly, Figure 4 shows the real variation between the explained and explanatory variables of the selected model. These graphs have been made on a 10% increase or decrease in dependent variable due to natural variation in explanatory variables. However, graphs 1, 2, 3, 4, and 5 are from IC, Tax, ET, FR, and GDP. 3.3. Robust Check by Autoregressive Distributive Lag (ARDL) estimator Table 11 demonstrates the estimated outcomes by the ARDL estimator to validate the finding of D-ARDL. In light of the estimated results, no more significant difference was found between the prior and current investigation for the specified model. However, according to the given value of cointEq(-1), there will need less than half a year to converge from the short to the long run. Moreover, due to a significant change in GDP, ET and IC would increase emissions by 0.391%, 0.317%, and 0.040%, respectively. Furthermore, 0.649% and 0.114% decline in emissions via a 1% increase in FR and environmental taxes in China. Table 11: Results of ARDL estimator Page 15/29 Variable Coefficient Std. Error T-Value P-value Short Run Results LGDP 0.1490* 0.0553 2.6925 0.013 LFR -0.6490* -0.6490 -4.3982 0.000 LET 0.0351 0.0377 0.9306 0.041 IC 0.0154* 0.0738 0.2088 0.051 LTax -0.1144*** 0.0659 -1.7358 0.097 CointEq(-1) -0.3802* 0.1237 -3.0733 0.005 Long Run Results LGDP 0.3919* 0.0450 8.6933 0.000 LFR -1.7068* 0.5564 -3.0676 0.005 LET 0.3178 0.1817 1.7483 0.050 IC 0.0405 0.2009 0.2017 0.000 LTAX -0.0618 0.1338 -0.4618 0.005 C 2.1543 0.2324 9.2669 0.000 R2 0.988 Adjusted R2 0.984 F-Statistics 235.686* (0.000) Note: * and ** shows the significance level at 1% and 5%, respectively. 3.5. BC- spectral Granger Causality Analysis It is exciting to run an advanced granger causality test, which employs the Breitung and Candelon causality test. Figure 5 shows the results of the granger causality test. The results show a red line for α = 5% and a green line for α = 10%. However, there exists a uni-directional causal association between GDP to carbon emissions. This infers that any significant change in economic progress would cause environmental pollution in China. Furthermore, there exists a one-way causal relationship between CO2 emissions to fertility rate, while the feedback hypothesis does not exist for FR to carbon emissions. Similarly, according to the given results, no causal association exists between RET and emissions level and vice versa. Moreover, a similar association has been observed in environmental taxes and international cooperation with emissions. 3.4. Dynamic Estimation of the Selected Model In order to robust check, this study also uses three techniques: FMOLS, DOLS, and CCR estimators. Table 12 does not show any variation concerning the association between dependent and independent variables. Once again, we can say GDP, ET, and international cooperation are increasing emissions, while vice versa for fertility rate and environmental taxes. In concluding remarks, all estimators show the same association. It would be a right direction for legislators to rethink implemented policies and try to achieve zero emissions by 2060. Table 12: Results of FMOLS, DOLS, and CCR estimators Page 16/29 Variable FMOLS DOLS CCR Coefficient Std. Error Coefficient Std. Error Coefficient Std. Error LGDP 0.4248* 0.0091 0.4248* 0.0112 0.4252* 0.0103 LFR -0.5833* 0.0813 -0.5788* 0.0822 -0.5781* 0.0746 LET 0.0715* 0.0191 0.0722* 0.0236 0.0717* 0.0281 IC 0.0731* 0.0290 0.0720* 0.0360 0.0745* 0.0353 LTAX -0.0099* 0.0148 -0.0105* 0.0182 -0.0108*** 0.0208 C 2.0930* 0.0434 2.0917* 0.0536 2.0890* 0.0539 R2 0.969 0.969 0.9689 Adjusted R2 0.962 0.963 0.9624 e: * and ** shows the significance level at 1% and 5% respectively Note: * and ** shows the significance level at 1% and 5%, respectively. Note: * and ** shows the significance level at 1% and 5%, respectively. 4- Conclusion And Policy Recommendations This paper aims to investigate the leading factors of carbon neutrality in China over the period of 1990 to 2020. Here we use a series of econometric techniques to analyze the proposed objectives. In a nutshell, GDP, environmental technologies, and international cooperation are increasing environmental damage. On the other hand, fertility rate and environmental taxes significantly contribute to environmental sustainability. Moreover, the current study also employs the Breitung and Candelon causality test to investigate the causal association among selected variables. The estimated results show the one-way causal association between GDP to CO2 and emissions to fertility rate in the selected region. Moreover, this study proposes some imperative policy implications for zero emissions. Firstly, economic growth has a positive association with CO2 emissions. From a policy perspective, there is need of time to Page 17/29 Page 17/29 focus more on green economic growth and allocate resources to eco-friendly sectors. However, investing in sustainable production and consumption patterns is crucial to secure sustainability. At this stage scale, effect is dominant, and there is a need to introduce advanced green technologies, which may reduce the level of emissions in China. Moreover, in this progress, skilled labor would be able to respond well. Moreover, the fertility rate performs well in reducing emissions, but there are some key suggestions to decline emissions further. It may be suitable for sustainability if higher authorities consider household consumption patterns and insist on consuming less emitter goods and services. Likewise, there is a need to improve the tax ratio on low and high-carbon products and invest revenue into clean and green projects. However, it would be a good policy if higher authorities regulated society’s development and tried to provide eco-friendly facilities for the new generation. In addition, there is a need to offer environment-related subjects, which may provide long-term sustainability to future generations. However, the outcomes for environment-related technologies (ERT) with emissions are shocking. China has not shifted its economic and human activities to clean and green inputs. Similarly, China needs proper to introduce eco-friendly technologies in businesses and other economic activities. Moreover, there is a need to allocate some budget to sustainable and green research and development activities that may produce skilled labor. Such activities must be designed in environmental technologies to attain a friendly environment in the long-run. Similarly, international cooperation (IC) does not show any satisfactory contribution to zero emissions. 4- Conclusion And Policy Recommendations However, numerous initiatives for environmental sustainability have been taken, such as Kyoto Protocol, Paris Agreement and SDGs, but the issue remains constant. Therefore, economies across the border should cooperate in fighting for zero emissions. At this time, every economy wants to achieve maximum growth at any cost of environmental damage. Simply put, only an agreement or commitment sig on a draft is not a solution for carbon neutrality. Therefore, countries must work together for environmental sustainability. Furthermore, environmental taxes (tax) also perform well in reducing carbon emissions and boosting sustainable development. Therefore, implementing a tax would cause zero emissions due to environmental awareness, green businesses, green production, resource allocation, and green growth in China. In addition, it is typical behavior of product suppliers; if higher authorities tax its wastage, this firm may use energy-efficient technologies and ultimately zero emissions. In contrast, there may be a chance in China that prices will go up due to heavy taxes and may slow economic progress. Thus, such economies should try to shift their production sector to clean and green energies. Page 18/29 However, this study is not free from limitations. Firstly, the current research does not include some critical factors like green energy. Because in five-year development plan green energy investment has taken a main place and China is investing in green energy projects. Therefore, future researchers should include renewable energy investment in their environmental models. Besides, some social and political factors have not been included in this analysis because of regression conditions. It would be good to investigate the heterogeneous impact on environmental quality if future studies included population aging, corruption, external conflicts, etc. Furthermore, there is another future direction that coming studies must consider the other proxies for environmental degradation except for emissions, which may produce different results for the same indicators. Finally yet importantly, future studies should investigate the Page 18/29 Page 18/29 same model in other emerging economies like India, Brazil, South Africa, and Russia and try to validate our outcomes. same model in other emerging economies like India, Brazil, South Africa, and Russia and try to validate our outcomes. Funding: We have not received any funding to execute this research study, the rigorous procedure of collecting data, and other associated processes to conduct this study. We have not received any funding to execute this research study, the rigorous procedure of collecting data, and other associated processes to conduct this study. Consent for publication: Consent approved by the authors for publication of this article. Consent for publication: Consent approved by the authors for publication of this article. Conflict of interest statement: The authors are well informed and declared no competing interests. Abbreviations CN: Carbon Neutrality, GDP: Gross domestic product, UN: the United Nations, FR: Fertility rate, CO2: Carbon emissions, ERT: Environmental-related technologies, IC: International cooperation, D-ARDL: Dynamic Auto-regression Distributive Lag model, FMOLS: Fully-Modified Ordinary Lest Square, DOLS: Dynamic Ordinary Least Square, CCR: Canonical Correlation Regression, VIF: Variance Inflation factor, PP: Phillips-Perron unit root, ADF: Augmented Dickey Fuller unit root, B-C test: Breitung and Candelon causality test. DECLARATION SECTION Data Availability Statement Availability of data and material: Data is available at the request from the corresponding author. Funding: References 1. Abbas, J., Zhang, Q., Hussain, I., Akram, S., Afaq, A., & Shad, M. A. (2020). Sustainable Innovation in Small Medium Enterprises: The Impact of Knowledge Management on Organizational Innovation through a Mediation Analysis by Using SEM Approach. 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https://openalex.org/W2797582353	http://www.vliz.be/imisdocs/publications/330085.pdf	English	null	Age and palaeoenvironment of the Utsira Formation in the northern North Sea based on marine palynology	Norwegian journal of geology	2,018	cc-by	16,650	NORWEGIAN JOURNAL OF GEOLOGY Vol 97 Nr. 4 https://dx.doi.org/10.17850/njg97-4-04 Age and palaeoenvironment of the Utsira Formation in the northern North Sea based on marine palynology Stijn De Schepper1,2 & Gunn Mangerud1 1 Department of Earth Science, University of Bergen, P.O. Box 7803, N–5020 Bergen, Norway. 2Uni Research Climate, Bjerknes Centre for Climate Research, Jahnebakken 5, N–5007 Bergen, Norway. E-mail corresponding author (Stijn De Schepper): stijn.deschepper@uni.no The Utsira Formation is a major player in the carbon capture and storage on the Norwegian Shelf. Although this northern North Sea unit has been subjected to several geological and monitoring studies, its lateral distribution and stratigraphic position are still not fully understood. This unit was considered to be late Neogene and deposited in neritic environments on the Norwegian Shelf, in an area from the Viking Graben to the Tampen Spur. Here, we present marine palynomorph (dinoflagellate cysts, acritarchs) data extracted mainly from cutting samples of eight industry wells that cover the entire distribution area of the Utsira Formation to provide an age and palaeoenvironmental reconstruction for this unit. We conclude that deposits classified as Utsira Formation are Late Miocene/Early Pliocene to Early Pleistocene in age. Early Pliocene sediments are found mainly in the Viking Graben area, whereas sediments with an Early Pleistocene age occur over the entire distribution area of the Utsira Formation. All sediments were deposited in neritic environments that gradually became shallower from the Early Pliocene to the Pleistocene. At the same time, the dinoflagellate cysts also indicate a cooling that corresponds well with late Neogene global cooling. Precise dating of the Utsira Formation is difficult, but this can be improved by (1) using samples from cored section (in contrast to using cuttings), (2) a clear and unquestionable lithological definition of the Utsira Formation and (3) a continuous, calibrated reference section in the Neogene North Sea for comparison of the bioevents. Keywords: Dinoflagellate cysts, biostratigraphy, palaeoenvironment, Pliocene, Utsira Formation, carbon dioxide storage Electronic Supplement 1: Sample information and palynological raw data. Electronic Supplement 2: Study sites (wells) information, sample position and dinoflagellate cyst and acritarch biostratigraphy Received 30. June 2017 / Accepted 17. November 2017 / Published online 1. January 2018 De Schepper, S. & Mangerud, G. 2017: Age and palaeoenvironment of the Utsira Formation in the northern North Sea based on marine palynology. Norwegian Journal of Geology 97, 305–325. https://dx.doi.org/10.17850/njg97-4-04. © Copyright the authors. This work is licensed under a Creative Commons Attribution 4.0 International License. © Copyright the authors. Introduction (Halland et al., 2011, 2014), seismic characterisation (Gregersen & Johannessen, 2007), biostratigraphic (e.g., Eidvin & Rundberg, 2001) and sequence-stratigraphic investigations (Galloway, 2002), and carbon dioxide accumulation (Bickle et al., 2007) and monitoring studies (Hermanrud et al., 2009). Several geological units on the Norwegian Shelf have been identified as potential reservoirs for carbon dioxide storage. In particular, the late Neogene Utsira Formation saline aquifer in the northern North Sea is considered as an excellent reservoir (e.g., Halland et al., 2014). As part of a carbon sequestration programme of Statoil ASA, carbon dioxide from Jurassic and Tertiary gas reservoirs has been captured and stored in the Utsira Formation, a half-open to fully open storage system. Since the start of carbon dioxide injection, the Utsira Formation has been the centre of attention for detailed geological study Although the Utsira Formation has been used for CCS since 1996, its stratigraphic position, lateral distribution and age remain a matter of debate (e.g., Eidvin et al., 2013). Originally, Isaksen & Tonstad (1989, p. 54–55) described the Utsira Formation in its type section (well 16/1–1, from 1064 to 644.5 m) as follows: “The formation consists of marine sandstones and claystones. 305 S. De Schepper & G. Mangerud 306 30/6-3 24/12-1 34/4-7 34/4-6 34/7-2 15/9-A-23 25/2-10S 34/7-4 16/1-1 Southern Utsira Mbr ? Central Utsira Mbr Northern Utsira Mbr Glauconitic Utsira Mbr ? ? Investigated wells Investigated wells, also proposed as type sections for Utsira Fm members (Eidvin et al. 2013) Stratigraphic division of the Utsira Fm according to Eidvin et al. (2013) Thickness of the Utsira Fm < 50 m 50–100 m 100–150 m 150–200 m 200–250 m 250–300 m 300–350 m >350 m Type well Cental Utsira Member is after Eidvin and Rundberg (2007) Tampen area United Kingdom Norway Bergen northern North Sea 6˚ 5˚ 4˚ 3˚ 2˚ 1˚ E 62˚ 61˚ 60˚ 59˚ 58˚ N HYDROCARBON FIELD SLEIPNER STATFJORD SNORRE VISUND TORDIS GULLFAKS VIGDIS OSEBERG 642 610 Study area 907 North Atlantic Iceland Sea Norwegian Sea Viking Graben area TROLL 1 2 3 4 62˚ 642 610 Study area 907 North Atlantic Iceland Sea Norwegian Sea 1 2 3 4 30/6-3 24/12-1 15/9-A-23 25/2-10S 16/1-1 Southern Utsira Mbr ? Central Utsira Mbr Utsira Mbr ? ? Investigated wells Investigated wells, also proposed as type sections for Utsira Fm members (Eidvin et al. 2013) Stratigraphic division of the Utsira Fm according to Eidvin et al. Introduction (2013) Thickness of the Utsira Fm < 50 m 50–100 m 100–150 m 150–200 m 200–250 m 250–300 m 300–350 m >350 m Type well Cental Utsira Member is after Eidvin and Rundberg (2007) Tampen area United Kingdom Norway Bergen northern North Sea 6˚ 5˚ 4˚ 3˚ 2˚ 1˚ E 61˚ 60˚ 59˚ 58˚ N HYDROCARBON FIELD SLEIPNER TORDIS GULLFAKS OSEBERG 610 Viking Graben area TROLL 2 3 4 Figure 1. Thickness map of the Utsira Formation including the location of the studied sites in the northern North Sea. (based on fig. 4–080 in the CO2 Storage Atlas of the Norwegian Petroleum Directorate at http://www.npd.no/Global/Norsk/3-Publikasjoner/Rapporter/Samleatlas/ Figurer-Figures/Chapter-4/Fig-4-080.pdf and Eidvin et al. 2014a). Distribution of the different members is approximate and based on Rundberg & Eidvin (2005), Eidvin & Rundberg (2007), Gregersen & Johannessen (2007) and Eidvin et al. (2013, 2014a). The inset map shows locations of Ocean Drilling Program (Sites 642, 907), Deep Sea Drilling Project (Site 610) and onshore sites mentioned in the text. 1 – Tjörnes Section (Iceland), 2 – St Erth Beds (SW England), 3 – crag deposits in East England, 4 – Antwerp Harbour (northern Belgium). Bergen Norway Thickness of the Utsira Fm Thickness of the Utsira Fm 60˚ 25/2-10S Figure 1. Thickness map of the Utsira Formation including the location of the studied sites in the northern North Sea. (based on fig. 4–080 in the CO2 Storage Atlas of the Norwegian Petroleum Directorate at http://www.npd.no/Global/Norsk/3-Publikasjoner/Rapporter/Samleatlas/ Figurer-Figures/Chapter-4/Fig-4-080.pdf and Eidvin et al. 2014a). Distribution of the different members is approximate and based on Rundberg & Eidvin (2005), Eidvin & Rundberg (2007), Gregersen & Johannessen (2007) and Eidvin et al. (2013, 2014a). The inset map shows locations of Ocean Drilling Program (Sites 642, 907), Deep Sea Drilling Project (Site 610) and onshore sites mentioned in the text. 1 – Tjörnes Section (Iceland), 2 – St Erth Beds (SW England), 3 – crag deposits in East England, 4 – Antwerp Harbour (northern Belgium). NORWEGIAN JOURNAL OF GEOLOGY Age and palaeoenvironment of the Utsira Formation in the northern North Sea based on marine palynology 307 Age and palaeoenvironment of the Utsira Formation in the northern North Sea based on marine palynology NORWEGIAN JOURNAL OF GEOLOGY The sandstones are clear to white, often light greenish and normally very fine to fine-grained, in places medium to very coarse grained. Occasionally rock fragments and lignite are found. Introduction The sandstones are separated by soft, plastic, light greenish claystones and minor siltstones. Glauconite and fossil fragments are common throughout.” In practice, the unit is generally identified in wells on the Norwegian Shelf based on gamma-ray logs. This follows the description by Isaksen & Tonstad (1989), who noted that the upper and lower boundaries can be identified based on, respectively, a decrease and an increase in the gamma-ray response in the type section. When the Skade Formation underlies the Utsira Formation, identification of the lower boundary of the Utsira Formation is more difficult, but is normally marked by a break on the velocity log (Isaksen & Tonstad, 1989, p. 55). However, Rundberg & Eidvin (2005) demonstrated an erroneous correlation between the Utsira and Skade formations between wells 16/1–1 and 24/12–1. This illustrates the difficulty in identifying the Utsira Formation using physical properties since diverse facies characterise the formation (Gregersen et al., 1997; Galloway, 2002; Rundberg & Eidvin, 2005). Notwithstanding, sandy units occurring within the Neogene to Quaternary on the Norwegian Shelf have been considered to belong to the Utsira Formation from the Viking Graben to the Tampen Spur (Fig. 1). Today, the Utsira Formation is considered to cover an area of at least 75 x 450 km2 with its main, sandy depocentre reaching 250–300 m thickness in the Viking Graben. The depocentre stretches out northwards to the Tampen Spur, pinching out between the Oseberg and Troll Fields (Fig. 1); but recently, an incident near the Tordis and Visund oil fields (Tampen Spur area), following injections into sediments that were interpreted as the Utsira Formation, questioned the presence of this formation there (Eidvin, 2009; Eidvin & Øverland, 2009). It can be concluded that the use of geophysical properties alone for identifying this unit may lead to erroneous correlation and identification. It is thus fair to say that, even today, the Utsira Formation remains poorly characterised in terms of lithology, geophysical properties, stratigraphy and lateral distribution unit within the Late Neogene of the Norwegian Shelf. Considering this background, it is not surprising that sediments considered to belong to the Utsira Formation have been dated using different methods to the Mid Miocene and Early Pliocene (Fig. 2). Introduction Several investigations have been undertaken to understand the stratigraphy and age of the Utsira Formation, and disentangle it from sandy deposits with similar geophysical properties on the Norwegian Shelf (e.g., Galloway, 2002; Eidvin & Rundberg, 2007; Gregersen & Johannessen, 2007; Eidvin et al., 2013, 2014a). In this study, we follow the subdivision of the Utsira Formation by Eidvin et al. (2013). These authors proposed to subdivide the Utsira Formation into a Southern, Northern and Glauconitic Utsira Member and presented new type well sections for each member (Figs. 1, 3, 4; Table 1). This stratigraphic division largely overlaps with Eidvin TimeScale Creator Epoch Age/Stage 3 4 5 6 7 8 9 10 11 12 13 14 15 Miocene Pliocene Langhian Serravallian Tortonian Messinian Zanclean Piacenzian Isaksen & Tonstad 1989 Eidvin & Rundberg 2001 Isaksen & Ledje 2001 Jordt et al. 1995 Piasecki et al. 2002 Head et al. 2004 Eidvin & Rundberg 2007 Gregersen & Johannessen 2007 AGE OF THE UTSIRA FORMATION Age (Ma) Pleistocene Gelasian 2 This study Halland et al. 2014 Rundberg & Smalley 1989 Smalley & Rundberg 1990 Figure 2. Age of the Utsira Formation as reported in the literature and in this study. This summary does not differentiate between the regions (Tampen area, Viking Graben), or between direct (Rundberg & Smalley, 1989; Smalley & Rundberg, 1990; Piasecki et al., 2002), indirect (Head et al., 2004) or inferred dating (Isaksen & Tonstad, 1989; Isaksen & Ledje, 2001; seismics study of Jordt et al., 1995; Halland et al., 2014), nor does it take into account whether the Utsira Formation was partially (cored section only in Piasecki et al., 2002) or entirely (e.g., Eidvin & Rundberg, 2001, 2007) dated. Epoch Age/Stage 3 4 5 6 7 8 9 10 11 12 13 14 15 Miocene Pliocene Langhian Serravallian Tortonian Messinian Zanclean Piacenzian Isaksen & Tonstad 1989 Eidvin & Rundberg 2001 Isaksen & Ledje 2001 Jordt et al. 1995 Piasecki et al. 2002 Head et al. 2004 Eidvin & Rundberg 2007 Gregersen & Johannessen 2007 AGE OF THE UTSIRA FORMATION Age (Ma) Pleistocene Gelasian 2 This study Halland et al. 2014 Rundberg & Smalley 1989 Smalley & Rundberg 1990 Figure 2. Age of the Utsira Formation as reported in the literature and in this study. Introduction This summary does not differentiate between the regions (Tampen area, Viking Graben), or between direct (Rundberg & Smalley, 1989; Smalley & Rundberg, 1990; Piasecki et al., 2002), indirect (Head et al., 2004) or inferred dating (Isaksen & Tonstad, 1989; Isaksen & Ledje, 2001; seismics study of Jordt et al., 1995; Halland et al., 2014), nor does it take into account whether the Utsira Formation was partially (cored section only in Piasecki et al., 2002) or entirely (e.g., Eidvin & Rundberg, 2001, 2007) dated. S. De Schepper & G. Mangerud 308 Skade Fm Skade Fm Utsira Fm Glauconitic Utsira Member Utsira Fm Northern Utsira Member Southern Utsira Member No Formal Name Isaksen & Tonstad (1989) Eidvin & Rundberg (2007) Central Utsira Member Eidvin et al. (2013, 2014a) Skade Fm Eir Fm Glauconitic Utsira Member Utsira Fm Northern Utsira Member Southern Utsira Member Unnamed Figure 3. Stratigraphic subdivision of late Neogene deposits according to Isaksen & Tonstad (1989) and Eidvin et al. (2013, 2014a). Seismic units of Rundberg & Eidvin (2005) LN-2 LN-1 UH-4 Viking Graben LATE NEOGENE STRATIGRAPHY OF THE NORTHERN NORTH SEA NORDLAND GROUP HORDALAND GROUP Group Fm 58° S 62° N NAUST Fm LARK FORMATION UTSIRA FORMATION Southern Utsira Mb Skade Fm Hiatus Eir Fm Hiatus Northern Utsira Mb Glauconitic Utsira Mb Tampen Spur Mb Hiatus Tampen Spur 15/9-A-23 24/12-1* 25/2-10S 30/6-3* 34/4-6* 34/4-7 34/7-4 34/7-2 Figure 4. Schematic representation of the late Neogene stratigraphy in the northern North Sea between 58˚ and 62˚N (based on Eidvin & Rundberg, 2007; Eidvin et al., 2013, 2014a), including the approximate position of the wells investigated in this study. Grey shading in each well represents the interval analysed for palynology. Asterisk () indicates type wells for the different Utsira Formation lithological units according to Eidvin et al. (2013). NORDLAND GROUP Northern Utsira Mb Figure 4. Schematic representation of the late Neogene stratigraphy in the northern North Sea between 58˚ and 62˚N (based on Eidvin & Rundberg, 2007; Eidvin et al., 2013, 2014a), including the approximate position of the wells investigated in this study. Grey shading in each well represents the interval analysed for palynology. Asterisk () indicates type wells for the different Utsira Formation lithological units according to Eidvin et al. (2013). & Rundberg (2007), where a Central Utsira Member was proposed in addition to a Southern, Northern and Glauconitic Utsira Member (Figs. 1 & 3). Introduction The Central Utsira Member of Eidvin & Rundberg (2007) separates a northern and a southern depocentre, and comprises an eastward-prograding sandy strandplain wedging out into mudstone-dominated facies to the east (Galloway, 2002; Rundberg & Eidvin, 2005). In the scheme of Eidvin et al. (2013), the Central Utsira Member is part of the Southern Utsira Member, meaning that well 24/12–1 belongs to the Southern Utsira Member. According to Eidvin & Rundberg (2007), this well would belong to the Central Utsira Member (Fig. 1). Eidvin et al. (2013, 2014a) also tentatively introduced the Eir Formation (Figs. 3 & 4), following the erroneous correlation between the Utsira and Skade formations between wells 16/1–1 and 24/12–1 (Isaksen & Tonstad, 1989; Rundberg & Eidvin, 2005). The Eir Formation represents sandy sections in the basal part of the Nordland Group and is found below the Utsira Formation and above the Skade Formation and the Mid Miocene unconformity. Finally, a potential type section for the Tampen Spur Member of the Naust Formation was suggested by Eidvin et al. (2013) (Fig. 4). In this study, we investigated the marine palynology (dinoflagellate cysts and acritarchs) in eight wells 309 WEGIAN JOURNAL OF GEOLOGY Age and palaeoenvironment of the Utsira Formation in the northern North Sea based on marine palynology Age and palaeoenvironment of the Utsira Formation in the northern North Sea based on marine palynology NORWEGIAN JOURNAL OF GEOLOGY Table 1. Lithostratigraphy, type well, depth and estimated age for the units investigated in this study (following Eidvin et al., 2013). Depth: m RKB – metres below rig floor. al., 2004; De Schepper et al., 2009; Verhoeven et al., 2011; Grøsfjeld et al., 2014). For dating, we can rely on relatively high-resolution, late Neogene, biozonation schemes and/ or calibrated stratigraphic ranges of dinoflagellate cysts and acritarchs for the Norwegian Sea (De Schepper et al., 2017), the North Sea (Munsterman & Brinkhuis, 2004; Dybkjær & Piasecki, 2010), the eastern North Atlantic (De Schepper & Head, 2008a, 2009) and the Iceland Sea (Schreck et al., 2012). Additionally, because the late Neogene dinoflagellate cyst record still contains several extant components, the modern (Marret & Zonneveld, 2003) as well as Pliocene (De Schepper et al., 2011) understanding of dinoflagellate cyst palaeoecology can be applied in the case of the Utsira Formation deposits. Formation Member Type well Depth (m RKB) Naust Fm. Tampen Spur Mbr. 34/4–7 1050–1090 Utsira Fm. Glauconitic Utsira Mbr. Introduction 34/4–6 1210–1250 Northern Utsira Mbr. 30/6–3 680–750 Southern Utsira Mbr. 24/12–1 495–730 Eir Fm. 25/2–10S 520–630 Methods Studied wells and samples Studied wells and samples to better understand the time of deposition and palaeoenvironment of Late Neogene sandy deposits on the Norwegian Shelf, including the Utsira Formation. The wells were specifically chosen to have a good spatial coverage of sediments belonging to the different members of the Utsira Formation as well as the Eir Formation and Tampen Spur Member (Table 1). Dinoflagellate cysts and acritarchs can provide detailed age control and palaeoenvironmental insights for sediments that have limited or coarse biostratigraphic control in the North Sea to sub-Arctic North Atlantic (e.g., Head, 1997; Louwye et Cuttings, side-wall core and core samples were collected from eight industry wells to investigate the Eir Formation, the different Utsira Formation members and the Tampen Spur Member (Naust Formation) at their proposed type wells (Table 2). Samples from well 25/2– 10S (Southern Utsira Member) and from Tampen area wells 34/7–2 (Glauconitic Utsira Member) and 34/7–4 (’Utsira Formation’) were also analysed for palynology. Three samples from well 15/9–A–23 were reinvestigated Table 2. Location of the eight industry wells investigated in this study with indicated the studied interval, number and type of samples. Samples were investigated from the Utsira Formation and its bounding lithological units. Depth: m RKB – metres below rig floor. SWC – sidewall core. Source: Norwegian Petroleum Directorate, factpages.npd.no. Asterisk () indicates type wells for the different Utsira Formation lithological units according to Eidvin et al. (2013). Well Field/Location Coordinates Total depth (m RKB) Water depth (m) Utsira Formation depth (m RKB) Investigated interval (depth m) Samples Number, type 15/9–A–23 Sleipner Øst 58˚ 22’ 2.05’’ N- 1˚ 54’ 32.06’’ E 5590 86 950–1350 1084.13–1084.99 3 Core 24/12–1 Gudrun Terrace 59˚ 2’ 29.8’’ N 1˚ 52’ 57.93’’ E 3966 113 497–732 440–750 11 Cutting 25/2–10S East of Øst Frigg 59˚ 53’ 11.8’’ N 2˚ 30’ 8.33’’ E 2967 120 475–520 470–570 6 Cutting 30/6–3* Oseberg 60˚ 34’ 52.9’’ N 2˚ 47’ 1.41’’ E 2940 102 656–892 710–740 3 Cutting 34/4–6* Snorre 61˚ 34’ 14.1’’ N 2˚ 13’ 19.9’’ E 3282 373.5 1128–1149 1220–1260 4 Cutting 34/4–7 Snorre 61˚ 31’ 9.8’’ N 2˚ 15’ 15.5’’ E 2950 354 1062–1097 1063–1190 8 Cutting & SWC 34/7–2 / 61˚ 17’ 57.2’’ N 2˚ 9’ 40.9’’ E 2475 245 1026–1035 990–1030 3 SWC 34/7–4 / 61˚ 29’ 4.4’’ N 2˚ 8’ 0.3’’ E 3115 319 1034–1115 980–1180 6 S. De Schepper & G. Studied wells and samples Mangerud 310 Well 25/2-10S Well 15/9-A-23 520 m 570 m 500 m 475 m Age Utsira Fm based on literature Middle Miocene to Zancelan Nordic Seas and eastern North Atlantic dinoflagellate cyst and acritarch events Age of the different studied lithological units Ma Period Epoch Age/Stage 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 Neogene Quaternary Miocene Pliocene Pleistocene Hol. Tortonian Messinian Zanclean Piacenzian Gelasian Calabrian 0 0 Tar. Ion. Cyst type I of de Vernal and Mudie (1989) F. filifera A. umbraculum Barssidinium spp. Well 24/12-1 570–580 m 720–730 m 440–450 m 520–530 m 740–750 m 500–510 m Well 30/6-3 Well 34/4-6 ? Well 34/4-7 Southern Utsira Mbr No Formal Name (NFN) Nordland Group LEGEND Eir Formation Glauconitic Utsira Mbr Northern Utsira Mbr Utsira Formation Tampen Spur Mbr (Nordland Group) most likely possible uncertain Well 34/7-2 Middle Miocene to Zancelan ? ? ? Polarity Norwegian Sea ODP Hole 642B North Atlantic DSDP Hole 610A O. tegillatum R. actinocoronata B. micropapillata complex Lavradosphaera crista O.? eirikinaum ? Utsira Formation ? ? ? ? Deposition either in Early Pliocene or Early Pleistocene Deposition either in Early Pliocene or Early Pleistocene Figure 5. Age estimate of the Utsira Formation, Eir Formation and Tampen Spur Member based on dinoflagellate cyst and acritarch stratigraphy. For comparison, the entire age range of the Utsira Formation as mentioned in the literature is also presented (see Fig. 2 for details). Age of the different studied lithological units Miocene Tortonian Figure 5. Age estimate of the Utsira Formation, Eir Formation and Tampen Spur Member based on dinoflagellate cyst and acritarch stratigraphy. For comparison, the entire age range of the Utsira Formation as mentioned in the literature is also presented (see Fig. 2 for details). 50% cold nitric acid (HNO3) and/or a short ultrasonic treatment was carried out on some samples (see Electronic Supplement 1). Before mounting, the residue was mixed with a 1% solution of polyvinyl alcohol (PVA) to prevent clotting, and stained with Safranin-O. It was then pipetted onto a 32 x 22 mm cover slip, which was mounted onto the glass microscope slide using glycerine jelly optical adhesive. to identify additional stratigraphic markers not reported in Piasecki et al. (2002). Studied wells and samples The sample depth of cutting samples in the wells must be considered as an approximate depth since they can represent an interval of up to 10 m of sediment (i.e., 740–750 m). Dinoflagellate cyst ranges in the Nordic Seas and North Atlantic Because there is no calibrated reference section in the North Sea, we used ranges of stratigraphic markers from independently calibrated (magnetostratigraphy, marine isotope stratigraphy) sections from nearby sites in the North Atlantic and Nordic Seas. It is important to make a comparison with both regions, because recent investigation has revealed the diachroneity of several stratigraphic markers between the North Atlantic, Iceland In this chapter and Table 3, we compile the stratigraphic ranges of several markers from the eastern North Atlantic Deep Sea Drilling Project (DSDP) Site 610, the Ocean Drilling Program (ODP) Site 907 in the Iceland Sea and Site 642 in the Norwegian Sea. Table 3. Compilation of the stratigraphic range of selected markers from the eastern North Atlantic Deep Sea Drilling Project (DSDP) Site 610, the Ocean Drilling Program (ODP) Site 907 in the Iceland Sea and Site 642 in the Norwegian Sea. Abbreviations: FA – first appearance, LA – last appearance Biostratigraphic event Norwegian Sea ODP Site 6421 Iceland Sea ODP Site 9072 eastern North Atlantic DSDP Site 6103,4 North Atlantic region compilation Dinoflagellate cysts LA Amiculosphaera umbraculum 1.44 Ma FA Ataxiodinium zevenboomii 5.3 Ma Not recorded LA Barssidinium pliocenicum/ Barssidinium graminosum ≤ 3 Ma 4.5 Ma 2.74 Ma 2.1–1.95 Ma in eastern England5 LA Batiacasphaera hirsuta 5.0 Ma (5.5 Ma?) 8.4 Ma / LA Batiacasphaera micropapillata complex 4.6 Ma 4.6 Ma 3.83 Ma LA Filisphaera filifera 1.44 Ma LA Invertocysta lacrymosa 3.27 Ma 4.5 Ma 2.74 Ma 2.7–2.8 Ma3 LA Operculodinium? eirikianum eirikianum ≤ 3 Ma 4.5 Ma 2.62 Ma LA Operculodinium tegillatum 4.6 Ma 4.5 Ma 3.71 Ma FA Operculodinium tegillatum 5.3 Ma 8.9 Ma continuously from 5.1 Ma >7.5 Ma in Belgium6 LA Reticulatosphaera actinocoronata 4.6 Ma 4.5 Ma 4.4 Ma7 LA Selenopemphix brevispinosa 4.6 Ma Late Pliocene e.g. 7,8 Acritarchs LA Cymatiosphaera? icenorum ≤3 Ma / 2.14 Ma 1.7 Ma at DSDP Site 6039 LA Lavradosphaera crista ≤3 Ma HO: 2.67 Ma,0 HCO: 3.00 Ma 2.9–3.0 Ma9 LA Leiosphaeridium rockhallensis 3.83 Ma Range Cyst type I of de Vernal & Mudie (1989) 4.7–4.6 Ma 4.7–4.2 Ma at DSDP Site 60310 LA "Veriplicidium franklinii" of Anstey (1992) 5.3 Ma ?5.3 Ma at ODP Site 98211 References: 1 – De Schepper et al. (2015, 2017), 2 – Schreck et al. (2012), 3 – De Schepper & Head (2008a), 4 – De Schepper & Head (2009), 5 – Head (1993), 6 – Louwye et al. Data storage Sea and Norwegian Sea (Fig. 5; De Schepper et al., 2015, 2017). The location of the Utsira Formation, roughly midway between the Norwegian Sea ODP Site 642 and eastern North Atlantic Site 610, makes the application of Nordic Seas and/or North Atlantic stratigraphic marker events sometimes difficult (see further). Sea and Norwegian Sea (Fig. 5; De Schepper et al., 2015, 2017). The location of the Utsira Formation, roughly midway between the Norwegian Sea ODP Site 642 and eastern North Atlantic Site 610, makes the application of Nordic Seas and/or North Atlantic stratigraphic marker events sometimes difficult (see further). All raw data are presented in Electronic Supplement 1. Palynological laboratory procedure and microscopy Dinoflagellate cysts and acritarchs were counted using a Zeiss AxioImager.A2 transmitted light microscope at the University of Bergen. Each slide was scanned at 400x magnification until about 200 or more dinoflagellate cyst specimens were counted or the entire slide was analysed. During this regular count, all encountered acritarchs, spores, pollen and freshwater algae were also enumerated. Consequently, at least five traverses were scanned at 200x magnification to look for rare taxa not seen during the regular count. Samples were processed by Palynological Laboratory Services Ltd (Holyhead, UK). One Lycopodium clavatum spore tablet (Batch no. 483.216, n = 18583 ± 1708 spores per tablet) was added to a specified weight of each sample prior to chemical degradation. Calcium carbonate and silica were removed by adding 50% hydrochloric acid (HCl) and 60% hydrofluoric acid (HF). In between and after the acid treatments, the sample was sieved through a 10-μm sieve cloth and collected. Oxidation using References: 1 – De Schepper et al. (2015, 2017), 2 – Schreck et al. (2012), 3 – De Schepper & Head (2008a), 4 – De Schepper & Head (2009), 5 – Head (1993), 6 – Louwye et al. )2007), 7 – Louwye et al. (2004), 8 – Louwye & De Schepper (2010), 9 – De Schepper & Head (2008b, 2014), 10 – M.J. Head, unpublished data, 11 – Van Ranst (2015). Biostratigraphic ranges of selected dinoflagellate cysts and acritarchs Dinoflagellate cyst ranges in the Nordic Seas and North Atlantic NORWEGIAN JOURNAL OF GEOLOGY Age and palaeoenvironment of the Utsira Formation in the northern North Sea based on marine palynology NORWEGIAN JOURNAL OF GEOLOGY Age and palaeoenvironment of the Utsira Formation in the northern North Sea based on marine palynology 311 NORWEGIAN JOURNAL OF GEOLOGY Age and palaeoenvironment of the Utsira Formation in the northern North Sea based on marine palynology Acritarch ranges in the Nordic Seas and North Atlantic Ocean Batiacasphaera micropapillata complex has a highest common occurrence (HCO) at 4.6 Ma in the Norwegian Sea, with occasional younger occurrences considered to be reworked (De Schepper et al., 2015, 2017). It has a HCO at 4.6 Ma in the Iceland Sea (Schreck et al., 2012), and a LA at 3.8 Ma in the North Atlantic (De Schepper & Head, 2008a). Cymatiosphaera? icenorum has a LA in the eastern North Atlantic at around 2.14 Ma and in the western North Atlantic at around 1.7 Ma (De Schepper & Head, 2014). Lavradosphaera crista has been recorded from the latest Messinian (c. 5.8 Ma) up to the Late Pliocene around 2.9–3.0 Ma (De Schepper & Head, 2014). Filisphaera filifera ranges up to c. 1.4 Ma in the eastern North Atlantic (De Schepper & Head, 2008a). Cyst type I of de Vernal & Mudie (1989) has a narrow stratigraphic range within the Zanclean of the Labrador Sea (de Vernal & Mudie, 1989) and in the Zanclean of the Norwegian Sea (4.6–4.7 Ma; De Schepper et al., 2017). In the western North Atlantic it occurs from 4.7 to 4.2 Ma (M.J. Head, unpublished data). Invertocysta lacrymosa has a relatively synchronous LA in the North Atlantic region at around 2.74 Ma (De Schepper & Head, 2008a). Its range top in the Nordic Seas is less well defined: in the Iceland Sea it ranges up to 4.5 Ma (Schreck et al., 2012), in the Norwegian Sea ODP Hole 642B up to 3.27 Ma (De Schepper et al., 2017) or possibly up to ~2.8 Ma (M. Smelror, unpublished data). “Veriplicidium franklinii” of Anstey (1992) was described for the first time from the Upper Miocene through possibly Lower Pliocene of Baffin Bay (Anstey, 1992). It is known from the Norwegian Sea ODP Hole 642B, where it occurs from the base of the studied interval at 5.9 Ma to near the Miocene–Pliocene boundary at 5.3 Ma (De Schepper et al., 2017). A single occurrence at 4.81 Ma can be considered as reworking. It is also possibly recorded between 8.2 and 5.3 Ma in the eastern North Atlantic ODP Site 982 (as Platycysta spp. in Van Ranst, 2015). Operculodinium? Dinoflagellate cyst ranges in the Nordic Seas and North Atlantic )2007), 7 – Louwye et al. (2004), 8 – Louwye & De Schepper (2010), 9 – De Schepper & Head (2008b, 2014), 10 – M.J. Head, unpublished data, 11 – Van Ranst (2015). Table 3. Compilation of the stratigraphic range of selected markers from the eastern North Atlantic Deep Sea Drilling Project (DSDP) Site 610, the Ocean Drilling Program (ODP) Site 907 in the Iceland Sea and Site 642 in the Norwegian Sea. Abbreviations: FA – first appearance, LA – last appearance Table 3. Compilation of the stratigraphic range of selected markers from the eastern North Atlantic Deep Sea Drilling Project (DSDP) Site 610, the Ocean Drilling Program (ODP) Site 907 in the Iceland Sea and Site 642 in the Norwegian Sea. Abbreviations: FA – first appearance, LA – last appearance References: 1 – De Schepper et al. (2015, 2017), 2 – Schreck et al. (2012), 3 – De Schepper & Head (2008a), 4 – De Schepper & Head (2009), 5 – Head (1993), 6 – Louwye et al. )2007), 7 – Louwye et al. (2004), 8 – Louwye & De Schepper (2010), 9 – De Schepper & Head (2008b, 2014), 10 – M.J. Head, unpublished data, 11 – Van Ranst (2015). 312 S. De Schepper & G. Mangerud Amiculosphaera umbraculum ranges up to c. 1.4 Ma in the eastern North Atlantic (De Schepper & Head, 2008a). A range top in the Nordic Seas is not available. Reticulatosphaera actinocoronata has a LA at 4.6 Ma in the Norwegian Sea (De Schepper et al., 2017) and at 4.5 Ma in the Iceland Sea (Schreck et al., 2012), consistent with its estimated range up to ~4.4 Ma in the North Atlantic (Louwye et al., 2004). Although this species can occur as reworked in Lower to Upper Pliocene deposits (e.g., Louwye & De Schepper, 2010), it can be considered as a relatively contemporaneous Nordic Seas–North Atlantic event (De Schepper et al., 2017). Ataxiodinium zevenboomii ranges from the Upper Miocene into the Pleistocene, but is recovered mainly from Pliocene deposits (e.g., Head, 1997; Louwye & Laga, 2008; De Schepper & Head, 2009). Its range in the Norwegian Sea starts at c. 5.3 Ma near the Miocene– Pliocene boundary (De Schepper et al., 2017). Dinoflagellate cyst ranges in the Nordic Seas and North Atlantic Selenopemphix armageddonensis is known from the Upper Miocene (de Verteuil & Norris, 1996; Louwye, 2002; Dybkjær & Piasecki, 2010) and may be reworked, but has been recorded in places in the Lower Pliocene of the North Sea (e.g., Louwye et al., 2004). Barssidinium graminosum and Barssidinium pliocenicum disappear from the eastern North Atlantic record at around 2.6 Ma (De Schepper & Head, 2008a, 2009), but have been reported from shallow deposits as young as 1.95 Ma in southern England (Head, 1993). Selenopemphix brevispinosa is a rare to common, persistent component of the palynological assemblage in the Norwegian Sea prior to the 4.6 Ma event (De Schepper et al., 2017). It also occurs in the Upper Pliocene of the North Sea (Louwye et al., 2004, 2007). Batiacasphaera hirsuta has a last appearance (LA) in the Iceland Sea at c. 8.4 Ma in the upper Tortonian (Schreck et al., 2012), but in the Norwegian Sea it has a persistent range up to 5.5 Ma, with spot occurrences up to 5.0 Ma (De Schepper et al., 2017). General considerations and limitations Downhole contamination or caving Because most investigated samples were (ditch) cutting samples from industry wells, downhole caving of younger sediments and their included palynomorphs presents a problem for dating the Utsira Formation deposits. Caving is the process where the in situ fossil assemblage is contaminated by stratigraphically younger fossils during the drilling operation. Especially the identification of first appearances (or lowest occurrences) can be hampered by downhole contamination. As a consequence, we have based our age estimates of the Utsira Formation on the extinctions or highest occurrences (known as first downhole occurrences in the industry) identified in the different wells. Using uncontaminated side-wall cores (e.g., in well 34/4–7, 34/7–2) or cored sections (well 15/9–A–23) has served as a quality control for the studied cuttings. Biostratigraphy and age assessment The dinoflagellate cysts and acritarch assemblage is most consistent with a Zanclean age for the studied interval, as Piasecki et al. (2002) also concluded. Nevertheless, our reinvestigation of three samples revealed previously unidentified stratigraphic markers including Batiacasphaera micropapillata complex, Operculodinium tegillatum, Operculodinium? eirikianum and Lavradosphaera spp. Together with Reticulatosphaera actinocoronata and the acritarch Cyst type I of de Vernal & Mudie (1989), this suggests an Early Pliocene age, older than 4.6 Ma when using the Nordic Seas bioevents. The range of Ataxiodinium zevenboomii in the Norwegian Sea starts at c. 5.3 Ma near the Miocene– Pliocene boundary, which provides a tentative maximum for the age of deposition. This is consistent with the first appearance of Operculodinium tegillatum in the Norwegian Sea at around 5.3 Ma. However, it must be noted that Operculodinium tegillatum is also known from the Upper Miocene in the North Atlantic realm (>7.5 Ma; Louwye et al., 2007; Schreck et al., 2012). Our study thus confirms and provides further detail on the Early Pliocene age proposed by Piasecki et al. (2002). We estimate the sediment to have been deposited between 5.3 and 4.6 Ma, possibly even entirely at around 4.7–4.6 Ma (Fig. 5). Age estimate limitations due to asynchrony of nearby extinction events We have compared the biostratigraphic events in the studied wells mainly with the Nordic Seas extinction events (ODP Sites 642 and 907) to estimate the ages in the studied wells. Acritarch ranges in the Nordic Seas and North Atlantic Ocean eirikianum eirikianum occurs well into the Upper Pliocene (≤ ~3 Ma) on the Vøring Plateau (De Schepper et al., 2017) and in the eastern North Atlantic (up to 2.6 Ma; De Schepper & Head, 2008a), but in the Iceland Sea it disappears already at c. 4.5 Ma (Schreck et al., 2012). Operculodinium tegillatum has a LA at 4.6 Ma in the Norwegian Sea (De Schepper et al., 2017), at 4.5 Ma in the Iceland Sea (Schreck et al., 2012), and at 3.7 Ma in the eastern North Atlantic (De Schepper & Head, 2008a). It first appears in the Norwegian Sea at around 5.3 Ma, but was recorded occasionally from c. 8.9 Ma and continuously from 5.1 Ma in the Iceland Sea (Schreck et al., 2012). It is also present in the southern North Sea Basin as Operculodinium antwerpensis in the Upper Miocene Diest Formation (>7.5 Ma; Louwye et al., 2007). NORWEGIAN JOURNAL OF GEOLOGY 313 NORWEGIAN JOURNAL OF GEOLOGY Age and palaeoenvironment of the Utsira Formation in the northern North Sea based on marine palynology Age and palaeoenvironment of the Utsira Formation in the northern North Sea based on marine palynology Results and interpretation israelianum/centrocarpum s.s. and Operculodinium? eirikianum eirikianum. Ataxiodinium zevenboomii and Selenopemphix brevispinosa were recorded in two samples, as well as several species of Batiacasphaera. One specimen of Selenopemphix armageddonensis in the lowermost investigated sample could be in situ, but may also be reworked. Reworking is proven by the presence of other Miocene taxa such as Cerebrocysta poulsenii, Hystrichosphaera obscura, Labyrinthodinium truncatum and Palaeocystodinium sp. General considerations and limitations This conservative approach results in older age estimates than when North Atlantic extinctions (DSDP Site 610) would have been used, due to the diachroneity of the North Atlantic and Nordic Seas extinction events (De Schepper et al., 2015, 2017). When calibrated extinction events were not available from the Nordic Seas (e.g., Amiculosphaera umbraculum, Filisphaera filifera), we had to rely on the North Atlantic bioevents. The Southern Utsira Member in well 15/9–A–23: a reinvestigation of Piasecki et al. (2002) The Southern Utsira Member in well 15/9–A–23: a reinvestigation of Piasecki et al. (2002) Limited reworking of Miocene taxa such as Cerebrocysta poulsenii, Hystrichosphaera obscura, Labyrinthodinium truncatum and Palaeocystodinium is observed. The occurrence of Selenopemphix armageddonensis, known from the Late Miocene but also recorded in the Early Pliocene of the North Sea (Louwye et al., 2004), can either be part of the in situ assemblage or reworked, as Piasecki et al. (2002) interpreted it. We reinvestigated three of the eight samples from the study by Piasecki et al. (2002) to look for previously unidentified stratigraphic markers. It is important to note that this was a cored section, implying that the palynological assemblages are not affected by caving. 1.1.1 Palynological assemblage Palaeoenvironment 1.1.1 Palynological assemblage The palynological assemblage is diverse, recording minimally 29 dinoflagellate cyst taxa as well as eight to eleven acritarch taxa per sample. Terrestrial palynomorphs (bisaccates, small pollen and spores) are as abundant, or even more abundant than marine palynomorphs. Concentrations could not be determined. The dominant dinoflagellate cyst species are Barssidinium pliocenicum, Barssidinium graminosum, Operculodinium tegillatum and Spiniferites/ Achomosphaera spp. indet. In places, common species are Filisphaera filifera, Habibacysta tectata, Operculodinium The sediments were most likely deposited on an open shelf, at some distance from the coast with almost no influence of riverine and open ocean waters. The dominance of Spiniferites/Achomosphaera and high numbers of Barssidinium and Operculodinium tegillatum in each sample suggest a neritic environment. High abundances of Operculodinium tegillatum are recorded from water depths of 30–50 m (Kattendijk Sands, southern North Sea Basin; Louwye et al., 2004; De Schepper et al., 2009), the outer shelf (ODP 642B, Vøring Plateau, water depth 1286 m; De Schepper et S. De Schepper & G. Mangerud 314 al., 2015, 2017) to the open ocean settings of the North Atlantic (ODP Hole 610A, water depth 2417 m; De Schepper & Head, 2008a) and Iceland Sea (ODP Hole 907A, water depth 2036 m; Schreck et al., 2012). The high abundance of terrestrial palynomorphs indicates a proximal coastline, but the (near-)absence of freshwater algae indicates no freshwater inflow via rivers. The single occurrence of Impagidinium patulum is the only evidence for an influence of warm, open ocean waters. Similarly, Piasecki et al. (2002) concluded with a deposition on the outer shelf, probably during a transgression, with limited influence of oceanic water. analytical noise, caving and/or reworking. It crucially illustrates the difficulty of using this dating method in the North Sea region (Eidvin et al., 2014b). Palynological assemblage Palynological assemblage Nine samples were studied from the Southern Utsira Member (= Central Utsira Member according to Eidvin & Rundberg, 2007), which likely correspond to the upper part of the Utsira Formation (Eidvin & Rundberg, 2007). One sample was studied from the underlying No Formal Name (NFN) unit and one from the overlying Nordland Group. The dinoflagellate cyst assemblage of the Central Utsira Member is moderately diverse, recording between 18 and 34 taxa per sample. The NFN unit recorded 22 dinoflagellate cyst taxa and the Nordland Group sample only 11 taxa. Dinoflagellate cysts and acritarchs are usually dominant, but terrestrial palynomorphs outnumber the marine palynomorphs in the upper part of the Utsira Formation. Also, four taxa of freshwater algae occur commonly throughout the studied section. Sea surface conditions were most likely temperate and warmer than at present. The dinoflagellate cyst assemblage is characterised mainly by extinct thermophilic taxa (e.g., Ataxiodinium zevenboomii, Bitectatodinium raedwaldii, Hystrichokolpoma rigaudiae), although cool-water tolerant taxa like Bitectatodinium tepikiense, Habibacysta tectata, Filisphaera filifera and Pyxidinopsis braboi also occur. The presence of Tuberculodinium vancampoae, Impagidinium patulum and Melitasphaeridium choanophorum indicate a sea surface temperature (SST) higher than present. Tuberculodinium vancampoae is today a (sub)tropical coastal species that is usually found in areas with winter SST higher than c. 13°C and summer SST above 14.5°C (Marret & Zonneveld, 2003). However, Piasecki et al. (2002) suggested that this species could be reworked. We cannot prove or disprove this suggestion, but the presence of Melitasphaeridium choanophorum is consistent with warmer conditions than at present. This species was continuously present in the eastern North Atlantic until ~3 Ma (De Schepper & Head, 2009) and until ~3.3 Ma in the Norwegian Sea (De Schepper et al., 2015, 2017). It has been discovered recently in surface sediments from the Gulf of Mexico, where summer SSTs are between 26 and 30°C, and winter SSTs are 18 to 25°C (Limoges et al., 2013). The dominant dinoflagellate cyst species in the Southern Utsira Member are Habibacysta tectata, Filisphaera filifera, Operculodinium centrocarpum sensu Wall & Dale (1966), Operculodinium? eirikianum, Operculodinium tegillatum and Spiniferites/Achomosphaera spp. Also, Barssidinium spp., Batiacasphaera spp., Nemato sphaeropsis labyrinthus, Operculodinium centrocarpum/ israelianum and Reticulatosphaera actinocoronata are common constituents of the assemblage. Reworked dinoflagellate cysts occur in every sample, but these were not identified further. Palaeoenvironment The palynological assemblages indicate a neritic (shelf) environment with low to moderate influence of fresh water (via rivers) and limited open ocean influence. The neritic environment is indicated by the dominance of autotrophic, spiniferate, dinoflagellate cysts. Several heterotrophic species were recorded, but never in high abundance. Marine palynomorphs (dinoflagellate cysts and acritarchs) are more abundant than the terrestrial palynomorphs in the lower part, while this is reversed in the upper part. Freshwater algae increase in abundance, and together with the high amount of pollen and spores indicate a closer proximity to the coast and a riverine influence in the upper part. The low occurrence of Impagidinium demonstrates a very limited influence of oceanic waters. The sample at 540 m (not shown) contains few dinoflagellate cysts and a major amount of pollen and spores, suggesting a shoaling of the environment to a very shallow, near-coastal environment. Stratigraphic dinoflagellate cyst markers such as Batiacasphaera micropapillata complex, Operculodinium tegillatum and Reticulatosphaera actinocoronata disappear from the record at 570 m. While the LA of the first two taxa in the eastern North Atlantic is recorded at around 3.7–3.8 Ma, all three taxa disappear simultaneously from the Nordic Seas at around 4.6 Ma. The range of the acritarch Cyst Type I of de Vernal & Mudie (1989) between 670 m and 570 m corroborates an Early Pliocene age, which may be close to 4.7–4.6 Ma. In the Nordic Seas, these taxa disappear as a consequence of changed oceanography in the Early Pliocene (De Schepper et al., 2015). In this well, we recorded also the HO of Operculodinium? eirikianum eirikianum and the acritarch Lavradosphaera crista at 570 m. Because both taxa occur well into the Upper Pliocene in the North Atlantic and Norwegian Sea, a hiatus and/or non-deposition due to a major shoaling environment is interpreted between 570 and 530 m in this well. Sample 550 m (not shown) is dominated by terrestrial palynomorphs and plant material in various stages of degradation but also records Spiniferites, Bitectatodinium, Filisphaera filifera, Habibacysta tectata and O. israelianum, together suggesting a shallow-marine environment. Stratigraphic markers were absent in this sample. The dinoflagellate cyst assemblage is characterised by typical Early Pliocene thermophilic species (e.g., Melitasphaeridium choanophorum, Operculodinium? eirikianum eirikianum, Operculodinium tegillatum) reflecting temperate conditions. Biostratigraphy and age assessment g p y g The assemblage in the single sample (740 m) from the NFN unit consists mainly of taxa that also occur in the overlying Southern Utsira Member. Because this sample was taken close to the boundary between the NFN and the Southern Utsira Member (730 m), caving could be responsible for the presence of Barssidinium pliocenicum, Batiacasphaera micropapillata complex, Operculodinium? eirikianum eirikianum and Reticulatosphaera actinocoronata in the NFN. If these species are nevertheless in situ, they are consistent with an Early Pliocene (older than 4.6 Ma) to Late Miocene age. The single specimen of Pentadinium laticinctum could be consistent with a Late Miocene to earliest Early Pliocene age, because this species has a highest occurrence (HO) in the Selenopemphix armageddonensis Zone (7.6–5.0 Ma) of Dybkjær & Piasecki (2010). Labyrinthodinium truncatum, with a HO in late Tortonian (Schreck et al., 2012), likely occurs as reworked. The nature of the samples and cuttings does allow the possibility that both Labyrinthodinium truncatum and Pentadinium laticinctum are in place, and thus imply a Miocene age. Comparison with foraminifers and Sr-isotopic data The marine palynology suggests a time of deposition during the Early Pliocene, overlapping partly with Late Miocene to Early Pliocene ages based on foraminifers (Wilkinson, 1999; Eidvin & Rundberg, 2007). Comparable to the outer-shelf environment concluded from the dinoflagellate cysts, foraminifers also indicate a middle- to outer-shelf environment with limited oceanic influence (Wilkinson, 1999). Strontium isotopic data were obtained from mollusc fragments, benthic foraminifera, and benthic and planktonic foraminifera in a core sample at 1080 m (Eidvin & Rundberg, 2007). Five 87/86Sr ages reported from in situ specimens exhibit a range between 1.35 and 6.46 Ma for the time of deposition. The oldest age appears too old for the Early Pliocene age assignment based on dinoflagellate cysts, whereas other 87/86Sr ages are much younger at <2.7 Ma. This wide range may be explained by high-frequency variation in seawater 87/86Sr, 315 NORWEGIAN JOURNAL OF GEOLOGY Age and palaeoenvironment of the Utsira Formation in the northern North Sea based on marine palynology Age and palaeoenvironment of the Utsira Formation in the northern North Sea based on marine palynology NORWEGIAN JOURNAL OF GEOLOGY Since these samples correspond to the upper part of the Utsira Formation (Rundberg & Eidvin, 2005; Eidvin et al., 2014a), the lower succession of the Utsira Formation in this southern part of the basin must be older. Biostratigraphy and age assessment eirikianum and the acritarch Lavradosphaera crista are absent. This indicates an age younger than ~3.0 Ma for the sediments above 520 m. Using the North Atlantic LA of Barssidinium graminosum and Barssidinium pliocenicum, a latest Pliocene to Early Pleistocene age of around 2.6 Ma, but possibly also as young as 1.95 Ma (Head, 1993), can be proposed for sample 520 m. The presence of Amiculosphaera umbraculum and Filisphaera filifera at 500 m and 440 m suggests that these samples are older than 1.4 Ma (De Schepper & Head, 2008a, 2009). Considering the single occurrences of Reticulatosphaera actinocoronata and Operculodinium? eirikianum as reworked, the sediment between 520 and 440 m was likely deposited between 3.0 and 1.4 Ma. The dinoflagellate cyst and acritarch assemblage of the Southern Utsira Member suggests that deposition occurred in two phases (Fig. 5; Electronic Supplements 1 & 2): a first depositional phase (720 to 570 m) took place in the Late Miocene to Early Pliocene (>4.6 Ma), followed by a second depositional phase between 3.0 and 1.4 Ma (520 to 500 m). The palynomorph assemblage in the Southern Utsira Member between 720 and 570 m differs from the assemblage in the overlying samples. A maximum age for the deposits between 720 m and 570 m can be proposed based on the presence of Operculodinium tegillatum. This indicates a maximum age as young as 5.3 Ma if only its Norwegian Sea range is considered. However, considering the entire known range of Operculodinium tegillatum, this lowermost part of the Southern Utsira Member could also be Late Miocene. A Late Miocene age is also supported by the occurrence of “Veriplicidium franklinii” of Anstey (1992) between 730 and 640 m, which in the Norwegian Sea ranges from >5.9 Ma to 5.3 Ma (De Schepper et al., 2015, 2017) and from 8.2 to 5.3 Ma in the eastern North Atlantic ODP Site 982 (as Platycysta spp. in Van Ranst, 2015). Palaeoenvironment Cool-water tolerant taxa Habibacysta tectata and Filisphaera filifera (Head, 1994; De Schepper et al., 2011) are also abundant and both show opposing trends: Habibacysta tectata is abundant at the base and decreases upward, whereas Filisphaera filifera only becomes very abundant in the upper three samples. These upper three samples furthermore do not contain extinct, warm-water Pliocene species (e.g., Melitasphaeridium choanophorum, Invertocysta lacrymosa) and record a decrease in species numbers compared to the other samples, suggesting a cooler environment. Comparison with foraminifer data and Strontium isotopic data The age estimates based on benthic foraminifer assemblages (Eidvin & Rundberg, 2007) partly overlap, but generally suggest an older age than the dinoflagellate cyst estimates. The benthic foraminifer assemblages between 750 and 550 m indicate a Mid Miocene to Early The assemblage in the upper two samples of the southern Utsira Member (520 and 500 m) and the Nordland Group sample does not contain any Lower Pliocene markers. As mentioned, also Operculodinium? eirikianum S. De Schepper & G. Mangerud 316 Pliocene age, whereas we conclude with a Late Miocene to Early Pliocene (>4.6 Ma) age. The uppermost part of the Southern Utsira Member between 500 and 520 m is Early Pliocene according to the benthic foraminifer assemblages. The dinoflagellate cysts here indicate a Late Pliocene (<3.0 Ma) to Pleistocene age. The Early Pleistocene benthic foraminifers in the lowermost part of the Nordland Group (480–500 m) correspond favourably with the dinoflagellate cysts, which indicate an Early Pleistocene age between 2.6–1.95 and 1.4 Ma. Biostratigraphy and age assessment The samples in the Eir Formation (570–520 m) may be Early Pliocene, older than 5.0 Ma, but can also be Late Miocene (Fig. 5; Electronic Supplements 1 & 2). Batiacasphaera hirsuta and Operculodinium tegillatum range up to 520 m. Although Batiacasphaera hirsuta has a LA of around 8.5 Ma in the Iceland Sea, its LA in the Norwegian Sea (Table 3) suggests an age older than 5.0 Ma. This is in correspondence with the known range of Operculodinium tegillatum in the Nordic Seas up to 4.6 Ma. When the Norwegian Sea range of Operculodinium tegillatum is considered, an Early Pliocene age can be inferred since this species has a lowest occurrence around 5.3 Ma. However, considering the entire known range of Operculodinium tegillatum (Table 3) and the fact that the samples are cuttings, the age could also be Late Miocene. Palaeoenvironment In fact, if Labyrinthodinium truncatum truncatum is in situ, the age of the Eir Formation would be >7.5 Ma. The comparison between strontium isotopic ages (table 2 in Eidvin & Rundberg, 2007) and dinoflagellate cyst biostratigraphy is problematic. The problem lies in the large scatter of the strontium isotope measurements in subsequent samples or even within the same sample. Hence, it is impossible to objectively determine whether the Sr-derived ages are accurate or affected by reworking or caving. For example, mollusc fragments in the sample at 650 m depth yield an age range between 2.41 and 8.96 Ma. Another example is the sample at 500 m, for which the Sr-isotope age (2.1 Ma) was considered too young and interpreted to be due to caving of the mollusc fragment (Eidvin & Rundberg, 2007). Compared to the Late Pliocene to Pleistocene age based on dinoflagellate cysts for the sediments above 520 m, a Sr-isotope age of 2.1 Ma at 500 m may, in hindsight, be an accurate age estimate. Samples investigated from the Southern Utsira Member (520–475 m) indicate an age between 4.6 and 3.0 Ma (Fig. 5). The HOs of Operculodinium tegillatum and Batiacasphaera hirsuta at 520 m suggest an age younger than 5.0 Ma and 4.6 Ma, respectively. The minimum age for this unit is not younger than c. 2.9–3.0 Ma based on the presence of the acritarch Lavradosphaera crista in all samples and occurrences of Operculodinium? eirikianum, Barssidinium spp. and Invertocysta lacrymosa. The Nordland Group sample (470 m) contains the same markers as the sediments below, and thus cannot be distinguished from the underlying Southern Utsira Member. The Glauconitic Utsira Member in well 34/4–6 The Northern Utsira Member in well 30/6–3 Palynological assemblage The three investigated cutting samples from the Northern Utsira Member are dominated by Filisphaera filifera, Habibacysta tectata and Spiniferites/Achomosphaera spp. indet. Palynological assemblage The reference well section for the Glauconitic Utsira Member is in well 34/4–6 from 1250 to 1210 m (Eidvin et al., 2013). In this interval, we analysed four samples and collected presence–absence data for three samples (1250, 1240 and 1230 m) and counted one sample at 1220 m. Palynological assemblage Sample 1220 m showed a dominance of Habibacysta tectata, Bitectatodinium spp. and Spiniferites spp. In total, 15 dinoflagellate cyst and 5 acritarch taxa were recorded. Terrestrial palynomorphs outnumber the dinoflagellate cysts, and also freshwater algae (Botryococcus, Pediastrum) were recorded. Palynological assemblage Biostratigraphy and age assessment Biostratigraphy and age assessment An age assessment based on the dinoflagellate cyst assemblage is open for interpretation (Fig. 5): the sediments could have been deposited in the Early Pliocene or Early Pleistocene (2.6–1.4 Ma). In each sample, Cyst Type I of de Vernal & Mudie (1989) is recorded (Electronic Supplement 2), which would favour an Early Pliocene age at around 4.6–4.7 Ma for this interval. This is consistent with the presence of other Early Pliocene palynomorphs Operculodinium tegillatum, Reticulatosphaera actinocoronata, Lavradosphaera crista, Cyst Type I of de Vernal & Mudie, 1989), but to arrive at this age the Late Miocene–earliest Pliocene Batiacasphaera hirsuta must be interpreted as reworked. Biostratigraphy and age assessment The lower two samples (1250 and 1240 m) likely demonstrate a diverse Miocene dinoflagellate cyst assemblage (including Apteodinium spiridoides, Cleistosphaeridium placacantha, Cordosphaeridium cantharellus, Distatodinium spp., Ennaedocysta spp., Glaphyrocysta spp.). An alternative interpretation that explains the presence of a mixture of markers is that all Upper Miocene to Lower Pliocene markers were reworked. This can be supported by the relatively high amounts of reworked palynomorphs in the samples. The high proportions of F. filifera and H. tectata (together 39–56%), could indicate that these are in situ and would be consistent with cooler conditions in the Early Pleistocene (compared to the generally warmer Early Pliocene). Using the stratigraphic ranges of those two species the deposits are older than 1.4 Ma. Finally, caving may also be responsible for the problematic interpretation. The Glauconitic Utsira Member in well 34/4–6 A substantial hiatus is apparent between the lower and upper two samples: the samples 1230 m and 1220 m yielded the Lower Pliocene markers Operculodinium tegillatum, Reticulatosphaera actinocoronata and Cyst Type I of de Vernal & Mudie (1989). If in situ, these taxa suggest a time of deposition during the Early Pliocene before 4.6 Ma. However, it is not unthinkable that these taxa were also reworked and that deposition took place during the Late Pliocene to Early Pleistocene between 3.0 and 1.4 Ma (Fig. 5). Evidence supporting a Pleistocene age is the dominance of cool-water taxa Filisphaera filifera and Habibacysta tectata, the absence of the acritarch genus Lavradosphaera and the low number (n = 15) of taxa recorded. This is almost half of the on average 29 taxa in the Early Pliocene Central and Southern Utsira Member of wells 24/12–1 and 15/9– A–23, and could correspond to the climatically cooler conditions during the Pleistocene. Palaeoenvironment NORWEGIAN JOURNAL OF GEOLOGY 317 Age and palaeoenvironment of the Utsira Formation in the northern North Sea based on marine palynology 317 AL OF GEOLOGY Age and palaeoenvironment of the Utsira Formation in the northern North Sea based on marine palynology 31 Palaeoenvironment Given the amount of reworking, potentially affecting the entire assemblage, palaeoenvironmental interpretations are speculative. Nevertheless, cool-water taxa F. filifera and H. tectata are dominant in the samples suggesting that these occur in situ. High concentrations of terrestrial palynomorphs and records of freshwater algae suggest that the coast was nearby. Most likely, cool conditions in shallow-water environments prevailed during deposition of the Northern Utsira Formation. Palaeoenvironment Palynological assemblage Six cutting samples between 570 and 470 m were studied. The samples at 570 and 540 m belong to the Eir Formation (Eidvin et al., 2013). The sample at 520 m is taken at the boundary of the Eir and Utsira formations. Because this is a cutting sample and the palynological assemblage is mostly similar to the Eir Formation assemblages (see Electronic Supplement 1), we consider the palynological assemblage to reflect the Eir Formation. The samples at 500 and 490 m are from the Utsira Formation and are likely part of the eastward prograding, sandy, strandplain facies (Galloway, 2002; Rundberg & Eidvin, 2005). Following Eidvin et al. (2013), we consider these samples to belong to the Southern Utsira Member (= Central Utsira Member according to Eidvin & Rundberg, 2007). The sample at 470 m is located within the basal Nordland Group. The palynological assemblages in all samples indicate deposition in a shallow shelf environment at some distance from the coast during temperate conditions. The marine and terrestrial palynomorphs suggest conditions comparable to the shallow-water Pliocene deposits that were deposited in proximity to the coast of the southern North Sea Basin (Louwye et al., 2004; De Schepper et al., 2009). In addition, Batiacasphaera hirsuta suggests a neritic environment with increased nutrients (Quaijtaal et al., 2014). Oceanic (Impagidinium) and freshwater algae are only occasionally recorded, indicating a very limited influence of oceanic waters and riverine input. Cool tolerant taxa (Filisphaera filifera, Habibacysta tectata) are a minor component of the assemblage, which is dominated by thermophilic, extinct taxa (e.g., Operculodinium tegillatum, Barssidinium spp.). Comparison with published age estimates In Eidvin et al. (2013), the interval between 520 and 475 m (Utsira Formation) is considered to be the Lower Pliocene and Eidvin et al. (2014a, fig. 22) place this interval in the Upper Miocene to Lower Pliocene, which is considerably older than our interpretation (4.6 to 3.0 Ma). Also, most 87/86Sr ages (5.41–4.09 Ma; (Eidvin et al., 2013) are slightly older than in our interpretation, although some overlap exists. The dominant dinoflagellate cyst species in all samples of well 25/2–10S are Operculodinium centrocarpum sensu Wall & Dale (1966) and Spiniferites/Achomosphaera spp. indet. Barssidinium spp. and Habibacysta tectata are common taxa in all samples, whereas Batiacasphaera hirsuta is common only in the Eir Formation. The Glauconitic Utsira and Tampen Spur mem- bers in well 34/4–7 Palynological assemblage Two samples (1190 and 1180 m) were investigated in the Utsira Formation. As the sediment is characterised as glauconitic sand (Eidvin & Rundberg, 2001), these samples can most likely be assigned to the Glauconitic Utsira Member. Two more samples were investigated from the immediately overlying Nordland Group sediments (1160 and 1140 m). In addition, three cuttings and one sidewall-core sample were investigated from the type section of the Tampen Spur Member of the Naust Formation (1090–1050 m; Eidvin et al., 2013). Comparison with published age estimates Eidvin et al. (2013) used planktonic and benthic foraminifera to estimate a Late Miocene to Early Pliocene age for the Utsira Formation, corroborating the Sr-isotope ages of 5.1 and 5.4 Ma (Eidvin & Rundberg, 2001). The dinoflagellate cysts indicate younger ages (4.6–3.0 Ma) for the upper sample (1180 m) in the Utsira Formation compared to the lower sample (1190 m, >4.6 Ma). The age for the lower sample is in agreement with the estimates of Eidvin et al. (2013), but the upper sample is thus considerably younger. The palynological assemblage is more diverse in the Utsira Formation than in the overlying deposits. Habibacysta tectata is common (5–16% of total assemblage) in the Utsira Formation and the basal Nordland Group, but is largely absent from the Tampen Spur Member (<1% of total assemblage in two samples). Filisphaera filifera, Operculodinium centrocarpum sensu Wall & Dale (1966) and Spiniferites/Achomosphaera spp. are dominant throughout. Palaeoenvironment The dinoflagellate cyst assemblages are dominated by Habibacysta tectata, Filisphaera filifera, Operculodinium centrocarpum sensu Wall & Dale (1966) and Spiniferites spp. indet. which points towards a shelf environment under cool-temperate conditions. Influence from the open ocean is minimal throughout, as shown by the occurrence of only a few specimens of Impagidinium. Continuous riverine input (presence of Botryococcus and Pediastrum) indicates the proximity of the coast during deposition of the Utsira Formation. The Tampen Spur Member was deposited even closer to the coast and near a strong riverine influence, since freshwater algae and terrestrial palynomorphs are more abundant. Palaeoenvironment Palaeoenvironment Cool-temperate conditions, in a shallow-water environment near the coast prevailed during deposition of the Glauconitic Utsira Member. Cool- water taxa Habibacysta tectata and Filisphaera filifera are abundant in the samples. High concentrations of terrestrial palynomorphs and records of freshwater algae indicate a proximity to the coast. Glauconitic deposits are commonly associated with transgressive phases (Sturrock, 1996). A high number of reworked dinoflagellate cysts from Miocene or older sediments is consistent with a transgression that may have eroded the Hordaland Group as well as Pliocene (Utsira Formation) sediments. Comparison with published age estimates Our age assessment could not conclude on an Early Pliocene or Pleistocene age for the interval between 710 and 740 m, although we favour the Early Pleistocene age. Eidvin et al. (2013) indicated an Early Pliocene age for the Utsira Formation between 740 and 680 m based on foraminifers. Sr-isotope ages (table 1 in Eidvin et al., 2013) indicate Quaternary (interpreted as caved) or Miocene ages (interpreted as reworked). Late Miocene ages of 6.99 Ma and 8.69 Ma were considered to be good estimates for the deposits between 710 and 740 m. Miocene ages are not supported by the dinoflagellate cysts. S. De Schepper & G. Mangerud 318 The Tampen Spur Member is older than 1.4 Ma based on the presence of Amiculosphaera umbraculum and Filisphaera filifera (Electronic Supplement 2). Comparison with published age estimates Foraminifers and Sr isotope ages indicate a Late Miocene to earliest Early Pliocene age according to Eidvin et al. (2013). Our analysis of the palynomorphs indicates a Miocene age for the interval 1250–1240 m and an Early Pliocene or a Pleistocene age for 1230–1220 m. The Glauconitic Utsira Member in well 34/7–2 in the Tampen area Biostratigraphy and age assessment The Utsira Formation (likely Glauconitic Utsira Member) in this well is older than 3.0 Ma, and the lowermost part may even be older than 4.6 Ma (Fig. 5; Electronic Supplement 2). The sample at 1190 m may be older than 4.6 Ma based on the presence of Reticulatosphaera actinocoronata and Operculodinium tegillatum. However, reworking of these specimens cannot be excluded which would suggest an age <4.6 Ma. The HO of Operculodinium? eirikianum, Melitasphaeridium choanophorum, Invertocysta lacrymosa, Heteraulacacysta sp. A of Costa & Downie (1979), Barssidinium spp. and the acritarch Lavradosphaera crista at 1180 m is consistent with an age older than 3.0 Ma. Biostratigraphy and age assessment Biostratigraphy and age assessment The different lithological units could not be differentiated using dinoflagellate cysts and acritarchs. The assemblages are characterised by few stratigraphic markers, substantial reworking and potentially also caving which makes an age assessment difficult and open to alternative interpretations. Firstly, considering the dominant species Operculodinium centrocarpum sensu Wall & Dale (1966) and Filisphaera filifera, a Pliocene to Pleistocene age can be proposed. Both taxa are an important component of the assemblages recorded in the Pliocene and Pleistocene of the other wells in this study. It follows that the deposits are not younger than 1.4 Ma, given the presence of Amiculosphaera umbraculum and the well-established LA of Filisphaera filifera. Secondly, the studied interval could be Late Miocene based on the occurrence of Cleistosphaeridium placacanthum in nearly all samples and an abundance peak in sample 1080 m. However, this species is most likely reworked since the overall assemblage is not characteristic for the Miocene. Thirdly, the studied interval could be older than 4.6 Ma based on the presence of Reticulatosphaera actinocoronata in almost all samples. However, Reticulatosphaera actinocoronata, Batiacasphaera minuta, Operculodinium? eirikianum and Operculodinium tegillatum have a near- contemporaneous LA in the Early Pliocene Nordic Seas, whereas in this well they have a HO at different depths in the studied interval. Furthermore, Operculodinium? eirikianum disappears first (sample 1180 m) in the studied interval, while it is known to range into the Upper Pliocene (Table 3). Finally, considering the occurrences of Operculodinium? eirikianum and Lavradosphaera crista in 1180 m as their HO, the samples between 1150 and 980 m could be younger than 3.0–2.6 Ma. This interpretation implies reworking of most of the Lower Pliocene and Miocene markers. Reworking does occur in the studied interval, and especially in sample 1080 m where several Miocene taxa (Cleistosphaeridium placacantha, Cordosphaeridium minimum, Distatodinium, Palaeocystodinium) were recorded. The Nordland Group sample is dominated by cool-water taxa (e.g., F. filifera) suggesting an age <2.6 Ma. At best, a Pliocene–Pleistocene age can be concluded based on the marine palynology, but a more precise dating remains speculative. is reworked implying an age younger than 4.6 Ma. This is corroborated by the absence of other Lower Pliocene markers (e.g., Operculodinium tegillatum). Given the absence of Lavradosphaera crista it may even be that the sample is younger than 3.0 Ma, although this is speculative and needs confirmation from analysing more samples. Palaeoenvironment Palaeoenvironment The Utsira Formation sample indicates outer shelf and warm temperate conditions. The overlying samples were deposited closer to the coast, in shallower environments, and during cool temperate conditions. Comparison with published age estimates Eidvin (2009) and Eidvin et al. (2013) estimated these deposits to be Late Miocene to Early Pliocene based on foraminifer assemblages. Two Sr-isotope ages of benthic foraminifers from sidewall core 1030 m (listed as 1130 m in table 1 on p. 4 in Eidvin, 2009 and in Eidvin et al., 2013) gave ages of 4.7 and 4.2 Ma. The Nordland Group sediments were dated to the Late Pliocene. For both the Utsira Formation and the Nordland Group sediments, our dinoflagellate cysts analysis suggests younger ages. Biostratigraphy and age assessment The overlying Nordland Group sediments are younger than 2.6 Ma, but older than 1.4 Ma given the presence of Amiculosphaera umbraculum and Filisphaera filifera. Palynological assemblage Palynological assemblage Three sidewall core samples were investigated from this well. One sample was from the Utsira Formation, and two from the overlying sediments of the Nordland Group. The assemblage in the Utsira Formation is dominated by Operculodinium? eirikianum, Bitectatodinium raedwaldii and Operculodinium centrocarpum sensu Wall & Dale (1966). The Nordland Group samples are dominated by Habibacysta tectata. Considerable reworking is identified in these samples with the occurrence of Cleistosphaeridium placacanthum, Hystrichosphaera obscura and Reticulatosphaera actinocoronata. Sediments of the Nordland Group underlying the Tampen Spur Member are younger than 2.6 Ma, possibly younger than 1.95 Ma. This is inferred from the absence of the Upper Pliocene markers Barssidinium spp., Operculodinium? eirikianum and Invertocysta lacrymosa (HOs at top Utsira Formation; Electronic Supplement 2). These species disappear from the North Atlantic at around 2.6–2.7 Ma, but Barssidinium spp. has been recorded up to 1.95–2.1 Ma in England (Head, 1993). Biostratigraphy and age assessment The Utsira Formation (1030 m) is likely older than 2.6 Ma (Fig. 5; Electronic Supplement 2) based on the abundant presence of Operculodinium? eirikianum in sample 1030 m. If Reticulatosphaera actinocoronata is in situ in this sample, the age could be older than 4.6 Ma. However, there is strong evidence for reworking in this sample, given the occurrence of several Miocene dinoflagellate cysts, even in high abundance (Cleistosphaeridium placacanthum) in the same sample. Most likely, Reticulatosphaera actinocoronata 319 NORWEGIAN JOURNAL OF GEOLOGY Age and palaeoenvironment of the Utsira Formation in the northern North Sea based on marine palynology Age and palaeoenvironment of the Utsira Formation in the northern North Sea based on marine palynology NORWEGIAN JOURNAL OF GEOLOGY Correlating the wells based on dinoflagellate cysts and dating the Utsira Formation The Glauconitic Utsira Member was deposited in the Pliocene (34/4–6) to Early Pleistocene (34/4–7; Figs. 5 & 6B). According to the palynology, the sediment intervals assigned to the Glauconitic Utsira Member (wells 34/4–6 and 34/4–7) were deposited at different times. In well 34/4–6, the interval between 1250 and 1210 m contains the reference section for the Glauconitic Utsira Member. Our palynological analyses revealed that this unit contains Miocene and Early Pliocene palynomorphs, which could indicate an age of deposition before 4.6 Ma. However, the Miocene and Early Pliocene palynomorphs could also be interpreted as reworked and as a consequence, the Glauconitic Utsira Member would then most likely have been deposited between 3.0 and 1.4 Ma. In well 34/4–7, this member appears to have been deposited between 4.6 Ma and 3.0 Ma. It follows that the Northern Utsira Member is older than the Glauconitic Utsira Member, in locations where the latter member stratigraphically overlies the Northern Utsira Member. The Utsira Formation is thickest in the Viking Graben (Fig. 1) and becomes much thinner towards the north in the Tampen area. Our palynological study indicates an Early Pliocene to Early Pleistocene age for the Southern, Northern and Glauconitic Utsira Members, with the different members showing a large spread in their age estimates. Three palynological events can be used to correlate the different wells and lithological units: the LA of a group of Early Pliocene taxa (Reticulatosphaera actinocoronata, Operculodinium tegillatum, Cyst type I of de Vernal & Mudie, 1989) at around 4.6 Ma, the LA of Lavradosphaera crista around 3.0 Ma and the LA of Filisphaera filifera at around 1.4 Ma (Fig. 6A, B). The type section for the Southern Utsira Member was proposed in well 24/12–1 by Eidvin et al. (2013) (Fig. 3). Our palynological analysis demonstrated that this section contains the most complete record of the Utsira Formation of all investigated wells (Fig. 5). It must be noted, however, that we have likely investigated only see the upper part of the Utsira Formation in well 24/12 (Rundberg & Eidvin, 2005; Eidvin et al., 2014a). This implies that the Southern Utsira Member (e.g., in wells 16/1–1, 16/2–1) may include deposits older than the Early Pliocene (age of the cored interval in well 15/9– A–23). In this well, we also find clear evidence for two distinct depositional phases possibly separated by a hiatus within the Southern Utsira Member. Palaeoenvironment Abundant freshwater algae and terrestrial palynomorphs in all samples suggest a shallow depositional environment close to the coast during cool temperate conditions (Filisphaera filifera, Habibacysta tectata). Well 34/7–4 in the Tampen area Palynological assemblage In the completion log from well 34/7–4 (www.npd.no), the sandy sediments between 1034 and 1115 m were assigned to the Utsira Sands. According to Løseth et al. (2012, 2013), this assignment is incorrect and they suggested that the sandy sediments are extrusive sands of possibly Paleocene age. In contrast, Eidvin & Rundberg (2001) and Rundberg & Eidvin (2005, 2015) considered these as Pleistocene turbiditic sands. Two samples (1180 and 1150 m) were studied below the sandy sediments (1034–1115 m), where three samples were investigated. Also, one sample (980 m) of the overlying Nordland Group was investigated. It is not possible to judge whether this sand is, in fact, extrusive sand (Løseth et al., 2012, 2013) or the result of turbiditic sedimentation (Eidvin & Rundberg, 2001; Rundberg & Eidvin, 2015) based on the palynological record. This is mainly due to the occurrence of both unspecified reworked and in situ palynomorphs, and also importantly, the potential of contamination via caving (cutting samples). Extrusion of sand may have occurred in pulses, with intermittent more quiet periods (Løseth et al., 2012). In such a scenario, both reworked (extruded) and in situ (Pleistocene) palynomorphs would be mixed and incorporated in the sediment. However, the palynomorph assemblage may equally reflect a Pleistocene palynological signature, where onshore The assemblages are dominated by Filisphaera filifera, Operculodinium centrocarpum sensu Wall & Dale (1966) and Spiniferites/Achomosphaera spp. indet., comparable to the assemblages in the other wells. A major difference with other wells is that all samples are characterised by considerable reworking. Reworking is substantial in sample 1080 m, where several Miocene species are recorded, amongst others Cleistosphaeridium placacanthum, Cordosphaeridium minimum and Hystrichosphaera obscura. S. De Schepper & G. Mangerud 320 320 The Northern Utsira Member could be Early Pleistocene in age although dating is speculative and can be questioned (Figs. 5 & 6B). The palynology of well 30/6–3, which contains the reference section of the Northern Utsira Member between 750 and 680 m, was not conclusive for an Early Pliocene, Late Pliocene and/or Early Pleistocene age. Similarly, the investigated sediments in well 34/7–2 were deposited in the Pliocene and/or Pleistocene, but a more precise age could not be given. or subsea erosion and/or remobilisation of sediments containing pre-Pleistocene palynomorphs contributed to the assemblage. Correlating the wells based on dinoflagellate cysts and dating the Utsira Formation The oldest sediments were deposited in the Late Miocene (≥5.3 Ma) to Early Pliocene (>4.6 Ma), whereas younger sediments were deposited in the Late Pliocene to Early Pleistocene between 3.0 and 1.95 Ma. Three samples from a 1-m interval investigated from well 15/9–A–23 corroborate the Early Pliocene age for the oldest phase of deposition of the Utsira Sands (see also Piasecki et al., 2002). The Southern Utsira Member in well 25/2–10S, which lies at the transition between the southern and northern depocentre (Fig. 1), consists of sediments dated between 3.0 and 4.6 Ma. This implies that deposition took place in between the oldest (> 4.6 Ma) and youngest (<3.0 Ma) depositional phase of the Southern Utsira Member in well 24/12–1. In summary, the Southern Utsira Member contains sediments deposited in the Early Pliocene as well as in the Pleistocene in its type well (24/12–1), and even during the Late Pliocene (25/2–10S; Figs. 5 & 6B). The discrepancy between the ages reported in the literature for the Utsira Formation and our palynological investigation (Figs. 2 & 5) shows the incompatibility of Sr-ages and foraminifer stratigraphy vs. dinoflagellate cyst stratigraphy. Sr-ages show a wide spread within the same sample and/or the same lithological unit, which suggests that these data should be treated with care (for discussion on Sr-ages in the North Sea, see Eidvin et al., 2014b). The foraminifer stratigraphy is based mainly on benthic assemblages, since planktonic foraminifers are scarce in the record. The disadvantage of benthic assemblages is that these may be diachronous over large distances. In contrast, dinoflagellate cysts are a diverse group of planktonic organisms that have a wide distribution. Several late Neogene biostratigraphic events have been calibrated to magnetostratigraphy and/or marine isotope stratigraphy in both nearby (Norwegian Sea) and farther afield regions (Iceland Sea, North Atlantic), with see clear indications of asynchrony between the Norwegian/Iceland seas and the North Atlantic (De Schepper et al., 2015, 2017). Notwithstanding this issue, it can be concluded from the palynostratigraphy that the Utsira Formation was deposited during the latest Miocene to Pleistocene, which is generally younger than previous age estimates (Figs. 4 & 5). Correlating the wells based on dinoflagellate cysts and dating the Utsira Formation NORWEGIAN JOURNAL OF GEOLOGY Age and palaeoenvironment of the Utsira Formation in the northern North Sea based on marine palynology 32 321 NORWEGIAN JOURNAL OF GEOLOGY Age and palaeoenvironment of the Utsira Formation in the northern North Sea based on marine palynology 321 Utsira Utsira Utsira 0 500 1500 1000 Depth (m) 0 500 1500 1000 Depth (m) Utsira NFN Skade NFN Nord- land GP Utsira Nord- land GP Eir Skade NFN <3.0 >1.4 S N 4.6–4.7 Utsira Skade Eir Nord- land GP G UTS Nord- land GP Horda- land GP TS Mb Nord- land GP TS Mb Horda- land GP G UTS Well 15/9-A-23 Well 24/12-1 Well 25/2-10S Well 30/6-3 Well 34/4-6 Well 34/4-7 Well 34/7-2 Nord- land GP Horda- land GP 4.6–4.7 1.95–~2.6 1.4–3.0 or >4.6 >3.0 1.4–1.7 >1.4 2.6–4.6 0 5 Age (Ma) 1.4 Ma 3.0 Ma 4.6 Ma Well 15/9-A-23 Well 24/12-1 Well 25/2-10S Well 30/6-3 Well 34/4-6 Well 34/4-7 Well 34/7-2 1 2 3 4 6 Utsira Utsira Utsira Utsira G UTS Utsira TS Mb G UTS <5.3? <2.6 >3.0 <4.6 <2.6 Eir HIATUS HIATUS ? ? ? ? ? ? ? Correlative event ~4.6 Ma (HO of Reticulatosphaera actinocoronata, Operculodinium tegillatum, Cyst type I of de Vernal and Mudie (1989) Correlative event ~3.0 Ma (HO of Lavradosphaerea crista) Correlative event ~1.4 Ma (HO of Filisphaera filifera) >5.0 <2.6 or 4.6–5.0 Miocene SWC sample Cuttings ? ? ? ? ? A) B) Utsira Formation G UTS ? ? ? Deposition either in Early Pliocene or Early Pleistocene Deposition either in Early Pliocene or Early Pleistocene <4.6? Southern Utsira Mbr No Formal Name (NFN) Nordland Group LEGEND Eir Formation Glauconitic Utsira Mbr Northern Utsira Mbr Utsira Formation Tampen Spur Mbr (Nordland Group) Figure 6. Correlating the wells based on dinoflagellate cysts and dating the Utsira Formation Depths in each well (A) and ages of deposition (B) for the different lithological units based on dinoflagellate cyst and acritarc h h d f h b f l f l h l f h h h h ll h b Utsira 0 500 1500 1000 Depth (m) 0 500 1500 1000 Depth (m) Utsira NFN Skade NFN Nord- land GP Utsira Nord- land GP Eir Skade NFN <3.0 >1.4 S N 4.6–4.7 Utsira Skade Eir Nord- land GP G UTS Nord- land GP Horda- land GP TS Mb Nord- land GP TS Mb Horda- land GP G UTS Well 15/9-A-23 Well 24/12-1 Well 25/2-10S Well 30/6-3 Well 34/4-6 Well 34/4-7 Well 34/7-2 Nord- land GP Horda- land GP 4.6–4.7 1.95–~2.6 1.4–3.0 or >4.6 >3.0 1.4–1.7 >1.4 2.6–4.6 <5.3? <2.6 >3.0 <4.6 <2.6 ? ? ? ? ? ? >5.0 <2.6 or 4.6–5.0 Miocene A) Utsira Formation <4.6? A) Well 25/2-10S Utsira Utsira 0 5 Age (Ma) 1.4 Ma 3.0 Ma 4.6 Ma Well 15/9-A-23 Well 24/12-1 Well 25/2-10S Well 30/6-3 Well 34/4-6 Well 34/4-7 Well 34/7-2 1 2 3 4 6 Utsira Utsira Utsira Utsira G UTS Utsira TS Mb G UTS Eir HIATUS HIATUS ? ? ? ? ? ? B) G UTS ? ? ? Deposition either in Early Pliocene or Early Pleistocene Deposition either in Early Pliocene or Early Pleistocene B) Correlative event ~4.6 Ma (HO of Reticulatosphaera actinocoronata, Operculodinium tegillatum, Cyst type I of de Vernal and Mudie (1989) Correlative event ~3.0 Ma (HO of Lavradosphaerea crista) Correlative event ~1.4 Ma (HO of Filisphaera filifera) SWC sample Cuttings Southern Utsira Mbr No Formal Name (NFN) Nordland Group LEGEND Eir Formation Glauconitic Utsira Mbr Northern Utsira Mbr Utsira Formation Tampen Spur Mbr (Nordland Group) re 6. Depths in each well (A) and ages of deposition (B) for the different lithological units based on dinoflagellate cyst and acritarch Figure 6. Depths in each well (A) and ages of deposition (B) for the different lithological units based on dinoflagellate cyst and acritarch stratigraphy, with indication of three bioevents useful for correlation in the Pliocene of the northern North Sea. The well stratigraphy is based on Eidvin & Rundberg (2007), Eidvin et al. (2013, 2014a). S. De Schepper & G. Correlating the wells based on dinoflagellate cysts and dating the Utsira Formation Mangerud 322 R T LGM MIS 6 MIS8 MIS12 MIS16 MIS22 MIS52 MIS58 Ge2 Ge1 Pia2 Pia1 Za2 Za1 Me2 Me1 R VP1 VP2 VP3 Age estimates for Miocene and Pliocene lithological units around the North Sea, based on dinoflagellate cyst biostratigraphy and sequence stratigraphy Luchtbal Sands Oorderen S. Merksem S. Kruisschans S. W. Belgium NW European sequences Hardenbol et al. (1998) Poederlee Fm Kasterlee Fm E. Belgium Diest Fm Kattendijk Formation Coralline Crag Red Crag St. Erth Beds England HIATUS ? ? Hörgi Fm Iceland Furuvik Fm HIATUS ? Tjörnes Beds DSDP Hole 610A Denmark ODP Hole 642B Ma Period Epoch Age/Stage 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 Neogene Quaternary Miocene Pliocene Pleistocene Hol. Tortonian Messinian Zanclean Piacenzian Gelasian Calabrian 0 0 Tar. Ion. RT7 RT5 RT6 RT4 RT3 RT2 RT1 RT8 I. multiplexum B. pliocenicum M. choanophorum S. armaggedonensis H. obscura Southern Utsira Mbr Glauconitic Utsira Mbr Nordland Group Tampen Spur Eir Formation Maximum age estimate for the lithological units in this study based on dinoflagellate cyst biostratigraphy HIATUS not zoned not zoned not zoned not zoned not zoned Dinoflagellate cyst biozones Polarity northern North Sea Utsira Formation Age Utsira Fm based on literature Middle Miocene to Zancelan HIATUS HIATUS HIATUS Verhoeven et al. (2011) Head (1993, 1996, 1997) De Schepper et al. (2009) HIATUS HIATUS HIATUS HIATUS HIATUS HIATUS HIATUS This study Louwye and De Schepper (2010) Diest Fm Northern Utsira Mbr? Northern Utsira Mbr? ? ? ? Figure 7. Comparison of the Utsira Formation, and its different members, to the late Neogene shallow-water deposits in the North Sea, North Atlantic and Iceland Sea. The NW European sequence-chronostratigraphic interpretation is after Hardenbol et al. (1998). T and R are transgressive and regressive facies cycles. R T LGM MIS 6 MIS8 MIS12 MIS16 MIS22 MIS52 MIS58 Ge2 Ge1 Pia2 Pia1 Za2 Za1 Me2 R Hardenbol et al. (1998) R R Messinian Miocene Figure 7. Comparison of the Utsira Formation, and its different members, to the late Neogene shallow-water deposits in the North Sea, North Atlantic and Iceland Sea. The NW European sequence-chronostratigraphic interpretation is after Hardenbol et al. (1998). T and R are transgressive and regressive facies cycles. Correlating the wells based on dinoflagellate cysts and dating the Utsira Formation Correlating the Utsira Formation with shallow- water Nordic Seas and North Sea deposits but it may also correspond to Late Pliocene deposits in Iceland (Furuvik Formation) and Belgium (Oorderen Sands and Poederlee Formation). The Northern Utsira Member could be Early Pliocene or Pleistocene, thus making correlations to other units difficult. The time of deposition of the Utsira Formation broadly corresponds to that of other shallow-water sediments in the North Sea and Iceland Sea (Fig. 7). The Early Pliocene deposition phase of the Southern Utsira Member was broadly contemporaneous with deposition of the Tjörnes Beds in Iceland, the Coralline Crag in eastern England and the Kattendijk Formation in Belgium. The second, younger depositional phase of the Southern Utsira Member occurs in the Pleistocene, when deposition had ceased in the Belgian part of the North Sea Basin. The St Erth Beds in western England and the Hörgi Formation in Iceland were deposited at around 2.0 Ma (Early Pleistocene). The dating of the Glauconitic Utsira Member and Northern Utsira Member is more uncertain, making correlations with other units more difficult. The Glauconitic Utsira Member could correspond to the Tjörnes Beds, Coralline Crag and Kattendijk Formation, Conclusions The definition of the Utsira Formation is based on identifying the unit boundaries on gamma-ray profiles, which has led to confusion between the Utsira and Skade formations (Isaksen & Tonstad, 1989; Rundberg & Eidvin, 2005; Eidvin et al., 2013). The Utsira Sands were reported in several industry wells in the Tampen area, but problems with injecting drilling muds resulted in incidents at the sea-floor and revealed a poor geological understanding of this unit (Eidvin & Øverland, 2009). The arising problems can be brought back to the loose sense in which this lithological unit has been identified across the Norwegian Shelf and the different facies included in the unit (e.g., Gregersen et al., 1997; Rundberg & Eidvin, 2005). As a consequence, Miocene, Early Pliocene and Pleistocene sediments have been grouped quite commonly within the Utsira Formation. One solution would be to more accurately define the Utsira Formation, with the introduction of different members (Eidvin & Rundberg, 2007; Eidvin et al., 2013, 2014a) being a good first step towards a better understanding of the Utsira Formation. This is crucial since the unit constitutes an important part of the carbon capture storage capacity on the Norwegian Shelf. 2 The ‘loose’ definition of the Utsira Formation. The definition of the Utsira Formation is based on identifying the unit boundaries on gamma-ray profiles, which has led to confusion between the Utsira and Skade formations (Isaksen & Tonstad, 1989; Rundberg & Eidvin, 2005; Eidvin et al., 2013). The Utsira Sands were reported in several industry wells in the Tampen area, but problems with injecting drilling muds resulted in incidents at the sea-floor and revealed a poor geological understanding of this unit (Eidvin & Øverland, 2009). The arising problems can be brought back to the loose sense in which this lithological unit has been identified across the Norwegian Shelf and the different facies included in the unit (e.g., Gregersen et al., 1997; Rundberg & Eidvin, 2005). As a consequence, Miocene, Early Pliocene and Pleistocene sediments have been grouped quite commonly within the Utsira Formation. One solution would be to more accurately define the Utsira Formation, with the introduction of different members (Eidvin & Rundberg, 2007; Eidvin et al., 2013, 2014a) being a good first step towards a better understanding of the Utsira Formation. This is crucial since the unit constitutes an important part of the carbon capture storage capacity on the Norwegian Shelf. Conclusions We have demonstrated that palynology is a valuable tool for detailed dating, palaeoenvironmental interpretation and understanding the depositional environment in the late Neogene of the North Sea. Specifically, we have provided a better understanding of the stratigraphy of sediments belonging to the Utsira Formation and their relationship with other late Neogene sedimentological units. The dinoflagellate cysts in the thickest part of the Utsira Formation (i.e., the southern distribution area) Age and palaeoenvironment of the Utsira Formation in the northern North Sea based on marine palynology 323 NORWEGIAN JOURNAL OF GEOLOGY those regions have revealed strong asynchrony in both the Early and Late Pliocene (De Schepper et al., 2015, 2017), hampering an unequivocal application of bioevents for dating sedimentary units in the North Sea. Therefore, a reference section in the (northern) North Sea is likely to be the only solution to ultimately clarify the stratigraphic position of the Utsira Formation and its different members. Such a site would become vitally important for establishing a North Sea, Atlantic–Nordic Seas, and even Arctic Ocean stratigraphy. are most consistent with a Late Miocene/Early Pliocene and Early Pleistocene age (Fig. 2), considerably younger than earlier age estimates based on foraminifera and Sr-isotope ages (e.g., Eidvin et al., 2014b). are most consistent with a Late Miocene/Early Pliocene and Early Pleistocene age (Fig. 2), considerably younger than earlier age estimates based on foraminifera and Sr-isotope ages (e.g., Eidvin et al., 2014b). The different ages indicate that the Utsira Formation sediments (or its different members) are not part of one depositional cycle. Likely, several depositional phases were interrupted by sea-level lowering as a result of (global) glaciation events during the Pliocene (Fig. 7). A more precise dating of the Utsira Formation is hampered by: 1 The use of cutting samples in this study. Due to the nature of cutting samples, it has not always been clear for each investigated sample whether specimens were caved, reworked or in situ. This has led to questionable age assignments especially in wells where the thickness of Utsira Formation sediments was limited (e.g., well 34/4–6, 34/4–7). As a result, the palynological assemblage of the Northern Utsira and Glauconitic Utsira members contained a mixture of reworked and in situ palynomorphs and dating of these units thus remains tentative. 2 The ‘loose’ definition of the Utsira Formation. Acknowledgements. Samples from wells 24/12–1, 25/2–10S, 30/6–3 and 34/4–6 were kindly provided by the Norwegian Petroleum Directorate and from wells 34/4–7, 34/7–2 and 34/7–4 by Statoil ASA. S. Piasecki (GEUS) kindly provided his palynological slides from well 15/9–A–23. Funding for sample preparations was provided by Statoil ASA. Malcolm J. Jones and Palynological Laboratory Services Ltd, Holyhead (UK) are thanked for the palynological preparations. A special thanks goes to Allex Cullum, Gitte V. Laursen, Bruce Tocher and Linn M. Johansen (Statoil ASA). References Anstey, C.E. 1992: Biostratigraphic and paleoenvironmental interpretation of upper middle Miocene through lower Pleistocene dinoflagellate cyst, acritarch, and other algal palynomorph assemblages from Ocean Drilling Program Leg 105, Site 645, Baffin Bay. MSc thesis, University of Toronto, 271 pp. Bickle, M., Chadwick, A., Huppert, H.E., Hallworth, M. & Lyle, S. 2007: Modelling carbon dioxide accumulation at Sleipner: Implications for underground carbon storage. Earth and Planetary Science Letters 255, 164–176. https://doi.org/10.1016/j.epsl.2006.12.013. Costa, L. & Downie, C. 1979: Cenozoic dinoflagellate cyst stratigraphy of Sites 403 to 406 (Rockall Plateau), IPOD, Leg 48. Deep Sea Drilling Project Initial Reports 48, 513–529. 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A continuously cored reference section in the (northern) North Sea with magneto stratigraphically calibrated biostratigraphy for both organic-walled palynomorphs and calcareous micro fossils is essential to avoid the use of potentially diachronous bioevents. For this study, we had to rely on the dinoflagellate cyst biostratigraphy from the Nordic Seas and eastern Atlantic Ocean drill sites. The dinoflagellate cyst and acritarch events from 3 The absence of a calibrated Neogene North Sea Basin biostratigraphy. A continuously cored reference section in the (northern) North Sea with magneto stratigraphically calibrated biostratigraphy for both organic-walled palynomorphs and calcareous micro fossils is essential to avoid the use of potentially diachronous bioevents. For this study, we had to rely on the dinoflagellate cyst biostratigraphy from the Nordic Seas and eastern Atlantic Ocean drill sites. 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https://openalex.org/W4313572829	https://tst.duit.in.ua/index.php/tst/article/download/338/307	Ukrainian	null	DEVELOPMENT OF THE PRINCIPLES OF BASALT FIBER MODIFICATION WITH THE PURPOSE OF INCREASING THE EFFICIENCY OF ITS APPLICATION IN TRANSPORT CONSTRUCTION	Zbìrnik naukovih pracʹ Deržavnogo unìversitetu ìnfrastrukturi ta tehnologìj. serìâ "Transportnì sistemi ì tehnologìï"	2,022	cc-by	5,153	УДК 666.972.16 УДК 666.972.16 Олександра Дорошенко1 1 Доцент, Кафедра залізничної колії та колійного господарства, Державний університет інфраструктури та технологій, вул. І. Огієнка, 19, м. Київ, 03049, Україна. ORCID: https://orcid.org/0000-0002-6724-0709 *Автор, відповідальний за листування: sane4kador@gmail.com Збірник наукових праць ДУІТ. Серія «Транспортні системи і технології», 2022. Вип. 0 ТЕХНІКА І ТЕХНОЛОГІЇ ТЕХНІКА І ТЕХНОЛОГІЇ DOI:10.32703/2617-9040-2022-40-2 DOI:10.32703/2617-9040-2022-40-2 РОЗРОБКА ПРИНЦИПІВ МОДИФІКАЦІЇ БАЗАЛЬТОВОГО ВОЛОКНА З МЕТОЮ ПІДВИЩЕННЯ ЙОГО ЕФЕКТИВНОСТІ Причиною передчасних пошкоджень покриттів, як правило, були порушення технології будівництва, дефіцит якісних цементів, використання низьких марок бетону, недостатнє повітрявтягування у бетон, надмірна дія сольових розчинів на ранній стадії твердіння бетону [3,4]. 70 роки в Радянському союзі почалося масове будівництво цементобетонних дорожніх покриттів із застосуванням машин із ковзною опалубкою на гусеничному ходу. Однак практика експлуатації таких покриттів у Радянському Союзі виявила низку суттєвих недоліків. З’явилися руйнування в температурних швах, лущення поверхневого шару бетону, відколи кромок плит і кутів, утворення вибоїн. Причиною передчасних пошкоджень покриттів, як правило, були порушення технології будівництва, дефіцит якісних цементів, використання низьких марок бетону, недостатнє повітрявтягування у бетон, надмірна дія сольових розчинів на ранній стадії твердіння бетону [3,4]. На відміну від Радянського Союзу, у багатьох країнах світу паралельно з будівництвом асфальтобетонних покриттів продовжувалося будівництво цементобетонних покриттів. Стабільне транспортно-експлуатаційні характеристики та висока довговічність довели їхню перевагу перед покриттями, побудованими із застосуванням органічних в’яжучих. Цементобетонні покриття мають високу розподільну здатність, незначне зношування при стиранні, високий коефіцієнт зчеплення, що мало залежить від зволоження покриття. Міцністні та деформативні характеристики цементобетону практично не змінюються при зміні температури, вологості та швидкості навантаження. Під час руху транспорту на дорогах з такими покриттями витрачається приблизно на 5…10 % менше палива, ніж на асфальтобетонних. Крім того, для виробництва цементобетонних покриттів є великі сировинні ресурси [5,6]. Сучасні технології будівництва цементобетонних покриттів припускають повну автоматизацію основних процесів з укладання та ущільнення бетонних сумішей, обробки поверхні бетону, влаштування температурних швів та догляду за бетоном. В останні роки спостерігається тенденція створення дорожніх бетонів підвищеної міцності та довговічності. Це досягається шляхом модифікації структури бетону хімічними добавками: пластифікуючими, повітрявтягуючими та газоутворюючими [7,6]. Розроблено нове покоління високоякісних бетонів (High performance concrete), що мають підвищену морозостійкість, ранню міцність (через 24 години не менше 35 МПа) та інші високі будівельно-технічні властивості. Подібні бетони використовують і в нашій країні, в тому числі на об’єктах транспортного будівництва. У промислово розвинених країнах (США, Канаді, Австралії, Великій Британії, Німеччині, Швеції, Норвегії, Іспанії та багатьох інших) розширюється будівництво покриттів автомобільних доріг із жорстких цементобетонних сумішей, що ущільнюються укочуванням [8,9]. Під бетоном, що укочується, мають на увазі жорсткий цементобетон, що транспортується великовантажними автосамоскидами або бетонозмішувачами на ділянку будівництва і ущільнюється котками різних типів. Такі бетони можливо використовувати при спорудженні дамб і підпірних стінок гребель. РОЗРОБКА ПРИНЦИПІВ МОДИФІКАЦІЇ БАЗАЛЬТОВОГО ВОЛОКНА З МЕТОЮ ПІДВИЩЕННЯ ЙОГО ЕФЕКТИВНОСТІ У статті розглядається обґрунтування використання базальтового волокна, як перспективного матеріалу, за допомогою якого можливо отримати новий клас будівельних матеріалів. У статті розглядається обґрунтування використання базальтового волокна, як перспективного матеріалу, за допомогою якого можливо отримати новий клас будівельних У результаті встановлено, що збільшення температури сприяє підвищенню лугостійкості волокна. Втрата маси при витримці в лужному розчині після 28 діб становить близько 5 %, тоді як початкове волокно за цей час втрачає понад 30 % маси волокна. Подальше збільшення температури ізотермічної витримки не ефективно через зниження лугостійкості волокон та економічної недоцільності. Для пояснення процесів, що відбуваються в структурі волокна в процесі його термічної обробки, були отримані рентгенограми і спектри вихідного і термообробленого волокна. 0 У процесі термообробки від 300 до 500 0С відбуваються процеси заліковування дефектів, зміни хімічного складу та структури поверхні волокон в результаті окислення Fe+2 Fе+3 та утворення залізокисневих тетраедрів [FеO4]Na із залученням на поверхню волокон лужних катіонів. Обґрунтовано доцільність термічної обробки базальтового волокна при температурі 500 0С, що приводить до ущільнення структури фібри та сприяють підвищенню її лугостійкості, що підтверджується морфоструктурними особливостями поверхні термообробленого базальтового волокна, витриманого у цементному розчині. Ключові слова: базальтове волокно, транспортне будівництво, термічна обробка, структура фібри, лугостійкості волокон, рентгенограми, спектри. Вступ. Серед характеристик фібробетону зазвичай виділяють його міцність при згинанні та стисканні. У фібробетону значення цього показника перевищує на 10...15% значення звичайного бетону. В конструкціях використовують тільки фібробетон або фібра та стрижнева або дротяна арматура. Вітчизняний та зарубіжний досвід показує, що фібробетон є універсальним матеріалом і знаходить все більше застосування в галузі транспортного будівництва. Аналіз останніх досліджень і постановка проблеми. Протяжність мережі доріг з цементобетонними покриттями у країнах, таких як Німеччина, становлять 31 %, США – 35 %, у Бельгії – 41 %. Середній термін служби цементобетонних покриттів становить понад 20 років. Навіть у тих країнах де асфальтобетонні покриття мають економічні переваги в порівнянні з цементобетонними, з метою вдосконалення та розвитку конкуруючої технології, за рахунок державних субсидій підтримується будівництво цементобетонних покриттів на рівні 10...20 % загального обсягу будівництва автомобільних доріг з капітальними типами покриттів [1,2]. У ірник наукових праць ДУІТ. Серія «Транспортні системи і технології», 2022. Вип. 0 14 ТЕХНІКА І ТЕХНОЛОГІЇ 70 роки в Радянському союзі почалося масове будівництво цементобетонних дорожніх покриттів із застосуванням машин із ковзною опалубкою на гусеничному ходу. Однак практика експлуатації таких покриттів у Радянському Союзі виявила низку суттєвих недоліків. З’явилися руйнування в температурних швах, лущення поверхневого шару бетону, відколи кромок плит і кутів, утворення вибоїн. Збірник наукових праць ДУІТ. Серія «Транспортні системи і технології», 2022. Вип. 0 РОЗРОБКА ПРИНЦИПІВ МОДИФІКАЦІЇ БАЗАЛЬТОВОГО ВОЛОКНА З МЕТОЮ ПІДВИЩЕННЯ ЙОГО ЕФЕКТИВНОСТІ У країнах Європи і Північної Америки бетон у транспортному будівництві використовують при влаштуванні шарів покриттів доріг, призначених для руху важких транспортних засобів (контейнеровози, великовантажних військових автомобілів, машин лісотехнічної промисловості); контейнерних терміналів, автостоянок; під’їзних, сільських, лісових доріг із високими транспортними навантаженнями; другорядних доріг та вулиць, місцевих доріг та автомагістралей, автомобільних під’їздів до аеропортів, злітно-посадкових смуг аеродромів; як шар посилення при реконструкції старих дорожніх покриттів [10]. Аналіз техніко-експлуатаційних показників всіх типів волокон, що використовуються в даний час у дорожньому будівництві, дозволяє говорити про те, що найбільш доцільно як армуючий елемент в цементобетон використовувати базальтове волокно [11,12,13]. р у р у В умовах дефіциту коштів назріла необхідність розробити прийнятну з фінансової та технічної точок зору програму будівництва магістральних доріг із використанням фібробетону у конструктивних шарах дорожнього одягу. Сучасні умови дорожнього будівництва диктують широке використання відходів промисловості – шлаки, горілі сланці відвалів кам’яновугільних шахт, золи, а також практично невичерпні ресурси Збірник наукових праць ДУІТ. Серія «Транспортні системи і технології», 2022. Вип. 15 ТЕХНІКА І ТЕХНОЛОГІЇ – крейдяні поклади та магматичні породи (базальти), які нині мають мале застосування в транспортному будівництві. Враховуючи умови та світовий досвід, доцільно прискорити розробки в галузі технології та розрахунку фібробетону, ширше застосовувати фібробетонні конструкції при проектуванні об’єктів транспортного будівництва, та використовувати як фібру, матеріали оптимальні за вартістю, а також за обсягом покладів в Україні. – крейдяні поклади та магматичні породи (базальти), які нині мають мале застосування в транспортному будівництві. Враховуючи умови та світовий досвід, доцільно прискорити розробки в галузі технології та розрахунку фібробетону, ширше застосовувати фібробетонні конструкції при проектуванні об’єктів транспортного будівництва, та використовувати як фібру, матеріали оптимальні за вартістю, а також за обсягом покладів в Україні. Базальтові волокна належать до сучасних ефективних матеріалів, що відрізняються високими фізико-механічними показниками і можуть використовуватися у великому діапазоні температур. Отримання фібробетону в сучасному будівництві на основі базальтового волокна є перспективним, оскільки вони працюють в агресивних середовищах (стійкість до кислот та лугів), мають гарні експлуатаційні, фізико-механічні характеристики. Всі ці властивості базальтових волокон спричиняють актуальність створення високоефективних будівельних матеріалів та виробів для різних напрямків будівельної галузі. [14] Встановлено [15], що волокно, що знаходиться в розчині протягом гідратації цементу, характеризується недостатньою лугостійкістю. Мета і завдання дослідження. Як зазначалося раніше [15], тривале зберігання фібри у розчині сприяє її розчиненню. У зв’язку з цим актуальна розробка способів модифікації поверхневого шару базальтового волокна, які забезпечать високу корозійну стійкість. р ру у р у Підвищити ефективність використання базальтового волокна у цементобетоні можна шляхом термічної обробки волокна. Збірник наукових праць ДУІТ. Серія «Транспортні системи і технології», 2022. Вип. 0 РОЗРОБКА ПРИНЦИПІВ МОДИФІКАЦІЇ БАЗАЛЬТОВОГО ВОЛОКНА З МЕТОЮ ПІДВИЩЕННЯ ЙОГО ЕФЕКТИВНОСТІ Матеріали та методи дослідження. Для вивчення впливу термообробки на властивості волокон фібру піддавали нагрівання в діапазоні температур від 300 до 700 0С з кроком 100 0С. Ізотермічна витримка становила 30 хв. Охолодження відбувалось за кімнатною температурою в повітряному середовищі (табл. 1). Таблиця 1. Волокно, модифіковане за градієнтом теплової обробки Виробник 3000С 4000С 5000С 6000С 7000С ТОВ Армбуд Колір нормальний Колір нормальний Колір освітлений Зміни в кольорі – більш червоний Зміни в кольорі – більш червоний Час температурної обробки був вибраний дослідним шляхом і склав 30 хв. У процесі проведення досліджень відзначено суттєву зміну кольору волокна залежно від температури обробки (табл. 1). Ймовірним поясненням цього факту є окислення заліза у структурі волокна, внаслідок цього волокно набуло червоного відтінку. Таблиця 1. Волокно, модифіковане за градієнтом теплової обробки Таблиця 1. Волокно, модифіковане за градієнтом теплової обробки Виробник 3000С 4000С 5000С 6000С 7000С ТОВ Армбуд Колір нормальний Колір нормальний Колір освітлений Зміни в кольорі – більш червоний Зміни в кольорі – більш червоний Час температурної обробки був вибраний дослідним шляхом і склав 30 хв. У процесі проведення досліджень відзначено суттєву зміну кольору волокна залежно від температури обробки (табл. 1). Ймовірним поясненням цього факту є окислення заліза у структурі волокна, внаслідок цього волокно набуло червоного відтінку. рник наукових праць ДУІТ. Серія «Транспортні системи і технології», 2022. Вип. 0 16 ТЕХНІКА І ТЕХНОЛОГІЇ Рис. 1. Втрата маси волокна при розчиненні залежно від часу випробування Рис. 2. Втрата маси при розчиненні залежно від температурної обробки після витримування в лужному середовищі протягом 28 діб Рис. 1. Втрата маси волокна при розчиненні залежно від часу випробування Рис. 1. Втрата маси волокна при розчиненні залежно від часу випробування Рис. 2. Втрата маси при розчиненні залежно від температурної обробки після витримування в лужному середовищі протягом 28 діб У результаті встановлено, що збільшення температури з 300 до 500 0С сприяє підвищенню лугостійкості волокна. Втрата маси при витримці в лужному розчині після 28 діб становить близько 5 %, тоді як початкове волокно за цей час втрачає понад 30 % маси волокна. Подальше збільшення температури ізотермічної витримки не ефективно через зниження лугостійкості волокон та економічної недоцільності. При аналізі мікроструктури як початкового, так і термообробленого армуючого компонента особливих змін немає (рис. 3). Це стосується як розміру (діаметра) (рис. 3 а, г), так і поверхні волокон (рис. 3 б, г). Діаметр окремих волокон коливається у широкому діапазоні – від 2 до 30 мкм. Збірник наукових праць ДУІТ. Серія «Транспортні системи і технології», 2022. Вип. 0 Збірник наукових праць ДУІТ. Серія «Транспортні системи і технології», 2022. Вип. 0 РОЗРОБКА ПРИНЦИПІВ МОДИФІКАЦІЇ БАЗАЛЬТОВОГО ВОЛОКНА З МЕТОЮ ПІДВИЩЕННЯ ЙОГО ЕФЕКТИВНОСТІ Поверхня фібри досить гладка, проте в окремих частинах її покривають глобулярні утворення, імовірно, продуктів карбонатизації, розміром до 1 мкм. Однак, якщо поверхня вихідної фібри досить сильно координована (рис. 4, а – в), то після термічної обробки вона покрита глобулярними утвореннями (рис. 4, г– е). На зразках вихідного анізотропного матеріалу є «виїдені» ділянки, поздовжні «борозни» у напрямку витягування волокон, що може говорити про їх внутрішню структуру (рис. 4, б, в). При цьому руйнування відбувається по найбільш слабкій частині – межі розділу, імовірно, окремих ниток волокна з алюмосилікатних ланцюгів. Якщо розглядати ділянку потверджену корозії, вона має чітку межу (рис. 4, б) і глибину вилуговування до 400 нм (рис. 4, в). Однак, реальна величина руйнування більша через корозію всього поверхневого шару невпорядкованого алюмосилікатного скла. На самих волокнах є глобулярна речовина, яку можна віднести як до рентгеноаморфного карбонату кальцію, так і до продуктів реакції лугу з алюмосилікатним матеріалом самої фібри. На волокнах після термічної обробки явних слідів вилуговування не виявлено (рис. 4, г –е). Привертає увагу відсутність поздовжніх «борозен», що є наслідком великої стійкості Збірник наукових праць ДУІТ. Серія «Транспортні системи і технології», 2022. Вип. 17 ТЕХНІКА І ТЕХНОЛОГІЇ модифікованої базальтової мікрофібри до лужного середовища. Загалом можна говорити, що морфологія поверхні волокна, підданого термічній обробці, не зазнає серйозних змін під дією агресивного середовища цементного молока (рис. 3). Низька лугостійкість початкового базальтового волокна може визначатися кількома факторами. В результаті різкого охолодження розплаву при формуванні волокон у них фіксується неоднорідна об'ємна та поверхнева пухка високотемпературна структура. При цьому волокно характеризуються наявністю дефектів поверхні (мікротріщини, мікропори, обриви зв’язків тощо). Усе це разом інтенсифікує процес розчинення силікатного каркаса. Рис. 3. Мікроструктура базальтового волокна: а, б – початкового; в, г – після термічної обробки при 500 0С Рис. 3. Мікроструктура базальтового волокна: р ру ур а, б – початкового; в, г – після термічної обробки при 500 0С а, б – початкового; в, г – після термічної обробки при 500 0С Рис. 4. Вплив лужного середовища модельної системи на мікроструктуру базальтового волокна: а – в початкового; г – e – після термічної обробки при 500 °С Рис. 4. Вплив лужного середовища модельної системи на мікроструктуру базальтового волокна: а – в початкового; г – e – після термічної обробки при 500 °С Збірник наукових праць ДУІТ. Серія «Транспортні системи і технології», 2022. Вип. 0 бірник наукових праць ДУІТ. Серія «Транспортні системи і технології», 2022. Вип. Збірник наукових праць ДУІТ. Серія «Транспортні системи і технології», 2022. Вип. 0 РОЗРОБКА ПРИНЦИПІВ МОДИФІКАЦІЇ БАЗАЛЬТОВОГО ВОЛОКНА З МЕТОЮ ПІДВИЩЕННЯ ЙОГО ЕФЕКТИВНОСТІ 0 18 ТЕХНІКА І ТЕХНОЛОГІЇ У процесі термообробки від 300 до 500 0С відбуваються процеси заліковування дефектів, зміни хімічного складу та структури поверхні волокон в результаті окислення Fe+2 Fе+3 та утворення залізокисневих тетраедрів [FеO4]Na із залученням на поверхню волокон лужних катіонів. Перелічені процеси найбільш активно проходять при 500 0С – температурі близької до температури склування (кордон переходу з твердого в пластичний стан). При більш високих температурах у базальтових волокнах, насамперед їх поверхні, починаються структурні зміни, пов’язані з передкристалізаційними процесами (600 0С) і кристалізацією (700 0С). Структурні перебудови, пов’язані з кристалізацією, супроводжуються утворенням різного роду дефектів, що роблять матеріал більш активним у хімічному відношенні та призводить до прискорення взаємодії з лугом. При цьому втрати маси волокон збільшуються. у Для пояснення процесів, що відбуваються в структурі волокна в процесі його термічної обробки, були отримані рентгенограми (рис. 5) і спектри (рис. 6) вихідного і термообробленого волокна. Для отримання уявлень про мікроструктурний стан початкового та термообробленого базальтового волокна використовувались дані рентгенівської дифракції. Рентгенограми базальтового волокна є типовими дифракційними картинами структурованих аморфних матеріалів з характерним розширеним профілем інтенсивності в області кутів близько 300 (рис. 5). Рентгенограми базальтового волокна є типовими дифракційними картинами структурованих аморфних матеріалів з характерним розширеним профілем інтенсивності в області кутів близько 300 (рис. 5). Рис. 5. Рентгенограма базальтового волокна до (1) і після (2) термічної обробки Рис. 5. Рентгенограма базальтового волокна до (1) і після (2) термічної обробки Характерною особливістю рентгенограм волокна є присутність одного відображення 32,50, причому найбільш інтенсивно це відображення проявляється на рентгенограмі початкового волокна. Виходячи із загальних уявлень про породоутворюючі мінеральні фази базальтів і на основі рентгенометричної діагностики виконаної із застосуванням дифракційної бази даних, це відображення може бути віднесено до Са(Al2Si2О8). Присутність на рентгенограмах відображення цього мінералу можна інтерпретувати як наслідок тонко пластичної форми кристалітів. Ці нанорозмірні мінерали на поверхні волокон відносять до новостворених фаз у процесі витяжки. При термообробці вони зникають з поверхі волокон через структурні перебудови. Порівняння ІЧ-спектрів базальтового волокна до і після термічної модифікації п видозміну профілів смуг поглинання алюмосилікатних груп області 1000... 300 см-1 (рис. Зокрема, термообробка викликає зменшення інтенсивностей смуг поглинання 1019 і 1052 см-1, характерних для валентних коливань зв'язків Si-O-Si (Al) у ланцюжкових аніонових Зокрема, термообробка викликає зменшення інтенсивностей смуг поглинання 1019 і 1052 см-1, характерних для валентних коливань зв'язків Si-O-Si (Al) у ланцюжкових аніонових 19 ТЕХНІКА І ТЕХНОЛОГІЇ угрупованнях (Q2) клинопіроксенів. З огляду на це відбувається зростання інтенсивностей смуг каркасних структурних фрагментів (Q4) у сфері хвильових чисел 1084…1300 див-1. аннях (Q2) клинопіроксенів. REFERENCES 1. Tolmachev S. N. (2013). Razvitie teorii razrusheniya i stojkosti dorozhnyh cementnyh betonov pri dejstvii agressivnyh faktorov [Development of the theory of destruction and resistance of road cement concrete under the action of aggressive factors]. Extended abstract of Doctor’s thesis. Harkov: Ukr. derzh. akad. zaliznichn. tr-ra [in Ukrainian]. 1. Tolmachev S. N. (2013). Razvitie teorii razrusheniya i stojkosti dorozhnyh cementnyh betonov pri dejstvii agressivnyh faktorov [Development of the theory of destruction and resistance of road cement concrete under the action of aggressive factors]. Extended abstract of Doctor’s thesis. Harkov: Ukr. derzh. akad. zaliznichn. tr-ra [in Ukrainian]. 1. Tolmachev S. N. (2013). Razvitie teorii razrusheniya i stojkosti dorozhnyh cementnyh betonov pri dejstvii agressivnyh faktorov [Development of the theory of destruction and resistance of road cement concrete under the action of aggressive factors]. Extended abstract of Doctor’s thesis. Harkov: Ukr. derzh. akad. zaliznichn. tr-ra [in Ukrainian]. 2. Brazhnik A. V. (2015). Monolitnye dorozhnye cementnye betony vysokoj morozostojkosti s organomineralnym kompleksom i fibroj [Monolithic road cement concretes of high frost resistance with organo-mineral complex and fiber]. Extended abstract of candidate’s thesis. Harkov: Ukr. derzh. akad. zaliznichn. tr-ra [in Ukrainian]. 2. Brazhnik A. V. (2015). Monolitnye dorozhnye cementnye betony vysokoj morozostojkosti s organomineralnym kompleksom i fibroj [Monolithic road cement concretes of high frost resistance with organo-mineral complex and fiber]. Extended abstract of candidate’s thesis. Harkov: Ukr. derzh. akad. zaliznichn. tr-ra [in Ukrainian]. f 3. L.Ferrante, J.Tirillò, F.Sarasini, F.Touchard, R.Ecault, M. A.Vidal Urriza, L.Chocinski-Arnault, & D.Mellier. (2015). Behaviour of woven hybrid basalt-carbon/epoxy composites subjected to laser shock wave testing: Preliminary results, Composites Part B. Engineering, 78, 162–173. https://doi.org/10.1016/j.compositesb.2015.03.084. 3. L.Ferrante, J.Tirillò, F.Sarasini, F.Touchard, R.Ecault, M. A.Vidal Urriza, L.Chocinski-Arnault, & D.Mellier. (2015). p g g, , p g j p 4. Milind V. (2015). Mohod Performance of Polypropylene Fibre Reinforced Concrete. IOSR Journal of Mechanical and Civil Engineering (IOSR-JMCE), 12(1), 28–36. https://doi.org/10.9790/1684-12112836. 5. Subagia, I. A., Kim, Y., Tijing, L. D., Kim, C. S., & Shon, H. K. (2014). Effect of stacking sequence on the flexural properties of hybrid composites reinforced with carbon and basalt fibers. Composites Part B: Engineering, 58, 251-258. https://doi.org/10.1016/j.compositesb.2013.10.027. 5. Subagia, I. A., Kim, Y., Tijing, L. D., Kim, C. S., & Shon, H. K. (2014). Effect of stacking sequence on the flexural properties of hybrid composites reinforced with carbon and basalt fibers. Composites Part B: Engineering, 58, 251-258. https://doi.org/10.1016/j.compositesb.2013.10.027. p g j p 6. Dorigato, A., & Pegoretti, A. (2014). ЛІТЕРАТУРА P. 815-833. https://doi.org/10.1177/1528083716674905 p g j p y 13. Overkamp T, Mahltig B, Kyosev Y. Strength of Basalt Fibers Influenced by Thermal and Chemical Tr Journal of Industrial Textiles, 2018. Vol. 47(5). P. 815-833. https://doi.org/10.1177/1528083716674905 14. Sudha. C, Mohan G. S. Behaviour of fibre reinforced concrete using basalt fibre in beam column joint under cyclic loading. ARPN Journal of Engineering and Applied Science, 2019. Vol. 14. P. 1463-1470. 14. Sudha. C, Mohan G. S. Behaviour of fibre reinforced concrete using basalt fibre in beam column joint under cyclic loading. ARPN Journal of Engineering and Applied Science, 2019. Vol. 14. P. 1463-1470. g g g pp 15. Дорошенко О.Ю. Обґрунтування можливості використання базальтового волокна як компонента цементо бетону для транспортного будівництва. Збірник наукових праць УкрДУЗТ Харків, 2021. вип.198. С. 22-29. 15. Дорошенко О.Ю. Обґрунтування можливості використання базальтового волокна як компонента цементо бетону для транспортного будівництва. Збірник наукових праць УкрДУЗТ Харків, 2021. вип.198. С. 22-29. РОЗРОБКА ПРИНЦИПІВ МОДИФІКАЦІЇ БАЗАЛЬТОВОГО ВОЛОКНА З МЕТОЮ ПІДВИЩЕННЯ ЙОГО ЕФЕКТИВНОСТІ З огляду на це відбувається зростання інтенсивностей смуг их структурних фрагментів (Q4) у сфері хвильових чисел 1084…1300 див-1. Рис. 6. Порівняння діаграм нормованих профілів ІЧ - спектрів початкового (а) та термообробленого (б) волокна Рис. 6. Порівняння діаграм нормованих профілів ІЧ - спектрів початкового (а) та термообробленого (б) волокна Зазначені обставини свідчать про збільшення рівня полімеризації, т.к. підвищення концентрації каркасних угруповань (Q4) відбувається за рахунок зменшення кількості ланцюжкових фрагментів (Q2). Завдяки цьому відбувається ущільнення структури базальтового волокна, що, зрештою, позитивно позначається на стійкості фібри до лужного впливу. Такі структурні зрушення стають можливими через перехід Fe+2 Fe+3 в результаті термообробки в окислювальних умовах, що відзначається багатьма дослідниками. Висновки. Обґрунтовано доцільність термічної обробки базальтового волокна при температурі 500 0С з подальшим охолодженням у повітряному середовищі при кімнатній температурі для підвищення його лужної стійкості. Запропоновано модель модифікації базальтового волокна при термічній обробці, засновану на переході Fe+2 Fe+3 в результаті термообробки в окислювальних умовах. Зміна координаційного оточення, що відбувається при цьому, ініціює утворення зв’язків між (Si2O6)-1 та їх трансформацію в каркасний структурний тип з утворенням залізовмісних плагіоклазових фаз. Дані процеси призводять до ущільнення структури фібри та сприяють підвищенню її лугостійкості, що підтверджується морфоструктурними особливостями поверхні термообробленого базальтового волокна, витриманого у цементному розчині. Збірник наукових праць ДУІТ. Серія «Транспортні системи і технології», 2022. Вип. 20 ТЕХНІКА І ТЕХНОЛОГІЇ ЛІТЕРАТУРА 1. Толмачев С. Н. Развитие теории разрушения и стойкости дорожных цементных бетонов при действии агрессивных факторов: автореф. дис. … д-ра техн. наук: 05.23.05 / Укр. держ. акад. залізн.. тр-ту. Харьков, 2013. 40 с. 1. Толмачев С. Н. Развитие теории разрушения и стойкости дорожных цементных бетонов при действии агрессивных факторов: автореф. дис. … д-ра техн. наук: 05.23.05 / Укр. держ. акад. залізн.. тр-ту. Харьков, 2013. 40 с. 2. Бражник А. В. Монолитные дорожные цементные бетоны высокой морозостойкости с органоминеральным комплексом и фиброй: автореф дис.… канд. техн. наук: 05.23.05 /Укр. держ. ун-т залізн. тр-та. Харьков, 2015. 26 с. 2. Бражник А. В. Монолитные дорожные цементные бетоны высокой морозостойкости с органоминеральным комплексом и фиброй: автореф дис.… канд. техн. наук: 05.23.05 /Укр. держ. ун-т залізн. тр-та. Харьков, 2015. 26 с. 3. 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(2021) Obgruntuvannya mozhlivosti vikoristannya bazaltovogo volokna yak komponenta cementobetonu dlya transportnogo budivnictva [Justification of the possibility of using basalt fiber as a component of cement concrete for transport construction]. Harkiv: Zbirniku naukovih prac UkrDUZT Harkiv, 198. 22-29 [in Ukrainian]. 15. Doroshenko, O.Yu. (2021) Obgruntuvannya mozhlivosti vikoristannya bazaltovogo volokna yak komponenta cementobetonu dlya transportnogo budivnictva [Justification of the possibility of using basalt fiber as a component of cement concrete for transport construction]. Harkiv: Zbirniku naukovih prac UkrDUZT Harkiv, 198. 22-29 [in Ukrainian]. Оleksandra Doroshenko Associate Professor, department of railway track and track economy State University of Infrastructure and Technology. St. I. Ohienko, 19, Kyiv, 03049, Ukraine. REFERENCES Flexural and impact behaviour of carbon/basalt fibers hybrid laminates. 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Effect of stacking sequence on the flexural and fracture properties of carbon/basalt/epoxy hybrid composites, Carbon Letters, 15(2), 125–128. https://doi.org/10.5714/CL.2014.15.2.125. p y y p p g 8. Khamees, S. S., Kadhum, M. M., & Nameer, A. A. (2020). Effects of steel fibers geometry on the mechanical properties of SIFCON concrete. Civil Engineering Journal, 6(1), 21-33. http://dx.doi.org/10.28991/cej-2020-03091450. 9 Ganesh A C Sowmiya K & Muthukannan M (2020 June) Investigation on the effect of steel fibers in p p g g , ( ), p g j 9. Ganesh, A. C., Sowmiya, K., & Muthukannan, M. (2020, June). Investigation on the effect of steel fibers in ірник наукових праць ДУІТ. Серія «Транспортні системи і технології», 2022. Вип. 0 21 ТЕХНІКА І ТЕХНОЛОГІЇ geopolymer concrete. In IOP Conference Series: Materials Science and Engineering (Vol. 872, No. 1, p. 012156). IOP Publishing. http://dx.doi.org/10.1088/1757-899X/872/1/012156. g p g 10. Chatiras, N., Georgiopoulos, P., Christopoulos, A., & Kontou, E. (2019). Thermomechanical characterization of basalt fiber reinforced biodegradable polymers. Polymer Composites, 40(11), 4340-4350. https://doi.org/10.1002/pc.25295. 11. Kytinou, V. K., Chalioris, C. E., & G. Karayannis, C. (2020). Analysis of residual flexural stiffness of steel fiber- reinforced concrete beams with steel reinforcement. Materials, 13(12), 2698. https://doi.org/10.3390/ma13122698. 12. Wang, G., Zhang, D., Wan, G., Li, B., & Zhao, G. (2019). Glass fiber reinforced PLA composite with enhanced g p g 10. Chatiras, N., Georgiopoulos, P., Christopoulos, A., & Kontou, E. (2019). Thermomechanical characterization of basalt fiber reinforced biodegradable polymers. Polymer Composites, 40(11), 4340-4350. https://doi.org/10.1002/pc.25295. 11 K ti V K Ch li i C E & G K i C (2020) A l i f id l fl l tiff f t l fib 10. Chatiras, N., Georgiopoulos, P., Christopoulos, A., & Kontou, E. (2019). words:: basalt fiber, transport construction, heat treatment, fiber structure, alkali resistance of -ray patterns, spectra. Збірник наукових праць ДУІТ. Серія «Транспортні системи і технології», 2022. Вип. 0 DEVELOPMENT OF THE PRINCIPLES OF BASALT FIBER MODIFICATION WITH THE PURPOSE OF INCREASING THE EFFICIENCY OF ITS APPLICATION IN TRANSPORT CONSTRUCTION DEVELOPMENT OF THE PRINCIPLES OF BASALT FIBER MODIFICATION WITH THE PURPOSE OF INCREASING THE EFFICIENCY OF ITS APPLICATION IN TRANSPORT CONSTRUCTION The article examines the experimental and theoretical justification of the use of basalt fiber, as a promising material, with the help of which it is possible to obtain a new class of building materials. As a result, it was established that an increase in temperature contributes to an increase in alkali resistance of the fiber. The mass loss during aging in an alkaline solution after 28 days is about 5%, while the initial fiber loses more than 30% of the fiber mass during this time. A further increase in the temperature of isothermal aging is not effective due to a decrease in alkali resistance of the fibers and economic impracticability. To explain the processes occurring in the structure of the fiber during its heat treatment, X-ray images and spectra of the original and heat-treated fiber were obtained. In the process of heat treatment from 300 to 500 0С, the processes of defect healing, changes in the chemical composition and surface structure of the fibers occur as a result of Fe+2 → Fe+3 oxidation and the formation of iron oxide tetrahedra [FeO4]Na with the attraction of alkaline cations to the surface of the fibers. 0 The expediency of thermal treatment of basalt fiber at a temperature of 500 0C is substantiated, which leads to compaction of the fiber structure and contributes to increasing its alkali resistance, which is confirmed by the morphostructural features of the surface of heat-treated basalt fiber aged in cement mortar. Keywords:: basalt fiber, transport construction, heat treatment, fiber structure, alkali resistance of fibers, X-ray patterns, spectra. бірник наукових праць ДУІТ. Серія «Транспортні системи і технології», 2022. Вип. 0 22
https://openalex.org/W2751360441	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0183092&type=printable	English	null	Characteristics of the King-Devick test in the assessment of concussed patients in the subacute and later stages after injury	PloS one	2,017	cc-by	7,557	Editor: Damir Janigro, Cleveland Clinic, UNITED STATES Copyright: © 2017 Subotic et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: Due to ethical restrictions imposed by the St. Michael’s Hospital Research Ethics Committee, the data underlying this study are available to interested, qualified researchers upon request to: Dr. David Mazer, c/o Sharon Freitag, St. Michael’s Hospital, 30 Bond Street, Toronto, Ontario, Canada, M5B 1W8. Arsenije Subotic1, Windsor Kwan-Chun Ting1, Michael D. Cusimano1,2* 1 Division of Neurosurgery, Department of Surgery, Injury Prevention Research Office, St. Michael’s Hospital, Toronto, Ontario, Canada, 2 University of Toronto, Toronto, Ontario, Canada * injuryprevention@smh.ca a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Subotic A, Ting WK-C, Cusimano MD (2017) Characteristics of the King-Devick test in the assessment of concussed patients in the subacute and later stages after injury. PLoS ONE 12(8): e0183092. https://doi.org/10.1371/journal. pone.0183092 Editor: Damir Janigro, Cleveland Clinic, UNITED STATES Editor: Damir Janigro, Cleveland Clinic, UNITED STATES RESEARCH ARTICLE a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Abstract Although the King-Devick (K-D) test has been used frequently in assessing sports related concussion early after injury, its characteristics over time after injury and in patients with pro- longed persistent symptoms are unknown. The purpose of this paper was to: evaluate the ability of the K-D Test to distinguish patients seen early after concussion from those with symptoms persisting more than 3 months compared to controls, assess changes in the K-D test times over time after concussion, and determine the relationship of K-D times to the Stroop Color and Word Test scores. We performed cross-sectional comparisons of patients with recent concussive brain injury (acute group) and those with symptoms persisting more than 3 months to healthy controls on the K-D test, the Sports Concussion Assessment Tool 3 (SCAT3), and the Stroop Color and Word Test. Longitudinal comparisons of the acute group over time within the first month after injury were also made. Post-concussive syn- drome (PCS) patients had significantly higher K-D times compared to controls (p = 0.01), while the acute group did not differ from controls(p = 0.33). K-D times at the second visit for the acute group were similar to those of controls (54.7 vs. 49.6, p = 0.31). While SCAT3 scores improved over time in the acute group, the K-D scores did not change between the first and second visit (55.2 vs. 54.7, p = 0.94). K-D scores correlated significantly with the Stroop scores for all three participant groups. The K-D test is likely useful very early after concussion in conjunction with baseline scores, and while scores in PCS patients remain elevated, they can be confounded by factors such as pre-morbid depression and medication use. High correlations with Stroop scores also suggest that performance on the K-D test can by proxy provide additional insight about cognitive function and predict performance on more cognitively demanding tasks. Characterizing the King-Devick test in the assessment of concussions (SCAT3), is frequently administered to concussed athletes to assess the number and degree of severity of symptoms pertaining to mTBI [1]. Symptoms on the SCAT3 can be counted and given a rating of severity, but these scores share the same limitations of all self-reported scales: they may be difficult to assess in the presence of preexisting symptoms and are open to poten- tial bias as athletes may under or over-report their symptoms. design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. y y Due to such limitations, other tests have been investigated that can help aid clinicians in the screening and diagnosis of those suspected of brain injury. There is increasing interest in the King-Devick test (K-D) as a screening tool for concussion and mTBI. The K-D test requires that participants read a series of three test cards of numbers, which become progressively more difficult to read, as quickly as they can [2]. The total time to complete all three test cards, and the number of errors committed are included in the total score. Studies have shown the K-D test to be a sensitive marker of brain injury by detecting attentional deficits and impaired sac- cadic eye movements, which have been associated with higher (worse) K-D scores among ath- letes [3–6]. The K-D test has become a popular sideline screening tool for concussions in sports, since it is easy to administer and usually takes less than two minutes to complete. Sev- eral studies have used it in hockey league cohorts [7–9], boxing and mixed martial arts (MMA) competitions [10,11], football games [6, 12–15], as well as rugby league competitions [5, 15– 18]. Despite its extensive use immediately following a suspected TBI, the efficacy of the K-D test in tracking symptom resolution longitudinally has been less widely investigated. Tjarks et al. [19] conducted a longitudinal study on concussed patients presenting at a sports clinic, but did not focus on patients affected by non-sports related injuries or those experiencing post-concussive syndrome (PCS) for more than 3 months. Most of the first visits after concus- sion occurred in the 6–10 day interval (40%) and after 30 days (26%), with 6% of the patients coming in the 1–5 day interval [19]. They found that K-D scores improved at each visit over the four-visit study period [19]. In addition, only Silverberg [20] and Benedict et al. [21] have examined the validity of the K-D test in assessing non-sports related TBI, but neither examined whether the K-D test could be used as an indicator of patient recovery over time after the injury. To our knowledge, previous studies have also not explored the option of correlating performance on the K-D test with tests assessing executive functions such as inhibitory control to see if the K-D test can by-proxy provide more understanding into cognitive function after head injuries. The purpose of this study was three-fold. First, we wanted to determine if the K-D test could accurately distinguish between non-injured healthy controls, acutely injured patients, and those with persistent post-concussive syndrome with symptoms lasting more than 3 months (PCS). We hypothesized that higher (worse) K-D test scores would be seen in the acutely injured group and PCS patients compared to healthy controls. Second, we aimed to determine if K-D test scores changed over time from the original injury among the acute group patients and if it correlated to symptom resolution over time. We hypothesized that K-D scores would correlate with self-report symptom scores and show improvement over time as symptoms resolved. Our third aim was to correlate and compare K-D scores with assessments of selective attention and processing speed, namely the Stroop Color and Word Test. Our hypothesis was that scores of the Stroop assessment would negatively correlate with K-D scores. Introduction Rising public concern regarding the occurrence of mild traumatic brain injuries (mTBIs) including concussion in sports has led to the development of tools that can help assist in the screening, diagnosis and follow-up of these injuries. The Sport Concussion Assessment Tool 3 Funding: We would like to thank the Canadian Institutes of Health Research MOP 123371 for the study funding. The funders had no role in the study 1 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0183092 August 31, 2017 Characterizing the King-Devick test in the assessment of concussions members or relatives of participants, as well as staff at SMH in the Emergency Department. Capacity for consent was determined by following the PATIENT Modified Aid to Capacity Evaluation (ACE) Screening Tool utilized by SMH. Participants must have been able to: com- municate, understand their current medical condition, understand the purpose of the research study, understand the option of declining to participate (with no impact on medical care), understand the risks of participating, and make a decision that is not substantially based upon hallucinations, delusions, or cognitive signs of depression. If any of these criteria were not met, we did not proceed to consent. After this, written informed consent was obtained, after which participants were assessed for eligibility according to the inclusion/exclusion criteria, outlined below. Inclusion/Exclusion criteria Acute group patients were defined as patients who had suffered a non-penetrating head injury and exhibited a GCS score of 13–15 at the time of recruitment. PCS patients were defined as those who had sustained a mTBI three or more months prior to their first testing visit, and were still experiencing ongoing symptoms. Inclusion criteria included: being the age of 16 or over, being able to provide informed written consent, and having sufficient fluency in English. Healthy controls were matched to mTBI patients according to age (±2.5years), sex, and years of education (±2.5 years). Controls participants must also not have had a history of prior head injuries. All participants were also asked to complete a screening form to ensure their eligibil- ity. Participants were excluded if they had the following medical conditions: history of multiple sclerosis, prior hydrocephalus, prior brain irradiation, prior stroke, comorbid early dementia, comorbid Parkinson’s disease, comorbid uncontrolled diabetes, comorbid eye disease causing strabismus, comorbid non-affective psychiatric illness, active substance abuse requiring treat- ment, comorbid alcohol related dementia, and comorbid immune-compromised (HIV/AIDS or taking immunosuppressive therapy). In addition, participants were excluded if they were unable to provide consent because of being medically unstable or intoxicated at the time of recruitment. At the time of screening we also asked for current medication use. Participant assessments PCS participants and controls were asked to come in for one visit, and those in the acute injury group were asked to come in for two visits. PCS and healthy controls were asked to come in at their earliest possible convenience. The first visit for acute participants was conducted within 10 days of the injury, while the second was conducted at 2–4 weeks post-injury (PI). The mini- mum time between the first and second visit was seven days. At each visit, participants com- pleted the K-D test, the SCAT3 Symptom Evaluation, and the Stroop Color and Word Test. The descriptions of these tests are outlined below. Methods The study was approved by the Research Ethics Board (REB) at St. Michael’s Hospital (SMH). Patients were recruited from the Emergency Department, Head Injury Clinic, and the inpa- tient Neurosurgery Ward. Healthy controls were recruited through word of mouth to family 2 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0183092 August 31, 2017 Participant characteristics In total, 17 acute, 28 PCS and 18 controls participants completed the K-D test. The demo- graphic data pertaining to each participant group are outlined in Table 1. Acute participants were assessed at a median of 5 days (Interquartile Range, IQR = 3–7) PI for their first visit. The second visit occurred at a median of 24 (IQR = 16–30) days PI. The sin- gle visit for the PCS patients occurred at a median of 366 days (IQR = 164–659) after injury. It was reported that 13/28 (44%) of PCS participants were using medication to relieve headache, anxiety and/or depression due to their injury, compared to 3/17 (18%) of acute participants (Table 1). Of these 12 participants in the PCS group, 3 were using benzodiazepines, 6 were using antidepressants, and 5 were using medications (ibuprofen, acetyl salicylic acid, acet- aminophen) to relieve headaches. SCAT3 symptom evaluation The symptom evaluation is composed of 22 different symptoms, each of which are rated on a scale of 0–6, with 0 indicating absence of symptoms and 6 being most severe [17, 21]. The total score is out of 22, with symptoms being counted towards the total score if they are non-zero values [17, 21]. The symptom severity score is obtained by summing the values of the individ- ual symptom scores, resulting in a maximum score of 132[17, 21]. King-Devick (K-D) test The K-D test is a saccadic eye measurement test that relies on the principle of rapid number naming [1–15]. The test contains a demonstration (practice) card and three test cards of vari- ably spaced single-digit numbers [1–3, 5]. Participants are asked to read out aloud the numbers from left to right as quick as they can without making any errors [1–15]. The time taken for each card as well as the number of errors was recorded and summed to provide the total K-D score [1–15]. The test usually takes less than two minutes to complete [3,5]. 3 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0183092 August 31, 2017 Characterizing the King-Devick test in the assessment of concussions Stroop Color and word test The Stroop Color and Word Test is a neuropsychological test used to measure executive con- trol and selective attention [22–25]. Participants are asked to name the colour of the words presented as fast as they can [22–25]. This becomes more difficult to do if the colour is incon- gruent with the word [23–25]. It is easier to name the word ‘red’ if it is printed in red than if it is green. In the latter situation, this conflict slows responding which leads to the ‘Stroop effect’ [23–25]. The scores on the task reflect how well participants can selectively direct attention to task relevant features while ignoring task-irrelevant features [23–25]. In our study, participants were asked to read the colour of words (blue, red, green) on a sheet of paper as fast as they can. There were 10 rows, each containing 10 words, resulting in a total of 100 words. The time limit was 45 seconds. If participants finished early, they were asked to read again from the begin- ning. The total number of words and number of errors are included in the final score. Statistical analyses Descriptive statistics were used to describe the participants. Differences in K-D Test, SCAT3, and the Stroop Color and Word Test scores between groups and time intervals were compared using one-way ANOVA. Post-hoc analysis using Fisher’s Least Significant Difference was per- formed after one-way ANOVA. Paired t-tests were used to do compare scores between the first and second visit for the acute group. Pearson correlation coefficients were used to calcu- late the correlation between the test scores. All analysis was conducted using Stata 13. Statisti- cal significance was set at α = 0.05. Characterizing the King-Devick test in the assessment of concussions 54.7 ± 15.1s, p = 0.94), and nor were they different from controls (p = 0.31). The total symptom score and symptom severity scores were higher for the acute group and PCS group compared to controls on the first visit (p<0.0001). For the acute group, symptom scores and severity Table 1. Basic demographic characteristics of acute mTBI, PCS, and control participant groups. Acute mtBI PCS Controls N First Visit 17 28 18 Second Visit 17 - - Age Mean ± SD 42.7 ±14.47 46.1 ±12.8 36.9 ±16.6 Sex, (%) Male 47% 18% 44% Female 53% 82% 56% Education Mean ± SD 14.9 ± 3.2 16.4 ± 3.2 15.4 ± 1.8 Mechanism of injury (%) Fall 47 36 - Sports 12 4 - Unintentional contact with an object 29 18 - MVC 6 38 - Intentional Assault 6 4 - Medication use, N (% of total population) Benzodiazepines 0 (0) 3 (11) 0 (0) Antidepressants 2 (12) 6 (21) 2 (11) Headache Relief 1 (6) 5 (18) 1 (6) Initial Glasgow Coma Scale (GCS) in ED, N 13 1 0 0 14 0 0 0 15 16 28 18 History of Concussion, (%) 47 50 - Abbreviations: SD, standard deviation; MVC, motor vehicle collision; ED, emergency department https://doi.org/10.1371/journal.pone.0183092.t001 Table 2. Summary of test scores by group and by visit. Acute mTBI PCS Controls K-D Times, Mean ± SD (s) First Visit 55.2 ± 12.5 64.1 ± 23.4* 49.6 ± 11.2 Second Visit 54.7 ± 15.1 - - Symptom Total Score, Mean ± SD First Visit 13.2±6.4 15.2 ± 6.06 1.4 ± 2.5 Second Visit 7.5 ± 5.9 - - Symptom Severity Score, Mean ± SD First Visit 32.6 ±19.2 44.4 ± 31.3 2.3 ± 4.2 Second Visit 13.4 ±12.3 - - Stroop Color and Word Test, Mean number of words ± SD First Visit 69.4 ± 15.9 72.3 ± 11.6 85.1 ± 12.4 Second Visit 70.5 ± 14.1 - - Post-hoc analysis. F-value = 4.66, P = 0.01. https://doi.org/10.1371/journal.pone.0183092.t002 Characterizing the King-Devick test in the assessment of concussions PLOS ONE \| https://doi.org/10.1371/journal.pone.0183092 August 31, 2017 5 / 13 Table 1. Basic demographic characteristics of acute mTBI, PCS, and control participant groups. K-D and SCAT3 score characteristics For the first visit, K-D mean scores were 55.2 ± 12.5s for acute group, 64.1 ± 23.4s for PCS, and 49.6 ± 11.2s for healthy controls (Table 2). PCS participants took significantly longer than con- trols(p = 0.01), but were not significantly different from the acute group (p = 0.13). K-D scores were not different from the first to second visit in the acute group (55.2 ± 12.5 s versus 4 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0183092 August 31, 2017 Acute mtBI PCS Controls N First Visit 17 28 18 Second Visit 17 - - Age Mean ± SD 42.7 ±14.47 46.1 ±12.8 36.9 ±16.6 Sex, (%) Male 47% 18% 44% Female 53% 82% 56% Education Mean ± SD 14.9 ± 3.2 16.4 ± 3.2 15.4 ± 1.8 Mechanism of injury (%) Fall 47 36 - Sports 12 4 - Unintentional contact with an object 29 18 - MVC 6 38 - Intentional Assault 6 4 - Medication use, N (% of total population) Benzodiazepines 0 (0) 3 (11) 0 (0) Antidepressants 2 (12) 6 (21) 2 (11) Headache Relief 1 (6) 5 (18) 1 (6) Initial Glasgow Coma Scale (GCS) in ED, N 13 1 0 0 14 0 0 0 15 16 28 18 History of Concussion, (%) 47 50 - Abbreviations: SD, standard deviation; MVC, motor vehicle collision; ED, emergency department 54.7 ± 15.1s, p = 0.94), and nor were they different from controls (p = 0.31). The total symptom score and symptom severity scores were higher for the acute group and PCS group compared to controls on the first visit (p<0.0001). For the acute group, symptom scores and severity 54.7 ± 15.1s, p = 0.94), and nor were they different from controls (p = 0.31). The total symptom score and symptom severity scores were higher for the acute group and PCS group compared to controls on the first visit (p<0.0001). For the acute group, symptom scores and severity Table 2. Summary of test scores by group and by visit. Acute mTBI PCS Controls K-D Times, Mean ± SD (s) First Visit 55.2 ± 12.5 64.1 ± 23.4 49.6 ± 11.2 Second Visit 54.7 ± 15.1 - - Symptom Total Score, Mean ± SD First Visit 13.2±6.4 15.2 ± 6.06 1.4 ± 2.5 Second Visit 7.5 ± 5.9 - - Symptom Severity Score, Mean ± SD First Visit 32.6 ±19.2 44.4 ± 31.3 2.3 ± 4.2 Second Visit 13.4 ±12.3 - - Stroop Color and Word Test, Mean number of words ± SD First Visit 69.4 ± 15.9 72.3 ± 11.6 85.1 ± 12.4 Second Visit 70.5 ± 14.1 - - *Post-hoc analysis. F-value = 4.66, P = 0.01. https://doi.org/10.1371/journal.pone.0183092.t002 PLOS ONE \| htt //d i /10 1371/j l 0183092 A t 31 2017 5 / 13 5 / 13 Characterizing the King-Devick test in the assessment of concussions Fig 1. Characterizing the King-Devick test in the assessment of concussions Table 3. Correlations of K-D test scores with other assessment scores. Symptom Total Score Symptom Severity Score Stroop Color and Word Test Acute: 1st Visit r -0.53 -0.43 -0.89 p 0.03 0.08 <0.0001 Acute: 2nd Visit r 0.05 0.15 -0.78 p 0.86 0.58 0.0002 PCS r 0.06 0.07 -0.75 p 0.76 0.71 <0.0001 Healthy Control r 0.30 0.22 -0.60 p 0.23 0.39 0.001 https://doi org/10 1371/journal pone 0183092 t003 Table 3. Correlations of K-D test scores with other assessment scores. Relationship between K-D scores and number of days between injury and test visit in the acute group. https://doi.org/10.1371/journal.pone.0183092.g001 Fig 1. Relationship between K-D scores and number of days between injury and test visit in the acute group. https://doi.org/10.1371/journal.pone.0183092.g001 https://doi.org/10.1371/journal.pone.0183092.g001 https://doi.org/10.1371/journal.pone.0183092.g001 scores improved significantly from the first visit to the second (13.2 ± 6.4 vs. 7.5 ± 5.9, p = 0.01) (32.6 ± 19.2 vs. 13.4 ± 12.3, p = 0.002) (Table 2). We also performed an analysis of K-D values over time to show the pattern of scores in the acute group. A scatterplot (Fig 1) showing K-D scores and the time between date of injury and test visits showed no significant correlation or relationship. In addition, we stratified the time between the date of injury and the first/second visit into four time intervals (Fig 2) to more precisely characterize K-D performance as a function of time. Again, there were no significant differences between each time interval in terms of K-D scores. Fig 2. Average K-D score for each time interval after injury in the acute group. Abbreviations: SD, standard deviation. Symbols: > =, greater than or equal to. https://doi.org/10.1371/journal.pone.0183092.g002 Fig 2. Average K-D score for each time interval after injury in the acute group. Abbreviations: SD, standard deviation. Symbols: > =, greater than or equal to. https://doi.org/10.1371/journal.pone.0183092.g002 6 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0183092 August 31, 2017 K-D and Stroop Color and word test characteristics For the Stroop Color and Word Test, the acute group at the first visit got fewer words correct than the controls (69.4 ± 15.9 versus 85.1 ± 12.4, p<0.0001, Table 2). Similarly, at the second visit, the acute group differed significantly from controls (p = 0.003), but did not exhibit an improvement from the first visit (p = 0.82). The PCS group also scored significantly lower than controls (p = 0.002). A strong, inverse correlation was found for all three groups (Table 3). PLOS ONE \| https://doi.org/10.1371/journal.pone.0183092 August 31, 2017 Characterizing the King-Devick test in the assessment of concussions between PCS and healthy controls at a median of 54 weeks PI. It is plausible that differences scores on the K-D test might already arise by 140 days PI and remain elevated past the 1 year mark. To our knowledge, no studies have so far administered the K-D test over multiple test visits in a span of multiple months to over a year PI. Future studies should focus on conducting more frequent visits over time, to better elucidate the pattern of performance on saccadic eye movement tests such as the K-D test among those suffering from chronic concussion. In terms of symptom scores, we found higher symptom and symptom severity scores among the PCS group compared to both controls and the acute mTBI, suggesting prolonga- tion of symptom burden in this group. A strong possibility is that symptoms related to PCS might not stem from concussion per se but are also influenced by psychological, personality, and psychosocial factors. Individuals react differently to injuries, and it is plausible that those suffering from PCS may develop a shaken sense of identity as concussion recovery takes longer than usual. This may lead to cognitive issues by suppressing attention, mental efficiency, learn- ing and memory, therefore creating symptoms that are unrelated to those caused by the con- cussion itself [34]. This can in turn lead to frustration and anxiety, leading to avoidance of anxiety provoking situations leading to a buildup of depression that builds over time, resulting in heightened symptom scores [34]. There is also the possibility that symptoms were not spe- cific to PCS, but rather a reaction to the trauma experienced by the head injury [35]. To better elucidate the precise effects of concussion in the development of symptoms in PCS, a control group consisting of patients suffering from non-head injuries would be useful to include in future analyses. In addition, as we also only conducted one study visit, we also cannot say con- clusively what the pattern of symptom scores over time in those with PCS are. There has not been sufficient literature conducted so far to provide enough evidence to characterize when elevated symptom scores compared with those with acute mTBI arise and when they subside. PLOS ONE \| https://doi.org/10.1371/journal.pone.0183092 August 31, 2017 Major findings We found that PCS patients performed significantly worse than controls and moderately worse than our acute group of patients on the K-D test. A number of potential explanations exist for this. Our PCS patients sustained their injuries more frequently in motor vehicle crashes (MVC) than the acute group and since MVC injuries are higher velocity events than those in sports, they are likely also linked with increased concussion severity and the prolonga- tion of symptom burden [26–31]. Those suffering from PCS may have experienced greater damage to cortical and subcortical structures in the prefrontal cortex, thus resulting in ongoing cerebral impairment and slowing of saccades beyond the usual 1-3-month period in which most decrements in cognitive function resolve [26, 32]. In addition, the PCS group was more medicated for conditions like depression or anxiety which can contribute to prolongation of K-D times and post-concussive symptoms. [30, 32]. Reilly [33] found that the usage of benzo- diazepines and other sedatives used to relieve anxiety have been associated with a decrease in saccadic acceleration and velocity and an increase in saccadic latency, or the time interval between two consecutive saccades, which would slow down saccadic eye movements [33]. However, it was also reported that the usage of antidepressants had no significant effect on sac- cadic eye movements [33]. So, it is difficult to be certain whether tests of saccadic eye move- ments can be used as sensitive and objective markers of brain dysfunction following injury, particularly in patients on confounding medications for depression or anxiety [33]. As we only conducted one test session for PCS participants, we were not able to determine at what point in time K-D scores worsened. A study by Heitger [26] showed saccadic eye movements among PCS patients to be significantly worse at 140 days PI compared with those with good recovery. Rizzo [32] also examined performance on the K-D test and found significant differences 7 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0183092 August 31, 2017 Limitations and future directions The interpretation of our study requires a consideration of its limitations. We did not control for the time during the day or the degree of sleep the participant had prior to testing, as they came at their earliest possible convenience. These factors could have produced variations in K-D scores or subjective self-report symptom questionnaires, as previous studies by Fransson [40] and Davies [41] have shown that sleep deprivation can adversely affect eye tracking and attention that is required by the K-D test. Our study design was such that participants could not be assessed immediately following their head injury, as some were admitted into the hospi- tal a couple of hours up until 2 days after their injury. As this was a voluntary study, many patients wanted to be assessed later as they wanted to leave the emergency room of the hospital as soon as their diagnosis was confirmed by a physician. Our mechanisms of injury were var- ied but when we stratified K-D times according to the mechanism of injury, we did not find any significant differences. Because of the confounding effects of medications, future studies, particularly with PCS patients should control for such medications. Future studies with larger sample sizes may wish to focus on performing an in-depth analysis on the effect of injury mechanism and time since injury, while controlling for a number of confounding factors on K-D times. Our PCS group, which was not designed to match non-injured controls, was also biased towards females. Our study is in line with previous literature which has reported that those suf- fering from PCS were more likely to be female, and that there are gender disparities in seeking treatment, which may be reflected in our study [30,34–35]. However, there has not been evi- dence suggesting that performance on visit based tests is influenced by gender, and that females with PCS score worse than males with PCS. Benedict et al. [21] examined the influence of gender and found it to be associated with increased symptom scores, but not performance on the K-D test. Therefore, although there is bias in gender in those who develop and report PCS, it is unlikely that saccadic performance is correlated with it. Characterizing the King-Devick test in the assessment of concussions patients with head injuries have no noticeable test differences from those of matched controls [38, 39]. Silverberg [20] found that the K-D test was not sensitive in differentiating acute mTBI patients and controls at a mean time of 31 hours after injury supporting our thesis that K-D times recover quickly after injury. Our findings therefore do not support the use of the K-D test as a screening test for those with acute mTBI without baseline scores. Our results also showed the novel and robust finding that K-D times were significantly neg- atively correlated with the number of words on the Stroop Color and Word Test among all three participant groups. This meant that injured and non-injured participants that completed the K-D test faster got more words correct in 45 seconds on the Stroop Color and Word Test. To date, the K-D has not been studied for its correlation with assessments like the Stroop Color and Word Test. These findings support the idea that the K-D test can also indirectly indicate the level of interference control and selective attention measured by the Stroop Color and Word Test, providing evidence of convergent validity for the K-D test as it also assesses mental abilities such as selective attention. Future studies should consider utilizing these two tests together as the Stroop Color and Word Task can capture additional deficits in attention due to the fact that more strenuous cognitive processes, such as interference control, are often helpful in uncovering signs of concussion in what might be a seemingly non-concussed indi- vidual [22–24]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0183092 August 31, 2017 Hou [36] did a study in which they found that there was no significant recovery in those with PCS from the 3 to 6-month period PI. McMahon et al. [37] also examined a cohort of individu- als with PCS and found that the number of symptoms increased from the 3 to 6 months and from 3 to 12 months PI. Our findings of heightened and persisting symptom scores at a median of 366 days after injury are therefore in line with some of the literature examining PCS, but more studies, examining more frequent visits and over a longer period of time are needed to better characterize symptom scores. Interestingly, we were not able to show a strong positive correlation between K-D scores and symptom scores. Even more striking is the fact that a statistically significant negative cor- relation was observed on the first test visit for acute participants. This suggests that the report- ing of subjective symptoms does not predict performance on visual based testing scores, and that performance on the K-D test does not predict who experiences greater number of symp- toms following injury. It is plausible that the pathways responsible for generating saccades in the brain are independent of those that contribute to behavioral, emotional, and cognitive states, and that both of these cognitive domains contribute to the screening for signs of concus- sion, albeit separately. Although we saw total symptom and severity scores improve between the first and second assessments in the acute group, we could not show an improvement in K-D times over the two visits. Previous studies have shown that the K-D test done on the sidelines in sporting contexts could detect abnormalities among affected athletes [1, 2–15]. These abnormalities included factors such as blurred vision or attentional deficits, and the K-D scores reflected this through an increase in scores from pre-injury baseline scores. [1, 2–15]. It could thus be that our acute patients had already recovered rapid eye movements by the time we performed the K-D tests (median 5 days post injury) and so they did not change by the second visit (median 24 days post injury). Neuropsychological tests done after the acute recovery period have found that PLOS ONE \| https://doi.org/10.1371/journal.pone.0183092 August 31, 2017 8 / 13 Acknowledgments The authors would like to thank the staff and students at the Injury Prevention Research Office for helping recruit and test participants as well as assisting with data entry. Special thanks to Dr. Rowan Jing and Ling Chen for helping conduct the statistical data analysis. We would also like to thank Dr. Francois Mathieu for providing insightful feedback on the manuscript. Conclusions We found that K-D times inversely correlate highly with the number of correct Stroop words regardless of whether participants had sustained an injury or not suggesting that the K-D test can by-proxy provide insightful information about more complex cerebral functions associ- ated with selective attention, such as response inhibition. We were not able to show any differ- ences in K-D times between acutely injured patients at a median of 5 days post injury and neither did we find times to improve over the subsequent two weeks. In contrast, we showed that PCS patients were slower on the K-D task than controls or acutely injured patients but they were also on multiple medications that might have confounded our results. Our findings do not refute the value of the K-D as a screening test very early after injury. Its use in PCS patients is confounded by factors such as depression frequently seen in patients with PCS. Fur- ther large scale studies of the test will better delineate its characteristics acutely and longitudinally. Characterizing the King-Devick test in the assessment of concussions the option of performing within-group and between-group analyses to better elucidate the effects that gender may play. Although factors such as motivation can play a role in neuropsychological assessments, we did not consider it necessary to have an effort test to assess motivation, as participants who consented were concerned and eager to participate and contribute to the improvement in treatments for concussion. It is difficult to quantify effort and motivation, and it is an inherent characteristic that varies from individual to individual. To our knowledge, no studies have so far been able to elucidate the effects of effort on performance on visual based assessment tests, nor has a reliable tool been developed for this purpose. Nonetheless, future research should aim to develop an effort test to more accurately control for this potentially confounding factor. Additionally, it is important to note that vision plays only one part in the examination of concussions. The K-D test, as a vision based test, should in appropriate cases, be used as a screening tool and as a compliment to a wider number of assessments in the treatment of con- cussion. We did not include physical assessments in our study, and future studies should con- sider using assessments of balance and other tests such as the vestibulo-ocular reflex in the examination of concussions as well in order to more adequately help clinicians screen and diagnose concussion [42]. Larger sample sizes will also allow us to capture acute patients who will develop PCS, to test hypotheses of the sensitivity and specificity of the K-D test in patients with concussion and mTBI over time. Limitations and future directions Due to our small sample size and difference in number of participants between the PCS and other groups, we are not in the position to draw definitive conclusions about gender and performance on the K-D test. Future studies, with larger sample sizes and with more balanced gender distributions, should explore PLOS ONE \| https://doi.org/10.1371/journal.pone.0183092 August 31, 2017 9 / 13 References 1. Galetta KM, Morganroth J, Moehringer N, Mueller B, Hasanaj L, Webb NATC, et al. Adding Vision to Concussion Testing: A Prospective Study of Sideline Testing in Youth and Collegiate Athletes. Journal of Neuro-Ophthalmology 2015 September; 35(3):235–241. https://doi.org/10.1097/WNO. 0000000000000226 PMID: 25742059 2. Oride M, Matrutani J, Rouse M, DeLand P. Reliability study of the Pierce and King–Devick saccade tests. Am J Optom Physiol Opt 1986; 63(6):419–24. PMID: 3728637 3. Galetta KM, Brandes LE, Maki K, Dziemianowicz MS, Laudano E, Allen M, et al. 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Neurology 2011 -04-26; 76(17):1456–1462. https://doi.org/10. 1212/WNL.0b013e31821184c9 PMID: 21288984 11. Leong DF, Balcer LJ, Galetta SL, Liu Z, Master CL. The King-Devick test as a concussion screening tool administered by sports parents. J Sports Med Phys Fitness 2014 Feb; 54(1):70–77. PMID: 24445547 12. Author Contributions Conceptualization: Arsenije Subotic, Windsor Kwan-Chun Ting, Michael D. Cusimano. Data curation: Arsenije Subotic, Windsor Kwan-Chun Ting, Michael D. Cusimano. Formal analysis: Arsenije Subotic, Windsor Kwan-Chun Ting. Funding acquisition: Michael D. Cusimano. Investigation: Arsenije Subotic, Windsor Kwan-Chun Ting, Michael D. Cusimano. Funding acquisition: Michael D. Cusimano. Investigation: Arsenije Subotic, Windsor Kwan-Chun Ting, Michael D. Cusimano. 10 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0183092 August 31, 2017 Characterizing the King-Devick test in the assessment of concussions Methodology: Arsenije Subotic, Windsor Kwan-Chun Ting, Michael D. Cusimano. Project administration: Windsor Kwan-Chun Ting, Michael D. Cusimano. Resources: Michael D. Cusimano. Supervision: Windsor Kwan-Chun Ting, Michael D. Cusimano. Validation: Michael D. Cusimano. Visualization: Arsenije Subotic, Michael D. Cusimano. Writing – original draft: Arsenije Subotic, Windsor Kwan-Chun Ting. Writing – review & editing: Arsenije Subotic, Michael D. Cusimano. Methodology: Arsenije Subotic, Windsor Kwan-Chun Ting, Michael D. Cusimano. Project administration: Windsor Kwan-Chun Ting, Michael D. Cusimano. Methodology: Arsenije Subotic, Windsor Kwan-Chun Ting, Michael D. Cusimano. Methodology: Arsenije Subotic, Windsor Kwan Chun Ting, Michael D. Cusiman Project administration: Windsor Kwan-Chun Ting, Michael D. Cusimano. 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https://openalex.org/W1562199142	https://mdsoar.org/bitstream/11603/11884/1/759.pd.pdf	English	null	A Semantic Approach to Cloud Security and Compliance	null	2,015	public-domain	3,384	A Semantic Approach to Cloud Security and Compliance Amit Hendre and Karuna Pande Joshi CSEE Department, University of Maryland Baltimore County Baltimore, MD, USA {hendre1, karuna.joshi}@umbc.edu} faced by cloud consumers and determined the compliance models and security controls that should be in place to manage the risk. We analyzed more than 20 security standards in cloud computing as well as in IT management. We also reviewed the security controls implemented by more than 100 cloud providers by studying the security related whitepapers on their websites. Second, based on this study, we have developed an ontology describing the cloud security controls, threats and compliances which is used to capture and store this information from standards and cloud providers in W3C standard semantic web languages. It provides us the capability in ongoing work to reason over it. Finally, we have developed a web-based application that can be used by consumer organization. It suggests, given the threats an organization faces, appropriate cloud security policies and providers that support them. This application classifies the threats faced by cloud users and determines the security and compliance policy controls that have to be activated for each threat. The application also displays the existing cloud providers that support the security policies. The focus of this paper is on the first and third contributions. Abstract— Cloud services are becoming an essential part of many organizations. Cloud providers have to adhere to security and privacy policies to ensure their users' data remains confidential and secure. Though there are some ongoing efforts on developing cloud security standards, most cloud providers are implementing a mish- mash of security and privacy controls. This has led to confusion among cloud consumers as to what security measures they should expect from the cloud services, and whether these measures would comply with their security and compliance requirements. We have conducted a comprehensive study to review the potential threats faced by cloud consumers and have determined the compliance models and security controls that should be in place to manage the risk. Based on this study, we have developed an ontology describing the cloud security controls, threats and compliances. We have also developed an application that classifies the security threats faced by cloud users and automatically determines the high level security and compliance policy controls that have to be activated for each threat. The application also displays existing cloud providers that support these security policies. Published in proceedings of IEEE Cloud 2015 conference, New York, June 2015 Published in proceedings of IEEE Cloud 2015 conference, New York, June 2015 A Semantic Approach to Cloud Security and Compliance Cloud consumers can use our system to formulate their security policies and find compliant providers even if they are not familiar with the underlying technology. In section III of this paper, we present our analysis of the various cloud security control models, compliance models and threats. The ontology we have developed for cloud security compliances and security standards is very briefly covered in section IV, and is not a focus of this paper. We describe our recommendation application in detail in section V and end with conclusions and future work. Keywords—Cloud computing, cloud security, Security compliance models, Cloud security models. II. RELATED WORK While cloud based solutions are attractive for their cost savings and rapid provisioning/scaling; privacy and security of cloud data remains a concern for most consumers [8] and a key barrier in adoption of the cloud. In recent years, various cloud security standards have been proposed or are being developed by standards bodies like Cloud Security Alliance (CSA) [9][10], International Organization for Standards (ISO) [14][15], National Institute for Standards and Technology (NIST)[18][19][20], etc. Most cloud providers are implementing a mish-mash of security and privacy controls. This has led to confusion and concern among consumers as to what security measures they should expect from the cloud services and what compliance policies to adopt for their enterprise data on the cloud. Previous studies [6][7][8][33] [34] have attempted to determine cloud security issues. Popović et al.’s [34] study on cloud security controls and standards has been focused primarily at the provider end and concentrated on cloud engineering. Subashini and Kavitha [6] present a survey of the different security risks to the cloud. This study is specific to the security issues due to the cloud service delivery models. Kamongi et. al. [33] have also developed a risk model for the cloud but haven’t tied it with existing compliance standards. How many cloud providers are adapting the cloud security standards in [2], [1] and are capable of handling potential threats remains an open question, and potential source of concerns to consumers who have to select between these providers. NIST’s cloud computing reference architecture [2][11] classifies security and privacy policies under the purview of the This work makes three key contributions. First, we have conducted a comprehensive study to review the potential threats 7. Security risk assessment and management: Cloud providers should implement the authorization and risk assessment for utilizing shared resources. Standards: STIG [13], ISO27002 [14], FedRAMP [5]. cloud provider. On the other hand, the security compliance model is applicable across all the roles in the reference architecture. Security controls used to protect a cloud environment are the same for all cloud delivery models. Compliance standards are applied on these security controls. 8. Privacy, electronic discovery and other legal issues: This focuses on managing the physical location of data and accessing it confidentially. To achieve this security control, documents, terms of services and privacy policies should be reviewed. Compliance model: EDRM- PSRRM [21]. A. Compliance Standards and cloud security controls. 11. Incident response: Cloud providers should develop a response plan in case of any incident like data breaches, data loss etc. Computer forensics has some different tools and techniques for incident response. Compliance standards: NIST 800-61 [30] and ISO 17799 [24]. In this section we discuss the key security controls that affect cloud security. We have referenced the NIST and CSA security documents [20][9][10]. We also co-relate them with compliance standards based on the description of controls. 1. Data encryption, key management: Data encryption and secure key management provides data confidentiality and integrity. Standards: FIPS 140-2 [19], Vaultive [12]. 12. Compliance, audit and accountability: After implementing the required compliances, regular audits should be conducted to ensure data security. Compliance standards: DMTF [25]. 2. Media protection: Media protection includes protection of entertainment content like music, movies and software. Compliance Standards: MPAA [28]. 13. Awareness and training: Cloud awareness and training programs, about threats and security controls, should be conducted for cloud consumers. Compliance standards: NIST 800-61[30] and ISO 17799 [24]. 13. Awareness and training: Cloud awareness and training programs, about threats and security controls, should be conducted for cloud consumers. Compliance standards: NIST 800-61[30] and ISO 17799 [24]. 3. Identification, authentication and authorization: Multi-tenancy requires that consumers share common resources in public domain. Identification of correct resources to authorized users is an important aspect of this security control. The users should be identified by key management and passwords. Cloud providers should also provide access controls to users, so that they can give rights to other authorized users. Compliance models: STIG [13], FedRAMP [5], Oauth and NIST 800-63. NIST classifies access control as a separate control supported by SOX [22] and Safe Harbor [16]. B. Threats to cloud computing and how to protect from threats by using security compliance models II. RELATED WORK The IT compliance model [3] focuses on electronic data processing, network and IT infrastructure. Compliance models implement rules and regulations across various components of IT to make them work harmoniously. Organizations often adopt a security control based on these compliance models. Transparency amongst the cloud service model, security controls and the compliance model will help consumers and end users achieve reliable cloud data protection. p 9. Contingency planning: The consumer should go over the provider’s contingency plans and service level agreements and make sure that provider meets their requirements. Compliance standards: HIPAA [17], NIST 800-34 10. Data center operations, maintenance: Security controls for data centers include configuration and personnel background check to allow entry into secured data center location, physical privacy of data center and authentication. Standards: PCI DSS [29], ISO27002 [14], HIPAA [17], NIST 800-16[26] and NIST 800-53 [18]. We used the Web Ontology Language (OWL) [32] to develop our ontology for Cloud security controls, threats and compliances, which is described briefly in section IV. V. CLOUD SECURITY POLICY RECOMMENDATION SYSTEM We have developed an application that can be used by cloud consumers to determine the cloud security and compliance policies that they want to enforce within their organization. This system helps users identify the cloud threats and the security and compliance models that protect against these threats. The application also lists the existing cloud providers who have implemented the standards in their services. Table 1: Recommendation of Security compliance model based on security threat For this application, we analyzed various security compliances, security policies/standards, and threats affecting cloud security. We next related these controls, standards and threats based on parameters like description of the security standards, the requirements of standard fulfillment, compliance description and also analysis of threats that affects the cloud security. This web-based application has been created by using PHP, HTML and AJAX web technology and MySQL database. Figure 2 shows the database architecture of the application. 6. Malicious Insiders: are people within the organization who can access and misuse the data. Legal action is advised for this type of threat. 7. Abuse of cloud services: Attackers can misuse the multi- tenancy feature of cloud to hack into other organizational data. Cloud providers should protect against consumers accessing other users’ data. 8. Insufficient due diligence: Currently many organizations are adopting cloud for cost savings without being aware of the other threats. Awareness programs should be developed so cloud consumers can understand cloud technologies. Figure 2: Database architecture for the application 9. Shared Technology vulnerabilities: Cloud providers deliver their service in scalable way by sharing the resources. This sharing strategy should be implemented in every domain in cloud computing and also for monitoring the system. B. Threats to cloud computing and how to protect from threats by using security compliance models We analyzed the security threats, identified in [1], [6] and other public documents from standards bodies, to determine the threats faced by cloud consumers. We related them to the security controls and compliance models that protect from these threats (Table 1). The key threats to cloud security include - 1. Data breaches: affect the confidentiality of data and eventually the organization. Data encrypted so that even if it is stolen, the attacker cannot use it. 4. Virtualization and resource abstraction: Virtualization introduces issues like inter virtual machine attacks, hypervisor security etc. Virtual machine setup should include firewall implementation. This security control is only supported by CSA. Compliance standards: DMTF- CADF [25] and PCI-DSS [29]. 2. Data loss: can happen due to hardware failure or malicious attacks on the system. Data backup policies should be implemented to overcome this type of threats. 3. Account or service traffic hijacking: affects the confidentiality and integrity of the users. Hackers can steal users’ personal data like bank credentials. Anti-phishing and fraud detection policies should be implemented to reduce these. 5. Portability and interoperability: The security standards implemented on cloud system should enable information sharing amongst the other system. Compliance standards: DMTF-CADF [25] and OASIS (SAML) [23] 4. Insecure interfaces and APIs: Users and providers communicate through interfaces and APIs. APIs should be able to encrypt the data and transfer through the interfaces. 6. Application security: Application security is overall security of the applications running on the cloud. It includes secured SDLC (software development lifecycle), authentication and authorization. Compliance standards: PCI DSS [29], ISO 27002 [14], SOX [22], HIPAA [17] 5. Denial of service: is to prevent valid users from accessing their data. The attacker can change the encryption key or can slow down the system to prevent users from using the service. To prevent this type of attacks, the users and cloud providers 5. Denial of service: is to prevent valid users from accessing their data. The attacker can change the encryption key or can slow down the system to prevent users from using the service. To prevent this type of attacks, the users and cloud providers should develop a mechanism so that the attackers cannot distinguish the patterns of communications. B. Threats to cloud computing and how to protect from threats by using security compliance models Table 1: Recommendation of Security compliance model based on security threat Type of threat Recommended Security Compliance Model Data breaches STIG [13], FedRAMP [5], DMTF- CADF[25], ISO-27001[15], FIPS 140-2[19], PCI DSS [29], ISO 27002[14], HIPAA[17], SOX[22] Data loss STIG [13], FedRAMP [5], DMTF- CADF [25], FIPS 140-2 [19], PCI DSS [29], ISO 27002[14], HIPAA[17], SOX[22] Account or service hijacking STIG [13], FedRAMP [5], DMTF-OVF [25], ISO- 27002[14], FIPS 140-2[19], NIST 800- 61[30], ISO 17799[24] Insecure interfaces/API OASIS and OVF, DMTF-CADF [25], ISO 27002[14], FIPS 140-2[19] Denial of services PCI DSS, ISO 27001[15], HIPAA[17], SOX[22], NIST 800-61[30], ISO 17799[24] Malicious insiders ISO 27002[14], FIPS 140-2[19], Vaultive[12], FedRAMP[5] Abuse of cloud services NIST 800-61[30], ISO 17799[24], NIST 800- 50 [27] Insufficient due diligence EDRM[21], NIST 800-61[30] Shared technology vulnerabilities ISO 27001[15], FIPS 140-2[19],PCI DSS, ISO 27002[14], HIPAA[17], SOX[22], MPAA [28] should develop a mechanism so that the attackers cannot distinguish the patterns of communications. Table 1: Recommendation of Security compliance model based on security threat Type of threat Recommended Security Compliance Model Data breaches STIG [13], FedRAMP [5], DMTF- CADF[25], ISO-27001[15], FIPS 140-2[19], PCI DSS [29], ISO 27002[14], HIPAA[17], SOX[22] Data loss STIG [13], FedRAMP [5], DMTF- CADF [25], FIPS 140-2 [19], PCI DSS [29], ISO 27002[14], HIPAA[17], SOX[22] Account or service hijacking STIG [13], FedRAMP [5], DMTF-OVF [25], ISO- 27002[14], FIPS 140-2[19], NIST 800- 61[30], ISO 17799[24] Insecure interfaces/API OASIS and OVF, DMTF-CADF [25], ISO 27002[14], FIPS 140-2[19] Denial of services PCI DSS, ISO 27001[15], HIPAA[17], SOX[22], NIST 800-61[30], ISO 17799[24] Malicious insiders ISO 27002[14], FIPS 140-2[19], Vaultive[12], FedRAMP[5] Abuse of cloud services NIST 800-61[30], ISO 17799[24], NIST 800- 50 [27] Insufficient due diligence EDRM[21], NIST 800-61[30] Shared technology vulnerabilities ISO 27001[15], FIPS 140-2[19],PCI DSS, ISO 27002[14], HIPAA[17], SOX[22], MPAA [28] the control elements listed in section III A. Each cloud security standard supports a compliance type. The ontology includes the relation between security standards and cloud security compliances listed in Table 1. The threats and its types, detailed in section III, are captured in our ontology. The ontology helped us determine the database design of our recommendation tool. Figure 1: Ontology describing relationship between Security Controls and security Compliance classes. Figure 1: Ontology describing relationship between Security Controls and security Compliance classes. IV. CLOUD SECURITY AND COMPLIANCE ONTOLOGY We have developed an OWL ontology [31] to capture the concepts of cloud security, threats and compliance controls. In this section we briefly describe this ontology; but it is outside the scope of this paper. The main classes of the ontology are cloud computing security (further divided into cloud security compliance models, cloud security controls and threats to cloud security) and cloud computing providers. Figure 2: Database architecture for the application Using our application, cloud consumers can get a list of all providers who support a compliance standard. If the consumers are not sure of the compliance standard to adhere to, they can also search on multiple security controls. When the user selects a particular security control, the system will display the Figure 1 describes the class cloud security compliances and its relation with security control class. The types of cloud security compliances, explained in section III, are represented in our ontology. The subclasses of the Cloud security control class are recommended security compliance model and then recommended cloud providers in the output. (Figure 3). Figure 3: Compliance and providers recommended based on Security Controls selected [4] SSAE16, The SSAE16 Auditing Standard , http://www.ssae-16.com/ [5] FedRAMP, http://www.gsa.gov/portal/category/102375 [6] S. Subashini, V. Kavitha, A survey on security issues in service delivery models of cloud computing, Journal of Network and Computer Applications, Volume 34, Issue 1, January 2011, Pages 1–11 [7] Ramgovind, S.; Eloff, M.M.; Smith, E., "The management of security in Cloud computing," Information Security for South Africa (ISSA), 2010 , vol., no., pp.1,7, 2-4 Aug. 2010 [8] T. Mather, S.Kumarswamy, S. Latif, Cloud Security and Privacy: An Enterprise Perspective on Risks and Compliance, O'Reilly Media, 2009 [9] CSA, Diana Kelley ,Understanding Cloud Controls Matrix v1.4.xls [10] CSA , Nov 14 2014, CSA security Guidance v3, [11] Mell, P. & Grance, t. (2011) The NIST Definition of Cloud Computing, (Special Publication 800-145). Figure 3: Compliance and providers recommended based on Security Controls selected [12] Vaultive, http://www.vaultive.com/technology/encryption-in-use/ [13] STIG, Application Security and Development STIG, 2014 This application also allows consumers to find security standards for the corresponding security threats. The system also lists the cloud providers that adhere to the standards, which help users to ensure the data security on cloud (Figure 4). Consumers can also get a list of all security standards supported by a cloud provider selected. IV. CLOUD SECURITY AND COMPLIANCE ONTOLOGY This application also allows consumers to find security standards for the corresponding security threats. The system also lists the cloud providers that adhere to the standards, which help users to ensure the data security on cloud (Figure 4). Consumers can also get a list of all security standards supported by a cloud provider selected. [14] Introduction to ISO 27002, http://www.standards.bz/iso-27002.html [15] ISO/IEC 27001, http://www.iso.org/iso/home/standards/management- standards/iso27001.htm [16] SAFE HARBOR, http://export.gov/safeharbor/eu/eg_main_018476.asp [17] HIPAA, may 2003,Summary of HIPAA privacy rules. http://www.hhs.gov/ocr/privacy/hipaa/understanding/summary/privacys ummary.pdf y p Figure 4: Recommendation of security compliance and Cloud providers based on security threat selected. [18] NIST SP 800-53 , Information security, http://csrc.nist.gov/publications/nistpubs/800-53-Rev3/sp800-53-rev3- final_updated-errata_05-01-2010.pdf [19] FIPS 140-2, Jan 2011, security requirement for cryptographic models, http://csrc.nist.gov/publications/fips/fips140-2/fips1402.pdf [20] NIST Recommended Security Controls for Federal Information Systems and Organizations., http://csrc.nist.gov/publications/nistpubs/800-53- Rev3/sp800-53-rev3-final_updated-errata_05-01-2010.pdf [21] EDRM-PSRRM, Privacy and Security risk reduction model, http://www.edrm.net/resources/psrrm [22] SOX , Sarbanes-Oxley Act, http://en.wikipedia.org/wiki/Sarbanes%E2%80%93Oxley_Act [23] Overview of OASIS SAML v2 , https://www.oasis-open.org/ committees/download.php/27819/sstc-saml-tech-overview-2.0-cd-02.pdf Figure 4: Recommendation of security compliance and Cloud providers based on security threat selected. [24] ISO 17799, International Standard ISO/IEC 17799:2000 Code of Practice for Information Security Management http://csrc.nist.gov/publications/secpubs/otherpubs/reviso-faq.pdf REFERENCES [1] Cloud Security Alliance ,2013, The Notorious Nine: Cloud Computing Top Threats in 2013, p8-p21. [2] NIST, NIST Cloud Computing Reference Architecture, 2011 [3] Privacy and data protection,Vol 7 Issue 4, IT compliance and IT security-Part 1, Dr. Jörg Hladjk, p 3-4 [3] Privacy and data protection,Vol 7 Issue 4, IT compliance and IT security-Part 1, Dr. Jörg Hladjk, p 3-4 VI. CONCLUSION AND ONGOING WORK [25] DMTF CADF, June 2012, Cloud Auditing Data Federation http://www.dmtf.org/sites/default/files/standards/documents/DSP2028_1 .0.0a.pdf We have conducted a comprehensive study to review the potential threats faced by cloud consumers and determined the compliance models and security controls that should be in place to manage the risk. We used this study to develop a semantically rich ontology to model the security threats, cloud security policies and controls and express the provider data in it. We have also developed an easy to use cloud security policy recommendation application for consumers who are planning to move their data to the cloud but are hesitant due to security concerns as they may not be aware of the security controls. As part of our ongoing work, we are further analyzing other IT compliance models that may be applicable in the cloud paradigm and determine if they should be incorporated into our cloud security application. We are also developing rules to reason over the ontology to better match compliant providers. [26] NIST 800-16, http://csrc.nist.gov/publications/drafts/800-16- rev1/draft_sp800_16_rev1_2nd-draft.pdf [27] NIST 800-50, for awareness and training , Oct 2003, program, http://csrc.nist.gov/publications/nistpubs/800-50/NIST-SP800-50.pdf [28] MPAA, entertainment content security and protection, http://www.fightfilmtheft.org/docs/2012_Annual_Trending_Report_Fin al.pdf, 2012 [29] PCI-DSS , oct 2010, requirement and security assessment , https://www.pcisecuritystandards.org/documents/pci_dss_v2.pdf [30] NIST 800-61, Computer Security Incident Handling Guide, Aug 2012, http://csrc.nist.gov/publications/nistpubs/800-61rev2/SP800-61rev2.pdf [31] A. Hendre, T. Finin, K.Joshi, Cloud Security and Compliance Ontology, July 2014, http://ebiquity.umbc.edu/resource/html/id/361/Cloud- Security-and-Compliance-Ontology [32] D. McGuinness, F. Van Harmelen, et al., OWL web ontology language overview, W3C recommendation, World Wide Web Consortium, 2004. [33] P Kamongi et. al., Nemesis: Automated Architecture for Threat Modeling and Risk Assessment for Cloud Computing, ASE 2014 [34] Popović, K.; Hocenski, Z., "Cloud computing security issues and challenges," MIPRO, 2010 Proceedings of the 33rd International Convention , vol., no., pp.344,349, 24-28 May 2010
https://openalex.org/W3090514339	http://ojs.sites.ufsc.br/index.php/mixsustentavel/article/download/3806/3206	Portuguese	null	ESTUDO DA ADIÇÃO DO LODO DE ETA - ESTAÇÃO DE TRATAMENTO DE ÁGUA EM ARGAMASSAS DE REVESTIMENTO	Mix Sustentável	2,020	cc-by	8,265	ARTIGOS ARTIGOS PALAVRAS CHAVE: Argamassa de Revestimento, Lodo de Estação de Tratamento de Água, Aproveitamento de Resíduos PALAVRAS CHAVE: Argamassa de Revestimento, Lodo de Estação de Tratamento de Água, Aproveitamento de Resíduos RESUMO No processo de tratamento de água, mais precisamente na lavagem dos filtros e descarga dos decantadores, é pro- duzido um resíduo conhecido como lodo. Este por sua vez, ainda é bastante negligenciado e na maioria dos casos, tem sua disposição final inadequada, sendo depositado em corpos hídricos ou em terrenos baldios, ocasionando a poluição destes locais. Nesta pesquisa, foram realizados testes com o resíduo coletado numa concessionária de abastecimento de água da região sul, caracterizando-os quanto as suas propriedades físico-químicas, e posterior- mente, produzindo argamassas com 0%, 3%, 5% e 10% de adição do lodo da ETA em relação à massa do agregado. Partindo de um planejamento experimental de misturas, verificou-se a influência de traço na resistência à tração na flexão, à compressão axial, na absorção de água por capilaridade e no módulo de elasticidade, após 28 dias de cura. Os resultados indicam que as propriedades mecânicas das argamassas sofrem uma diminuição com a adi- ção do lodo de ETA, em relação à argamassa de referência, com a sua incorporação as argamassas apresentaram menores valores de módulo de elasticidade, o que indica que estas argamassas possuem uma boa capacidade de sofrer deformações, e a adição do lodo de ETA possibilitou a diminuição da absorção de água por capilaridade das argamassas, deixando-as mais resistentes contra agentes deletérios que ocasionam patologias nas mesmas. ABSTRACT In the process of water treatment, more precisely in the washing of the filters and discharge from the decanters, a residue known as sludge is produced. This, in turn, is still largely neglected and in most cases, this sludge has its final inadequate dis- posal being deposited in water bodies or in waste land, causing pollution of these places. In this research, tests were carried out with the waste collected in a water supply concessionaire in the southern region, characterizing its physical and chemical properties, and later, producing mortars with 0%, 3%, 5% and 10% addition of the sludge from the ETA in relation to the aggre- gate mass. Starting from an experimental planning of mixtures, the influence of trace on the tensile strength in flexion, axial compression, water absorption by capillarity and elasticity modulus after 28 days of curing was verified. The results indicate that the mechanical properties of mortars suffer a decrease with the addition of water treatment sludge in relation to the refer- ence mortar, with its incorporation the mortars showed lower values of modulus of elasticity, which indicates that these mor- tars have a good capacity to suffer deformations, and the addition of water treatment sludge enabled the reduction of water absorption by capillarity of mortars, leaving them more resistant against deleterious agents that cause pathologies in them. KEY WORDS: Coating Mortar, Water Treatment Plant Sludge, Waste Utilization http://dx.doi.org/10.29183/2447-3073.MIX2020.v6.n4.19-28 ISSN: 2447-0899 (IMPRESSA) \| 2447-3073 (ONLINE) http://dx.doi.org/10.29183/2447-3073.MIX2020.v6.n4.19-28 ISSN: 2447-0899 (IMPRESSA) \| 2447-3073 (ONLINE) Mix Sustentável \| Florianópolis \| v.6 \| n.4 \| p.19-28 \| ago. \| 2020 Mix Sustentável \| Florianópolis \| v.6 \| n.4 \| p.19-28 \| ago. \| 2020 Estudo da adição do lodo de ETA \| S. S. Laurindo, E. G. P. Antunes, A. E. Savi, J. A. Speck & J. H. Piva https://doi.org/10.29183/2447-3073.MIX2020.v6.n4.19-28 1. INTRODUÇÃO Estes resíduos são gerados nas etapas de decantação e fi ltração (ACHON; BARROSO; CORDEIRO, 2013). Dessa maneira, este trabalho teve como objetivo ana- lisar as características físicas e propriedades mecânicas de argamassas produzidas com adição do lodo da ETA. Para tal, fez-se necessária a caracterização físico-química do lodo da ETA e, posteriormente, a avaliação da sua infl uência nas resistências à tração na fl exão, compressão axial, mó- dulo de elasticidade e absorção de água por capilaridade e coefi ciente de capilaridade das argamassas produzidas. 2. MATERIAIS E MÉTODOS Segundo Teixeira et al. (2006), os lodos gerados em ETA’s são classifi cados como resíduos sólidos devendo ser devidamente tratados e dispostos sem que provoquem da- nos ao meio ambiente. Ao sair da ETA, o lodo contém um grande percentual de umidade e deve, preferencialmente, passar por um processo de desidratação, diminuindo esse percentual e aumentando a concentração de sólidos. As principais etapas do procedimento experimental adota- do neste trabalho encontram-se no fl uxograma da Figura 1. Figura 01 - Fluxograma: Etapas do trabalho. Fonte: Autores, 2018. 20 20 A Lei Federal 11.445 de 5 de janeiro de 2007, estabele- ce as diretrizes nacionais para o saneamento básico. No inciso III do artigo 2º, traz em seus princípios fundamen- tais o abastecimento de água, esgotamento sanitário, lim- peza urbana e manejo dos resíduos sólidos realizados de formas adequadas à saúde pública e à proteção do meio ambiente (BRASIL, 2007). Almeida, Carvalho e Passig (2010) afi rmam que no país, quase todas as ETA’s não possuem qualquer forma de tra- tamento para esses resíduos, na maioria eles são lançados em corpos d´agua, causando efeitos ao meio ambiente e não cumprindo as legislações ambientais. De acordo com Megda, Soares e Achon (2005) em várias partes do mundo, o tratamento e a disposição de lodos de ETA’s vêm sendo tratados como oportunidade de aumento de receita e, principalmente, redução de custos e de impactos ambientais em empresas e siste- mas autônomos de saneamento básico. Uma alternati- va para a disposição do lodo é o seu reaproveitamento, incorporando-o na argamassa de revestimento utilizada na construção civil. Figura 01 - Fluxograma: Etapas do trabalho. Fonte: Autores, 2018. Mix Sustentável \| Florianópolis \| v.6 \| n.4 \| p.19-28 \| ago. \| 2020 1. INTRODUÇÃO aglomerante(s) inorgânico(s) e água, contendo ou não aditivos, com propriedades de aderência e endureci- mento, podendo ser dosada em obra ou em instalação própria (argamassa industrializada). As Estações de Tratamento de Água (ETA) possuem o papel de fornecer água potável a população, de forma que atenda os padrões conforme exigido pela Portaria de Consolidação n° 5/2017 do Ministério da Saúde. Para que ocorra a transfor- mação da água bruta (sem tratamento e imprópria ao con- sumo humano), em uma água potável, as estações de trata- mento comumente adotam o sistema de tratamento de ciclo completo, que inclui os processos de: captação, coagulação, fl oculação, decantação, fi ltração e cloração (COMUSA, 2017). As Estações de Tratamento de Água (ETA) possuem o papel de fornecer água potável a população, de forma que atenda os padrões conforme exigido pela Portaria de Consolidação n° 5/2017 do Ministério da Saúde. Para que ocorra a transfor- mação da água bruta (sem tratamento e imprópria ao con- sumo humano), em uma água potável, as estações de trata- mento comumente adotam o sistema de tratamento de ciclo completo, que inclui os processos de: captação, coagulação, fl oculação, decantação, fi ltração e cloração (COMUSA, 2017). Para a retirada das impurezas contidas na água bruta neste sistema de tratamento são utilizados produtos quí- micos, que fazem a coagulação das impurezas e estas fi cam retidas nos fi ltros ou depositadas em decantadores. Após um período de funcionamento, essas unidades devem ser lavadas para a retirada do acúmulo dessas impurezas, ge- rando resíduos que são o lodo e a água de lavagem de fi l- tros. Estes resíduos são gerados nas etapas de decantação e fi ltração (ACHON; BARROSO; CORDEIRO, 2013). Santos (2008) afi rma que os revestimentos argamas- sados são fundamentais para a durabilidade e preser- vação de uma edifi cação, tendo como função proteger os elementos de vedação da ação direta dos agentes agressivos, regularizar e servir de base para aplicação de outros revestimentos. Para a retirada das impurezas contidas na água bruta neste sistema de tratamento são utilizados produtos quí- micos, que fazem a coagulação das impurezas e estas fi cam retidas nos fi ltros ou depositadas em decantadores. Após um período de funcionamento, essas unidades devem ser lavadas para a retirada do acúmulo dessas impurezas, ge- rando resíduos que são o lodo e a água de lavagem de fi l- tros. 2.2. 1 Lodo de ETA O estudo foi realizado na Estação de Tratamento de Água (ETA) de Criciúma, sul do estado de Santa Catarina, Brasil, que faz captação de 1.000 litros de água por segundo. A ETA é do tipo convencional, com a correção do pH feita através de adição do geocálcio e a coagulação pelo rea- gente policloreto de alumínio (PAC). O lodo foi coletado diretamente da lagoa de decanta- ção na ETA, e transferido para fôrmas metálicas que foram levadas à estufa e mantidas a temperatura de 100º C por 24 h. Assim que as fôrmas foram retiradas da estufa, os sólidos desidratados foram cominuídos com o auxílio de um moinho de martelos. 21 Figura 02 - (a) Lodo na ETA (b) após secagem (c) após cominuição. Fonte: Autores, 2018. Para especifi car a composição química foi adotada a técnica de espectrometria de fl uorescência de Raios X (FRX), utilizando o espectrômetro de raios X por dispersão de comprimento de onda - WDXRF - Axios Max Panalytical. Para especifi car a composição química foi adotada a técnica de espectrometria de fl uorescência de Raios X (FRX), utilizando o espectrômetro de raios X por dispersão de comprimento de onda - WDXRF - Axios Max Panalytical. Para determinar as fases cristalinas realizou-se en- saios em difratômetro de raios X, marca Shimadzu, modelo XRD-6000, goniômetro theta theta, radiação k-α com tubo de cobre de comprimento de onda (λ) de 1,5406 Å. O passo realizado nas análises foi de 2°/min. O range de medida foi de 3 a 80°, com 25 kV de voltagem e 25 mA de corrente elétrica. 2.1. Materiais Utilizou-se cimento do tipo Portland, CP II–Z–32, confor- me a classifi cação da ABNT NBR 11578:1991, com massa específi ca de 2,96 g/cm³. A ABNT NBR 13281:2005 defi ne argamassa como uma mistura homogênea de agregado(s) miúdo(s), A cal hidratada pertence à classe CH III, atendendo aos requisitos da ABNT NBR 7175:2003, e foi utilizada visando Estudo da adição do lodo de ETA \| S. S. Laurindo, E. G. P. Antunes, A. E. Savi, J. A. Speck & J. H. Piva https://doi.org/10.29183/2447-3073.MIX2020.v6.n4.19-28 Estudo da adição do lodo de ETA \| S. S. Laurindo, E. G. P. Antunes, A. E. Savi, J. A. Speck & J. H. Piva https://doi.org/10.29183/2447-3073.MIX2020.v6.n4.19-28 Figura 02 - (a) Lodo na ETA (b) após secagem (c) após cominuição. Fonte: Autores, 2018. (a) (b) (c) a melhoria na trabalhabilidade e retenção de água das argamassas produzidas. Para a produção de alguns traços de argamassa, uti- lizou-se aditivo plastifi cante com intuito de se manter a relação água/aglomerante. O aditivo é composto por resi- nas naturais, trata-se de um líquido escuro, isento de clo- retos e com densidade de 1,01 g/cm³. O agregado miúdo utilizado foi a areia média lavada com módulo de fi nura de 2,37 e diâmetro máximo carac- terístico de 2,4 mm, verifi cados de acordo com a ABNT NBR NM 248:2003. A massa específi ca é de 2,36 g/cm³, com valor determinado conforme a ABNT NBR NM 52:2009. A água empregada em todas as etapas do trabalho foi obtida diretamente da rede de abastecimento de água da concessionária local, e segue as prescrições da ABNT NBR 15900-1:2009. Mix Sustentável \| Florianópolis \| v.6 \| n.4 \| p.19-28 \| ago. \| 2020 2.2. Metodologia A retenção de água foi avaliada segundo a ABNT NBR 13277:2005, que consiste na medida da massa de água reti- da pela argamassa após a sucção, realizada por meio de uma bomba de vácuo a baixa pressão em um funil de filtragem. Para a determinação da densidade de massa, utilizou- -se as orientações da ABNT NBR 13278:2005. O procedi- mento foi realizado para todas as misturas. Denominaram-se as argamassas de acordo com o per- centual de adição, por exemplo, o traço com 3% de adi- ção é chamado de T3 (traço com adição de 3% de lodo em relação a areia) e assim, respectivamente para os demais, conforme apresenta a Tabela 01. Neste artigo, para a análise de variância nos ensaios de resistência a compressão axial, a tração na flexão e mó- dulo de elasticidade foi utilizada a ANOVA, que consiste numa técnica estatística em que três ou mais amostras distintas são comparadas, analisando assim se há uma diferença significativa entre as médias e se os fatores in- fluenciam alguma variável dependente. Por fim, o teste de Tukey avaliou em quais traços havia diferenças esta- tisticamente significativas quando ao teor de lodo de ETA adicionado. Estes testes têm nível de confiança de 95%. E para se constatar que há diferença significativa entre as variáveis, o valor P deve ser inferior a 0,05. Os testes foram executados no software Microsoft Excel. Amostra Traço (Cimento: Cal: Areia: Lodo) % de adição T0 1:0,5:5:0 0 T3 1:0,5:5:0,03 3 T5 1:0,5:5:0,05 5 T10 1:0,5:5:0,10 10 Tabela 01 - Caracterização das argamassas produzidas. Fonte: Autores, 2018. Amostra Traço (Cimento: Cal: Areia: Lodo) % de adição T0 1:0,5:5:0 0 T3 1:0,5:5:0,03 3 T5 1:0,5:5:0,05 5 T10 1:0,5:5:0,10 10 Tabela 01 - Caracterização das argamassas produzidas. Fonte: Autores, 2018. 22 Os ensaios de resistência à compressão e à tração na flexão foram realizados conforme estabelece a ABNT NBR 13279:2005. Moldaram-se três corpos-de-prova prismá- ticos com seção transversal de 4 cm x 4 cm e 16 cm de comprimento para cada tipo de mistura, e respeitou-se o tempo de cura de 28 dias. Para a realização do ensaio de compressão, foram utilizadas as metades dos corpos de prova resultantes do ensaio de tração na flexão, conforme descreve a ABNT NBR 13279:2005. 2.2. Metodologia Para a execução dos ensaios laboratoriais, foram pro- duzidas quatro argamassas de revestimento seguindo os parâmetros estabelecidos pela ABNT NBR 7200:1998. O traço adotado para todas as argamassas foi de 1:0,5:5 (cimento: cal: areia), sendo um deles o de referência e os demais com diferentes teores de adição de lodo de ETA, como agregado. Os teores de adição foram defi nidos com base em pesquisas na área relacionadas a incorporação do lodo da ETA em argamassa de revestimento e con- creto, por Tafarel et al. (2016), Ribeiro (2012) e Hoppen et al. (2005). De acordo com as pesquisas, os teores oscilam entre 4% e 15%, e acima de 10%. Nesse sentido, os resul- tados obtidos não foram satisfatórios, sendo adotados os teores de 3%, 5% e 10% para a pesquisa. Para determinar as fases cristalinas realizou-se en- saios em difratômetro de raios X, marca Shimadzu, modelo XRD-6000, goniômetro theta theta, radiação k-α com tubo de cobre de comprimento de onda (λ) de 1,5406 Å. O passo realizado nas análises foi de 2°/min. O range de medida foi de 3 a 80°, com 25 kV de voltagem e 25 mA de corrente elétrica. A determinação da massa específi ca do lodo foi reali- zada conforme a ABNT NBR NM 52:2009, e para obter o va- lor de massa unitária utilizou-se a ABNT NBR NM 45:2006. Devido a origem do material, foi necessária a determi- nação de impurezas orgânicas, o ensaio foi realizado se- guindo a norma ABNT NBR NM 49:2001. A determinação da massa específi ca do lodo foi reali- zada conforme a ABNT NBR NM 52:2009, e para obter o va- lor de massa unitária utilizou-se a ABNT NBR NM 45:2006. Devido a origem do material, foi necessária a determi- nação de impurezas orgânicas, o ensaio foi realizado se- guindo a norma ABNT NBR NM 49:2001. Além destes quesitos, cabe salientar que a fi m de me- lhorar a composição granulométrica da areia utilizada como agregado nas argamassas produzidas, foi defi nido Com o auxílio de um microscópio metalográfi co foi analisado a microestrutura dos grãos do lodo de ETA. Mix Sustentável \| Florianópolis \| v.6 \| n.4 \| p.19-28 \| ago. \| 2020 Mix Sustentável \| Florianópolis \| v.6 \| n.4 \| p.19-28 \| ago. \| 2020 Estudo da adição do lodo de ETA \| S. S. Laurindo, E. G. P. Antunes, A. E. Savi, J. A. 2.2. Metodologia Speck & J. H. Piva https://doi.org/10.29183/2447-3073.MIX2020.v6.n4.19-28 Adaptações estas relativas aos ciclos de carga e descarga. Foram confeccionados 3 corpos de prova cilíndricos com 5 cm de diâmetro e 10 cm de comprimento, para cada tipo de argamassa produzida, e após os 28 dias de cura executado o ensaio. O equipamento utilizado para este ensaio foi uma prensa hidráulica da marca EMIC PC200CS, juntamente com um extensômetro elétrico. Adaptações estas relativas aos ciclos de carga e descarga. Foram confeccionados 3 corpos de prova cilíndricos com 5 cm de diâmetro e 10 cm de comprimento, para cada tipo de argamassa produzida, e após os 28 dias de cura executado o ensaio. O equipamento utilizado para este ensaio foi uma prensa hidráulica da marca EMIC PC200CS, juntamente com um extensômetro elétrico. uma faixa granulométrica para a incorporação do lodo. Essa utilizou-se do material passante na peneira de malha 2,36 mm e retido na peneira de malha 1,18 mm, pois era a faixa que possuía maior deficiência de material retido na areia utilizada. Buscou-se atingir uma distribuição granu- lométrica que se enquadrasse na zona ótima, estabeleci- da na Tabela 2 da NBR ABNT 7211:2005. O ensaio para determinação da absorção de água por capilaridade e do coeficiente de capilaridade seguiram os procedimentos descritos na ABNT NBR 15259:2005. Foram utilizados três corpos de prova para cada dosagem defini- da. Após a determinação da massa inicial (m0) em gramas, para cada corpo de prova, os mesmos prova foram posi- cionados com sua face quadrada no local do ensaio. O ní- vel de água permaneceu constante a (5 ± 1) mm acima da face em contato com a água, determinando-se a massa, em gramas, de cada corpo de prova aos 10 minutos (m10) e aos 90 minutos (m90). A absorção de água por capilari- dade, calculada para cada tempo, foi expressa em gramas por centímetro quadrado (g/cm²), consistindo na razão entre a variação de massa pela área da seção transversal do corpo de prova em contato com a água. Foi verificado o índice de consistência conforme pres- creve a ABNT NBR 13276:2016, que recomenda consistência de 260 ± 5 mm, e assim obtido a relação água/aglomerante. Mix Sustentável \| Florianópolis \| v.6 \| n.4 \| p.19-28 \| ago. \| 2020 3.1. Caracterização do Resíduo Do ponto de vista químico, o resíduo é constituído em sua maior parte por SiO2 (dióxido de silício), Al2O3 (óxido de alumínio) e Fe2O3 (óxido de ferro) que correspondem à cerca de 66,38%. É possível perceber uma elevada porcen- tagem de Perda ao Fogo (PF), segundo Paschoal (2004 apud MÁS, 2002) a elevada perda ao fogo se caracteriza pela ele- vada quantidade de matéria orgânica presente no material. Amostra Relação água/ aglomerante Aditivo (g) Consistência (mm) T0 0,62 0 263,00 T3 0,62 0 261,00 T5 0,62 0,15 255,00 T10 0,62 0,84 263,00 Tabela 03 - Relação água/aglomerante e índice de consistência. Fonte: Autores, 2018. As características dos resíduos das ETA’s variam de uma estação para outra, pois dependem das característi- cas do manancial, dos produtos químicos utilizados e do tipo de processo de tratamento empregado, mas de for- ma geral os lodos são compostos por substâncias sólidas, orgânicas e inorgânicas, provenientes da água bruta e de coagulantes e floculantes utilizados no tratamento, sendo os coagulantes mais utilizados no Brasil os sais de alumí- nio e ferro (BITTENCOURT et al., 2012). Tabela 03 - Relação água/aglomerante e índice de consistência. Fonte: Autores, 2018. A análise microscópica do grão do lodo de ETA indicou que o mesmo se trata de um material poroso, e segundo Neville e Brooks (2013) o material fino não deve estar pre- sente em grande quantidade, pois devido a sua finura e consequentemente grande área superficial, aumentam a demanda de agua necessária para a molhagem de todas as partículas da mistura. Na Figura 03, encontra-se o resultado do ensaio de im- purezas orgânicas. A análise desse ensaio se faz por meio da comparação entre a coloração da solução após conta- to com o material utilizado e a solução padrão de acordo com a ABNT NBR NM 49:2001. A cor mais escura que a so- lução padrão indica a possibilidade do agregado ser por- tador de compostos orgânicos nocivos. A Tabela 04 apresenta os valores de retenção de água e densidade de massa no estado fresco das argamassas produzidas. Amostra Retenção de Água (%) Densidade (g/cm³) T0 84 2.13 T3 98 2.11 T5 93 2.03 T10 95 1.86 Tabela 04 - Valores de Retenção de Água e Densidade. Fonte: Autores, 2018. 3.1. Caracterização do Resíduo Com a caracterização físico-química do lodo de ETA, foi possível definir os parâmetros para a sua incorporação na argamassa. Realizados os ensaios de massa unitária solta e massa específica, obtiveram-se os valores de 0,82 g/cm³ e 2,59 g/cm³, respectivamente. As características químicas do lodo da ETA são descritas na Tabela 2. O equipamento utilizado no ensaio de compressão para romper os corpos de prova foi a prensa EMIC, mode- lo PC200CS, aplicando uma velocidade de carregamento de (0,25 ± 0,05) MPa/s, e para o ensaio de tração na flexão foi a prensa EMIC, modelo DL10000, com uma velocidade de (0,05 ± 0,02) MPa/s. Elementos Teor (%) Elementos Teor (%) SiO2 31,76 K2O 1,35 Al2O3 24,97 CaO 0,64 O módulo estático de elasticidade à compressão foi realizado conforme prescreve a ABNT NBR 8522:2008, po- rém o ensaio realizado foi adaptado para as argamassas. Mix Sustentável \| Florianópolis \| v.6 \| n.4 \| p.19-28 \| ago. \| 2020 Estudo da adição do lodo de ETA \| S. S. Laurindo, E. G. P. Antunes, A. E. Savi, J. A. Speck & J. H. Piva https://doi.org/10.29183/2447-3073.MIX2020.v6.n4.19-28 Estudo da adição do lodo de ETA \| S. S. Laurindo, E. G. P. Antunes, A. E. Savi, J. A. Speck & J. H. Piva https://doi.org/10.29183/2447-3073.MIX2020.v6.n4.19-28 Estudo da adição do lodo de ETA \| S. S. Laurindo, E. G. P. Antunes, A. E. Savi, J. A. Speck & J. H. Piva https://doi.org/10.29183/2447-3073.MIX2020.v6.n4.19-28 Fe2O3 9,65 Na2O 0,46 TiO2 0,39 SO3 1,49 MnO 0,22 P2O5 0,29 MgO 0,28 PF 28,50 Tabela 02 - Análise química do lodo da ETA. Fonte: Autores, 2018. Fe2O3 9,65 Na2O 0,46 TiO2 0,39 SO3 1,49 MnO 0,22 P2O5 0,29 MgO 0,28 PF 28,50 Tabela 02 - Análise química do lodo da ETA. Fonte: Autores, 2018. A Tabela 03 demostra que o índice de consistência das argamassas produzidas com percentuais 0% e 3% de adição do lodo de ETA mantiveram-se constante, com os percentuais de 5% e 10% foi encontrado dificuldade de atingir a consistência especificada pela norma sem adi- cionar mais água ao traço, porém com o intuito de man- ter a relação água/aglomerante para todas as argamassas iguais, optou-se pelo uso de um aditivo plastificante, e assim atender a consistência sem adicionar mais água. Mix Sustentável \| Florianópolis \| v.6 \| n.4 \| p.19-28 \| ago. \| 2020 3.1. Caracterização do Resíduo Amostra Retenção de Água (%) Densidade (g/cm³) T0 84 2.13 T3 98 2.11 T5 93 2.03 T10 95 1.86 Figura 03 - Determinação de impurezas orgânicas, (a) Solução após contato com o lodo de ETA (b) Solução padrão. Fonte: Autores, 2018. Tabela 04 - Valores de Retenção de Água e Densidade. Fonte: Autores, 2018. Tabela 04 - Valores de Retenção de Água e Densidade. Fonte: Autores, 2018. A retenção de água é uma propriedade que está asso- ciada à capacidade da argamassa fresca manter a sua tra- balhabilidade quando sujeita a solicitações que provocam perda de água de amassamento, seja por evaporação, seja pela absorção de água da base (CARASEK, 2007). A reten- ção permite que as reações de endurecimento da argamas- sa se tornem mais gradativa, promovendo a adequada hi- dratação do cimento e consequente ganho de resistência. Figura 03 - Determinação de impurezas orgânicas, (a) Solução após contato com o lodo de ETA (b) Solução padrão. Fonte: Autores, 2018. 3.2. Propriedades no estado fresco da argamassa O traço que necessitou de menor quantidade de água para atender a consistência especificada pela ABNT NBR 13276:2016 de 260 ± 5 mm, sem aditivo, foi o T0, a partir dele foi fixada a relação água/aglomerante em 0,62. Nota-se que com a incorporação do lodo na arga- massa, os valores de retenção de água aumentaram, em relação à argamassa de referência (T0), utilizando 3% de Mix Sustentável \| Florianópolis \| v.6 \| n.4 \| p.19-28 \| ago. \| 2020 Mix Sustentável \| Florianópolis \| v.6 \| n.4 \| p.19-28 \| ago. \| 2020 Estudo da adição do lodo de ETA \| S. S. Laurindo, E. G. P. Antunes, A. E. Savi, J. A. Speck & J. H. Piva https://doi.org/10.29183/2447-3073.MIX2020.v6.n4.19-28 Estudo da adição do lodo de ETA \| S. S. Laurindo, E. G. P. Antunes, A. E. Savi, J. A. Speck & J. H. Piva https://doi.org/10.29183/2447-3073.MIX2020.v6.n4.19-28 adição do lodo de ETA (T3) a retenção de água aumentou 15%, a T5 resultou em um acréscimo de 10% e a T10 um aumento de 12%. Com a análise estatística de variância (ANOVA) para os valores de resistência a tração por flexão, foi possível comprovar que há uma diferença significativa entre as amostras, obteve-se valor de P= 2,7196 E-5 < 0,05. 3.1. Caracterização do Resíduo Esse é um aspecto positivo da adição do lodo de ETA na argamassa de revestimento, sendo esse aumento pos- sivelmente ocasionado pela porosidade dos grãos do lodo de ETA. No momento da mistura, esses vazios são preenchidos com água, saturando-os e fazendo com que a argamassa diminua sua retenção visto que possui água enclausurada nela. O teste de Tukey apontou que as médias de T3 e T5 não diferem significativamente entre si, isso demonstra que ao adicionar 3% ou 5% de lodo de ETA na argamassa, ela não altera a sua resistência tração na flexão, entre os outros grupos há disparidades significativas. Com base nos valores obtidos pelo ensaio de compressão axial, quando comparada à média das amostras observa-se uma disparidade expressiva entre as amostras. Em relação à argamassa de referência (T0), a T3 diminuiu 25,21%, a T5 sofre uma queda de 28,31% e a T10 um decréscimo de 45,86%. Em relação à densidade de massa, cabe ressaltar que a partir dos valores obtidos, existe uma tendência de quan- to maior a incorporação do lodo de ETA, menor os valores de densidade de massa. Quanto mais leve for a argamassa, mais trabalhável será em longo prazo, o que reduz o esfor- ço do operário na sua aplicação, resultando em um aumen- to de produtividade ao final da jornada de trabalho. Por meio da análise de variância (ANOVA), com os va- lores de resistência à compressão axial foi comprovada que existe uma diferença existente entre os dados das amostras, ao obter-se um valor de P= 1,4478 E-6 <0,05, porém somente com o Teste de Tukey que é possível afir- mar quais as amostras diferem ou não entre si, e por meio dele constatou-se que as amostras T3 e T5 não possuem diferença significativa entre si para resistência à compres- são axial, nos demais grupos há diferença. 3.3. Propriedades no estado endurecido da argamassa 3.3.1 Resistência à tração na flexão e à compressão Através dos resultados obtidos, pode-se observar que a adição do lodo de ETA nas argamassas influencia de for- ma similar nas resistências à tração na flexão e compres- são axial, ocasionando uma redução nas mesmas. Os valores médios de resistência à tração por flexão e à compressão axial estão situados na Tabela 05. 24 As médias deste ensaio para cada percentual de adição do lodo de ETA demonstram que a argamassa sofreu redu- ções de suas propriedades mecânicas conforme o aumen- to do teor adicionado, sofrendo uma queda acentuada em sua resistência à tração por flexão e à compressão axial. A diminuição das propriedades mecânicas da argamas- sa pode estar diretamente ligada com a grande quantida- de de matéria orgânica presente no lodo de ETA. Segundo Neville e Brooks (2013) os agregados naturais podem ser suficientemente fortes e resistentes ao desgaste e mesmo assim, não serem adequados para a produção do material cimentício caso contenham impurezas orgânicas que inter- firam no processo de hidratação do mesmo. Resistência (28 dias) Tração por flexão Compressão Axial Amostras Média ± DP* (MPa) Média ± DP* (MPa) T0 3.45a** ± 0,15 12.72a ± 0,58 T3 2.51b ± 0,25 9.51b ± 0,51 T5 2.41b ± 0,07 9.12b ± 0,07 T10 1.99c ± 0,12 6.89c ± 0,37 Desvio Padrão * médias seguidas pela mesma letra, em uma mesma coluna, não apresentam diferenças significantes, ao nível de significância de 5%, pelo Teste de Tukey. Tabela 05 - Valores de resistência de tração na flexão e à compressão axial. Fonte: Autores, 2018. 3.3.2 Módulo de elasticidade Os resultados obtidos no ensaio de Módulo de Elasticidade das argamassas produzidas encontram-se na Tabela 06. Amostra Módulo de Elasticidade ± DP* (GPa) T0 18,95a** ± 3,28 T3 12,29b ± 1,62 T5 13,32b ± 1,15 T10 12,40b ± 2,24 Desvio Padrão * médias seguidas pela mesma letra, em uma mesma coluna, não apresentam diferenças significantes, ao nível de significância de 5%, pelo Teste de Tukey. Tabela 06 - Valores de Módulo de Elasticidade. Fonte: Autores, 2018. Tabela 05 - Valores de resistência de tração na flexão e à compressão axial. Fonte: Autores, 2018. Ao analisar os resultados obtidos na tração por flexão, entre as amostras com 0% de adição do lodo de ETA (T0) e com 3% (T3), houve um decréscimo de 27,25%, entre a T0 e T5 uma queda de 30,14% e entre T0 e T10 a diminuição foi de 42,31%, sendo a mais significativa entre elas. Mix Sustentável \| Florianópolis \| v.6 \| n.4 \| p.19-28 \| ago. \| 2020 Estudo da adição do lodo de ETA \| S. S. Laurindo, E. G. P. Antunes, A. E. Savi, J. A. Speck & J. H. Piva https://doi.org/10.29183/2447-3073.MIX2020.v6.n4.19-28 Estudo da adição do lodo de ETA \| S. S. Laurindo, E. G. P. Antunes, A. E. Savi, J. A. Speck & J. H. Piva https://doi.org/10.29183/2447-3073.MIX2020.v6.n4.19-28 De acordo com Silva (2014), a propriedade mecânica que quantifica a capacidade de uma argamassa em se deformar sem que ocorra ruptura, é o módulo de elasticidade. Trata- se de uma propriedade mecânica fundamental no estudo da fissuração de revestimentos argamassados, sendo desejável um módulo de elasticidade menor em relação a outros mate- riais cimentícios tradicionais, de forma a minimizar a possibi- lidade de ocorrência de fissuração. Por outro lado, se a arga- massa apresentar um módulo de elasticidade muito inferior ao do suporte, poderá afetar a durabilidade do revestimento. De acordo com Dias e Carasek (2003), a maior incidência de manifestações patológicas nos revestimentos argamas- sados é a umidade, destacando a importância da análise de absorção de água e da permeabilidade para os mesmos. 4. CONCLUSÕES A partir dos resultados obtidos com o estudo da incorpo- ração do lodo de ETA na argamassa de revestimento, foi possível analisar a aplicabilidade desse tipo de argamassa. Em relação à argamassa de referência (T0), a amos- tra T3 teve uma queda de 35,14%, a amostra T5 reduziu 29,71% enquanto a T10 obteve um decréscimo de 34,56%. A adição do lodo de ETA nas argamassas contribuiu com o aumento da sua retenção de água, e com a redução da absorção de água por capilaridade, comparada com a argamassa convencional. Esses resultados demonstram que o lodo de ETA traz benefícios para a argamassa de re- vestimento, tornando-a mais estanque e resistente contra agentes deletérios que possam causar a sua deterioração. Por meio da análise estatística de variância (ANOVA), foi comprovado que existe diferença significativa entre as mé- dias com diferentes percentuais de adição, resultando num valor de P= 0,018 < 0,05. O Teste de Tukey demonstrou que as médias de T3, T5 e T10 não possuem diferença significa- tiva entre si, somente o T0 em relação aos outros grupos. Pode-se observar que as argamassas com menor resis- tência mecânica apresentam menores módulos de elasti- cidade, e consequentemente maior capacidade de absor- ver deformações, sendo um aspecto proveitoso para as argamassas com adição do lodo de ETA. Para os ensaios de tração na flexão, compressão axial e módulo de elasticidade, a adição do lodo da ETA nas ar- gamassas resultou em uma diminuição nas propriedades das mesmas. Porém não foi descartado a sua aplicabilida- de devido à ênfase da pesquisa não ter sido a melhoria das mesmas. Para os mesmos, o teste de ANOVA e teste de Tukey comprovou esta disparidade entre amostras com diferentes teores de adição do lodo de ETA. 25 3.3.2 Módulo de elasticidade Os resultados encontrados demonstraram que com a in- corporação do lodo na argamassa, a absorção de agua por capilaridade e o coeficiente de capilaridade decrescem, re- forçando o que foi constado no ensaio de retenção de água, isso pode ocorrer devido a porosidade dos grãos do lodo de ETA, esses poros já preenchidos com água, diminuem a ab- sorção quando comparados com a argamassa convencional. Ao analisar a Tabela 06, é possível perceber que com a adição do lodo de ETA na argamassa, o módulo de elas- ticidade diminui, quando comparado a argamassa sem adição do lodo de ETA. 3.3.3 Absorção de água por capilaridade e do coeficiente de capilaridade Ao considerar todas as propriedades estudadas pode se verificar que uma porcentagem de adição de 3% e 5% do lodo de ETA contribui satisfatoriamente para a argamassa de revestimento, além de trazer sustentabilidade, destinan- do o resíduo que seria descartado no meio ambiente. A absorção de água capilar é medida pelo coeficiente de capilaridade, que é descrito como a capacidade que a ar- gamassa endurecida tem de absorver água. A determina- ção deste coeficiente define a velocidade com que a água é absorvida pelo material (PASCOA, 2012). Quanto menor a taxa de absorção de água, mais re- sistente será o material frente à entrada de agentes de- letérios do meio ambiente, que podem levar o material (concreto ou argamassa) a sofrer com problemas de dura- bilidade (KIRCHHEIM et al., 2004). REFERÊNCIAS ACHON, Cali Laguna; BARROSO, Marcelo Melo; CORDEIRO, João Sérgio. Resíduos de estações de trata- mento de água e a ISO 24512: desafio do saneamento brasileiro. Eng. Sanit. Ambient., São Paulo, v. 18, n. 2, p.115-122, jun. 2013. Disponível em: http://www.scielo. br/pdf/esa/v18n2/a03v18n2. Acesso em: 24 jun. 2018. ALMEIDA, Ana Paula Verona de; CARVALHO, Karina Querne de; PASSIG, Fernando Hermes. Caracterização Quantitativa do Lodo Gerado na Estação de Tratamento de Água de Campo Mourão PR. Revista Técnico-científica do IFSC, Florianópolis, v. 1, n. 1, O ensaio demonstrou que a amostra T10 apresentou o resultado mais satisfatório, com uma redução no teor de absorção de 52,94% aos 10 minutos e 56,41% aos 90 minu- tos em relação ao traço de referência T0, seguido do traço T5 que obteve uma redução de 35,29% aos 10 minutos e 38,46% aos 90 minutos e a amostra T3 que não sofreu alteração da absorção aos 10 minutos e aos 90 minutos teve uma queda de 7,69%. Mix Sustentável \| Florianópolis \| v.6 \| n.4 \| p.19-28 \| ago. \| 2020 Estudo da adição do lodo de ETA \| S. S. Laurindo, E. G. P. Antunes, A. E. Savi, J. A. Speck & J. H. Piva https://doi.org/10.29183/2447-3073.MIX2020.v6.n4.19-28 p.36-41, 5 jul. 2010. Disponível em: https://periodicos. ifsc.edu.br/index.php/rtc/article/viewFile/206/149. Acesso em: 11 set. 2017. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. NBR 13281: Argamassa para assentamento e revestimento de paredes e tetos – Requisitos. Rio de Janeiro, 2005. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. NBR 13276: Argamassa para assentamento e revestimento de paredes e tetos – Determinação do índice de con- sistência. Rio de Janeiro, 2016. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. NBR 13277: Argamassa para assentamento e revestimento de paredes e tetos - Determinação da retenção de água. Rio de Janeiro, 2005. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. NBR 13278: Argamassa para assentamento e revestimento de paredes e tetos - Determinação da densidade de massa e do teor de ar incorporado. Rio de Janeiro, 2005. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. NBR 13279: Argamassa para assentamento e revestimento de paredes e tetos – Determinação da resistência à tração na flexão e à compressão. Rio de Janeiro, 2005. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. NBR NM 52: Agregado miúdo – Determinação da massa es- pecífica e massa específica aparente. Rio de Janeiro, 2009. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. NBR 11578: Cimento Portland composto – Especificação. Rio de Janeiro, 1991. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. REFERÊNCIAS NBR 7175: Cal hidratada para argamassas – Requisitos. Rio de Janeiro, 2003. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. NBR NM 248: Agregados – Determinação da composição granulométrica. Rio de Janeiro, 2003. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. NBR 15900-1: Água para amassamento do concreto – Parte 1: Requisitos. Rio de Janeiro, 2009. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. NBR 7200: Execução de revestimento de paredes e tetos de argamassas inorgânicas – Procedimento. Rio de água por capilaridade e do coeficiente de capilarida- de. Rio de Janeiro, 2005. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. NBR 7211: Agregados para concreto – Especificação. Rio de Janeiro, 2005. BITTENCOURT, S; SERRAT, B.M; AISSE, M.M, et al; Aplicação de lodos de estações de tratamento de água e de tratamento de esgoto em solo degradado. Engenharia Sanitária e Ambiental. vol.17 no.3 Rio de Janeiro. Julho/Setembro. 2012. Disponível em: http:// www.scielo.br/pdf/esa/v17n3/v17n3a08. Acesso em: 24/06/2018. p.36-41, 5 jul. 2010. Disponível em: https://periodicos. ifsc.edu.br/index.php/rtc/article/viewFile/206/149. Acesso em: 11 set. 2017. água por capilaridade e do coeficiente de capilarida- de. Rio de Janeiro, 2005. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. NBR 7211: Agregados para concreto – Especificação. Rio de Janeiro, 2005. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. NBR 13281: Argamassa para assentamento e revestimento de paredes e tetos – Requisitos. Rio de Janeiro, 2005. BITTENCOURT, S; SERRAT, B.M; AISSE, M.M, et al; Aplicação de lodos de estações de tratamento de água e de tratamento de esgoto em solo degradado. Engenharia Sanitária e Ambiental. vol.17 no.3 Rio de Janeiro. Julho/Setembro. 2012. Disponível em: http:// www.scielo.br/pdf/esa/v17n3/v17n3a08. Acesso em: 24/06/2018. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. NBR 13276: Argamassa para assentamento e revestimento de paredes e tetos – Determinação do índice de con- sistência. Rio de Janeiro, 2016. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. NBR 13277: Argamassa para assentamento e revestimento de paredes e tetos - Determinação da retenção de água. Rio de Janeiro, 2005. BRASIL. Congresso. Câmara dos Deputados. Lei nº 11445, de 5 de janeiro de 2007. Brasília, Disponível em: http://www2.camara.leg.br/legin/fed/lei/2007/ lei-11445-5-janeiro-2007-549031-normaatualizada-pl. pdf. Acesso em: 24 jun. 2018. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. NBR 13278: Argamassa para assentamento e revestimento de paredes e tetos - Determinação da densidade de massa e do teor de ar incorporado. Rio de Janeiro, 2005. CARASEK, H. Argamassas. In: ISAIA, Geraldo Cechella. Materiais de Construção Civil e Princípios de Ciência e Engenharia dos Materiais. São Paulo: Arte Interativa, 2007. Cap. 26, p. 863-904. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. REFERÊNCIAS NBR 13279: Argamassa para assentamento e revestimento de paredes e tetos – Determinação da resistência à tração na flexão e à compressão. Rio de Janeiro, 2005. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. NBR NM 52: Agregado miúdo – Determinação da massa es- pecífica e massa específica aparente. Rio de Janeiro, 2009. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. NBR 13279: Argamassa para assentamento e revestimento de paredes e tetos – Determinação da resistência à tração na flexão e à compressão. Rio de Janeiro, 2005. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. NBR NM 52: Agregado miúdo – Determinação da massa es- pecífica e massa específica aparente. Rio de Janeiro, 2009. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. NBR 13279: Argamassa para assentamento e revestimento de paredes e tetos – Determinação da resistência à tração na flexão e à compressão. Rio de Janeiro, 2005. COMUSA - Serviços de Água e Esgoto de Novo Hamburgo (Rio Grande do Sul). Tratamento de Água. 2017. Disponível em: http://www.comusa.rs.gov.br/in- dex.php/saneamento/tratamentoagua. Acesso em: 15 maio 2018. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. NBR NM 52: Agregado miúdo – Determinação da massa es- pecífica e massa específica aparente. Rio de Janeiro, 2009. 26 26 DIAS, Letícia Almeida; CARASEK, Helena. Avaliação da permeabilidade e da absorção de água de revestimen- tos de argamassa pelo método do cachimbo. V simpó- sio brasileiro de tecnologia. P. 543-555. 2003. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. NBR 11578: Cimento Portland composto – Especificação. Rio de Janeiro, 1991. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. NBR 11578: Cimento Portland composto – Especificação. Rio de Janeiro, 1991. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. NBR 7175: Cal hidratada para argamassas – Requisitos. Rio de Janeiro, 2003. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. NBR 7175: Cal hidratada para argamassas – Requisitos. Rio de Janeiro, 2003. TEIXEIRA, S.R.; de SOUZA, S.A.; de SOUZA, N.R.; ALÉSSIO, P.; SANTOS, G.T.A. Efeito da Adição de Lodo de Estação de Tratamento de Água (ETA) nas Propriedades de Material Cerâmico Estrutural. Cerâmica. on-line., v.52, n.323, pp. 215-220. 2006. Disponível em: http://dx.doi. org/10.1590/S0366-69132006000300016. Acesso em: 03 abr. 2012. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. NBR NM 248: Agregados – Determinação da composição granulométrica. Rio de Janeiro, 2003. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. NBR NM 248: Agregados – Determinação da composição granulométrica. Rio de Janeiro, 2003. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. NBR 15900-1: Água para amassamento do concreto – Parte 1: Requisitos. Rio de Janeiro, 2009. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. REFERÊNCIAS NBR 15900-1: Água para amassamento do concreto – Parte 1: Requisitos. Rio de Janeiro, 2009. HOPPEN, Cinthya et al. Uso de Lodo de Estação de Tratamento de Água Centrifugado em Matriz de Concreto de Cimento Portland para reduzir o Impacto Ambiental. Química Nova, São Paulo, v. 29, n. 1, p.79- 84, 08 set. 2005. Disponível em: http://www.scielo.br/ pdf/qn/v29n1/27861.pdf. Acesso em: 29 maio 2018. SANTOS, Heraldo Barbosa dos. Ensaio de Aderência das Argamassas de Revestimento. 2008. 50 f. Monografia (Especialização) - Curso de Especialização em Construção Civil, Universidade Federal de Minas Gerais, Belo Horizonte, 2008. Disponível em: http://pos. demc.ufmg.br/novocecc/trabalhos/pg1/Monografia HOPPEN, Cinthya et al. Uso de Lodo de Estação de Tratamento de Água Centrifugado em Matriz de Concreto de Cimento Portland para reduzir o Impacto Ambiental. Química Nova, São Paulo, v. 29, n. 1, p.79- 84, 08 set. 2005. Disponível em: http://www.scielo.br/ pdf/qn/v29n1/27861.pdf. Acesso em: 29 maio 2018. SANTOS, Heraldo Barbosa dos. Ensaio de Aderência das Argamassas de Revestimento. 2008. 50 f. Monografia (Especialização) - Curso de Especialização em Construção Civil, Universidade Federal de Minas Gerais, Belo Horizonte, 2008. Disponível em: http://pos. demc.ufmg.br/novocecc/trabalhos/pg1/Monografia HOPPEN, Cinthya et al. Uso de Lodo de Estação de Tratamento de Água Centrifugado em Matriz de Concreto de Cimento Portland para reduzir o Impacto Ambiental. Química Nova, São Paulo, v. 29, n. 1, p.79- 84, 08 set. 2005. Disponível em: http://www.scielo.br/ pdf/qn/v29n1/27861.pdf. Acesso em: 29 maio 2018. HOPPEN, Cinthya et al. Uso de Lodo de Estação de Tratamento de Água Centrifugado em Matriz de Concreto de Cimento Portland para reduzir o Impacto Ambiental. Química Nova, São Paulo, v. 29, n. 1, p.79- 84, 08 set. 2005. Disponível em: http://www.scielo.br/ pdf/qn/v29n1/27861.pdf. Acesso em: 29 maio 2018. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. NBR 7200: Execução de revestimento de paredes e tetos de argamassas inorgânicas – Procedimento. Rio de Janeiro, 1998. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. NBR 8522: Concreto – Determinação do módulo estático de elasticidade à compressão. Rio de Janeiro, 2008. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. NBR 15259: Argamassa para assentamento e revestimento de paredes e tetos – Determinação da absorção de SANTOS, Heraldo Barbosa dos. Ensaio de Aderência das Argamassas de Revestimento. 2008. 50 f. Monografia (Especialização) - Curso de Especialização em Construção Civil, Universidade Federal de Minas Gerais, Belo Horizonte, 2008. Disponível em: http://pos. demc.ufmg.br/novocecc/trabalhos/pg1/Monografia ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. NBR 8522: Concreto – Determinação do módulo estático de elasticidade à compressão. Rio de Janeiro, 2008. AUTORES Programa de Pós-graduação em Construção Civil da Universidade Federal de São Carlos, São Carlos. ORCID https://orcid.org/0000-0001-5785-5913 JAISON ARAUJO SPECK, M.Sc. \| Universidade do Extremo Sul Catarinense \| Curso de Engenharia Civil \| Criciúma, Santa Catarina, Brasil \| Correspondência para: Av. Universitária, 1105 - Bairro Universitário CEP: 88806-000 - Criciúma-SC \| Email: jspeck@casan.com.br 27 RIBEIRO, Rodolfo Faquini. Estudo de dosagem de lodo de Estação de Tratamento de Água (ETA) em argamassa. 2012. 61 f. TCC (Graduação) - Curso de Engenharia Ambiental, Universidade Tecnológica Federal do Paraná, Campo Mourão, 2012. Disponível em: http://repositorio.roca.utfpr.edu.br/jspui/bitstre- am/1/1890/1/CM_COEAM_2012_1_16 .pdf. Acesso em: 29 maio 2018. RIBEIRO, Rodolfo Faquini. Estudo de dosagem de lodo de Estação de Tratamento de Água (ETA) em argamassa. 2012. 61 f. TCC (Graduação) - Curso de Engenharia Ambiental, Universidade Tecnológica Federal do Paraná, Campo Mourão, 2012. Disponível em: http://repositorio.roca.utfpr.edu.br/jspui/bitstre- am/1/1890/1/CM_COEAM_2012_1_16 .pdf. Acesso em: 29 maio 2018. ORCID https://orcid.org/0000-0002-1753-4944 JORGE HENRIQUE PIVA, M.Sc. \| Universidade do Extremo Sul Catarinense \| Curso de Engenharia Civil \| Criciúma, Santa Catarina, Brasil \| Correspondência para: Av. Universitária, 1105 - Bairro Universitário CEP: 88806-000 - Criciúma-SC \| Email: jhpiva@unesc.net ORCID https://orcid.org/0000-0002-1753-4944 JORGE HENRIQUE PIVA, M.Sc. \| Universidade do Extremo Sul Catarinense \| Curso de Engenharia Civil \| Criciúma, Santa Catarina, Brasil \| Correspondência para: Av. Universitária, 1105 - Bairro Universitário CEP: 88806-000 - Criciúma-SC \| Email: jhpiva@unesc.net SILVA, Tiago Rodrigues da. Comportamento Mecânico de Argamassas de Reboco com Regranulado Negro de Cortiça.2014. 147 f. Dissertação (Mestrado) - Curso de Engenharia Civil, Técnico Lisboa, Lisboa, 2014. Disponível em: https://fenix.tecnico.ulisboa.pt/down- loadFile/563345090412722/Dissertacao.pdf. Acesso em: 13 jun. 2018. AUTORES Heraldo Barbosa.pdf. Acesso em: 11 set. 2017. MEGDA, Cláudia Regina; SOARES, Leonardo Vieira; ACHON, Cali Laguna. Propostas de Aproveitamento de Lodos Gerados em ETA’s. In: Congresso Brasileiro de Engenharia Sanitária e Ambiental, 23, 2005, Campo Grande. Disponível em: http://www.bvsde.paho.org/ ORCID https://orcid.org/0000-0002-4074-5985 SIBELE SILVEIRA LAURINDO \| Universidade do Extremo Sul Catarinense \| Curso de Engenharia Civil \| Criciúma, Santa Catarina, Brasil \| Correspondência para: Av. Universitária, 1105 - Bairro Universitário CEP: 88806-000 - Criciúma-SC \| Email: sibeleslaurindo@gmail.com bvsacd/abes23/I-019.pdf. Acesso em: 11 set. 2017. KIRCHHEIM, Ana Paula et al. Estudo da Absorção Capilar em Argamassas de Cimento Portland Branco Estrutural com Diferentes Teores de Adições Pozolânicas. In: Conferência Latino-Americana de Construção Sustentável X Encontro Nacional de Tecnologia do Ambiente Construído, 2004, Porto Alegre. Anais. São Paulo: Antac - Associação Nacional de Tecnologia no Ambiente Construído, 2004. p. 1 - 11. Disponível em: ftp://ip20017719.eng.ufjf.br/Public/ AnaisEventosCientificos/ENTAC_2004/trabalhos/ PAP0940d.pdf. Acesso em: 13 jun. 2018. bvsacd/abes23/I-019.pdf. Acesso em: 11 set. 2017. KIRCHHEIM, Ana Paula et al. Estudo da Absorção Capilar em Argamassas de Cimento Portland Branco Estrutural com Diferentes Teores de Adições Pozolânicas. In: Conferência Latino-Americana de Construção Sustentável X Encontro Nacional de Tecnologia do Ambiente Construído, 2004, Porto Alegre. Anais. São Paulo: Antac - Associação Nacional de Tecnologia no Ambiente Construído, 2004. p. 1 - 11. Disponível em: ftp://ip20017719.eng.ufjf.br/Public/ AnaisEventosCientificos/ENTAC_2004/trabalhos/ PAP0940d.pdf. Acesso em: 13 jun. 2018. ORCID https://orcid.org/0000-0002-9698-1100 ELAINE GUGLIELMI PAVEI ANTUNES, Dra. \| Universidade do Extremo Sul Catarinense \| Curso de Engenharia Civil \| Criciúma, Santa Catarina, Brasil \| Correspondência para: Av. Universitária, 1105 - Bairro Universitário CEP: 88806- 000 - Criciúma-SC \| Email: elainegpa@unesc.net ORCID https://orcid.org/0000-0002-6114-8725 ALINE EYNG SAVI, Dra. \| Universidade do Extremo Sul Catarinense \| Curso de Arquitetura e Urbanismo \| Criciúma, Santa Catarina, Brasil \| Correspondência para: Av. Universitária, 1105 - Bairro Universitário CEP: 88806- 000 - Criciúma-SC \| Email: arquiteta.alinesavi@gmail.com NEVILLE, A. M.; BROOKS, J. J.. Tecnologia do Concreto. 2. ed. Porto Alegre: Bookman Editora, 2013. 466 p. NEVILLE, A. M.; BROOKS, J. J.. Tecnologia do Concreto. 2. ed. Porto Alegre: Bookman Editora, 2013. 466 p. PASCHOAL, J.A.A. Estudos de parâmetros de qualidade para a cerâmica estrutural vermelha. 2004. 188 p. Tese de Doutorado. Dissertação (Mestrado). Programa de Pós-graduação em Construção Civil da Universidade Federal de São Carlos, São Carlos. PASCHOAL, J.A.A. Estudos de parâmetros de qualidade para a cerâmica estrutural vermelha. 2004. 188 p. Tese de Doutorado. Dissertação (Mestrado). REFERÊNCIAS ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. NBR 15259: Argamassa para assentamento e revestimento de paredes e tetos – Determinação da absorção de ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. NBR 15259: Argamassa para assentamento e revestimento de paredes e tetos – Determinação da absorção de Mix Sustentável \| Florianópolis \| v.6 \| n.4 \| p.19-28 \| ago. \| 2020 Estudo da adição do lodo de ETA \| S. S. Laurindo, E. G. P. Antunes, A. E. Savi, J. A. Speck & J. H. Piva https://doi.org/10.29183/2447-3073.MIX2020.v6.n4.19-28 Estudo da adição do lodo de ETA \| S. S. Laurindo, E. G. P. Antunes, A. E. Savi, J. A. Speck & J. H. Piva https://doi.org/10.29183/2447-3073.MIX2020.v6.n4.19-28 Heraldo Barbosa.pdf. Acesso em: 11 set. 2017. MEGDA, Cláudia Regina; SOARES, Leonardo Vieira; ACHON, Cali Laguna. Propostas de Aproveitamento de Lodos Gerados em ETA’s. In: Congresso Brasileiro de Engenharia Sanitária e Ambiental, 23, 2005, Campo Grande. Disponível em: http://www.bvsde.paho.org/ bvsacd/abes23/I-019.pdf. Acesso em: 11 set. 2017. KIRCHHEIM, Ana Paula et al. Estudo da Absorção Capilar em Argamassas de Cimento Portland Branco Estrutural com Diferentes Teores de Adições Pozolânicas. In: Conferência Latino-Americana de Construção Sustentável X Encontro Nacional de Tecnologia do Ambiente Construído, 2004, Porto Alegre. Anais. São Paulo: Antac - Associação Nacional de Tecnologia no Ambiente Construído, 2004. p. 1 - 11. Disponível em: ftp://ip20017719.eng.ufjf.br/Public/ AnaisEventosCientificos/ENTAC_2004/trabalhos/ PAP0940d.pdf. Acesso em: 13 jun. 2018. COMO CITAR ESTE ARTIGO LAURINDO, Sibele Silveira; ANTUNES, Elaine Guglielmi Pavei; SAVI, Aline Eyng; SPECK, Jaison Araujo; PIVA, Jorge Henrique. Estudo da Adição do Lodo de ETA - Estação de Tratamento de Água em Argamassas de Revestimento. MIX Sustentável, [S.l.], v. 6, n. 4, p. 19-28, ago. 2020. ISSN 24473073. Disponível em:<http://www.nexos.ufsc.br/index.php/mixsus- tentavel>. Acesso em: dia mês. ano. doi:https://doi. org/10.29183/2447-3073.MIX2020.v6.n4.19-28. TAFAREL, Nathann Francisco et al. Avaliação das propriedades do concreto devido à incorporação de lodo de estação de tratamento de água. Revista Matéria, Rio de Janeiro, v. 21, n. 4, p.974-986, dez. 2016. Disponível em: http://www.scielo.br/pdf/rmat/v21n4/ 1517-7076-rmat-21-04-00974.pdf. Acesso em: 29 maio 2018. TAFAREL, Nathann Francisco et al. Avaliação das propriedades do concreto devido à incorporação de lodo de estação de tratamento de água. Revista Matéria, Rio de Janeiro, v. 21, n. 4, p.974-986, dez. 2016. Disponível em: http://www.scielo.br/pdf/rmat/v21n4/ 1517-7076-rmat-21-04-00974.pdf. Acesso em: 29 maio 2018. DATA DE ENVIO: 14/11/2019 DATA DE ACEITE: 02/06/2020 DATA DE ENVIO: 14/11/2019 DATA DE ACEITE: 02/06/2020 Mix Sustentável \| Florianópolis \| v.6 \| n.4 \| p.19-28 \| ago. \| 2020 Mix Sustentável \| Florianópolis \| v.6 \| n.4 \| p.19-28 \| ago. \| 2020
https://openalex.org/W3113375075	https://abqarijournal.usim.edu.my/index.php/abqari/article/download/336/211	Indonesian	null	Metodologi Dakwah Menurut Abdul Karim Zaidan (1917-2014): dalam Bukunya Usul Da’wah	al-'Abqari : journal of Islamic social sciences and humanities/al-'Abqari	2,020	cc-by	6,861	Abstrak Artikel ini membicarakan tentang sumber metodologi dakwah, pendekatan dakwah menurut pandangan Abdul Karim Zaidan. Menurutnya, sumber metodologi dakwah ialah al-Quran, al-Sunnah, sejarah hidup para Sahabat, pendapat dan pandangan para fuqaha serta pengalaman pendakwah itu sendiri. Artikel ini menggunakan kaedah kajian perpustakaan. Objektif artikel ini ialah memperkenalkan biografi Abdul Karim Zaidan. Di samping itu menjelaskan manhaj dakwahnya iaitu berasaskan pratik yang pernah dilakukan oleh Rasulullah dan para sahabat. Rasulullah telah menggunakan pendekatan pengkisahan, dialog, penyelesaian masalah, pengajian ilmu, khutbah dan ceramah serta penulisan dengan menulis surat kepada ketua-ketua negara. Buku Usul al-Da’wahnya telah menjadi rujukan utama kepada pendakwah di seluruh dunia. Artikel ini mendapati penggunaan manhaj dakwah yang betul akan memberi impak positif kepada kejayaan perkembangan dakwah Islam. Kata kunci: Biografi Abdul Karim, manhaj Dakwah, Uslub dan Wasilah dakwah. Penyakit dakwah. Dakwah Methodology According to Abdul Karim Zaidan's View (1917-2014): In His Book Usul Da’wah Mohd Rumaizuddin Ghazali Universiti Sains Islam Malaysia Mohd Rumaizuddin Ghazali Universiti Sains Islam Malaysia VOL. 23 (1) 2020: 235-249 Journal of Islamic Social Sciences and Humanities مجلة الثقافة اإلسالمية واإلنسانية VOL. 23 (1) 2020: 235-249 Journal of Islamic Social Sciences and Humanities مجلة الثقافة اإلسالمية واإلنسانية Abstract The article discusses the sources of da'wah methodology, da'wah approaches according to Abdul Karim Zaidan's view. According to him, the sources of da'wah methodology are the Qur'an, al-Sunnah, the history of Sahabah, the opinions and views of the fuqaha and the experiences of the dai’e themselves. This article employs library research. The objective of this article is to introduce the biography of Abdul Karim Zaidan. It also describes his dakwah methodology, which is based on the practices carried out by the Prophet and his Companions. The Prophet has utilised 235 العبقري جملة Vol. 23 (1), 2020 Journal al-‘Abqari various methods such as narrations, dialogues, problem solving, focussed deliberations of knowledge, sermons and lectures as well as writings through correspondences with various heads of state. Through his own book, Usul al-Da’wah, he has been regarded as the source of reference to the Muslim preachers the world over. This article found that proper adoption of preaching methodologies will contribute positive impacts on the successful dissemination of the Islamic dakwah. Keywords: Biography of Abdul Karim, Manhaj Dakwah, Uslub and Wasilah, Preaching. Preaching Plight. PENDAHULUAN Artikel ini membicarakan secara ringkas buograji Andul Karim Zaidan dan pendekatan manhaj dakwah yang berkesan. Di samping turut menjelaskan penyakit- penyakit dakwah dan wasilah dakwah. Perkara yang dimaksudkan wasilah ialah segala yang membantu para pendakwah dalam menyampaikan dakwah secara berkesan. NAMA DAN PENDIDIKAN Nama penuhnya ialah Abdul Karim Zaidan Bahij al-‘Ani. Bahij adalah nama datuknya, al-‘Ani nisbah kepada kepada perkampungan Anah yang terletak dalam daerah Al-Anbar yang merupakan tempat kelahiran bapa dan datuknya. Sementara Abdul Karim dilahirkan di kawasan Karakh berdekatan Suq Hamadah, di Baghdad pada tahun 1917 dalam rekod pendaftaran rasmi. Menurutnya, tarikh kelahirannya sebenarnya pada tahun 1921. Hal ini disebabkan kelewatan pendaftaran kelahiran yang hanya dilakukan selepas jangka masa yang panjang. Oleh itu, kerana pendaftaran kelahiran tersebut hanya melihat berdasarkan kepada ketinggian dan besar kanak- kanak di hadapan mereka. Bapanya telah meninggal dunia ketika umurnya berusia tiga tahun dan ibunya pula meninggal ketika beliau telah meningkat dewasa. Ibu dan kakak-kakaknya sangat menyayanginya sehingga ia tidak merasai sebagai anak yatim (drzedan.com). Beliau belajar al-Quran di Kuttab di kampungnya. Setelah itu, abang sulung beliau memasukkan beliau ke sekolah rendah di Karakh di mana beliau belajar selama enam tahun. Beliau kemudiannya menyambung pelajaran di peringkat pertengahan selama tiga tahun dan di peringkat menengah selama dua tahun menerusi aliran sastera. Setelah berjaya mendapat sijil peringkat menengah, beliau mengajar di sekolah peringkat rendah untuk menyara keluarga. Pada pertengahan tahun 1930-an beliau mengajar di daerah Diyali selama empat tahun, di Sekolah Abi Saida, Maqdiah dan akhirnya berpindah ke sekolah rendah di Baghdad (drzedan.com). 236 Metodologi Dakwah Menurut Abdul Karim Zaidan (1917-2014): Dalam Bukunya Usul Da’wah Pada awal tahun 1946, Kementerian Pendidikan telah mengeluarkan keputusan menerima pelajar-pelajar yang tamat peringkat menengah untuk memasuki universiti. Ini melayakkan beliau memasuki fakulti undang-undang. Fakulti ini mempunyai peranan istimewa kepada dirinya kerana di fakulti itulah beliau belajar sehingga bergelar graduan dan kemudiannya menjadi tempat beliau mengajar. Antara subjek yang diajarnya ialah Makhal ila Syariat al-Islamiah, Nikah dan Talak, Pewarisan dan Fara’id, Usul Fiqh dan subjek muamalat dalam mazhab hanafi. Beliau berada di fakulti ini sehingga tahun 1950. Setelah tamat pengajian, beliau mulai mengajar agama di Sekolah Karakh sebuah sekolah menengah. Pada tahun 1953, beliau dilantik menjadi pengetua di sekolah menengah al-Najibiah dan nazir di Kementerian Pendidikan. Setelah itu, beliau dilantik menjadi pengarah pendidikan di daerah Diyali dan kemudian pengarah bahagian pendidikan agama (drzedan.com). Kemudian beliau memasuki kolej syariah di bawah Fakulti Undang-Undang, Universiti Kaherah. Beliau lulus setelah dua tahun pengajian dengan mengemukakan tesis bertajuk “- Athru al-Qusud fi Tasarufat wa al-Uqud pada tahun 1958. Pada tahun 1960, Abdul Karim menjadi pensyarah di Fakulti Undang-Undang dengan mengajar subjek syariah Islamiah dan Usul Fiqh. NAMA DAN PENDIDIKAN Beliau kemudiannya menyambung pengajian di peringkat kedoktoran dengan tajuk أحكام الذميين والمستأمنين في دار اإلسالم Ahkam al-Zimmiyyin wa al-Musta’minin fi dar al-Islam - (Hukum Ahli Zimmah dan warga yang Diberi Keamanan dalam Negara Islam) dengan pangkat cemerlang di bawah penyeliaan Dr Abdul Salam Madkhur (drzedan.com). Antara guru-guru beliau semasa di Universiti Kaherah ialah Syeikh Muhammad Abu Zahrah, Syeikh Ali Khafif dan Syeikh Muhammad Zafzaf. Abdul Karim Zaidan juga telah ditukarkan dari Fakulti Undang-Undang ke bahagian agama di Fakulti Sastera, Universiti Baghdad. Di sini, beliau dinaikkan pangkat dari tutor menjadi pensyarah, professor madya dan profesor. Pada tahun 1978, beliau telah dianugerah Profesor Emiritus di atas kecemerlangan akademik beliau. Pada tahun 1950an, beliau telah menunaikan haji dan semasa pulang beliau singgah di al-Quds dan Syria lalu berjumpa dengan Nasruddin al-Albani. Dari segi akademik, beliau telah memegang jawatan profesor Syariah Islamiah dan pernah menjadi Ketua Jabatan Syariah di Fakulti Undang-Undang, Ketua Jabatan Agama di Fakulti Sastera dan juga Dekan Pengajian Islam di Universiti Baghdad. Beliau juga terlibat dengan Majma’ Fiqh al-Islami di bawah kelolaan Rabitah al-Alam al-Islami dan menjadi ahli majlis tertinggi Universiti al-Iman sejak tahun 1999. Beliau juga telah menerima Anugerah Raja Faisal Antarabangsa pada tahun 1997 di atas usaha penulisan al- Mufassal fi al-Ahkam al-Mar’ah sebanyak 11 jilid. MENGAJAR DI SANA’A DAN MENINGGAL DUNIA Pada tahun 1992, beliau telah pergi ke Sana’a dan menjadi pensyarah di Bahagian Pengajian Islam, Fakulti Sastera, Universiti Sana’a dan di Universiti al-Iman Kebanyakan subjek yang diajar menggunakan buku karangan beliau seperti al-Wajiz fi Usaul Fiqh, Nizam al-Qada dan Madkhal Ila Syariah al-Islamiah. Beliau meninggal dunia pada hari Isnin 27 Januari 2014/ 25 Rabiul Awwal 1435H dalam usia 97 tahun di Sana’an dan dikebumikan di Baghdad, Iraq. MENGHADIRI SEMINAR Beliau telah membentangkan kertas kerja dalam pelbagai seminar di dalam dan luar negara. Antaranya beliau menyampaikan ceramah umum Syariah al-Islamiah di 237 العبقري جملة Vol. 23 (1), 2020 Journal al-‘Abqari Kuwait pada tahun 1960-an dan terlibat dalam penerbitan Ensiklopedia Fikah bersama Kerajaan Kuwait. Pada tahun 1969, beliau membentangkan kertas kerja tajuk “Undang-Undang Antarabangsa dalam Syariah Islamiah” dalam seminar Undang- Undang dan Sains Politik Ketiga yang dianjurkan oleh Universiti Liga Arab di Baghdad. Beliau turut menghadiri Muktamar Fekah di Riyadh pada tahun 1970-an dan menyampaikan siri syarahan mengenai Fekah al-Islami di Qatar pada tahun 1995 sehingga 1996. Beliau juga terlibat dengan seminar Perbankan Islam pada tahun 1997 yang diadakan di Sana’a, Yaman. Kuwait pada tahun 1960-an dan terlibat dalam penerbitan Ensiklopedia Fikah bersama Kerajaan Kuwait. Pada tahun 1969, beliau membentangkan kertas kerja tajuk “Undang-Undang Antarabangsa dalam Syariah Islamiah” dalam seminar Undang- Undang dan Sains Politik Ketiga yang dianjurkan oleh Universiti Liga Arab di Baghdad. Beliau turut menghadiri Muktamar Fekah di Riyadh pada tahun 1970-an dan menyampaikan siri syarahan mengenai Fekah al-Islami di Qatar pada tahun 1995 sehingga 1996. Beliau juga terlibat dengan seminar Perbankan Islam pada tahun 1997 yang diadakan di Sana’a, Yaman. JAWATAN-JAWATAN AKADEMIK a. Profesor Syariah Islamiyyah dan Ketua Fakulti Undang-Undang di Universiti Baghdad b. Profesor Syariah Islamiyyah dan Ketua Bahagian Agama di Fakulti Sastera, Universiti Baghdad c. Profesor Syariah Islamiyyah di Fakulti Pengajian Islam d. Profesor Fiqh dan Usul Fiqh di Universiti al-Iman, Sana’a e. Profesor Syariah Islamiyyah di Bahagian Pengajian Islam dan pengajian peringkat Sarjana dan Doktor Falsafah di Universiti Sana’a sehingga beliau meninggal dunia. e. Profesor Syariah Islamiyyah di Bahagian Pengajian Islam dan pengajian peringkat Sarjana dan Doktor Falsafah di Universiti Sana’a sehingga beliau meninggal dunia. GURU DAN RAKAN-RAKAN Antara guru yang banyak mempengaruhi kehidupannya ialah Syeikh Amjad al- Zahawi. Beliau dilahirkan pada tahun 1300H/1883M di Baghdad. Mendapat pendidikan awal di Madrasah al-Rasyidah di Baghdad. Setelah itu, beliau menyambung pelajaran ke Istanbul selama enam tahun di kuliah kehakiman. Setelah berjaya, beliau kembali ke Baghdad dan menjadi hakim di sana. Setelah tamat dari pekerjaan rasmi, beliau berkecimpung dalam dakwah pada tahun 1946 dan menubuhkan persatuan-persatuan Islam di Iraq seperti Persatuan al-Adab al-Islamiah dan al-Ukhuwah al-Islamiah. Beliau juga ketua merupakan ketua Liga Ulama Iraq dan sentiasa memperjuangkan isu Palestin. Beliau meninggal dunia pada hari Jumaat 14 Syaaban 1387/1967. Abdul Karim mengakui terpengaruh dengan gurunya ini terutama dari sifat keikhlasan, ketakwaan dan keprihatinan mengenai isu-isu umat Islam. Dalam keprihatinan isu-isu umat Islam, beliau akan menghadiri setiap perhimpunan yang membincang isu-isu tersebut dan melakukan lawatan ke negara- negara umat Islam untuk mengetahui keadaan dan membela nasib mereka. Abdul Karim Zaidan telah mendampingi gurunya (Suhbah). Ia bukan sekadar hubungan murid dengan guru tetapi sentiasa berdampingi dengan mempelajari dalam pengajiannya secara umum dan khusus, semasa sihat dan sakit serta mengiringinya ketika bermusafir. Abdul Karim Zaidan menyatakan kecintaan kepada gurunya seperti kecintaan murid kepada guru dan kecintaan anak kepada ayahnya kerana Allah (al- Aqil, 2002:328). Antara rakannya ialah Syeikh Muhammad Mahmud al-Sawaf yang dikenali sebagai Abu Mujahid. Beliau dilahirkan di bandar Mawsol pada I Syawal 1333H/1915 dan 238 Metodologi Dakwah Menurut Abdul Karim Zaidan (1917-2014): Dalam Bukunya Usul Da’wah dibesarkan dalam persekitaran yang kuat beragama. Mendapat didikan dari Syeikh Abdullah al-Ni’mat, Syeikh Soleh al-Jawadi dan Syeikh Amjad al-Zahiri. Beliau belajar di Madrasah al-Fisaliah dan kemudian menyambung ke al-Azhar. Semasa di Mesir, beliau terpengaruh dengan idea-idea pembaruan yang dibawa oleh Hasan al- Banna. Apabila pulang ke Iraq, beliau menjadi Ketua Ikhwan al-Muslimin Iraq dan sepenuh masa dalam aktiviti dakwah. Beliau bersama–sama dengan Syeikh Amjad al- Zahiri menubuhkan al-Jam’iyyah al-Ukhuwah al-Islamiah. Pada tahun 1958, berlaku Revolusi tentera yang mana golongan komunis telah mengambil alih pemerintahan dan menangkap serta memenjarakan mereka. Pada tahun 1962, beliau berhijrah ke Arab Saudi dengan menjadi guru di Kuliah Syariah. Beliau juga menjadi ahli Kesatuan Liga Fikah dan antara pengasas Rabitah al-‘Alam al-Islami dan pegawai di Kementerian Pelajaran Arab Saudi. Beliau meninggal dunia pada hari Jumaat 13 Rabiul Akhir 1413 (Oktober 1992) di lapangan terbang Istanbul ketika hendak pulang ke Arab Saudi. Jenazahnya di sembahyang di Masjidil Haram dan dikebumikan di Mekkah (al-Aqil, 2002:210). 2. Majlis-Majlis Akademik a. Ahli Majlis Ulama Universiti Islam sejak tahun 1970-an a. Ahli Majlis Ulama Universiti Islam sejak tahun 1970-an b. Ahli Majlis Fiqh di Rabitah al-‘Alam al-Islami sejak tahun 2000 b. Ahli Majlis Fiqh di Rabitah al-‘Alam al-Islami sejak tahun 2000 c. Ahli Majlis Fiqh di Universiti al-Iman, Sana’a MENGANGGOTAI JAMAAH IKHWAN AL-MUSLIMUN MENGANGGOTAI JAMAAH IKHWAN AL-MUSLIMUN MENGANGGOTAI JAMAAH IKHWAN AL-MUSLIMUN Ikhwan al-Muslimun Iraq ditubuhkan oleh Muhamad Mahmud Sawaf yang mendapat didikan dari Hasan al-Banna ketika beliau menuntut di al-Azhar. Beliau telah pergi Mesir pada tahun 1939 untuk menyambung pelajaran di al-Azhar. Apabila pulang di Iraq pada tahun 1946, beliau menjadi tenaga pengajar dan pendakwah yang menyebarkan pemikiran Ikhwan al-Muslimun terutama dalam majalah al-Ukhuwwah al-Islamiah dimana Sawaf menjadi ketua editor dan Abdul Karim Zaidan juga menjadi penulisnya. 239 العبقري جملة Vol. 23 (1), 2020 Vol. 23 (1), 2020 Journal al-‘Abqari Dalam bidang dakwah, Abdul Karim Zaidan terlibat dengan Ikhwan al-Muslimun sejak tahun 1950-an dan menjadi pimpinan pergerakan tersebut di Iraq pada tahun 1958. Program dakwah yang dilakukannya adalah melalui pengajian–pengajian agama di masjid-masjid dan juga ulasan khutbah selepas solat Jumaat. Di samping itu, beliau menulis artikel di majalah pendidikan Islam, Majalah al-Ukhuwwah al- Islamiah dan majalah al-Muslimun. Beliau menganggotai Ikhwan al-Muslimun pada musim panas tahun 1950 setelah tamat dari Fakulti Undang-Undang. Peristiwa ini terjadi setelah saudara Mustafa Wahab memberi buku mengenai Ikhwan dan Metodologi Dakwah yang membuatkan beliau merasa kagum dan tertarik dengan pergerakan ini. Lalu beliau berjumpa dengan Muhamad Mahmud Sawaf dan menganggotai pergerakan tersebut. Pada tahun 1959, apabila Muhamad Mahmud Sawaf berpindah ke Arab Saudi kerana ugutan bunuh dari pemerintah yang berideologi komunis, pihak pimpinan Tertinggi Ikhwan telah melantik Abdul Karim Zaidan menjadi Muraqib (Ketua) Ikhwan al-Muslimun di Iraq sejak tahun 1960 sehingga tahun 1990-an. Namun oleh kerana keadaan politik di Iraq dan tekanan yang kuat dari pemerintah, strategi dakwah Ikhwan Iraq telah bertukar kepada bentuk dakwah individu dan rahsia. Menurut Abdul Karim Zaidan, strategi ini bukan bermaksud mengberhentikan dakwah Ikhwan tetapi lebih kepada pertukaran pendekatan mengikut keadaan semasa. Pihak Ikhwan al-Muslimun Iraq telah membubarkan pertubuhan Ikhwan Iraq dan mereka bergerak secara senyap kerana tekanan pemerintah yang menangkap dan menghukum gantung mereka yang terlibat. Menurut Abdul Karim Zaidan lagi, keadaan politik di Iraq yang tidak stabil serta ketiadaan kebebasan individu telah menyebabkan beliau mengambil keputusan untuk berpindah ke Yaman sehinggalah beliau meninggal dunia (Taufik al-Wa’iyy, 2006: 296-298, www.zedan.com/ www.qaradawi.net). Menurut Ustaz Hashim Jasin, pergerakan Ikhwan di Iraq lebih berupayat memperkembangkan idea-ideanya di kalangan mahasiswa walaupun dilakukan secara rahsia. Pendedahan buku-buku tokoh Ikhwan mudah didapati daripada Mesir. Hal ini dibuktikan dengan aktiviti sepuluh pelajar Malaysia yang dihantar ke Iraq oleh almarhum Yusuf Rawa ke sana untuk mendapat didikan usrah Ikhwan. MENGANGGOTAI JAMAAH IKHWAN AL-MUSLIMUN Abdul Karim Zaidan sentiasa memantau perkembangan pelajar-pelajar ini melalui naqib yang telah dilantiknya bagi mengurus urusan pelajar-pelajar tersebut (Temubual Hashim Jasin,4 Julai 2016). GABUNGAN MANHAJ FIKAH DAN DAKWAH Walaupun bermazhab Hanafi Abdul Karim Zaidan mengajar fikah dengan pelbagai mazhab. Beliau kemudiannya membuat pertimbangan dan memilih pendapat yang lebih sahih dan rajih. Beliau menggabungkan fikah dengan elemen-elemen dakwah. Tidak semua ahli fikah berpersepsi sebagai pendakwah. Hal ini adalah kerana sesetengah ahli fikah hanya pakar dalam permasalahan fikah sahaja tetapi tidak 240 Metodologi Dakwah Menurut Abdul Karim Zaidan (1917-2014): Dalam Bukunya Usul Da’wah mampu berkhutbah atau berceramah dan menyampaikan pengajaran umum. Abdul Karim Zaidan mempunyai buku khusus membicarakan asas-asas dakwah iaitu Usul al-Da’wah. Keistimewaan buku ini dan buku-buku karangannya ialah ia menggabungkan fikah dan dakwah. Abdul Karim Zaidan adalah seorang yang fakih dan pendakwah yang hebat, Buku-buku beliau menjadi silibus di universiti-universiti di seluruh dunia. Beliau juga seorang yang terkenal berpendirian tegas dalam menyatakan kebenaran dan melatih umat Islam agar sentiasa berjihad menentang kezaliman. Beliau meniupkan semangat jihad di Iraq, di Palestin dan di Yaman. Jihad semuanya bukanlah jihad senjata tetapi jihad dalam menyatakan kebenaran kepada pemerintah (al-Qaradawi.net). Yusuf al-Qaradawi menyebutkan bahawa Abdul Kariam Zaidan amat prihatin terhadap isu umat Islam terutama dalam isu Iraq dan mengikuti setiap peristiwa yang berlaku. Menurut al-Qaradawi, walaupun usia Abdul Karim telah melangkaui 90 tahun, beliau tetap gigih berkhidmat untuk dakwah dan Islam. Di dalam dirinya terdapat sifat ketokohan, keilmuan, kefakihan dan pendakwah serta semangat jihad yang tinggi (www. al-qaradawi.net). PENULISAN اا PENULISAN اا ENULISAN 1. أحكام الذميين والمستأمنين في دار االسالم -Ahkam al-Zimmiyyin wa al-Musta’minin fi Dar al-Islam 2. الكفالة والحوالة في الفقه المقارن.- Al-Kifalah wa al-Hiwalah fi al-Fiqh al-Maqaran 3. اصول الدعوة- Kitab al-Usul al-Da’wah 4. الفرد والدولة في الشريعة - Al-Fard wa ad-Dawlah fi al-Syari’ah 5. المفصل في أحكام المرأة والبيت المسلم - Al-Mufassal fi Ahkam al-Mar’ah wa Bayt al-Muslim 6. الوجيز في أصول الفقه - Al-Wajiz fi Usul al-Fiqh 7. . الوجيز في شرح القواعد الفقهية - Al-Wajiz fi Syarh al-Qawa’id al-Fiqhiyyah 8. ال مدخل لدراسة الشريعة االسالمية –Madkhal li Dirasah Syari’ah al-Islamiyyah 9. نظرات في الشريعة االسالمية والقوانين الوضعية- Nazarat fi al-Syari’ah al-Islamiyyah wa Qawanin al-wada’iyah 10. المستفاد من قصص القرآن للدعوة والدعاة - Al-Mustafat min Qasas al-Quran al-Karim lil Da’wah wa al-Da’ah 11. السنن اإللهية في األمم والجماعات واألفراد في الشريعة اإلسالمية- Al-Sunan al-Ilahiyyah fi al-Umam wa al-Ifrad wa al-Jama’at 12. بحوث فقهية معاصرة –Buhuth Fiqhiyyah Mu’asarah 13. نظام القضاء في الشريعة اإلسالمية-Nizam al-Qada’ Fi al-Syari’ah al-Islamiyyah 14. أثر القصود في التصرفات والعقود - Athru al-Qusud fi tasarufat wa al-‘Uqud 15. حالة الضرورة في الشريعة اإلسالمية- Halah al-Darurah fi al-Syari’ah al-Islamiyyah 16. االختالف في الشريعة اإلسالمية – al-Ikhtilaf fi fi al-Syari’ah al-Islamiyyah 17. حقو ق األفراد في دار اإلسالم-Huquq al-Afrad fi Dar al-Islam 18. اإليمان بالقضاء والقدر- al-Iman bi al-Qada wa al-Qadar NULISAN 1. أحكام الذميين والمستأمنين في دار االسالم -Ahkam al-Zimmiyyin wa al-Musta’minin fi Dar al-Islam 2. الكفالة والحوالة في الفقه المقارن.- Al-Kifalah wa al-Hiwalah fi al-Fiqh al-Maqaran 3. اصول الدعوة- Kitab al-Usul al-Da’wah 4. الفرد والدولة في الشريعة - Al-Fard wa ad-Dawlah fi al-Syari’ah 5. المفصل في أحكام المرأة والبيت المسلم - Al-Mufassal fi Ahkam al-Mar’ah wa Bayt al-Muslim 6. الوجيز في أصول الفقه - Al-Wajiz fi Usul al-Fiqh 7. . الوجيز في شرح القواعد الفقهية - Al-Wajiz fi Syarh al-Qawa’id al-Fiqhiyyah 8. ال مدخل لدراسة الشريعة االسالمية –Madkhal li Dirasah Syari’ah al-Islamiyyah 9. نظرات في الشريعة االسالمية والقوانين الوضعية- Nazarat fi al-Syari’ah al-Islamiyyah wa Qawanin al-wada’iyah 10. المستفاد من قصص القرآن للدعوة والدعاة - Al-Mustafat min Qasas al-Quran al-Karim lil Da’wah wa al-Da’ah 11. السنن اإللهية في األمم والجماعات واألفراد في الشريعة اإلسالمية- Al-Sunan al-Ilahiyyah fi al-Umam wa al-Ifrad wa al-Jama’at 12. بحوث فقهية معاصرة –Buhuth Fiqhiyyah Mu’asarah 13. نظام القضاء في الشريعة اإلسالمية-Nizam al-Qada’ Fi al-Syari’ah al-Islamiyyah 14. أثر القصود في التصرفات والعقود - Athru al-Qusud fi tasarufat wa al-‘Uqud 15. حالة الضرورة في الشريعة اإلسالمية- Halah al-Darurah fi al-Syari’ah al-Islamiyyah 16. PENULISAN اا االختالف في الشريعة اإلسالمية – al-Ikhtilaf fi fi al-Syari’ah al-Islamiyyah 17. حقو ق األفراد في دار اإلسالم-Huquq al-Afrad fi Dar al-Islam 18. اإليمان بالقضاء والقدر- al-Iman bi al-Qada wa al-Qadar ENULISAN 1. أحكام الذميين والمستأمنين في دار االسالم -Ahkam al-Zimmiyyin wa al-Musta’minin fi Dar al-Islam 2. الكفالة والحوالة في الفقه المقارن.- Al-Kifalah wa al-Hiwalah fi al-Fiqh al-Maqaran 3. اصول الدعوة- Kitab al-Usul al-Da’wah 4. الفرد والدولة في الشريعة - Al-Fard wa ad-Dawlah fi al-Syari’ah 5. المفصل في أحكام المرأة والبيت المسلم - Al-Mufassal fi Ahkam al-Mar’ah wa Bayt al-Muslim 6. الوجيز في أصول الفقه - Al-Wajiz fi Usul al-Fiqh 7. . الوجيز في شرح القواعد الفقهية - Al-Wajiz fi Syarh al-Qawa’id al-Fiqhiyyah 8. ال مدخل لدراسة الشريعة االسالمية –Madkhal li Dirasah Syari’ah al-Islamiyyah 9. نظرات في الشريعة االسالمية والقوانين الوضعية- Nazarat fi al-Syari’ah al-Islamiyyah wa Qawanin al-wada’iyah 10. المستفاد من قصص القرآن للدعوة والدعاة - Al-Mustafat min Qasas al-Quran al-Karim lil Da’wah wa al-Da’ah 11. السنن اإللهية في األمم والجماعات واألفراد في الشريعة اإلسالمية- Al-Sunan al-Ilahiyyah fi al-Umam wa al-Ifrad wa al-Jama’at 12. بحوث فقهية معاصرة –Buhuth Fiqhiyyah Mu’asarah 13. نظام القضاء في الشريعة اإلسالمية-Nizam al-Qada’ Fi al-Syari’ah al-Islamiyyah 14. أثر القصود في التصرفات والعقود - Athru al-Qusud fi tasarufat wa al-‘Uqud 15. حالة الضرورة في الشريعة اإلسالمية- Halah al-Darurah fi al-Syari’ah al-Islamiyyah 16. االختالف في الشريعة اإلسالمية – al-Ikhtilaf fi fi al-Syari’ah al-Islamiyyah 17. حقو ق األفراد في دار اإلسالم-Huquq al-Afrad fi Dar al-Islam 18. اإليمان بالقضاء والقدر- al-Iman bi al-Qada wa al-Qadar ح يj f y Q q yy 8. ال مدخل لدراسة الشريعة االسالمية –Madkhal li Dirasah Syari’ah al-Islamiy 9. نظرات في الشريعة االسالمية والقوانين الوضعية- Nazarat fi al-Syari’ah al-Islamiyyah wa Qawanin al-wada’iyah 10. المستفاد من قصص القرآن للدعوة والدعاة - Al-Mustafat min Qasas al-Quran al-Karim lil Da’wah wa al-Da’ah اإ لأ لأإ 12. بحوث فقهية معاصرة –Buhuth Fiqhiyyah Mu asarah 13. نظام القضاء في الشريعة اإلسالمية-Nizam al-Qada’ Fi al-Syari’ah al-Islamiyyah 14. أثر القصود في التصرفات والعقود - Athru al-Qusud fi tasarufat wa al-‘Uqud 15. حالة الضرورة في الشريعة اإلسالمية- Halah al-Darurah fi al-Syari’ah al-Islamiyyah 16. االختالف في الشريعة اإلسالمية – al-Ikhtilaf fi fi al-Syari’ah al-Islamiyyah 17. حقو ق األفراد في دار اإلسالم-Huquq al-Afrad fi Dar al-Islam 18. اإليمان بالقضاء والقدر- al-Iman bi al-Qada wa al-Qadar 241 Journal al-‘Abqari العبقري جملة Vol. PENULISAN اا 23 (1), 2020 Journal al-‘Abqari Dalam buku Usul al-Da’wah tersebut, Abdul Karim Zaidan membicarakan tentang sumber metodologi dakwah, pendekatan dan penyakit dakwah serta mengenai jalan- jalan (wasilah) dakwah. 3. Sejarah hidup para Sahabat. Dalam sejarah hidup para sahabat yang besar dan tabi’in telah memberi contoh yang baik dan berguna kepada para pendakwah. Mereka adalah orang yang lebih memahami tentang ajaran Islam dan dakwah Islamiah. Sepatutnya, ahli ilmu mengambil manfaat daripada mereka. Dalam kehidupan mereka penuh dengan kisah-kisah, SUMBER METODOLOGI DAKWAH Sumber metodologi dakwah ialah al-Quran, al-Sunnah, sejarah hidup para Sahabat, pendapat dan pandangan para fuqaha, serta pengalaman pendakwah itu sendiri. 1. Al-Quran. Dalam al-Quran terdapat banyak ayat-ayat mengenai kisah para nabi dengan kaumnya yang perlu diambil pengajaran dan metodologi yang telah digunakan dalam menghadapi mereka. Al-Quran juga memerintahkan manusia untuk mengikut manhaj dakwah Rasulullah SAW. Firman Allah: 1. Al-Quran. Dalam al-Quran terdapat banyak ayat-ayat mengenai kisah para nabi dengan kaumnya yang perlu diambil pengajaran dan metodologi yang telah digunakan dalam menghadapi mereka. Al-Quran juga memerintahkan manusia untuk mengikut manhaj dakwah Rasulullah SAW. Firman Allah: ٰٓ أُوْل َٰٓئِك ۡۗۡٱلَّذِين ه د ى ٱّللَّ ُُۖ ف بِهُد ى هُمُ ٱقۡت دِه Mereka (nabi-nabi) itulah, orang-orang yang telah diberi petunjuk oleh Allah maka turutilah oleh kamu (wahai Muhammad) akan petunjuk mereka (al-An’am: 90) Dalam al-Quran terdapat pelbagai pendekatan yang digunakan dalam berdakwah. Al-Quran berbicara mengenai dakwah para nabi terutama nabi Ulul Azmi kerana Allah sendiri yang mendidik dan memberi panduan kepada mereka. Al-Quran menyebut pendekatan pengkisahan, dialog, perumpamaan dan akhlak para pendakwah. 2. Sunnah Nabawiyyah. Dalam Sunnah terdapat banyak hadis yang berkaitan dengan dakwah serta pendekatannya. Begitu juga dalam sejarah hidup nabi SAW sama ada di Makkah atau Madinah telah memberi contoh dalam pendekatan dakwah. Para pendakwah perlu memahami cara dan pendekatan yang telah dilakukan oleh Rasulullah SAW kerana Rasulullah SAW telah mempraktikkannya sesuai dengan apa yang diperintahkan Allah dan menyampaikan risalah Islam dengan sempurna. Rasulullah SAW telah menggunakan pendekatan pengkisahan, dialog dan penyelesaian masalah. Di samping dakwah yang dilakukan melalui pengajian, khutbah dan ceramah serta penulisan dengan menulis surat kepada ketua-ketua negara. 2. Sunnah Nabawiyyah. Dalam Sunnah terdapat banyak hadis yang berkaitan dengan dakwah serta pendekatannya. Begitu juga dalam sejarah hidup nabi SAW sama ada di Makkah atau Madinah telah memberi contoh dalam pendekatan dakwah. Para pendakwah perlu memahami cara dan pendekatan yang telah dilakukan oleh Rasulullah SAW kerana Rasulullah SAW telah mempraktikkannya sesuai dengan apa yang diperintahkan Allah dan menyampaikan risalah Islam dengan sempurna. Rasulullah SAW telah menggunakan pendekatan pengkisahan, dialog dan penyelesaian masalah. Di samping dakwah yang dilakukan melalui pengajian, khutbah dan ceramah serta penulisan dengan menulis surat kepada ketua-ketua negara. 2. Sunnah Nabawiyyah. Dalam Sunnah terdapat banyak hadis yang berkaitan dengan dakwah serta pendekatannya. Begitu juga dalam sejarah hidup nabi SAW sama ada di Makkah atau Madinah telah memberi contoh dalam pendekatan dakwah. SUMBER METODOLOGI DAKWAH Para pendakwah perlu memahami cara dan pendekatan yang telah dilakukan oleh Rasulullah SAW kerana Rasulullah SAW telah mempraktikkannya sesuai dengan apa yang diperintahkan Allah dan menyampaikan risalah Islam dengan sempurna. Rasulullah SAW telah menggunakan pendekatan pengkisahan, dialog dan penyelesaian masalah. Di samping dakwah yang dilakukan melalui pengajian, khutbah dan ceramah serta penulisan dengan menulis surat kepada ketua-ketua negara. 3. Sejarah hidup para Sahabat. Dalam sejarah hidup para sahabat yang besar dan tabi’in telah memberi contoh yang baik dan berguna kepada para pendakwah. Mereka adalah orang yang lebih memahami tentang ajaran Islam dan dakwah Islamiah. Sepatutnya, ahli ilmu mengambil manfaat daripada mereka. Dalam kehidupan mereka penuh dengan kisah-kisah, 242 Metodologi Dakwah Menurut Abdul Karim Zaidan (1917-2014): Dalam Bukunya Usul Da’wah qudwah, ijtihad, fatwa, kesabaran dan musyawarah yang perlu diambil iktibar. Metodologi Dakwah Menurut Abdul Karim Zaidan (1917-2014): Dalam Bukunya Usul Da’wah qudwah, ijtihad, fatwa, kesabaran dan musyawarah yang perlu diambil iktibar. 4. Pendapat para fuqaha. Fuqaha ialah orang yang terlibat dalam menggali hukum yang praktis dari sumber-sumber dan dalil-dalil agama. Di antara hukum yang berkaitan dengan dakwah seperti hukum amar-ma’ruf dan nahi mungkar, jihad, hisbah dan semua ini mereka susun dalam suatu bab tertentu dalam kitab –kitab fikah. Oleh itu, para pendakwah harus mengambil pandangan mereka terutama hukum yang berkaitan ibadat dan muamalat. 4. Pendapat para fuqaha. Fuqaha ialah orang yang terlibat dalam menggali hukum yang praktis dari sumber-sumber dan dalil-dalil agama. Di antara hukum yang berkaitan dengan dakwah seperti hukum amar-ma’ruf dan nahi mungkar, jihad, hisbah dan semua ini mereka susun dalam suatu bab tertentu dalam kitab –kitab fikah. Oleh itu, para pendakwah harus mengambil pandangan mereka terutama hukum yang berkaitan ibadat dan muamalat. 5. Pengalaman. Pengalaman adalah guru terbaik bagi mereka yang selalu bergaul dengan masyarakat. Pengalaman pendakwah bergaul dengan orang ramai merupakan teori-teori yang difahami dari sumber-sumber terdahulu dan memperbetulkan kesalahan berdasarkan pengalaman yang lalu agar tidak berulang. Oleh itu, pendakwah perlu mengambil manfaat dari pengalamannya sendiri dan juga pengalaman orang lain dalam pendekatan berdakwah (Abdul Karim Zaidan, 1992: 413). Abdul Karim Zaidan juga menjelaskan perkara yang dapat memudahkan pendakwah beriltizam dengan manhaj dan metode dakwah ialah: i. Ilmu dan kefahaman yang mendalam terhadap manhaj dengan selalu memerhati, memikir dan memahami manhaj yang disebut dalam sumber- sumber yang telah diterangkan sebelum ini. i. Ilmu dan kefahaman yang mendalam terhadap manhaj dengan selalu memerhati, memikir dan memahami manhaj yang disebut dalam sumber- sumber yang telah diterangkan sebelum ini. ii. SUMBER METODOLOGI DAKWAH Ketakwaan kerana takwa dapat menyinari hati pendakwah mencerdaskan fikiran dalam memandang yang hak itu dengan lebih jelas dan nyata. iii. Sentiasa menyerahkan diri kepada Allah dan memohon agar diberi ilmu pengetahuan dan fikiran yang cerdas ii. Ketakwaan kerana takwa dapat menyinari hati pendakwah mencerdaskan fikiran dalam memandang yang hak itu dengan lebih jelas dan nyata. iii. Sentiasa menyerahkan diri kepada Allah dan memohon agar diberi ilmu pengetahuan dan fikiran yang cerdas. iv. Membersihkan diri dari segala penyakit riya’ dan menggantikannya dengan sifat ikhlas kepada Allah sehingga tidak ada di dalam hatinya keinginan kepada manusia, pangkat dan kemegahan (Zaidan, 1992: 418). Sementera itu, Abdul Karim Zaidan menjelaskan uslub dan pendekatan dakwah seperti berikut : Sementera itu, Abdul Karim Zaidan menjelaskan uslub dan pendekatan dakwah seperti berikut : i. Mengenal pasti penyakit penerima dakwah dan mengenal ubat bagi penyakit tersebut. Penyakit utama manusia ialah jahilnya mengenai Tuhan, ingkar kepada Allah dan tidak mengakui dirinya sebagai hamba Allah dan tidak mengikuti ajaran yang dibawa nabi Muhammad SAW. Oleh itu, ubatnya ialah menjelaskan akidah yang sebenar, menjelaskan asas-asas ajaran agama dan melakukan amar makruf dan nahi mungkar. g ii. Menghilangkan syubhat yang timbul sama ada syubhat yang berhubung dengan diri peribadi pendakwah dengan tuduhan dan fitnah sebagai sesat, g ii. Menghilangkan syubhat yang timbul sama ada syubhat yang berhubung dengan diri peribadi pendakwah dengan tuduhan dan fitnah sebagai sesat, 243 العبقري جملة Vol. 23 (1), 2020 Journal al-‘Abqari bodoh dan gila; syubhat yang berkaitan dengan dakwah dengan mengatakan tuduhan membuat sesuatu yang baru dan keluar dari adat nenek moyang; syubhat yang dengan penerima dakwah dengan membangkitkan rasa benci kepada dakwah, mementingkan kepentingan diri dan menanamkan keyakinan kepada agama nenek moyang mereka. bodoh dan gila; syubhat yang berkaitan dengan dakwah dengan mengatakan tuduhan membuat sesuatu yang baru dan keluar dari adat nenek moyang; syubhat yang dengan penerima dakwah dengan membangkitkan rasa benci kepada dakwah, mementingkan kepentingan diri dan menanamkan keyakinan kepada agama nenek moyang mereka. Targhib dan tarhib. Perkara yang dimaksudkan dengan targhib (memberi kegembiraan) ialah semua perkara yang dapat menarik penerima dakwah untuk menerima dakwah dan tetap dengan kebenaran sementara tarhib (memberi peringatan) ialah segala yang dapat menimbulkan rasa takut sekiranya tidak menerima atau menolak kebenaran atau tidak istiqamah setelah menerima kebenaran. Antara bentuk targhib dan tarhib ialah memperingatkan orang dengan nikmat yang telah diperolehi kerana itu hendaklah mereka berterima kasih dan mentaati Allah. SUMBER METODOLOGI DAKWAH Antara bentuk kewaspadaan ialah waspada dari kemaksiatan, waspada terhadap keluarga 244 Metodologi Dakwah Menurut Abdul Karim Zaidan (1917-2014): Dalam Bukunya Usul Da’wah dan anak, waspada mengikuti hawa nafsu, waspada terhadap orang yang kafir dan munafik. Metodologi Dakwah Menurut Abdul Karim Zaidan (1917-2014): Dalam Bukunya Usul Da’wah dan anak, waspada mengikuti hawa nafsu, waspada terhadap orang yang kafir dan munafik. dan anak, waspada mengikuti hawa nafsu, waspada terhadap orang yang kafir dan munafik. ii. Meminta bantuan orang lain. Para pendakwah diharuskan meminta bantuan kepada orang yang berpengalaman dan petah bercakap dalam menyampaikan dakwah. Begitu juga para pendakwah harus meminta bantuan dari kaum muslimin untuk memelihara dari gangguan orang yang ingin mengancamnya dan mencegahnya dari menyampaikan dakwah. Pendakwah juga dibenarkan meminta bantuan dari orang bukan Islam untuk memberi perlidungan dan keamanan kepada dirinya, menyingkirkan segala gangguan dan memberi kesempatan untuk melaksanakan tugas dakwah. ii. Meminta bantuan orang lain. Para pendakwah diharuskan meminta bantuan kepada orang yang berpengalaman dan petah bercakap dalam menyampaikan dakwah. Begitu juga para pendakwah harus meminta bantuan dari kaum muslimin untuk memelihara dari gangguan orang yang ingin mengancamnya dan mencegahnya dari menyampaikan dakwah. Pendakwah juga dibenarkan meminta bantuan dari orang bukan Islam untuk memberi perlidungan dan keamanan kepada dirinya, menyingkirkan segala gangguan dan memberi kesempatan untuk melaksanakan tugas dakwah. iii. iii. Peraturan dan Disiplin. Para pendakwah amat memerlukan kepada peraturan dan disiplin agar dapat menyampaikan dakwah dengan lebih baik. Pendakwah perlu berdisiplin dengan waktu dengan membahagikan masa kepada beberapa waktu dan menetapkan waktu tersebut untuk dirinya, keluarga, masyarakat dan dakwah. Begitu juga apabila dakwah dilakukan secara jamaah dan organisasi, maka ia memerlukan peraturan dan disiplin. Antara asas-asas peraturan organisasi yang perlu dipatuhi oleh pendakwah ialah berkaitan kepimpinan ketua dan ketaatan kepadanya dan sentiasa bermusyawarah (Zaidan,1992: 448-468) Wasilah dalaman ialah menyampaikan dakwah kepada Allah melalui perkataan, perbuatan dan akhlak yang mulia. Wasilah dalaman ialah menyampaikan dakwah kepada Allah melalui perkataan, perbuatan dan akhlak yang mulia. Wasilah dalaman ialah menyampaikan dakwah kepada Allah melalui perkataan, perbuatan dan akhlak yang mulia. 1. Dakwah dengan perkataan. Ia merupakan asas dalam berdakwah kerana al- Quran itu sendiri mengandungi makna-makna dakwah yang merupakan kata- kata Allah yang telah diturunkan kepada nabi Muhammad SAW. Antara syarat-syarat dalam dakwah dengan perkataan dan pembicaraannya ialah hendaklah perkataan itu jelas dan terang serta difahami oleh pendengar. Kata- katanya tidak terlalu cepat, sentiasa cermat dan perlahan agar pendengar memahami maksud yang hendak disampaikan. SUMBER METODOLOGI DAKWAH Tarhib pula dengan mengancam mereka bahawa nikmat itu akan dicabut apabila mereka enggan dan tidak menerima dan kufur dengan nikmat Allah. Di samping itu akan mendapat azab seksaan yang pedih di Akhirat. iii. Targhib dan tarhib. Perkara yang dimaksudkan dengan targhib (memberi kegembiraan) ialah semua perkara yang dapat menarik penerima dakwah untuk menerima dakwah dan tetap dengan kebenaran sementara tarhib (memberi peringatan) ialah segala yang dapat menimbulkan rasa takut sekiranya tidak menerima atau menolak kebenaran atau tidak istiqamah setelah menerima kebenaran. Antara bentuk targhib dan tarhib ialah memperingatkan orang dengan nikmat yang telah diperolehi kerana itu hendaklah mereka berterima kasih dan mentaati Allah. Tarhib pula dengan mengancam mereka bahawa nikmat itu akan dicabut apabila mereka enggan dan tidak menerima dan kufur dengan nikmat Allah. Di samping itu akan mendapat azab seksaan yang pedih di Akhirat. iv. Pendidikan dan Pembelajaran. Apabila penerima dakwah telah menerima dakwah dan mendapat hidayah, maka kewajipan pendakwah mendidik dan mengajar mereka dengan asas-asas ajaran agama kerana di dalam diri mereka masih banyak penyakit yang perlu dibersihkan seperti penyakit syirik dan sebagainya. Para pendakwah bukan sahaja mendidik tetapi juga memberi pengajaran yang mendorong mereka untuk melaksanakan ajaran agama. Oleh itu, pendidikan agama merupakan perkara yang amat diperlukan dalam memahami ajaran Islam (Zaidan, 1992: 421-447). Seterusnya Abdul Karim Zaidan membicarakan wasilah dakwah. Perkara yang dimaksudkan wasilah ialah segala yang membantu para pendakwah dalam menyampaikan dakwah secara berkesan. Ia terbahagi kepada dua iaitu wasilah luaran dan wasilah dalaman. Wasilah luaran mempunyai tiga aspek iaitu kewaspadaan, memohon bantuan orang lain dan disiplin. Sementara wasilah dalaman merangkumi dakwah dengan perkataan, dakwah dengan perbuatan dan dakwah dengan akhlak yang baik. Wasilah luaran terbahagi kepada: i. Kewaspadaan iaitu seorang yang teliti atau berhati-hati serta bersiap sedia menghadapi segala cabaran dan ancaman yang akan menimpa dirinya dari orang yang benci kepadanya. Para pendakwah harus waspada dalam menyampaikan dakwah di tengah-tengah masyarakat agar tidak mendatangkan kebinasaan dan menghalang jalan dakwahnya. Antara bentuk kewaspadaan ialah waspada dari kemaksiatan, waspada terhadap keluarga i. Kewaspadaan iaitu seorang yang teliti atau berhati-hati serta bersiap sedia menghadapi segala cabaran dan ancaman yang akan menimpa dirinya dari orang yang benci kepadanya. Para pendakwah harus waspada dalam menyampaikan dakwah di tengah-tengah masyarakat agar tidak mendatangkan kebinasaan dan menghalang jalan dakwahnya. SUMBER METODOLOGI DAKWAH Pendakwah juga harus menjauhi kata-kata yang mengandungi ancaman, pemaksaan dan penghinaan kepada penerima dakwah sebaliknya menggunakan kata-kata yang lemah lembut serta mudah difahami. Antara bentuk perkataan seperti khutbah dan pidato, pengajian, ceramah dan diskusi serta dialog. Antara kaedah dalam pelaksanaan amar ma’aruf dan nahi munkar ialah: i. Pendakwah harus mengetahui ilmu dan hakikat maaruf dan mencegah mungkar terlebih dahulu sebelum melaksanakan tanggungjawab tersebut. ii. Melakukan dengan cara yang lemah lembut dan bukan dengan cara yang kasar. iii. Melihat tentang kebaikan dan keburukan hasil dari pelaksanaan tersebut. Umpamanya sekiranya mencegah 245 Journal al-‘Abqari العبقري جملة Vol. 23 (1), 2020 Journal al-‘Abqari keburukan akan menimbulkan keburukan yang lebih besar lagi, maka tidaklah wajib bagi pendakwah melarangnya keburukan akan menimbulkan keburukan yang lebih besar lagi, maka tidaklah wajib bagi pendakwah melarangnya keburukan akan menimbulkan keburukan yang lebih besar lagi, maka tidaklah wajib bagi pendakwah melarangnya iv. Apabila kebaikan dan kemungkaran itu bercampur dalam diri seseorang atau jamaah, maka pendakwah harus melihat yang mana yang lebih utama sama ada menyuruh melakukan kebaikan atau mencegah mereka berbuat mungkar v. Pendakwah melakukan sekadar kuasanya dan tidaklah dibebankan melainkan apa yang mampu dilakukan. 2. Berdakwah dengan perbuatan iaitu menyingkir perkara mungkar dengan perbuatan atau mendorong orang berbuat baik seperti mendirikan masjid, sekolah dan sebagainya atau sesuatu perbuatan yang dapat menunjang pelaksanaan syariat Islam. Antara syarat untuk melakukan pencegahan kemungkaran itu ialah mengenal pasti kebaikan dan kejahatan sebelum mengambil tindakan dan memperhitungkan akibat-akibat yang mungkin timbul berikutan tindakan tersebut. Di samping itu, ia harus memiliki kemampuan dan kesanggupan diri untuk mengubahnya. Pendakwah juga harus mempunyai sikap benci terhadap perkara mungkar dan melakukannya sesuai dengan perintah agama. 3. Akhlak yang baik dari pendakwah yang dapat memikat hati-hati penerima dakwah. Akhlak yang baik yang menjadi contoh kerpada orang lain apabila ia telah memenuhi dua syarat iaitu akhlak yang baik dan bersesuian dengan perkataan dan perbuatan. Apabila dua dasar ini ada dalam diri pendakwah, maka segala tindak tanduk dan perbuatan merupakan pendekatan terbaik dalam menarik orang lain kepada agama Islam (Zaidan, 1992: 470-486). Ada juga yang membahagikan pendekatan dakwah kepada penulisan, pendengaran dan penglihatan. Perkara yang dimaksudkan dengan dakwah melalui penulisan merangkumi penulisan buku, artikel, penyelidikan, buletin dan pengkisahan. Sementara pendekatan dakwah dengan percakapan dan pendengaran merangkumi seminar, ceramah, forum, khutbah, pengajian dan dialog. Sementara pendekatan dakwah dengan percakapan, endengaran dan penglihatan adalah dakwah melalui televisyen dan komunikasi baru seperti melalui internet, filem dan cinema. SUMBER METODOLOGI DAKWAH Bentuk dakwah juga boleh dilakukan melalui aktiviti teater, minggu dakwah, penubuhan perpustakaan, radio Islam, penerbitan akhbar dan majalah Islam dan organisasi dakwah. Kini para pendakwah perlu memanfaatkan teknologi baru dalam penyampaian dakwah melalui media baru seperti laman web, facebook dan instagram. Hal ini kerana dalam dunia ICT, elemen media baru memberi ruang dan peluang yang cukup besar dalam berdakwah dengan lebih pantas. 246 Metodologi Dakwah Menurut Abdul Karim Zaidan (1917-2014): Dalam Bukunya Usul Da’wah Penguasaan yang dimaksudkan ialah kemampuan para pendakwah untuk menarik orang ramai dan memperkembangkan keupayaan mereka sekalipun mereka itu terdiri dari berbagai bentuk mentaliti, kecenderungan, status dan penguasaan ilmu yang tidak sama. Manusia secara umum tidak sama dalam semua perkara, sama ada dalam cara berfikir, status kehidupan, kecenderungan, kecerdasan otak dan kemampuan emosi dan imaginasi. Pendakwah yang berjaya ialah pendakwah yang mampu mengharung dan mempengaruhi manusia dengan fikrah dan dakwahnya, sekalipun mereka itu terdiri dari berbagai tabiat dan status. Pendakwah yang berjaya ialah pendakwah yang mampu menarik ramai orang dan menguasai mereka dari sudut fikrah dan harakah (Fathi, 1992: 9). Dengan penjelasan ini, "penguasaan" membawa pengertian kemampuan peribadi, keahlian di sudut akhlak, sifat imaniah dan anugerah Allah yang dapat menolong para pendakwah dan menjadikan mereka sebagai menara hidayah dalam masyarakat mereka. Ia juga dapat menjadikan mereka pusat tumpuan bagi orang yang berada di sekeliling mereka dan mampu menarik orang ramai. Pada hakikatnya kemampuan untuk "menguasai" dianggap sebagai syarat kelayakan yang pertama dan terpenting bagi peribadi seorang pendakwah. Tanpa kemampuan "penguasaan" ini pendakwah belum wujud dan dakwah juga belum berlaku (Fathi, 1992:10). PENGUASAAN LUARAN DAN DALAMAN Penguasaan luaran ialah menguasai mereka yang di luar dakwah, gerakan dan organisasi iaitu tahap sebelum bergabung ke dalam organisasi. Manakala penguasaan dalaman pula membawa erti menguasai mereka yang telah berada dalam organisasi iaitu menguasai mereka yang telah diorganisasikan dan telah menggabungkan diri ke dalam amal-amal Islam dan gerakan. Kedua-dua bidang penguasaan ini saling menyempurnakan antara satu sama lain. Kedua-duanya amat penting dan memerlukan kemampuan penguasaan yang tinggi. Kejayaan dakwah amat bergantung kepada syarat memiliki penguasaan kedua-dua bidang ini kerana tidak ada ertinya penguasaan luaran jika tidak disertai dengan penguasaan dalaman (Fathi, 1992. :14). Penguasaan luaran bermaksud membawa dakwah kepada orang ramai dan menjadikan mereka mengimani, mempercayai dan terpengaruh dengannya yang kemudian akan mendorong mereka menggabungkan diri ke dalamnya, bekerja, berjihad dan berkorban merupakan proses yang terlalu sukar dan susah. la memerlukan kemampuan dan tuntutan-tuntutan yang berbagai bentuk. Siapa yang memiliki sebahagian besar darinya, beliau adalah pendakwah yang mendapat taufik, berjaya dan mampu menguasai orang ramai dan menarik mereka satu demi satu ke dalam Islam dan ke dalam dakwah. Para pendakwah yang memiliki syarat-syarat penguasaan ini akan memberi peluang yang luas untuk menguasai orang ramai dan pengaruh dakwah terhadap mereka semakin banyak (Fathi, 1992:15). Antara ciri-ciri dan syarat-syarat seperti berikut: 247 العبقري جملة Vol. 23 (1), 2020 Vol. 23 (1), 2020 Journal al-‘Abqari العبقري جملة i. Memahami Agama Allah. ii. Qudwah Hasanah (Keteladanan yang ba iii. Sabar iv. Lemah Lembut dan Belas Kasihan. v. Memudahkan bukan Menyusahkan. vi. Tawaduk dan Merendahkan Diri. vii. Manis Muka dan Manis Tutur kata viii. Memberi khidmat kepada orang lain i. Memahami Agama Allah. iv. Lemah Lembut dan Belas Kasihan. v. Memudahkan bukan Menyusahkan. vi. Tawaduk dan Merendahkan Diri. vii. Manis Muka dan Manis Tutur kata viii. Memberi khidmat kepada orang lain KESIMPULAN Abdul Karim Zaidan telah merumuskan manhaj yang perlu ada pada diri para pendakwah agar dakwah yang dilakukan itu berkesan dan berjaya menarik orang ramai kepada Islam. Sumber metodologi dakwah ialah al-Quran, al-Sunnah, sejarah hidup para Sahabat, pendapat dan pandangan para fuqaha, serta pengalaman pendakwah itu sendiri. Perkara yang dimaksudkan wasilah ialah segala yang membantu para pendakwah dalam menyampaikan dakwah secara berkesan. Ia terbahagi kepada dua iaitu wasilah luaran dan wasilah dalaman. Wasilah luaran mempunyai tiga aspek iaitu kewaspadaan, memohon bantuan orang lain dan disiplin. Sementara wasilah dalaman merangkumi dakwah dengan perkataan, dakwah dengan perbuatan dan dakwah dengan akhlak yang baik. Oleh itu, para pendakwah yang berjaya perlu memahami asas-asas, manhaj serta wasilah dakwah yang yang telah dikemukakan oleh Abdul Karim Zaidan dalam bukunya ini. RUJUKAN Abu Daud, Sulaiman bin As’ath al-Sajastani. (T.th). Sunan Abu Daud. Beirut: Dar al- Kitab al-Arabi Abu Faris, Muhamammd Abdul Qadir.(1992). Usus fi al-da’wah wa wasa’il nashriha. Amman: Dar al-Furqan. RUJUKAN Abu Daud, Sulaiman bin As’ath al-Sajastani. (T.th). Sunan Abu Daud. Beirut: Dar al- Kitab al-Arabi Kitab al-Arabi Abu Faris, Muhamammd Abdul Qadir.(1992). Usus fi al-da’wah wa wasa’il nashriha. Amman: Dar al-Furqan. Abu Faris, Muhamammd Abdul Qadir.(1992). Usus fi al-da’wah wa wasa’il nashriha. Amman: Dar al-Furqan. Al-Aqil, Abdullah Sulaiman. (2002). Min ‘alam al-da’wah wa harakah al-Islamiah al-mu’asarah. Kaherah: Dar al-Tauzi’ wa Nashr al-Islamiyyah. Al-Bukhari, Muhammad bin Ismail Abu Abdullah. (1987). Al-Jami’ al-sahih al- mukhtasar (al-Jami’ al-sahih). (Peny.Mustafa al-Bugha). Beirut: Dar Ibn Kathir. Al-Buti, Muhammad Said Ramadan. (1990). Kubra al-yaqiniyyat al-kawniyyat. Damsyik: Dar al-Fikr ________. (2011). al-Hubb fi al-Quran. Damsyik: Dar al-Fikr. Al-Ghazali, Muhamad. (1992). Kayfa Nata‘amal ma‘a al-Qur’an. Kaherah: Dar al- Wafa’, Kaherah. Al-Humaidi, Muhammad bin Futuh. (2002). al-Jam’u bayn al-Bukhari wa Muslim. Beirut: Dar Ibn Hazm. Al-Qaradawi, (1989). Al-Sabr fi al-Qur’an. Kaherah: Maktabah Wahbah. , ( ) y Beirut: Dar Ibn Hazm. Al-Qaradawi, (1989). Al-Sabr fi al-Qur’an. Kaherah: Maktabah Wahbah. 248 odologi Dakwah Menurut Abdul Karim Zaidan (1917-2014): Dalam Bukunya Usul Da’wa (1992) A l i t l h k h l I l i h fl l h l h Metodologi Dakwah Menurut Abdul Karim Zaidan (1917-2014): Dalam Bukunya Usul Da wah ________. (1992). Awlawiyat al-harakah al-Islamiah fl al-marhalah ________. (1992). Awlawiyat al-harakah al-Islamiah fl al-marhalah _____. (1992). Awlawiyat al-harakah al-Islamiah fl al-marhalah al- qadimah. Kaherah: Maktabah Wahbah. ________. (2009). Thaqafah al-Da‘iyyah. Kaherah: Maktabah Wahbah. al qadimah. Kaherah: Maktabah Wahbah. ________. (2009). Thaqafah al-Da‘iyyah. Kaherah: Maktabah Wahbah. q . (2009). Thaqafah al-Da‘iyyah. Kaherah: Maktabah Wahbah. Al-Turmidhi, Muhammad bin Isa (peny. Ahmad Muhammad Syakir dan al-Albani). (T.th). al-Jami’ al-sahih sunan al-Turmidhi. Beirut: Dar al-Ihya’ al-Turath. Al-Wa’iyy, Taufiq Yusuf. (2006). Kubra al-jamaat al-Islamiyyat al-islahiyat fi al-‘alam al-mu’asir. Mansurah: Mu’assasah al-Shuruq. al-‘alam al-mu’asir. Mansurah: Mu’assasah al-Shuruq. Humaidi, Muhammad Futuh. (2002). Jam’u bayn al-sahian al-Bukhari wa Muslim. Beirut: Dar Ibn Hazam. Ibn Majah. (T.th). Sunan Ibn Majah (Pen. Muhamad Fuad Abd Baqi dan al-Albani). Beirut: Dar al-Fikr. Kalimat al-‘Alamah al-Qaradawi fi Ta’bin ‘Abd al-Karim Zaydan. Retrieved from https://www.al-qaradawi.net/node/857 (akses 20 Ogos 2018). Muslim, Abu Husein.t.t. Al-Jami’ al-sahih al-musamma sahih Muslim.Beirut: Dar al- Jil. Sirah al-Zatiyyah al-Shaykh. Retrieved from https://drzedan.com/content.php?id=24 (akses 10 Ogos 2018). Ulwan, Nasih. (2010). Salsilah madrasah al-du’at. Kaherah: Dar al-Salam. Yakan, Fathi. (1988). Madha ya’ni intima’ li al-Islam. Beirut: Mu’assasah al- Risalah. ________. (2010). Tasawwur asasi gerakan Islam. Shah Alam: Dewan Pustaka Islam. Islam. Zaidan, Abdul Karim. (1986).Majmu’ah buhuth fiqhiah. Beirut: Muassasah al- Risalah. ________. (1987). Al-Wajiz fi usul al-fiqh. RUJUKAN Beirut: Mu’assasah al-Risalah. ________. 1988). Al-Ahkam al-Zimmiyyin wa Musta’minin fi Dar al-Islam. Beirut : Mu’assasah al-Risalah. ________. 1990). Madkhal li dirasat al-syari’at al-Islamiah. Beirut: Mu’assasah al- Risalah. ________. (1992). Usul al-da’wah. Kaherah: Dar al-Wafa’ ________. 1994). Mufassal fi ahkam al-mar’ah wa a-bayt al-Muslim. 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https://openalex.org/W3211392239	https://europepmc.org/articles/pmc8575958?pdf=render	English	null	Sensing small interaction forces through proprioception	Scientific reports	2,021	cc-by	9,465	OPEN Fazlur Rashid1, Devin Burns2 & Yun Seong Song1* Understanding the human motor control strategy during physical interaction tasks is crucial for developing future robots for physical human–robot interaction (pHRI). In physical human–human interaction (pHHI), small interaction forces are known to convey their intent between the partners for effective motor communication. The aim of this work is to investigate what affects the human’s sensitivity to the externally applied interaction forces. The hypothesis is that one way the small interaction forces are sensed is through the movement of the arm and the resulting proprioceptive signals. A pHRI setup was used to provide small interaction forces to the hand of seated participants in one of four directions, while the participants were asked to identify the direction of the push while blindfolded. The result shows that participants’ ability to correctly report the direction of the interaction force was lower with low interaction force as well as with high muscle contraction. The sensitivity to the interaction force direction increased with the radial displacement of the participant’s hand from the initial position: the further they moved the more correct their responses were. It was also observed that the estimated stiffness of the arm varies with the level of muscle contraction and robot interaction force. Beyond traditional robots that perform isolated tasks away from human operators1–4 , future robots are expected to be physically closer to the users and perform interactive tasks5–8. In particular, robots that can physically inter- act with humans through direct contact have the potential to assist the human workforce in various scenarios, such as in healthcare, manufacturing, or education9–12. For example, the foreseen shortage of physical therapists and nurses amplifies the necessity for the development of effective and intuitive physical Human–Robot Inter- action (pHRI). Robots may provide physical assistance to patients like human therapists would for effective movement assistance and rehabilitation10–15.if In order to advance pHRI, however, it is crucial to first understand the underlying mechanism of effective physical interaction from the perspective of human users16.Indeed, humans are experts of physical human–human interaction (pHHI) such as while hand-shaking17,18, walking together16,19,20, or jointly carrying loads21,22. In many pHHI tasks, humans coordinate their movements together, not through verbal communication or visual feed- back, but through the interaction forces acting at their arms and hands20. www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports \| (2021) 11:21829 1Department of Mechanical and Aerospace Engineering, Missouri University of Science and Technology, Rolla, MO 65401, USA. 2Department of Psychological Science, Missouri University of Science and Technology, Rolla, MO 65401, USA. email: songyun@mst.edu OPEN This physical communication between partners can lead to improved performance in the absence of explicitly shared motor goals19,23–27, a distinction of skill levels20 or roles28, or even motor adaptation29–31. These information-carrying interaction forces are typically 20 N or less20,32, are usually kept below 10 N33,34, and can sometimes be as low as 1 N35. It would then be required of the humans in physical interaction tasks to be sensitive to the small changes in the interaction forces for better motor communication with the partner. p How, then, do human partners remain sensitive to small interaction forces during physical interaction tasks? One possibility is through the mechanoreceptors distributed at the site of the physical coupling, typically through holding of hands16,19,23,25. However, these receptors may not be suitable for detecting subtle changes in the interac- tion forces due to the high preload of grip forces that is crucial to maintain a stable physical coupling36,37. That is, the small changes in the interaction forces could be below the Weber fraction (< 10 %) of the pre-existing stimuli on the pressor receptors (grip force), making them unreliable for detecting interaction forces38,39. If they relied only upon these mechanoreceptors, humans would have to loosen their grip for reliable motor communication at the cost of unreliable physical coupling. p y p g Alternatively, proprioceptors on muscles, tendons, and joints may help detect the small interaction forces through the resulting kinematic displacements of the arm40,41.The interaction force at the hand will create the corresponding movement of the upper and lower arm, which then creates length changes in the muscles and tendons that are sensed by muscle spindles and/or Golgi tendon organs (GTOs)42,43. Even in certain interaction tasks where there is little arm movement (such as in20), small movements can create muscle length changes above 1Department of Mechanical and Aerospace Engineering, Missouri University of Science and Technology, Rolla, MO 65401, USA. 2Department of Psychological Science, Missouri University of Science and Technology, Rolla, MO 65401, USA. email: songyun@mst.edu Scientific Reports \| (2021) 11:21829 \| https://doi.org/10.1038/s41598-021-01112-w www.nature.com/scientificreports/ Figure 1. Percentage of correct responses varies with the level of muscle contraction where correctness was maximum (~ 100%) at high robot force with low muscle contraction (RHML) condition (a) experimental results (different colors represent different participants) (b) ANOVA analysis of the percentage of correct responses. Figure 1. OPEN Percentage of correct responses varies with the level of muscle contraction where correctness was maximum (~ 100%) at high robot force with low muscle contraction (RHML) condition (a) experimental results (different colors represent different participants) (b) ANOVA analysis of the percentage of correct responses. Table 1. Fixed effects of the percentage of correct responses using linear mixed model fit by REML and t tests use Satterthwaite’s method (Response ~ Robot.forceMuscle.contraction + (1 \| Participants)). p < 0.05, p < 0.01, p < 0.001. Estimate Std. Error df (degree of freedom) t value Pr(>\|t\|) Intercept 0.9896 0.02 102.92 45.51 p < 0.001 RL − 0.1188 0.03 137.00 − 4.17 p < 0.001* MH − 0.1917 0.03 137.00 − 6.73 p < 0.001*** RLMH − 0.3313 0.04 137.00 − 8.23 p < 0.001** Table 1. Fixed effects of the percentage of correct responses using linear mixed model fit by REML and t tests use Satterthwaite’s method (Response ~ Robot.forceMuscle.contraction + (1 \| Participants)). p < 0.05, p < 0.01, p < 0.001. the Weber fraction. In this view, small changes in the interaction forces may be detected by the proprioceptors, as long as the arm stiffness is low enough to allow detectable movement in response to the small interaction force. To this end, this work is aimed at investigating the physical interaction strategy in humans for effective motor i i h h ll i i f I i l hi k i i h ff f h f h the Weber fraction. In this view, small changes in the interaction forces may be detected by the proprioceptors, as long as the arm stiffness is low enough to allow detectable movement in response to the small interaction force. f To this end, this work is aimed at investigating the physical interaction strategy in humans for effective motor communication through small interaction forces. In particular, this work investigates the effect of the state of the arm in the sensitivity to the information provided by the interaction force from an external source. The hypothesis is that humans are more sensitive to the direction of the subtle push on their palm when the arm is displaced more as a consequence of the push. Supporting observations may indicate the presence of a specific pHHI/pHRI strategy that the humans modulate their arm stiffness to improve sensitivity to small interaction forces. Results Sensitivity to interaction forces is affected by the robot force as well as muscle contraction levels. Out of a total of 1920 trials among 20 participants, the number of correct responses was 1443 and the number of incorrect responses was 477 trials (including the number of no-response: 255). Sensitivity to interaction forces is affected by the robot force as well as muscle contraction levels. Out of a total of 1920 trials among 20 participants, the number of correct responses was 1443 and the number of incorrect responses was 477 trials (including the number of no-response: 255). p ( g p ) Among the 4 possible combinations of conditions, the sensitivity to the interaction force direction was high- est when the applied force was high and the muscle contraction was low (RHML, average 99.0%), and lowest in RLMH (average 34.8%, Fig. 1a). It was observed that with a low level of robot interaction force and low level of muscle contraction (RLML) condition, the percentage of correct responses was comparable (average 87.1%) with high robot interaction force and high muscle contraction (RHMH, average 79.8%) condition, where in both RLML and RHMH, the sensitivity was higher than in RLMH and lower than in RHML. These trends were statistically significant where applying lower robot interaction force (RLMH and RLML conditions) decreased the sensitivity by 11.88% (p < 0.001), whereas high muscle contraction (RHMH and RLMH condi- tions) decreased the sensitivity by 19.17% (p < 0.001, Fig. 1b). The combined effect of the low robot interaction force (RL) and the high muscle contraction (MH) was also significant, decreasing the sensitivity of the interaction force by an additional 33.13% (p < 0.001). These results are summarized in Table 1. High radial displacement of the hand increases the sensitivity to small interaction forces. The radial displacement of the hand from the center (initial position) in each trial was strongly correlated with the sensitivity of the interaction force direction (Fig. 2a). The radial displacement was the highest during the RHML condition (red), during which the chance to make correct responses was also the highest. As the radial displace- ments are lower in RLML and RHMH trials, the chance of correct responses was also lower. The radial dis- placement was the smallest in the RLMH condition where the least correct responses were made. Linear regres- https://doi.org/10.1038/s41598-021-01112-w Scientific Reports \| (2021) 11:21829 \| www.nature.com/scientificreports/ (a) (b) Figure 2. (a) Percentage of correct responses increases with radial displacement from the initial position and was highest during high robot interaction force with low muscle contraction (RHML, red) condition, during which the radial displacement was also high. Results Sensitivity to interaction forces is affected by the robot force as well as muscle contraction levels. Out of a total of 1920 trials among 20 participants, the number of correct responses was 1443 and the number of incorrect responses was 477 trials (including the number of no-response: 255). Including participant vari- ability, the logarithmic radial displacement of all trials with correct and incorrect responses was 2.270 ± 0.430 mm (out of 1443 trials) and 1.876 ± 0.302 mm (out of 477 trials), respectively. Paired sample t test showed a difference of 0.40 ± 0.26 mm with a large effect size (Cohen’s D = 1.54).hff sion showed a correlation of R2 = 0.228 between the percentage of correct responses and the radial displacement in a logarithmic scale. In addition, the radial displacement was higher in trials with correct responses than in tri- als with incorrect responses after removing participant variability (p < 0.001, Fig. 2b). Including participant vari- ability, the logarithmic radial displacement of all trials with correct and incorrect responses was 2.270 ± 0.430 mm (out of 1443 trials) and 1.876 ± 0.302 mm (out of 477 trials), respectively. Paired sample t test showed a difference of 0.40 ± 0.26 mm with a large effect size (Cohen’s D = 1.54).hff f gf ( ) Then, a generalized linear mixed-effects model was used to analyze the items that affect the correctness of the response in a trial-by-trial manner, where the binomial outcome measure was whether participants responded correctly on that particular trial. These tests were conducted using the "lmerTest’’ package for R.44 .We included fixed effects for the robot interaction force (two levels), muscle contraction (two levels), the direction of the push from the robot (one variable for X/Z and another for +/−), and logarithmic radial displacement on that particular trial, with a random intercept for participants (allowing for different overall sensitivity) and by-participant ran- dom slopes for muscle contraction (allowing the effect of contraction to differ across participants). Table 2 shows that the sensitivity to the direction of the force was reduced by low robot interaction force (RL, p < 0.001), high muscle contraction (MH, p < 0.001), and with Z-direction movement (DZ, p = 0.04), as indicated by the negative estimates and the corresponding odds ratios below 1 (where {odd ratio} = exp (estimate)). The participants were only 12% as likely to be correct in RL trials than in RH trials, only 6% as likely to be correct in MH trials than in ML trials, and only 77% as likely to be correct in the Z direction movements than in the X direction. Results Sensitivity to interaction forces is affected by the robot force as well as muscle contraction levels. Out of a total of 1920 trials among 20 participants, the number of correct responses was 1443 and the number of incorrect responses was 477 trials (including the number of no-response: 255). Linear regression fit gives R2 = 0.228 (b) mean and standard deviation of logarithmic radial displacement of all correct response trials (c) was higher than incorrect response trials (I) (p < 0.001). (b) (a) (b) Figure 2. (a) Percentage of correct responses increases with radial displacement from the initial position and was highest during high robot interaction force with low muscle contraction (RHML, red) condition, during which the radial displacement was also high. Linear regression fit gives R2 = 0.228 (b) mean and standard deviation of logarithmic radial displacement of all correct response trials (c) was higher than incorrect respons trials (I) (p < 0.001). (a) (b) (a) Figure 2. (a) Percentage of correct responses increases with radial displacement from the initial position and was highest during high robot interaction force with low muscle contraction (RHML, red) condition, during which the radial displacement was also high. Linear regression fit gives R2 = 0.228 (b) mean and standard deviation of logarithmic radial displacement of all correct response trials (c) was higher than incorrect response trials (I) (p < 0.001). Table 2. Fixed effects of all parameters using generalized linear mixed model fit by maximum likelihood in a trial-by-trial manner (Response ~ Robot.force + Muscle. contraction+Direction+Dimension+LogDisplacement+(Muscle contraction \| Participant)) p < 0 05 p < Estimate Std. Error z value Odds ratio Pr(>\|z\|) (Intercept) 3.65 0.42 8.76 p < 0.001 RL − 2.10 0.15 − 14.06 0.12 p < 0.001* MH − 2.85 0.30 − 9.54 0.06 p < 0.001* Dir+ 0.52 0.13 3.91 1.68 p < 0.001* DZ − 0.27 0.13 − 2.03 0.77 0.04* LogD 0.27 0.11 2.36 1.31 0.02* Table 2. Fixed effects of all parameters using generalized linear mixed model fit by maximum likelihood in a trial-by-trial manner (Response ~ Robot.force + Muscle. contraction + Direction + Dimension + LogDisplacement + (Muscle.contraction \| Participant)). p < 0.05, p < 0.01, *p < 0.001. y p contraction + Direction + Dimension + LogDisplacement + (Muscle.contraction \| Participant)). p < 0.05, p < 0.01, p < 0.001. sion showed a correlation of R2 = 0.228 between the percentage of correct responses and the radial displacement in a logarithmic scale. In addition, the radial displacement was higher in trials with correct responses than in tri- als with incorrect responses after removing participant variability (p < 0.001, Fig. 2b). Results Sensitivity to interaction forces is affected by the robot force as well as muscle contraction levels. Out of a total of 1920 trials among 20 participants, the number of correct responses was 1443 and the number of incorrect responses was 477 trials (including the number of no-response: 255). On the other hand, the sensitivity was increased by positive direction pushes (Dir+, in X+ or Z+ directions, p < 0.001) and larger radial displacement (LogD, p=0.02), as indicated by the positive estimates and the corresponding odds ratios above 1. The participants were 1.68 times more likely to be correct in X+ or Z+ directions than in X− or Z− directions, and were 1.31 times more likely to be correct for every unit increase (1 log (mm)) in the loga- rithmic radial displacement. The number of observations in this analysis was 1920 (20 participants x 96 trials). Unlike the ANOVA analysis presented in Table 1, the interaction between the robot force and the muscle contraction was not significant in this analysis (and thus excluded from the final model), suggesting that it may ph y p p Unlike the ANOVA analysis presented in Table 1, the interaction between the robot force and the muscle contraction was not significant in this analysis (and thus excluded from the final model), suggesting that it may have been a statistical artifact caused by the ceiling effect of near-perfect accuracy in the RHML condition. https://doi.org/10.1038/s41598-021-01112-w Scientific Reports \| (2021) 11:21829 \| www.nature.com/scientificreports/ Estimate Std. Error df (degree of freedom) t value Pr( >\|t\|) Intercept 245.26 25.26 34.44 9.71 p < 0.001* RL − 58.47 18.70 58.00 − 3.13 0.003 MH 181.56 18.70 58.00 9.71 p < 0.001*** Table 3. Fixed effects of stiffness norm using linear mixed model fit by REML and t tests use Satterthwaite’s method (Stiffness ~ Robot.force + Muscle.contraction + (1 \| Participant)). p < 0.05, p < 0.01, *p < 0.001. Table 3. Fixed effects of stiffness norm using linear mixed model fit by REML and t tests use Satterthwaite’s method (Stiffness ~ Robot.force + Muscle.contraction + (1 \| Participant)). p < 0.05, p < 0.01, p < 0.001. Table 4. Estimated average stiffness values of all 20 participants. Stiffness (N/m) Conditions RHMH RHML RLMH RLML Kxx 312.81 205.11 263.67 156.11 Kxz − 8.36 −17.13 17.72 − 8.02 Kzx 21.86 − 20.67 26.30 − 10.93 Kzz 371.85 207.15 305.01 158.98 Table 4. Estimated average stiffness values of all 20 participants. Figure 3. Norm of the arm stiffness increases with the increase of muscle contraction and robot interaction force. Results Sensitivity to interaction forces is affected by the robot force as well as muscle contraction levels. Out of a total of 1920 trials among 20 participants, the number of correct responses was 1443 and the number of incorrect responses was 477 trials (including the number of no-response: 255). The average stiffness was highest during high robot interaction force with high muscle contraction (RHMH) condition (a) experimental results (different colors represent different participants) (b) ANOVA analysis of the stiffness norm. Figure 3. Norm of the arm stiffness increases with the increase of muscle contraction and robot interaction force. The average stiffness was highest during high robot interaction force with high muscle contraction (RHMH) condition (a) experimental results (different colors represent different participants) (b) ANOVA analysis of the stiffness norm. Estimated arm stiffnesses depend on both robot force and muscle contraction levels. The estimated arm stiffness was dependent on the experimental conditions (Fig. 3a). The norms of the 2 × 2 stiffness matrices computed from the force–displacement relationship after 3 s were averaged across trials and participants for the four conditions. The average arm stiffness norm was the lowest in the RLML condition (167.13 N/m) and highest in the RHMH condition (372.95 N/m), with intermediate values in RHML (225.07 N/m) and RLMH conditions (314.57 N/m). The stiffness was higher in RH than in RL, and in MH than in ML. These trends were statistically significant (p < 0.001, Fig. 3b and Table 3). The low robot interaction force (RL) reduced the arm stiffness norm by − 58.47 N/m, whereas the high muscle contraction (MH) increased the arm stiffness norm by 181.56 N/m. The estimated 2 × 2 stiffness matrices are provided in Table 4, which shows the character- istics of typical arm stiffness matrices with low off-diagonal terms36,45. www.nature.com/scientificreports/ In contrast, during low muscle contraction (0~20% MVC) grip force was less than 5 N, so 1 N of applied force should be at least a 20% change. However, detecting a change in cutaneous pressure is not the only way to sense the applied force. As force is applied to the hand, the joints in the arms are displaced as a result, unless the human body is completely rigid which is impossible. This displacement is picked up by the proprioception on muscles and tendons (ex. the Golgi-tendon organs and/or the muscle spindle), which are known to sense kinematics. That is, forces at the hands may be sensed as the arm is displaced as a result, regardless of whether the applied force is above the detec- tion threshold of the cutaneous pressure receptors. To our knowledge, this work is the first to provide evidence for this alternative force sensing through proprioception. This is especially relevant in scenarios in which the handgrip must be tight to ensure the security of the mechanical coupling between the two partners (ex. providing balance assistance during walking). Indeed, our experiment suggests that the participants may be utilizing these kinematic sensors as an effective force sensor. It was observed that the sensitivity of the interaction force direction was higher when the radial displacement/movement of human arms were larger. In addition, the sensitivity of the interaction force was higher when the muscle contraction was low that reduced the pressure on the palm. In this view, participants may have sensed the direction of the interaction force by sensing the displacement/ movement of their arms and/or from the changes in the pressure on their palm when the grip was not tight (ML conditions). When the grip was very tight (MH), however, the hand-robot handle coupling between the human and the robot served mainly as a mechanical connection that allowed the interaction force to generate arm displacements which are eventually sensed by the proprioceptors. This view is consistent with the recent observation that the muscle spindles may encode forces during stretch49. Although there are two parallel sources of information for force sensitivity (cutaneous force sensor and proprioception), this experiment was designed to emphasize a scenario where the mechanoreceptors on the skin may not be effective due to tight handgrip and thus proprioception would be more important (ex. overground physical interaction tasks). www.nature.com/scientificreports/ However, vari- ous other scenarios could exist where the relative importance of these channels would vary (or even negatively interfere). To further investigate this, future experiments may alter the sensations through the two channels. For example, the arm could be fixed to oblige the participants to use the cutaneous pressure receptors on the palm, allow softer grip, provide steeper changes in the interaction forces, or allow self-selection of arm postures (ex.44) to study the effect of joint configurations to force sensitivity. yf ji g y In this experiment, all participants were asked to sense the direction of interaction force through the senses in their arms and hands without any visual feedback. In all four combinations of conditions, the participant’s arms were displaced from the initial position in X and Z-directions due to the applied interaction force from the robot handle. Participants were stiffer in the Z-direction (Kzz > Kxx) than X-direction and correspondingly, the sensitivity was lower in the Z-directions (+Z, −Z) than X-directions (+X, −X). The observation that the stiff- ness in Z direction was higher than in the X direction is consistent with prior findings45. Participants were also more sensitive in the positive directions (+X, +Z) than in the negative directions (-X, −Z). While this effect is significant, the biomechanical basis for this observation is unknown. It was observed that human arm displace- ment was higher during low muscle contraction conditions (ML) in which the arms are estimated to be less stiff than in the high muscle contraction conditions (MH) as shown in Fig. 3b. This implies a relationship between the arm stiffness and the force sensitivity50. The arm displacement was the smallest in the low robot interaction force and high muscle contraction (RLMH) condition. The movement of human arms increased at a higher robot interaction force than low robot interaction force for the same level of muscle contraction. This observation is analogous to the results in51 where bigger force is easier to discriminate and promote haptic communication between humans. On the other hand, participants’ ability to sense the interaction force direction was high when the arm dis- placement was also high - which occurred when the estimated arm stiffness was low. For example, the sensitivity of the direction of interaction force was higher in the RLML condition than the RLMH condition. Discussion Forces applied to the hand may be sensed by the respective force sensors at the hand, such as the cutaneous pressure receptors on the palm. However, when the pressure on the palm is high due to a strong hand grip, the cutaneous pressure sensors on the skin may suffer from decreased sensitivity to small changes in the pressure, since our ability to detect a change in pressure (the Just Noticeable Difference, or JND) tends to be about 8-10% of the current stimulus intensity. This makes the detection of small interaction forces to be less effective through the pressure sensors on the hands in our MH conditions where the co-contraction of the forearm muscles increases the grip force. Indeed, the approximate grip force for the high muscle contraction (70-80% MVC) was 20–30 N36,46–48, meaning that a 1 N applied force is an increase of only 3-5%, likely below the threshold for detection. Scientific Reports \| (2021) 11:21829 \| https://doi.org/10.1038/s41598-021-01112-w www.nature.com/scientificreports/ www.nature.com/scientificreports/ However, it is emphasized once again that a direct measure of the interaction force was not available in this research, and thus the reported arm stiffness is not a direct measurement. Therefore, further research is required to find the variation of endpoint stiffness with different levels of the interaction force.f iff Better pHRI may be possible by lowering the stiffness of the robot arm to mimic the characteristics of the human arms. Effective pHRI begins from a better understanding of how human participants communicate movement intentions with their partners through the physical coupling. It has been suggested that humans can effectively guide other humans by hand by using interaction forces to communicate intentions during walking16,19,20,handshaking17,18, etc. In this work, it was suggested that humans are more sensitive to the interac- tion forces when their arm stiffness is lower. Humans may expect their partner’s arm stiffness to be lower because it is natural and advantageous for them to communicate through interaction forces. If so, in pHRI, the humans may also expect their robot partners to have a compliant, low-stiffness arm, rather than a stiff and sluggish arm. A low-stiffness robot arm may be regarded as more human-like. Additionally, compliant participants (low arm stiffness) were more sensitive than stiffer participants (high arm stiffness) for the same level of interaction forces (RH or RL)50.Therefore, it could be speculated that this is at the basis of reduced sensitivity in participants with a pathologically high level of limb stiffness, as for spastic patients.hift f There are a number of valuable additional benefits of a low-stiffness robot arm. For example, a soft, easy-to- manipulate robot arm is less likely to be a safety threat to a human partner52,53 and may help the subjective quality of the pHRI to improve. This may be especially important in healthcare applications where a robot may interact with frail populations. Also, to provide low stiffness, a robot arm may be designed using smaller actuators or power sources to reduce development cost and the overall size of the robot.fh Note, however, that robots do not require low stiffness for increased force sensitivity. Their sensors (elec- tromechanical transducers) do not suffer from the same reduced sensitivity at a higher force that is common in human perception. Hence, if all the robot needs is to sense the interaction force from the human partner, its arm impedance is irrelevant. Experimental setup. 20 Experimental setup. 20 healthy young adults (19 males and 1 female), 18 to 35 years of age (22.1 ± 4.025 years) without a self-reported history of neuromuscular injuries or disorders participated in this study. All par- ticipants reported themselves to be right-handed. The experimental protocol and procedure were approved and in accordance with relevant guidelines and regulations of the institutional review board (IRB) of the University of Missouri. All participants gave their written informed consent and were free to withdraw their participation at any time. The hypothesis and the experiment design are preregistered in the open science foundation (OSF: osf.io/qr785).h q The experiment involved externally applied interaction forces to the hand of a seated participant as he/she relaxed or contracted their lower arm muscles. All participants were seated in a rigid chair to keep their back against the chair at all times. Shoulder straps were used to help maintain their posture as depicted in Fig. 4a,b 55. Using their right hand, participants grabbed the handle of a haptic robot (Phantom Premium 1.5/6 DOF-HF, 3D Systems, Rock Hill, SC, USA) in front of them as shown in Fig. 4b. The right arm was posed such that the distance from their sternum to the right hand was approximately 30% of their arm length, with the shoulder abduction angle of ~71°, shoulder horizontal flexion of ~45°, elbow flexion angle of ~ 90°, and the forearm and wrist in its neutral position (~0°)55,56. The strength of the grip was inferred by the level of activity of the hand- grip muscles on the forearm46,57–59 using single-channel electromyography (Muscle SpikerShield Bundle model #V2.61, Backyard Brains, Inc. MI, USA) above the forearm flexor muscle group. A high grip force was identified as 70–80% of the maximum voluntary contraction (MVC) of the forearm muscles, whereas a Low grip force was identified as 0~20% of MVC in such a way that there was a clear distinction between high and low grip forces. i Because the participants were asked to maintain their posture at all times, the contraction of the forearm flexor muscle groups was accompanied by a co-contraction of the whole forearm muscles. A haptic robot was used to apply interaction force in two different magnitudes in one of four directions to a seated participant as shown in Fig. 4. www.nature.com/scientificreports/ The low arm stiffness of the robot would be for the benefit of the human partner, and not as much for itself. However, the role of arm stiffness is not restricted to a single goal. It is well known that humans increase their arm stiffness to improve stability45,54. For tasks that require both high sensitivity to interaction forces (low stiffness) as well as good stability (high stiffness), sophisticated modulation of arm stiff- ness may be required. The central nervous system (CNS) may make swiftly switching decisions on when to stiffen or loosen an arm, depending on the state of the interaction at that moment. Neither consistently high nor low arm stiffness may be ideal for any given scenario, which warrants further research on arm stiffness modulation on complex tasks.hf p This work was mainly inspired by the necessity to implement intuitive and effective pHRI. It is suggested that low muscle contraction may help increase the sensitivity to the small interaction forces, which may contain movement intentions of the partner, by allowing higher arm displacements to occur. The results of this work imply that the lower robot arm stiffness or human arm muscle contraction may be the desired characteristics of pHRI and pHHI. The findings of this experiment can be used to guide the design of a robot for physical interac- tion tasks with a human. www.nature.com/scientificreports/ Hence, with the same level of robot interaction force in the same specific posture of the human arm, the sensitivity of the interaction force direction varied depending on the level of muscle contraction/stiffness/muscle activation level of the human arm. In addition, for the same specific posture, experimental trials where the arm was stiffer (less displacement) were less likely to be correct than trials with low arm stiffness (high displacement). Overall, human arm movement was related to the correctness of the interaction force/sensitivity of the interaction force direction. Hence, humans may benefit from lowering their arm stiffness as it would help them to increase the displacement of the arm, allowing even small interaction forces to be detected.hf The estimated magnitudes (norms) of the 2 × 2 stiffness matrices are smaller when the robot force was low (RL) and higher in RH, despite the fact that the muscle contraction levels were kept similar, as shown in Fig. 3b and Table 3 (p < 0.003). A possible explanation for this phenomenon is that it is due to the well-known nonlinear force-displacement relationship of skeletal muscles. Given a nonlinear force-displacement curve originating from a specific level of muscle contraction and posture, the slope (stiffness) of the curve is high for high robot interac- tion force and low for lower robot interaction force. As a consequence, a linear approximation of the arm stiffness would be lower with low robot interaction force. That is, even if the participants did not modulate their muscle contraction level (%MVC), the arm stiffness may be estimated differently depending on the applied force level. f yf y g Nonetheless, there still is a possibility of voluntary modulations of the muscle contraction by the participant, due to the inherently variable muscle activity recordings that cannot completely rule out such cases. In this regard, a possible alternative explanation to the lower stiffness in RL conditions is that there may be an unmeasured lowering of muscle contraction in RL conditions, intentionally or otherwise, so as to be more sensitive to the low level of interaction force. This lowering of the arm stiffness may not have occurred as prominently in the RH conditions since the higher interaction forces are easier to detect even without lowering the arm stiffness to take advantage of the proprioception. https://doi.org/10.1038/s41598-021-01112-w Scientific Reports \| (2021) 11:21829 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 4. Seated human posture during the experiment with a haptic robot (a) experimental setup (b) top view of the experimental setup (c) applied robot interaction force profile for a trial of ~ 5 s. Figure 4. Seated human posture during the experiment with a haptic robot (a) experimental setup (b) top view of the experimental setup (c) applied robot interaction force profile for a trial of ~ 5 s. the intensity of that information. The magnitude of the force was controlled in an open-loop manner where the appropriate motor torque profiles were commanded to the robot by the experimenter. The position of the robot handle (which is also the position of the participant’s hand) was measured by the encoders of the robot joints. The resolution of the handle position sensing at the configuration shown in Fig. 4a was ~10−6 m. There was no dead zone in the system. the intensity of that information. The magnitude of the force was controlled in an open-loop manner where the appropriate motor torque profiles were commanded to the robot by the experimenter. The position of the robot handle (which is also the position of the participant’s hand) was measured by the encoders of the robot joints. The resolution of the handle position sensing at the configuration shown in Fig. 4a was ~10−6 m. There was no dead zone in the system. Experimental protocol. The aim of this study was to find what affects the sensitivity of the interaction force during physical interactions in humans. In this experiment, participants were asked to identify if the direc- tion of push as the robot provided the interaction force at the hands. Participants were blindfolded to encourage them to focus on the sensation at their hands to identify the direction of the push. y p At each trial, the robot-provided interaction force was either high (2 N) or low (1 N), and the grip on the robot handle was either high (70~80% of MVC) or low (0~20% of MVC). It can be considered a low robot interaction force as RL, high robot interaction force as RH, low muscle contraction as ML, and high muscle contraction as MH. www.nature.com/scientificreports/ Therefore, there were four different conditions in the experiment such as high robot interaction force with high muscle contraction (RHMH), high robot interaction force with low muscle contraction (RHML), low robot interaction force with high muscle contraction (RLMH), and low robot interaction force with low muscle contraction (RLML). Participants had no knowledge of the intensity setting of the interaction force in any particular trial. Each time the force was applied, participants were asked to identify whether the direction of the interaction force towards them (+Z), away from them (−Z), to their right (+X), or left (−X), while maintaining their pose (maintained the initial abduction angle, shoulder horizontal flexion angle, elbow flexion angle, and the forearm and wrist posi- tion). Indeed, the participant’s hand moved in response to the applied robot interaction force. They were allowed to give the response at any time during the 5-second period of the force application. Participants’ responses were recorded as correct, incorrect, or no-response, where they either declared that they could not identify the direction correctly or if they failed to provide a response within 5-s. For each of the four conditions (RHMH, RHML, RLMH, and RLML), there were 6 pushes in each of the four directions (+Z, −Z, +X, and −X), with a total of 96 trials in each experiment session. All 96 trials were equally randomized in the directions and intensity of the interaction forces as well as in the levels of muscle contraction. To avoid muscle fatigue, the randomized sequence of trials was checked to ensure that there were no more than four consecutive high-MVC trials. Also, mandatory ~1 min breaks were provided during the experiment (after each of the four consecutive MH trials). Each trial lasted approximately 10 s. In addition to the correctness of the response, the radial displacement of the hand as a result of the interaction force was recorded throughout the 5-s in all trials. Data processing and analysis. For each trial, the measurement included the response (correct, incorrect, or no-response) and the maximum radial displacement (1) R = max dx(t)2 + dz(t)2 , t = [0, 5] (1) where dx and dz are the displacements of the handle in the X and Z directions with respect to its initial position t t = 0.f Human arm stiffness was also estimated in the experiment by considering the applied robot interaction force and the resulting hand displacement. Experimental setup. 20 The robot applied a force that gradually increased from 0 → 1 N (Low) or 0 → 2 N (High) over a 5-second duration in such a way that the maximum level of interaction force was reached ~3 s (Fig. 4c). The slow increase in the force was to avoid stretch reflex56. After ~3 s the level of interaction force remained maxi- mum constant value (2N or 1N) until ~5 s when a single trial was ended as presented in Fig. 4c. Then, the level of interaction force remained constant until 5 s. The robot provided this force to the hand in one of the four directions on a horizontal plane (+X, −X, +Z, or −Z directions, Fig. 4b). The direction of the interaction force was the target information that the robot provided to the human, whereas the level of the interaction force was https://doi.org/10.1038/s41598-021-01112-w Scientific Reports \| (2021) 11:21829 \| References 1. Hartley, J., Robots at Work: A practical Guide for Engineers and Managers (North Holland, 1983) 1. Hartley, J., Robots at Work: A practical Guide for Engineers and Managers (North Holland, 1983) S l T l f C l k f S l b l d G d b O S 1. 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Nishihara, S. et al. www.nature.com/scientificreports/ While direct measurement of the interaction force was not available, the commanded robot interaction force was used as an approximation of the interaction force value, from which the 2-dimensional endpoint stiffness of the arm was estimated through the following procedure36,45,60:hf f The quasi-static stiffness of the arm is related to the interaction forces and the hand displacement such that Scientific Reports \| (2021) 11:21829 \| https://doi.org/10.1038/s41598-021-01112-w www.nature.com/scientificreports/ Fx Fz = Kxx Kxz Kzx Kzz dx(t) dz(t) , t = 3 sec (2) where Fx and Fz are the robot interaction force in the X and Z-direction; dx (t) and dz (t) are the displacements in X and Z, respectively, Kxx, Kxz, Kzx, and Kzz are the elements of the 2-dimensional stiffness matrix. To avoid dynamic effects, measurements at 3 s were used. Then, the stiffness elements Kxx, Kxz, Kzx, and Kzz were deter- mined for each participant for each of the four conditions (RHMH, RHML, RLMH, and RLML) by using the linear least square regression method. However, the magnitude of the velocities of the hand (=robot handle) at ~3 s for all 1960 trials was small (Vx: 1.39 ± 1.23 mm/sec and Vz: 1.49 ± 1.21 mm/s). Therefore, we can assume that at ~3 s, the hand is quasi-static and only the stiffness component of the arm dynamics is present at that time.if f A two-way measure of analysis of variance (ANOVA) was used to find the effect of the robot interaction force and muscle contractions on the measurement of the sensitivity of the interaction force direction. A generalized linear mixed-effects model was also used to analyze the data in a trial-by-trial manner, where the binomial outcome measure was whether participants responded correctly on that particular trial. For this, no-response was considered as an incorrect response. This analysis included fixed effects of robot interaction force, muscle contraction, motion direction (X/Z and +/−), and the logarithm of the maximum radial displacement on that particular trial, with a random intercept for participant and by-participant random slopes for muscle contraction. The maximum radial displacement was transformed to its logarithmic value due to the skewness and kurtosis of the raw data sets. Received: 31 January 2021; Accepted: 22 October 2021 Received: 31 January 2021; Accepted: 22 October 2021 References Clinical accuracy evaluation of femoral canal preparation using the ROBODOC system. J. Orthop. Sci. 9(5) 452–461 (2004). 2. Honl, M. et al. Comparison of robotic-assisted and manual implantation of a primary total hip replacement: A prospective study JBJS 85(8), 1470–1478 (2003). 13. Dukelow, S. P. et al. Quantitative assessment of limb position sense following stroke. Neurorehabil. Neural Repair 24(2), 178–187 (2010). 14. Farris, R. J. et al. 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https://openalex.org/W2464563061	https://link.springer.com/content/pdf/10.1007/s10433-016-0388-x.pdf	English	null	Comparing two frailty concepts among older people with intellectual disabilities	European journal of ageing	2,016	cc-by	11,821	Eur J Ageing (2017) 14:63–79 DOI 10.1007/s10433-016-0388-x Eur J Ageing (2017) 14:63–79 DOI 10.1007/s10433-016-0388-x ORIGINAL INVESTIGATION Comparing two frailty concepts among older people with intellectual disabilities Josje D. Schoufour1,2 • Michael A. Echteld1 • Heleen M. Evenhuis1 Published online: 24 June 2016 The Author(s) 2016. This article is published with open access at Springerlink.com Abstract In general, disabilities are considered a conse- quence of frailty rather than a cause of frailty, whereas in people with intellectual disabilities (ID), disabilities are often lifelong, which could have consequences for the feasibility and validity of frailty instruments. To better understand frailty in people with ID, we compared two broadly used concepts: the frailty phenotype (FP) and the frailty index (FI) taking into account their feasibility (e.g., percentage of participants able to complete the frailty assessments), agreement, validity (based on 5-year mor- tality risk), inﬂuence of motor disability, and the relation between single frailty variables and mortality. The FI and an adapted version of the FP were applied to a represen- tative dataset of 1050 people with ID, aged 50 years and over. The FI was feasible in a larger part of the dataset (94 %) than the adapted FP: 29 % for all ﬁve items, and 81 % for at least three items. There was a slight agreement between the approaches (j = 0.3). However deﬁned, frailty was related with mortality, but the FI showed higher discriminative ability and a stronger relation with mortal- ity, especially when adjusted for motor disabilities. Con- cluding, these results imply that the used FI is a stronger predictor for mortality and has higher feasibility than our adaptation of the FP, in older people with ID. Possible explanations of our ﬁndings are that we did not use the exact FP variables or that the FI includes multiple health domains, and the variables of the FI have lower sensitivity to lifelong disabilities and are less determined by mobility. Keywords Frailty Frailty index Frailty phenotype Survival Intellectual disability Keywords Frailty Frailty index Frailty phenotype Survival Intellectual disability & Josje D. Schoufour j.schoufour@erasmusmc.nl Responsible editor: D. J. H. Deeg. 2 Department of Epidemiology, Erasmus Medical Center, PO box 2040, 3000 CA Rotterdam, The Netherlands 1 Intellectual Disability Medicine, Department of General Practice, Erasmus Medical Center, PO box 2040, 3000 CA Rotterdam, The Netherlands Introduction Frailty is a complex cascade that involves several age-re- lated physiological alterations, eventually leading to loss of function and failure to respond to a stressor event (Clegg et al. 2013). The frailty ‘phenotype’ by Fried et al. (2001), and the frailty index (FI) developed by Rockwood and Mitnitski (Mitnitski et al. 2001; Rockwood and Mitnitski 2007) are the most evaluated and most frequently used measures, representing two different concepts and deﬁni- tions of frailty (Bouillon et al. 2013). y The frailty phenotype was operationalized as a biolog- ical syndrome, based on a cluster of symptoms that are commonly observed in frail older people including unin- tended weight loss, low grip strength, exhaustion, slow gait speed, and low physical activity (Fried et al. 2001). The underlying concept is the cycle of frailty, based on age- related physiological changes, including low energy expenditure, nutritional deﬁciencies, and sarcopenia. Because the frailty phenotype has a clear underlying eti- ology, it is, although correlated to, distinct from disabilities and chronic disease (Fried et al. 2004). The frailty phe- notype distinguishes between three different frailty states, based on the number of symptoms present in a person. If none of the symptoms are present, a person is classiﬁed as non-frail, one or two marks a person as pre-frail, and frailty is deﬁned as the presence of three or more. The frailty Responsible editor: D. J. H. Deeg. Responsible editor: D. J. H. Deeg. & Josje D. Schoufour j.schoufour@erasmusmc.nl 1 Intellectual Disability Medicine, Department of General Practice, Erasmus Medical Center, PO box 2040, 3000 CA Rotterdam, The Netherlands 2 Department of Epidemiology, Erasmus Medical Center, PO box 2040, 3000 CA Rotterdam, The Netherlands 12 Eur J Ageing (2017) 14:63–79 64 phenotype can relatively easily be used as a clinical assessment for frailty, using clearly described cut-off values. to complete the frailty assessments. Second, validity was assessed. Because there is no gold standard for frailty, validity is usually based on criterion validity, in this case the relation with adverse health outcomes. We therefore calculated the relation between frailty and 5-year survival. Third, in addition to general feasibility and criterion validity, we were interested in the predictive value of the single items that are part of the frailty phenotype and the FI in order to ﬁnd risk factors that highly contribute to the mortality risk of people with ID. Study design and participants This study was part of the HA-ID study. This study addressed the health of 1050 older people with ID in the Netherlands. Details about the recruitment and selection process have been described elsewhere (Hilgenkamp et al. 2011). Brieﬂy, the study sample consisted of clients, aged 50 years and over, from three Dutch care provider services offering a broad spectrum of care and support to people with ID. All clients aged 50 years and over (N = 2322) were invited to participate. Eventually 1050 clients, or their legal representatives, provided informed consent, forming a nearly representative study sample for the Dutch popu- lation of older adults (aged 50 and above) with ID who use formal care, albeit with a slight underrepresentation of men, people aged 80 and over, and people living inde- pendently. Ethical clearance was provided by the Medical Ethics Committee of the Erasmus Medical Center Rotter- dam (MEC 2008-234) and by the ethics committees of the participating care organizations. The study followed the guidelines of the Declaration of Helsinki. The two approaches agree that, as a consequence of multisystem deterioration, frailty is an age-related state of vulnerability to adverse health outcomes (Theou and Rockwood 2015). Nevertheless, because of different underlying conceptualizations and operationalizations, it has been suggested not to compare the two instruments but rather consider them as complementary to one other (Ce- sari et al. 2014). Even so, to better understand frailty in populations with different characteristics, it can be helpful to compare different concepts. Previous comparisons showed that the FI has a somewhat stronger relation with negative health outcomes than the phenotype approach (Theou and Rockwood 2015). In people with intellectual disabilities (ID), insights into how frailty originates, develops, and affects health out- comes are currently speculative. Results from the general population cannot directly be applied to this group, because of their lifelong disabilities (cognitive, motor, and sensory) and chronic comorbidity, which may inﬂuence both the development and the consequences of frailty. Moreover, muscle function and mobility play a central role in the frailty phenotype, whereas these frailty characteristics could be lifelong in people with ID and highly determine the frailty status (Evenhuis et al. 2013). Introduction Last, because lifelong motor disabilities can highly determine the frailty status, we evaluated the inﬂuence of motor disability on the frailty status. The FI operationalizes frailty quantitatively as an aspeciﬁc accumulation of health deﬁcits in multiple domains, and can include health variables such as signs, diseases, disabilities, laboratory abnormalities, and symp- toms as long as they are not too common or too rare, generally increase with age, and together cover several health domains. In order to ﬁnd an FI that best captures the risk for adverse health outcomes, it has been suggested to also include disabilities and diseases (Theou et al. 2012). The FI-score is a continuous value between 0 and 1. Because of the continuous nature of the FI, it is possible to study individual changes over time, as frailty is usually an irregular trajectory inﬂuenced by stress and recovery (Mitnitski et al. 2012). On the other hand, on a population level, the FI shows very regular characteristics including an exponential relation with age (Mitnitski et al. 2013). 123 Data collection Baseline data were collected between February 2009 and July 2010 within three main themes: (1) physical activity and ﬁtness, (2) nutrition and nutritional state, and (3) mood and anxiety. Within these themes the participants under- went an extensive diagnostic assessment including a physical assessment, a ﬁtness test battery, several ques- tionnaires (regarding, e.g., nutrition, depression, disabili- ties), and laboratory tests in addition to the collection of health record data. Data on age, gender, and residential status were collected through the care provider services. Level of ID was obtained from the scores determined by psychologists or test assistants from available IQ tests. The A better understanding of the different concepts of frailty in people with ID can help improve the under- standing of frailty in people with ID and better understand how people with ID age. Furthermore, it provides insight and direction for a future-screening instrument for frailty in this lifelong disabled population. The main aim of our study was therefore to compare the feasibility and validity of the frailty phenotype and the frailty index in older adults with ID. First, feasibility was assessed by evaluating the percentage of participants able 123 65 Eur J Ageing (2017) 14:63–79 falling, listless, grip strength, HDL cholesterol, and knowing which year it is. An overview of all the deﬁcits included in the FI is provided in Table 4. All items were recorded between 1 (presence of the deﬁcit) and 0 (absence of the deﬁcit). The FI-score was calculated as the total number of deﬁcits present as a proportion of those counted (e.g., 12 deﬁcits in a 51-item FI results in an FI of 12/51 = 0.24). In the case of missing data, the deﬁcit was removed from both the numerator and the denominator, but at least 30 deﬁcits were required per individual. For the sake of direct comparisons with Fried phenotype, we used several previously identiﬁed cut points. Because using cut points for the FI is generally not advised and cut-offs are arbitrary, we applied three different, previously used, cut- offs for the FI. First of all, an FI of less than 0.2 was considered as non-frail or ‘robust,’ a score between 0.2 and 0.35 as ‘pre-frail,’ and a score above 0.35 as ‘frail’ (Kul- minski et al. 2008; Rockwood et al. 2007). Data collection Second, an FI- score of B0.08 was considered non-frail, a score between 0.08 and 0.25 as pre-frail and a score equal to or higher than 0.25 as frail, in accord with prior studies (Rockwood et al. 2007; Rockwood et al. 2004; Song et al. 2010). Third, we classiﬁed participants as non-frail if FI B0.10, frail if FI C0.21, and pre-frail if the score was between 0.10 and 0.21, identiﬁed using stratum speciﬁc likelihood ratios (Hoover et al. 2013). diagnosis of Down syndrome was retrieved from medical ﬁles. Up to March 2015, all-cause mortality data (time of death) were collected through the care organizations. Frailty phenotype Previously, an adapted version of the frailty phenotype was applied using the criteria of the Cardiovascular Health Study (Evenhuis et al. 2012; Fried et al. 2001). According to the original criteria, an individual should be classiﬁed as frail if at least three of the following ﬁve are present: weight loss, weakness, slowness, low physical activity, and poor endurance or exhaustion. Brieﬂy, weight loss was deﬁned as losing more than 3 kg within 3 months. Weak- ness was assessed using a handgrip dynamometer. Slow- ness was assessed using comfortable walking speed, measured as the average of three recordings of the time to complete a distance of 5 m. Participants in a wheelchair and participants unable to perform the walking test due to physical limitations were also classiﬁed as having a slow walking speed. Low physical activity was deﬁned as walking fewer than 5000 steps/day measured with pedometers (NL-1000; New Lifestyles, Lees Summit, MO). Participants in a wheelchair and participants unable to perform the test due to physical limitations were also classiﬁed as having low physical activity (less than 5000 steps/day). Exhaustion was deﬁned as answering ‘moderate problem’ or ‘severe problem’ to the ‘lacks energy’ item from the Anxiety, Depression, and Mood Scale (Esbensen et al. 2003). Additional information about the used frailty phenotype variables and the originally intended frailty phenotype criteria are provided in Table 5. Individuals with one or two criteria present were classiﬁed as pre-frail. Individuals with no criteria present were classiﬁed as non-frail or ‘robust.’ At least three out of ﬁve criteria needed to be known before the frailty phenotype could be applied. Statistical analysis First, baseline characteristics (gender, age, level of ID, presence of Down syndrome), the mean FI-score and the percentage of non-frail, pre-frail, and frail participants were provided as the percentage for categorical variables and the mean (with SD) for continuous variables. The prevalence of each item of the phenotype was provided. Second, the feasibility of the instruments was analyzed by calculating the percentage of participants able to complete the frailty assessments. A non-response analysis was per- formed to compare the participants with and without completed data for frailty, using a Pearson Chi-square for categorical data and ANOVA for continuous data. Third, the Cohen’s Kappa statistic was used to estimate agreement between the instruments. For this analysis, the categorized FI was compared with the frailty phenotype (e.g., non-frail, pre-frail, frail). Agreement was considered as poor for Kappa values lower than 0.21, slight for 0.21–0.40, mod- erate for 0.41–0.60, good for 0.61–0.80, and excellent for values 0.81–1 (Cohen 1960). Fourth, the ability to predict 5-year all-cause mortality was calculated for both instru- ments and compared to each other. The hazard ratio’s (HR) for mortality were calculated for the frailty phenotype and for the categorized FI in separate Cox regression models. Dummy variables were composed for the pre-frail and frail Frailty index Because we used a stepwise entry of the variables, leading to different predictor sets for the various imputation sets, pooling of the results was impossible. We therefore provide the results of the 10th imputation set. Fifth, the inﬂuence of motor disability was assessed by including motor disability into a Cox regression model. Feasibility Less than a third of the participants (n = 307, 29 %) could complete the full frailty phenotype assessment as intended. 40 % (n = 419) had four completed assessments, 12 % (n = 122) had three completed assessments, and 19 % (n = 202) had less than three completed assessments. By including all participants with at least three known frailty phenotype criteria, the frailty phenotype could be applied to 848 (81 %) participants. Table 1 provides an overview of the feasibility of the single frailty phenotype variables. The 202 excluded participants were on average more intellectually disabled (X2 = 32.8, p \ 0.001), and had on average a higher FI-score (M = 0.31, SD = 0.12) than those included ([M = 0.27, SD = 0.13], t(982) = 3.28, p = 0.001). For other baseline characteristics, no signiﬁ- cant differences between the included and excluded par- ticipants were found. For 167 participants (17.2 %), all 51 included deﬁcits were known. In 68 (6.4 %) participants, there was too much missing data to calculate an FI. There were no signiﬁcant associations between the number of missing data and the FI-score or between the participants with a known FI-score (n = 982, 94 %) and those without, with respect to gender, age, level of ID, and Down syndrome. For all survival analyses, the data on participants who were lost to follow-up were censored and the proportional Hazards assumption was tested with the scaled Schoenfeld residuals. Statistical analyses were performed using SPSS version 20.0 and R version 3.0.0. A two-sided p value of \0.05 was considered signiﬁcant. Agreement For 838 participants, the frailty phenotype and the FI were known. The Cohen’s Kappa agreement between the three categorized FIs and the frailty phenotype ranged between 0.10 and 0.30, corresponding with poor till fair agreement (Table 2). From the three applied FI cut-off values, only the ﬁrst (non-frail \0.2; frail [0.35) showed a fair agree- ment with the frailty phenotype (Kappa agreement 0.3). The two other applied FI cut-off points showed poor agreement with the frailty phenotype (Kappa agreement 0.10 and 0.11). Each frailty phenotype variable was inde- pendently of age, level of ID, gender, and Down syndrome signiﬁcantly associated with the FI (Table 1). Frailty index We therefore provide the results of the 10th imputation set. Fifth, the inﬂuence of motor disability was assessed by including motor disability into a Cox regression model. (13 %) as frail. The mean FI-score was 0.27 (SD = 0.13). Using the ﬁrst deﬁned cut-off (non-frail \ 0.2, frail[0.35), 325 (33.1 %) participants were classiﬁed as non-frail, 392 as pre-frail (37.3 %), and 265 as frail (25.2 %) according to the FI. According to the second used cut-off (non-frail B0.08, frail C0.25), 33 (3.4 %) were non-frail, 445 (45 %) pre-frail, and 504 (51 %) frail. The third applied cut-off (non-frail B0.10, frail C0.21) classiﬁed 65 (6.6 %) par- ticipants as non-frail, 285 (29 %) as pre-frail, and 632 (64 %) as frail. groups to compare their mortality risk with the non-frail group. Additionally, the p for trend was provided. A comparative analysis was performed by including the two frailty instruments in one Cox regression model. This analysis was repeated with an FI that excluded the criteria that were also used for the frailty phenotype. In other words, the deﬁcits’ walking speed, grip strength, fatigue, and weight loss were excluded from the FI. All models were adjusted for age (years), level of ID (with dummy variables for moderate and severe/profound), gender and the presence of Down syndrome. A receiver operating characteristics (ROC) curve was constructed and the area under the ROC curve (AUC) was calculated to measure the discriminative ability of the instruments in relation to survival. These calculations were based on the Nearest Neighbor Estimator, which uses time-dependent ROC and AUC, to account for censoring (Heagerty et al. 2000). In order to ﬁnd the frailty variables that explained most variance in survival time, we calculated the HR for each FI item and for each frailty phenotype item. Additionally, we added all available frailty measures (frailty phenotype and FI) into a forward Cox Regression model. Full case anal- ysis resulted in a small and very selective group. Therefore, for this analysis, we used a multiple imputation procedure using fully conditional speciﬁcation (Markov chain Monte Carlo method) with a maximum of 100 iterations. In total, we created 10 imputed datasets using all the frailty mea- sures as predictors in addition to the baseline characteris- tics—Down syndrome, age, gender, and level of ID. Frailty index An FI was previously created with 51 baseline items from the HA-ID study (Schoufour et al. 2013). A standardized procedure was followed to develop the FI (Searle et al. 2008): all items were (1) related to health, (2) positively associated with age, (3) frequently but not too often present in the population ([5 %, \80 %), and (4) measured in at least 70 % of the participants. Furthermore, the items did not correlate too strongly with each other (r \ 0.7), and together the items covered a range of health problems (physical, psychological, and social). Deﬁcits included are, for example, mobility, calf circumferences, bathing, 12 3 Eur J Ageing (2017) 14:63–79 66 groups to compare their mortality risk with the non-frail group. Additionally, the p for trend was provided. A comparative analysis was performed by including the two frailty instruments in one Cox regression model. This analysis was repeated with an FI that excluded the criteria that were also used for the frailty phenotype. In other words, the deﬁcits’ walking speed, grip strength, fatigue, and weight loss were excluded from the FI. All models were adjusted for age (years), level of ID (with dummy variables for moderate and severe/profound), gender and the presence of Down syndrome. A receiver operating characteristics (ROC) curve was constructed and the area under the ROC curve (AUC) was calculated to measure the discriminative ability of the instruments in relation to survival. These calculations were based on the Nearest Neighbor Estimator, which uses time-dependent ROC and AUC, to account for censoring (Heagerty et al. 2000). In order to ﬁnd the frailty variables that explained most variance in survival time, we calculated the HR for each FI item and for each frailty phenotype item. Additionally, we added all available frailty measures (frailty phenotype and FI) into a forward Cox Regression model. Full case anal- ysis resulted in a small and very selective group. Therefore, for this analysis, we used a multiple imputation procedure using fully conditional speciﬁcation (Markov chain Monte Carlo method) with a maximum of 100 iterations. In total, we created 10 imputed datasets using all the frailty mea- sures as predictors in addition to the baseline characteris- tics—Down syndrome, age, gender, and level of ID. Because we used a stepwise entry of the variables, leading to different predictor sets for the various imputation sets, pooling of the results was impossible. Sample characteristics The mean age of the study sample (n = 1050) was 61.6 (SD = 8.0). Nearly half were female (n = 511, 49 %), nearly half had a moderate level of ID (n = 506, 48 %), and 14 % (n = 149) was diagnosed with Down syndrome. According to the frailty phenotype, 230 (27 %) were classiﬁed as non-frail, 508 (60 %) as pre-frail, and 110 123 Eur J Ageing (2017) 14:63–79 67 Table 1 The variables of the frailty phenotype, feasibility, and association with survival and the frailty index Association with Feasible (%) Classiﬁed as frail for this item n (%) Mortality HR (95 % CI)a Frailty index B (SE)a Grip strength 977 (93) 46 (4.4) 2.06 (1.13–3.74) 0.07 (0.06–0.09) Weight loss 725 (69) 384 (53) 1.93 (1.13–3.31) 0.08 (0.05–0.11) Exhaustion 975 (92) 171 (18) 1.95 (1.36–2.81) 0.11 (0.09–0.13) Slow walking speed 818 (78) 271 (33) 3.64 (2.31–5.75) 0.15 (0.14–0.17) Physical inactivity 422 (10) 255 (60) 5.43 (2.07–14.3) 0.14 (0.11–0.16) a HR and B are adjusted for age, gender, level of ID, and the presence of Down syndrome. The regression coefﬁcient B represents differences in absolute frailty index score (and corresponding 95 % conﬁdence interval) e, gender, level of ID, and the presence of Down syndrome. The regression coefﬁcient B represents differences in d corresponding 95 % conﬁdence interval) Table 2 Agreement among the frailty index (using different cut-off values) and the frailty phenotype based on three frailty categories n = 838a Frailty phenotype Non-frail Pre-frail Frail Total Agreement Frailty index Non-frail \0.2 151 146 2 299 0.30 Pre-frail 0.2–0.35 68 232 27 327 Frail [0.35 5 126 81 212 Total 224 504 110 838 Frailty index Non-frail B0.08 23 5 0 28 0.10 Pre-frail 0.08–0.25 162 235 8 405 Frail C0.25 39 264 102 405 Total 224 504 110 838 Frailty index Non-frail B0.10 45 12 0 57 0.11 Pre-frail 0.10–0.21 110 151 2 263 Frail C0.21 69 341 108 518 Total 224 504 110 838 a From the total HA-ID population (n = 1050), 838 had a known frailty phenotype and a frailty index score a From the total HA-ID population (n = 1050), 838 had a known frailty phenotype and a frailty index score was no association with mortality in the pre-frail and frail participants compared to the reference group. The p for trend was highly signiﬁcant. Sample characteristics Repeating the analysis with all participants with a known FI (n = 982) revealed somewhat stronger associations between the FI and mortality (Table 6). The ROC curve showed that the ﬁrst categorized FI (robust \0.25, frail [0.35) had a higher discriminative ability in relation to all-cause mortality (AUC = 0.78) than the frailty phenotype (AUC = 0.64). The second (robust B0.08, frail C0.25) and third cut values (robust B0.10, frail C0.21) had an AUC of 0.69 and 0.66, respectively. Survival Of the total HA-ID cohort (n = 1050), 207 participants died during the follow-up. Table 3 shows the HR’s for pre- frail and frail individuals, using the non-frail state as a reference group, and the p for trend across all categories. However deﬁned, frailty was signiﬁcantly related to mor- tality. Those classiﬁed as pre-frail or frail using the frailty phenotype were, respectively, 2.04 and 4.20 times more likely to die during the follow-up period than those clas- siﬁed as non-frail. Those classiﬁed as pre-frail or frail with the FI (using the\0.2 to deﬁne robust, and[0.35 to deﬁne frail participants) were, respectively, 2.27 and 10.3 times more likely to die than the non-frail group. If both instru- ments were included in one Cox regression model, the frailty phenotype no longer predicted mortality, whereas the FI did. If all frailty phenotype items were excluded from the FI, virtually the same results were obtained. Although the HR for the frailty phenotype groups slightly increased, they remained not signiﬁcant (data not shown). For the two FI scores that used the lowest cut-off of 0.08 or 0.10, two participants died in the reference group. There Almost all single frailty items were associated with survival (Tables 1, 4). Mobility-related items of the frailty phenotype (e.g., walking speed and physical activity) were more strongly associated with both mor- tality and the FI than the other items (e.g., grip strength, weight loss, and exhaustion). The forward regression analysis showed that a broad range of variables, includ- ing walking stairs, present at the day care center, panic attacks, asthma/COPD and hemoglobin, and fast fatigue, were selected as independent predictors for survival (Table 7). Survival 12 3 Eur J Ageing (2017) 14:63–79 68 Table 3 Hazard ratio’s for 5-year all-cause mortality according to the three level frailty index, using three sets of cut-off values, and the frailty phenotype Table 3 Hazard ratio’s for 5-year all-cause mortality according to the three level frailty index, using three sets of cut-off values, and the frailty phenotype Frailty measure Status N = 818 = 164 Single frailty instrument Both frailty instruments Motor disability n cat cat HR (95 % CI) Wald p HR (95 % CI) Wald p HR (95 % CI) Wald p Frailty phenotype Non-frail 221 19 Reference Trend: \0.001 Reference Trend: 0.90 Reference Trend: 0.03 Pre-frail 488 100 2.04 (1.23–3.37) 7.74 0.005 1.12 (0.65–1.92) 0.17 0.68 1.70 (1.01–2.84) 4.05 0.04 Frail 109 45 4.20 (2.39–7.39) 24.9 \0.001 1.16 (0.62–2.19) 0.22 0.64 2.31 (1.24–4.32) 6.96 0.008 Frailty index \0.2 295 19 Reference Trend: \0.001 Reference \0.001 Reference \0.001 0.2–0.35 317 44 2.27 (1.31–3.93) 8.52 0.005 2.19 (1.24–3.87) 7.20 0.007 2.22 (1.28–3.85) 7.96 0.005 [0.35 206 101 10.3 (5.97–17.9) 69.6 \0.001 9.66 (5.23–17.8) 52.5 \0.001 8.53 (4.69–15.5) 49.4 \0.001 Frailty phenotype Non-frail 221 19 Reference Trend: \0.001 Reference Trend: 0.019 Reference Trend: 0.03 Pre-frail 488 100 2.04 (1.23–3.37) 7.74 0.005 1.42 (0.84–2.38) 1.71 0.008 1.70 (1.01–2.84) 4.05 0.04 Frail 109 45 4.20 (2.39–7.39) 24.9 \0.001 2.21 (1.23–4.00) 6.93 0.02 2.31 (1.24–4.32) 6.96 0.008 Frailty index B0.08 28 2 Reference Trend: \0.001 Reference Trend: \0.001 Reference Trend: \0.001 0.08–0.25 398 32 0.94 (0.22–3.99) 0.01 0.94 0.84 (0.20–3.58) 0.06 0.81 0.96 (0.23–4.08) 0.00 0.96 C0.25 392 130 3.98 (0.95–16.7) 3.55 0.06 2.93 (0.67–12.7) 2.06 0.15 3.15 (0.74–13.4) 2.42 0.12 Frailty phenotype Non-frail 221 19 Reference Trend: \0.001 Reference Trend: 0.001 Reference Trend: 0.03 Pre-frail 488 100 2.04 (1.23–3.37) 7.74 0.005 1.46 (0.87–2.43) 2.05 0.54 1.70 (1.01–2.84) 4.05 0.04 Frail 109 45 4.20 (2.39–7.39) 24.9 \0.001 2.56 (1.44–4.56) 10.2 0.03 2.31 (1.24–4.32) 6.96 0.008 Frailty index B0.10 56 2 Reference Trend: \0.001 Reference Trend: \0.001 Reference Trend: \0.001 0.10–0.21 260 19 1.79 (0.41–7.75) 0.61 0.44 1.59 (0.36–6.96) 0.39 0.15 1.86 (0.43–8.05) 0.69 0.41 C0.21 502 143 6.55 (1.58–27.2) 6.72 0.01 4.79 (1.12–20.4) 4.48 0.001 5.31 (1.27–22.2) 5.25 0.02 Note HR hazard ratio, CI conﬁdence interval, the total number of deceased participants, the non-frail state was used a reference category for each frailty instrument. All models were adjusted for age, gender, level of ID, and Down syndrome. Survival Participants were excluded if they had missing data on one of the frailty instruments, motor disabilities, or other covariates (n = 232). The model motor disability was adjusted for the level of motor impairment, ‘no walking impairment’ was used as a reference category; the model for motor disability included only the frailty phenotype or the frailty index Note HR hazard ratio, CI conﬁdence interval, the total number of deceased participants, the non-frail state was used a reference category for each frailty instrument. All models were adjusted for age, gender, level of ID, and Down syndrome. Participants were excluded if they had missing data on one of the frailty instruments, motor disabilities, or other covariates (n = 232). The model motor disability was adjusted for the level of motor impairment, ‘no walking impairment’ was used as a reference category; the model for motor disability included only the frailty phenotype or the frailty index Discussion The FI could be calculated for 94 % of the participants, whereas the frailty phenotype was feasible in 81 %. For less than a third of the participants (29 %), all frailty phenotype criteria could be measured. This is in agreement with results from studies among assisted-living participants, where nearly 40 % could not complete the assessment (de la Rica- Escuin et al. 2014; Hogan et al. 2012). This dropout was mainly caused by more severe cognitive impairment and chronic comorbidity. This result is in line with results observed in the general population: persons in whom the phenotype cannot be measured completely are signiﬁcantly more disabled, have more chronic diseases, are more likely to die, and have a higher FI-score (Collerton et al. 2012; Ravindrarajah et al. 2013). On the other hand, dropout for the FI appeared to be random. The agreement between the two instruments was lower compared to other studies (Theou and Rockwood 2015) and the associations between each single frailty phenotype measure and the FI were rather weak, in contrast to others (Hoogendijk et al. 2015). Fourth, we were unable to apply the exact parameters as those proposed in the Cardiovascular Health Study to measure the frailty phenotype. This could have led to an unknown shift in its predictive validity (Theou et al. 2015). In addition, the analyses were applied to participants with at least three elements of the frailty phenotype measured. It is likely that this caused an underestimation of the true frailty prevalence. Measurements that are more feasible for the ID population might have increased the predictive validity of the frailty phenotype. For example, it is known that physical activity is hard to measure with pedometers in people with ID (Hilgenkamp et al. 2012). Using an instrument such as the StepWatch or GPS could have led to more valid results for the element ‘physical activity’ (van Schijndel-Speet et al. submitted). In accordance with ﬁndings in the general population, we found that the predictive value and thereby the criterion validity for the FI is stronger than that of the frailty phenotype (Blodgettetal.2015;Hoganetal.2012;Kulminskietal.2008; Rockwood etal. 2007; Theou and Rockwood2015; Woo et al. 2012). There are several explanations for our results. Nevertheless, overall the frailty phenotype showed a strong relation with mortality, indicating that physical ﬁt- ness and mobility are important to lengthen the lifespan. Discussion explain part of the variance. Indeed, our forward regression analysis implies that, although physical variables are extremely important, disabilities, diseases, and cognition independently add to the explained variance of the model. In this prospective population-based study, we compared two concepts of frailty in older people with ID: the frailty phenotype and the FI. The FI was more often feasible in the ID population than the frailty phenotype. Both instruments were valid in terms of predicted value for survival; partici- pants classiﬁed as frail by either instrument had increased 5-year mortality risks. Even so, people designated as frail by the FI were more likely to decease than those designated as frail by the frailty phenotype. However, the CIs for the FI were wider than the CIs observed for the frailty phenotype, indicating a larger uncertainty in the estimation. Motor dis- abilities are an important risk factor for mortality. After adjusting the survival models for motor disability, both, but mainly the frailty phenotype, lost predictive value. Previ- ously, we suggested that the FI might be a more suit- able concept for this population because of lifelong disabilities (Evenhuis et al. 2013). The current results con- ﬁrm this suggestion. Second, and in line with the ﬁrst suggestion, the frailty phenotype seems to be too determined by mobility limi- tations. Indeed, in our study the frailty phenotype had only limited additional predictive value to motor disabilities alone. This limits the predictive value of the frailty phe- notype, because motor disabilities appear to be less strong predictors for mortality in our ID sample than observed in the general population (Feeny et al. 2012; Majer et al. 2011). Lifelong or early motor impairment, which is common in this population, is likely to be less predictive than motor impairment acquired in later life. Third, the phenotype approach has the advantage that it focuses on ﬁve core clinical features, that are, in theory, easy to measure. Nevertheless, these pre-deﬁned elements are not measurable in all individuals with an ID. This appears less of a problem with the FI approach, which does not require the use of a pre-deﬁned set of variables or even the same number of variables (Rockwood et al. 2006). We were therefore able to design an FI for the ID population, whereas the elements of the frailty phenotype are designed for the general population. Motor disability and frailty dependent were 4.10 (95 % CI = 2.83–5.96) times, more likely to have deceased during the follow-up compared to those who walked independently. The last column in Table 3 shows the relation of the two frailty approaches with survival, independent of motor disability at baseline. Although both approaches remain signiﬁcantly related with mortality, the frailty phenotype loses much of its predictive value. Information on mobility was known for 989 participants. At baseline, 731 (74 %) participants walked independently, 151 (15 %)walkedwithsupport,and107(11 %)werewheelchair dependent. Those who walked with support were 2.03 (95 % CI = 1.40–2.97) times, and those who were wheelchair 123 Eur J Ageing (2017) 14:63–79 69 Discussion Speciﬁcally, the mobility-related frailty phenotype items (e.g., walking speed and physical inactivity) were most strongly associated with both mortality and the FI. The group with low physical activity and low walking speed also includes those bound to a wheelchair. These results indicate that even though mobility impairment is less predictive for mortality than observed in the general pop- ulation, it is a very important risk factor for mortality and overall health (e.g., the FI). It has been shown in the HA-ID First, the FI has a much broader approach than the frailty phenotype. It includes all factors that are considered important for frailty (e.g., nutritional status, physical activity, energy, cognition) (de Vries et al. 2011; Gobbens et al. 2010). In contrast, the frailty phenotype focuses on physical frailty only. It appears that, among the highly heterogeneous ID population, physical parameters do only 12 3 Eur J Ageing (2017) 14:63–79 70 study that elements from the frailty phenotype (e.g., grip strength, walking speed) predict disability in mobility and activities of daily living (Oppewal et al. 2014). In the general population, physical activity and ﬁtness can reduce or prevent frailty (Liu and Fielding 2011; Theou et al. 2011). Whether increased physical ﬁtness and activity will also reduce or delay frailty in people with ID needs to be investigated. observed. Using the robust group as a reference group was therefore complicated and resulted in underpowered HRs. We therefore placed most emphasis on the ﬁrst applied cut value (robust if FI was below 0.20). In order to better understand the agreement and validation of different cut points, a longer follow-up and/or more participants are required. The two frailty concepts used in our study have a dif- ferent purpose and different underlying justiﬁcation. Nev- ertheless, by comparing the two different concepts, we tried to improve the understanding of frailty in people with ID. The cycle of frailty, which serves as the biological basis of the frailty phenotype, might not be the only rele- vant aspect in the ID population. Mainly because in this cycle of age-related decline, it is supposed that its indi- vidual components are associated with each other and with further physiological losses, disability, dependency, and eventually death. In contrast, in our population motor dis- abilities can be lifelong and congenital and childhood disabilities are more likely to contribute to frailty than the other way around. Discussion For example, it was observed that, according to the frailty phenotype, people with motor disabilities were very likely to also be frail or pre-frail; only 8 % of the participants using a walking aid or wheelchair were classiﬁed as robust (Evenhuis et al., 2012). The FI, within clearly deﬁned borders, simply counts how many things are wrong with an individual. Even though the FI also includes lifelong disabilities, it seems that these lifelong problems less inﬂuence its validity. Nevertheless, also in people with ID, disabilities increase as a consequence of aging and frailty (Schoufour et al. 2014, 2015). Therefore, identifying frail individuals can assist clinicians in identifying people at risk for adverse health outcomes, who may thereafter beneﬁt from interventions. The main strength of our study is its large-scale and prospective population-based design, in which we used standardized and internationally accepted methods to measure frailty. Nevertheless, several limitations need to be taken into account. First, although the population was near-representative, older people with ID using specialized support, living independently or with relatives were slightly underrepresented in the HA-ID study. Because of the high correlation between frailty and more severe ID, this underrepresentation might have caused slightly higher prevalence of frailty (Hilgenkamp et al. 2011). Second, we did not take into account time and costs as feasibility aspects. It is very likely that regarding costs, the frailty phenotype is more feasible for clinical practice. Never- theless, with this study we mainly wanted to better understand frailty and its consequences in this population. For the clinical implementation of any frailty instrument, time and cost should be taken into account. Third, we studied the relation between frailty and survival because mortality is an easily veriﬁable, dichotomous, and non- arbitrary outcome. Nevertheless, other health outcomes including care need, hospitalization, and disabilities are needed to obtain full insight into the negative consequences of frailty in people with ID. Fourth, the frailty phenotype and the FI are the two most commonly applied concepts. Nevertheless, there are other concepts and frailty instru- ments that were not included in this study. These measures were chosen because they allow objective measurements, which are needed in a population where only about 25 % is capable of reliable, self-report. In addition, the baseline data of the HA-ID study were already collected before frailty became of interest. Therefore, we were limited to frailty instruments that could be constructed using the available data. Discussion 57000003, 314030302). Further support was provided by the three participating care organizations (Abrona, Ipse de Bruggen, and Amarant). With this study, we aimed to better understand frailty in people with ID by applying two different frailty instru- ments. Our results imply that the used FI is a stronger pre- dictor for mortality than our adaptation of the phenotype in the population of older people with ID. Possible explana- tions of our ﬁndings are that we did not use the exact frailty phenotype variables or that the FI includes multiple health domains. The differences between the two frailty approa- ches may also be caused by the FI being less determined by lifelong disability and mobility, compared to the frailty phenotype. We suggest that future studies on frailty in people with ID take into account that the feasibility of frailty instruments can be hampered, and adapted instruments are required. Furthermore, lifelong disabilities, such as mobility impairment, could inﬂuence the prevalence of frailty and the validity of frailty instruments. Although we acknowl- edge mobility impairment as a very important aspect of frailty, we suggest using multiple frailty domains, in order to capture the risk for mortality the best. Future research needs to focus on the clinical feasibility of the FI. Particu- larly, it should be studied whether routinely collected data can be used to construct an FI for people with ID. Compliance with ethical standards Conﬂict of interests The authors declare that there is no conﬂict of interest. Ethics approval This study was approved by the Ethics Committee of the Erasmus Medical Center Rotterdam (MEC-2008-234) and the ethics committees of the participating care organizations (Abrona, Ipse de Bruggen, and Amarant). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://crea tivecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Discussion In addition, there is value using the two most commonly used measurements in connecting to a large body of published work in order to compare popu- lation characteristics. Last, it has been advised to use the FI as a continuous scale, and not apply cut points. Even so, for the sake of comparing the FI with the three frailty strata, proposed by Fried et al., we created three frailty groups applying three different cut points. Nevertheless, the cut points that classiﬁed individuals as robust if the FI was below 0.10 or 0.08 resulted in small groups of robust participants and, in line with expectations regarding these robust individuals, limited number of deaths were Although efforts have been made, there is not yet a validated frailty screening instrument for the ID population (Brehmer-Rinderer et al. 2013). Screening and monitoring the health status of people with ID can potentially have great beneﬁcial effects because recovering from a frail state is complicated, putting more emphasis on early detection and prevention (Rockwood et al. 2011). As the FI provides the highest feasibility and the highest predictive validity, we advise to screen for frailty using an FI like approach. Nevertheless, several steps need to be taken into account before the FI can be applicable to clinical practice. The original FI is composed of 51 items, of which some are not applicable to clinical practice (for example, the block test to measure manual dexterity and the DDS questionnaire to diagnose dysphagia). It should be studied whether the FI remains valid after the removal of less clinically applicable measures. Also, the stability of the FI should be tested by determining the test–retest reliability. Additionally, it is yet 123 123 Eur J Ageing (2017) 14:63–79 71 unclear if the FI is sensitive to changes over time. In the long run, routinely collected data might be used to calcu- late an FI and monitor frailty status over time. Funding This study was supported by a grant from the National Care for the Elderly Programme (NPO) which is part of the Netherlands Organisation for Health Research and Development (ZonMW; Nos. 57000003, 314030302). Further support was provided by the three participating care organizations (Abrona, Ipse de Bruggen, and Amarant). Funding This study was supported by a grant from the National Care for the Elderly Programme (NPO) which is part of the Netherlands Organisation for Health Research and Development (ZonMW; Nos. Appendix See Tables 4, 5, 6, and 7. Table 4 Overview of deﬁcits included in the frailty index (n = 982) Table 4 Overview of deﬁcits included in the frailty index (n = 982) # Deﬁcit Additional information Cut-off values and FI scores Percentage per category HR (95 % CI)a p (for trend) 1 Bladder control ADL, Completed by professional caregivers of the participants Incontinent = 1 23.1 3.86 (2.70–5.52) \0.001 Sometimes continent = 0.5 24.1 1.39 (0.92–2.08) Continent = 0 52.7 – 2 Dressing ADL, Completed by professional caregivers of the participants Needs help = 1 19.0 5.48 (3.70-8.13) \0.001 Partly with help = 0.5 25.8 2.04 (1.38–3.02) No help = 0 55.2 – 3 Walking stairs ADL, Completed by professional caregivers of the participants Needs help = 1 27.6 4.53 (3.12–6.58) \0.001 Partly with help = 0.5 20.7 1.75 (1.12–2.73) No help = 0 51.7 – 4 Bathing ADL, Completed by professional caregivers of the participants With help = 1 64.3 2.50 (1.65–3.80) \0.001 No help = 0 35.7 – 5 Transfer bed to chair ADL, Completed by professional caregivers of the participants Unable, no sitting balance = 1 9.4 4.79 (3.22–7.12) \0.001 Major help = 0.66 3.0 5.17 (3.00–8.90) Minor help = 0.33 13.2 2.71 (1.82–4.01) No help = 0 74.4 – 6 Groceries IADL, completed by professional caregivers of the participants Not independently = 1 51.2 3.08 (1.93–4.93) \0.001 With help = 0.5 20.9 1.12 (0.63–1.98) Can do groceries = 0 27.9 – 7 Housekeeping IADL, completed by professional caregivers of the participants Not independently = 1 74.6 3.75 (1.51–9.29) 0.001 With help = 0.5 15.2 1.58 (0.56–4.51) Can do housekeeping = 0 10.2 – 12 123 Eur J Ageing (2017) 14:63–79 72 Table 4 continued # Deﬁcit Additional information Cut-off values and FI scores Percentage per category HR (95 % CI)a p (for trend) 8 Falling Number of falls in the last three months. Appendix Information gathered via the professional care giver [11 falls = 1 1.3 2.61 (1.07–6.41) 0.016 6–10 falls = 0.75 1.0 1.60 (0.39–6.56) 3–5 falls = 0.5 3.8 2.27 (1.27–4.06) 1–2 falls = 0.25 17.4 1.31 (0.91–1.87) 0 falls = 0 76.4 – 9 Present at the care center (max 10 shifts per week) Information gathered via the professional care giver \3 visits a week = 1 15.7 1.82 (1.27–2.59) 0.001 C3 visits a week = 0 84.3 – 10 Fatigued ADESS (Dutch translation of the Anxiety, Depression And Mood Scale) over the past six months. Completed by professional caregivers Very often = 1 6.0 3.36 (3.36–2.06) \0.001 Often = 0.66 17.3 1.85 (1.85–1.24) Sometimes = 0.33 30.9 1.67 (1.67–1.17) Never = 0 45.8 – 11 Listless ADESS (Dutch translation of the Anxiety, Depression And Mood Scale) over the past six months. Completed by professional caregivers Very often = 1 3.2 3.54 (2.05–6.13) \0.001 Often = 0.66 8.6 1.87 (1.19–2.95) Sometimes = 0.33 23.7 1.46 (1.04–2.04) Never = 0 64.5 – 12 Panic attacks ADESS (Dutch translation of the Anxiety, Depression And Mood Scale) over the past six months. Completed by professional caregivers Very often = 1 3.4 3.47 (2.00–6.06) \0.001 Often = 0.66 6.8 1.44 (0.85–2.43) Sometimes = 0.33 13.7 1.71 (1.17–2.50) Never = 0 76.1 – 13 Decreased food intake, due to loss of appetite, digestive problems, chewing of swallowing difﬁculties Mini Nutritional Assessment (MNA) over the past three months. Completed by professional caregivers Severe decrease in food intake = 1 4.3 2.61 (1.61–4.22) \0.001 Moderate decrease in food intake = 0.5 9.5 1.94 (1.31–2.87) No decrease in food intake = 0 86.2 – 14 Weight loss Mini Nutritional Assessment (MNA) over the past three months. Completed by professional caregivers Weight loss greater than 3 kg = 1 Does not know = 0.5 4.7 – 0.023 Weight loss 1–3 kg = 0.5 24.2 2.05 (1.20–3.52) No weight loss = 0 71.0 1.24 (0.90–1.73) 15 Fluid intake per day (water, juice, coffee, tea, milk) Mini Nutritional Assessment (MNA) over the past three months. Appendix Completed by professional caregivers Less than 3 cups = 1 0.5 10.9 (3.94–30.0) \0.001 1 to 5 cups = 0.5 14.8 1.13 (0.77–1.65) [5 cups = 0 84.6 – 16 Calf circumference (CC) in cm Mini Nutritional Assessment (MNA) Completed by professional caregivers CC \ 31 = 1 21.3 1.92 (1.39–2.67) \0.001 CC C 31 = 0 78.7 – 17 Only eats selected types of food (e.g., pudding, rice) Screening Tool of Eating Problems (STEP) over the last month. Completed by professional caregivers [ 10 times = 1 4.5 1.73 (1.04–2.90) \0.001 Between 1–10 times = 0.5 3.9 2.84 (1.68–4.79) Not at all/not a problem = 0 91.6 – 18 Only eats small amounts of the presented food Screening Tool of Eating Problems (STEP) over the last month. Completed by professional caregivers [ 10 times = 1 4.2 1.95 (1.12–3.41) 0.025 Between 1–10 times = 0.5 12.2 1.39 (0.95–2.04) Not at all/not a problem = 0 83.6 – 12 3 Eur J Ageing (2017) 14:63–79 73 Table 4 continued # Deﬁcit Additional information Cut-off values and FI scores Percentage per category HR (95 % CI)a p (for trend 19 Only eats foods of certain textures Screening Tool of Eating Problems (STEP) over the last month. Appendix Completed by professional caregivers [ 10 times = 1 5.4 2.61 (1.65–4.14) \0.001 Between 1–10 times = 0.5 2.5 2.36 (1.26–4.39) Not at all/not a problem = 0 92.1 – 20 Mobility Provided by professional caregivers Wheelchair = 1 10.9 4.10 (2.83–5.96) \0.001 Walks with support = 0.5 15.3 2.04 (1.40–2.97) Walks independently = 0 73.8 – 21 CVA Medical ﬁle, last 24 months Yes = 1 94.0 1.57 (0.95–2.59) 0.080 No = 0 6.0 – 22 Coronary heart diseases/heart failure/cardiac dysrhythmia/ pacemaker Medical ﬁle, last 24 months Yes = 1 9.2 2.26 (1.51–3.38) \0.001 No = 0 90.8 – 23 Cancer Medical ﬁle, entire life Yes = 1 4.9 1.27 (0.70–2.31) 0.43 No = 0 95.1 – 24 Asthma/COPD Medical ﬁle, last 24 months, mediation Yes = 1 13.2 2.27 (1.60–3.24) \0.001 No = 0 86.8 – 25 GERD Medical ﬁle, last 24 months Yes = 1 20.0 1.52 (1.07–2.15) 0.02 No = 0 80.0 – 26 Obstipation Medical ﬁle, last 24 months, medication Yes = 1 39.7 2.02 (1.48–2.76) \0.001 No = 0 60.3 – 27 Risk for Diabetes Mellitus (DM) or known DM Medical ﬁle, blood glucose levels, medication DM according to medical ﬁle or taking drugs for DM and/or serum glucose C 7 mmol/ l = 1 12.4 1.17 (0.76–1.80) 0.75 No DM according to medial ﬁle, no DM drugs and blood glucose 6.1–6.9 = 0.5 2.7 0.90 (0.33–2.43) No DM according to medial ﬁle, no DM drugs and blood glucose \ 6.1 = 0 84.8 – 28 Scoliosis Medical ﬁle Yes = 1 10.6 1.27 (0.81–1.99) 0.30 No = 0 89.4 – 29 Visual/Hearing impairments (V/H impairment) Medical ﬁle At least one severe V/H impairment = 1 24.6 1.76 (1.16–2.67) 0.023 Two moderate V/H impairment = 1 One moderate V/H impairment = 0.5 29.9 1.56 (1.04–2.33) No V/H impairment = 0 45.5 – 30 Medication use (polypharmacy) Medical ﬁle C 7 drugs = 1 19.8 3.12 (2.13–4.67) \0.001 4–6 drugs = 0.5 31.2 1.75 (1.21–2.52) 0–3 drugs = 0 48.9 – 31 Over or under weight Medical examination BMI \ 18.5 OR [ 30 = 1 27.7 1.44 (0.93–2.23) 0.15 BMI 18.5–20 OR 25–30 = 0.5 41.0 1.01 (0.68–1.51) BMI 20–25 = 0 31.3 – 32 High blood pressure Medical ﬁle Yes = 1 21.5 0.94 (0.64–1.39) 0.76 No = 0 78.5 – 33 Peripheral atherosclerosis Medical examination Ankle Arm index 0.005 [0.9 = 1 9.9 1.90 (1.15–3.15) 0.8–0.9 = 0.5 12.2 1.86 (1.17–2.97) \0.8 = 0 78.0 – 12 23 Eur J Ageing (2017) 14:63–79 74 Table 4 continued # Deﬁcit Additional information Cut-off values and FI scores Percentage per category HR (95 % CI)a p (for tren 34 Osteoporosis (t-score) Medical examination \2.5 = 1 32.7 1.14 (0.72–1.81) 0.109 -1 till -2.5 = 0.5 38.8 0.72 (0.45–1.14) [-1 = 0 28.6 – 35 Manual Dexterity (BBT) Fitness assessment The participants were asked to move as many colored blocks as possible in one minute. Hypercholesterolemia Medical registry Appendix The blocks were 2.5 cm3 and needed to be moved from one side of a wooden box to the other side Lowest quartile = 1 26.8 2.75 (1.51–5.00) \0.001 Second quartile = 0.66 21.6 1.04 (0.55–1.97) Third quartile = 0.33 26.1 0.83 (0.45–1.53) Highest quartile = 0 25.7 – 36 Walking speed Fitness assessment Comfortable walking speed was measured by the average of three records of the time needed to complete 5 meters after 3 meters for acceleration Slow walking speed was Stratiﬁed for height and gender 64.7 3.27 (2.27–4.71) \0.001 Male height B 173 cm C 7 s = 1 Male height [ 173 cm C 6 s = 1 Females height B 159 cm C 7 s = 1 Females [ 159 cm C 6 s = 1 Faster = 0 35.3 – Participant who were not able to succeed the walking speed assessment due to physical limitations were scored positive (score 1) as well Slow walking speed was Stratiﬁed for height and gender 37 Grip strength Fitness assessment Measured with a Jamar Hand Dynamometer (#5030J1, Sammons Preston Rolyan, USA) Grip strength was stratiﬁed for gender and BMI 52.9 47.1 1.32 (0.86–2.03) 0.21 Below cut-off values = 1 Male BMI B24: C29 kg = 0 BMI 24.1–26: C30 kg = 0 BMI 26.1–28: C30 kg = 0 BMI [28: C32 kg = 0 Female BMI B23: C17 kg = 0 BMI 23.1–26: C17.3 kg = 0 BMI 26.1–29: C18 kg = 0 BMI [29: C21 kg = 0 Participant who were not able to succeed the grip strength assessment due to physical limitations were scored positive (score 1) as well 38 Hypercholesterolemia Medical registry Yes = 1 10.0 0.58 (0.30–1.11) 0.58 No = 0 90.0 – BMI B24: C29 kg = 0 BMI 24.1–26: C30 kg = 0 BMI 26.1–28: C30 kg = 0 BMI [28: C32 kg = 0 38 123 123 75 Eur J Ageing (2017) 14:63–79 Eur J Ageing (2017) 14:63–79 Table 4 continued # Deﬁcit Additional information Cut-off values and FI scores Percentage per category HR (95 % CI)a p (for trend) 39 HDL Blood examination HDL was stratiﬁed for gender 24.6 1.17 (0.69–1.98) 0.49 Male 59.0 0.92 (0.56–1.50) 0–0.9 mmol/l = 1 16.4 – 0.9–1.55 mmol/L = 0.5 [1.55 mmol/L = 0 Female 0–1.1 mmol/l = 1 1.1–1.55 mmol/L = 0.5 [1.55 mmol/L = 0 40 Hemoglobin Blood examination Stratiﬁed for gender 23.8 2.67 (1.87–3.08) \0.001 Male 76.2 – 8.6–10.5 mmol/L = 0 \8.6 OR [ 10.5 mmol/L = 1 Female 7.5–9.5 mmol/L = 0 \7.5 OR [ 9.5 mmol/L = 1 41 Dysphagia Diagnosis via DDS questionnaire Severe dysphagia = 1 52.0 2.10 (1.29–3.42) 0.006 Moderate dysphagia = 0.5 26.1 1.52 (0.88–2.63) No Dysphagia = 0 21.8 – 42 Hospitalization Asked in informed consent form. Appendix Hospitalization is the past 12 months [2 = 1 0.6 8.57 (2.59–28.3) \0.001 1–2 = 0.5 11.0 1.74 (1.16–2.62) No = 0 88.4 – 43 Makes a sad/ depressing impression SDZ, completed by professional caregivers Last three months Often = 1 4.6 4.54 (2.48–7.94) \0.001 Several times = 0.66 12.9 1.97 (1.23–3.15) Sometimes = 0.33 36.9 1.87 (1.31–2.68) Never/very rare = 0 45.6 – 44 Has fun and interest in daily activities SDZ, completed by professional caregivers Last three months Never/very rare = 1 4.7 3.06 (1.68–5.57) 0.001 Sometimes = 0.66 19.2 1.99 (1.30–3.06) Several times = 0.33 39.1 1.40 (0.94–2.08) Often = 0 36.9 – 45 Sleeps more than regularly (trouble getting out of bed, falls asleep during the day) SDZ, completed by professional caregivers Last three months Often = 1 4.5 4.55 (2.68–7.72) \0.001 Several times = 0.66 9.8 2.85 (1.84–4.42) Sometimes = 0.33 22.8 1.96 (1.35–2.85) Never/very rare = 0 62.9 – 46 Fast fatigued/listless SDZ, completed by professional caregivers Last three months Often = 1 6.4 4.57 (2.82–7.39) \0.001 Several times = 0.66 13.5 2.03 (1.28–3.21) Sometimes = 0.33 35.3 1.62 (1.11–2.37) Never/very rare = 0 44.8 – 47 Is slow or passive in his/her movements SDZ, completed by professional caregivers Last three months Never/very rare = 0 Sometimes = 0.33 Several times = 0.66 Often = 1 6.5 12.1 26.5 54.9 4.66 (2.89–7.50) 2.73 (1.78–4.20) 1.64 (1.11–2.42) \0.001 48 Knowing which year it is The Dementia Questionnaire for Mentally Retarded Persons (DMR) Normally No = 1 57.4 1.55 (1.04–2.27) 0.09 Sometimes = 0.5 5.3 1.18 (0.57–2.45) Normally Yes = 0 37.2 – 49 Knowing the way to familiar places The Dementia Questionnaire for Mentally Retarded Persons (DMR) Normally No = 1 12.6 3.43 (2.36–5.00) \0.001 Sometimes = 0.5 7.6 2.44 (1.56–3.97) Normally Yes = 0 79.8 – 12 123 Eur J Ageing (2017) 14:63–79 76 Table 4 continued # Deﬁcit Additional information Cut-off values and FI scores Percentage per category HR (95 % CI)a p (for trend) 50 Is seeing group mates The Dementia Questionnaire for Mentally Retarded Persons (DMR) Normally No = 1 36.2 2.50 (1.59–3.91) \0.001 Sometimes = 0.5 36.5 1.59 (1.03–2.46) Normally Yes = 0 27.3 – 51 Knowing that today is a weekend or a week day The Dementia Questionnaire for Mentally Retarded Persons (DMR) Normally No = 1 23.5 3.56 (2.46–5.16) \0.001 Sometimes = 0.5 9.7 2.06 (1.28–3.30) Normally Yes = 0 66.8 – a HR Hazard ratio, calculated for each provided category using the ‘healthiest’ option as a reference group; HR are adjusted for age, gender, level of ID, and the presence of Down syndrome a HR Hazard ratio, calculated for each provided category using the ‘healthiest’ option as a reference group; HR are adjusted for age, gender, level of ID, and the presence of Down syndrome Table 5 Frailty phenotype variables as originally intended by Fried et al. Appendix (2001, 2012) and the adapted frailty phenotype by Evenhuis et al. (2012) O i i l t A li d t th HA ID t d Table 5 Frailty phenotype variables as originally intended by Fried et al. (2001, 2012) and the adapted frailty phenotype by Evenhuis et al. (2012) Original measurement Applied to the HA-ID study Weakness Grip strength: lowest 20 % (by gender, body mass index) As originally suggested using the Jamar Hand Dynamometer [#5030J1, Sammons Preston Rolyan, Dolgeville, NY] Shrinking: Weight loss [10 lbs (4.54 kg) lost unintentionally in prior year An item of the Mini Nutritional Assessment, weight loss during the past 3 months was assessed on a 4-point rating scale. Losses [3 kg were scored Exhaustion Exhaustion by self-report Exhaustion was estimated using the item ‘‘Lacks energy’’ of the Anxiety, Depression and Mood Scale, using a 4-point rating scale. No exhaustion was classiﬁed as no problems or mild problems and exhaustion was classiﬁed as moderate problem and severe problem. Because self-report is difﬁcult for a large part of the intellectual disabled population, proxy-based answers were used Slowness Walking time/15 ft: slowest 20 % (by gender, height) As originally suggested. Appendix In addition, all participants in a wheelchair and all participants who could not engage in the walking speed assessment because of physical limitations were classiﬁed as ‘slow’ Low activity kcal/week: lowest 20 % males: \383 kcal/week females: \270 kcal/week All participants walking fewer than 5000 steps/day (sedentary lifestyle) were scored as ‘low activity,’ as were all participants in a wheelchair and all participants who could not engage in the walking speed assessment because of physical limitations 123 123 123 Eur J Ageing (2017) 14:63–79 77 Table 6 Hazard ratio’s for 5-year all-cause mortality according to the three level frailty index and frailty phenotype for all participants Frailty measure Status n cat cat Single frailty instrument Both frailty instruments Motor disability HR (95 % CI) Wald p HR (95 % CI) Wald p HR (95 % CI) Wald p Frailty phenotype Non-frail 227 20 Reference Trend: \0.001 Reference Trend: 0.90 Reference Trend: 0.04 Pre-frail 492 100 1.96 (1.10–3.21) 7.21 0.007 1.12 (0.65–1.92) 0.17 0.68 1.62 (0.98–2.68) 3.52 0.06 Frail 109 45 4.09 (2.35–7.13) 24.8 \0.001 1.16 (0.62–2.19) 0.22 0.64 2.23 (1.20–4.12) 6.48 0.011 Frailty Index \0.2 320 19 Reference Trend: \0.001 Reference \0.001 Reference \0.001 0.2–0.35 382 52 2.48 (1.45–4.24) 11.0 \0.001 2.19 (1.24–3.87) 7.20 0.007 2.40 (1.40–4.12) 10.2 \0.001 [0.35 259 122 11.0 (6.49–18.9) 78.3 \0.001 9.66 (5.23–17.8) 52.5 \0.001 9.13 (5.18–16.1) 58.5 \0.001 Frailty phenotype Non-frail 227 20 Reference Trend: \0.001 Reference Trend: 0.019 Reference Trend: 0.04 Pre-frail 492 100 1.96 (1.10–3.21) 7.21 0.007 1.42 (0.84–2.38) 1.71 0.008 1.62 (0.98–2.68) 3.52 0.06 Frail 109 45 4.09 (2.35–7.13) 24.8 \0.001 2.21 (1.23–4.00) 6.93 0.02 2.23 (1.20–4.12) 6.48 0.011 Frailty Index B0.08 33 2 Reference Trend: \0.001 Reference Trend: \0.001 Reference Trend: \0.001 0.08–0.25 437 33 1.09 (0.26–4.60) 0.02 0.90 0.84 (0.20–3.58) 0.06 0.81 1.09 (0.26–4.59) 0.01 0.91 C0.25 491 158 5.07 (1.22–21.0) 5.01 0.025 2.93 (0.67–12.7) 2.06 0.15 4.02 (0.96–16.8) 3.64 0.06 Frailty phenotype Non-frail 227 20 Reference Trend: \0.001 Reference Trend: 0.001 Reference Trend: 0.04 Pre-frail 492 100 1.96 (1.10–3.21) 7.21 0.007 1.46 (0.87–2.43) 2.05 0.54 1.62 (0.98–2.68) 3.52 0.06 Frail 109 45 4.09 (2.35–7.13) 24.8 \0.001 2.56 (1.44–4.56) 10.2 0.03 2.23 (1.20–4.12) 6.48 0.011 Frailty index B0.10 64 2 Reference Trend: \0.001 Reference Trend: \0.001 Reference Trend: \0.001 0.10–0.21 281 19 1.94 (0.45–8.38) 0.79 0.37 1.59 (0.36–6.96) 0.39 0.15 1.97 (0.46–8.51) 0.83 0.36 C0.21 616 172 7.80 (1.90–32.0) 8.12 0.004 4.79 (1.12–20.4) 4.48 0.001 6.28 (1.52–26.0) 6.44 0.01 Note HR hazard ratio, CI conﬁdence interval, the total number of deceased participants, the non-frail state was used a reference category for each frailty instrument. 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https://openalex.org/W3171181876	https://link.springer.com/content/pdf/10.1007/s00209-022-03074-9.pdf	English	null	A note on Bridgeland moduli spaces and moduli spaces of sheaves on $$X_{14}$$ and $$Y_3$$	Mathematische Zeitschrift	2,022	cc-by	24,407	Mathematische Zeitschrift (2022) 302:803–837 https://doi.org/10.1007/s00209-022-03074-9 Mathematische Zeitschrift (2022) 302:803–837 https://doi.org/10.1007/s00209-022-03074-9 Mathematische Zeitschrift This note is part of the Undergraduate Mathematcial Research Project of Zhiyu Liu mentored by Shizhuo Zhang. Shizhuo Zhang is supported by ERC Consolidator Grant WallCrossAG, no. 819864. A note on Bridgeland moduli spaces and moduli spaces of sheaves on X14 and Y3 Zhiyu Liu1 · Shizhuo Zhang2 Received: 11 July 2021 / Accepted: 9 June 2022 / Published online: 20 July 2022 © The Author(s) 2022 B Shizhuo Zhang Shizhuo.Zhang@ed.ac.uk Zhiyu Liu zhiyuliu@stu.scu.edu.cn 1 Department of Mathematics, Sichuan University, Chengdu 610064, Sichuan , People’s Republic of China 2 School of Mathematics, The University of Edinburgh, JCMB Building, Kings Building, Edinburgh EH9 3FD, UK B Shizhuo Zhang Shizhuo.Zhang@ed.ac.uk Zhiyu Liu zhiyuliu@stu.scu.edu.cn 1 Department of Mathematics, Sichuan University, Chengdu 610064, Sichuan , People’s Republic of China 2 School of Mathematics, The University of Edinburgh, JCMB Building, Kings Building, Edinburgh EH9 3FD, UK Mathematics Subject Classiﬁcation Primary 14F05; Secondary 14J45 · 14D20 · 14D23 1.1 Background The notion of stability on a triangulated category was introduced by Bridgeland in [14]. It enables us to construct moduli spaces of semistable objects in a general triangulated category, which provides a powerful machinery to study the geometry of classical moduli spaces. One of the most striking progress is made in [4, 5], where the authors construct moduli space of Bridgeland stable objects on a K3 surface S with respect to a stability condition σ in the stability manifold Stab(S). They realized each birational model of a moduli space of stable sheaves on such a K3 surface S as Bridgeland moduli space. They showed that the minimal model program for this moduli space corresponds to a wall-crossing in Stab(S) in a precise way. On the other hand, it has been widely accepted that a non-trivial semi-orthogonal component Ku(X) (called the Kuznetsov component) of a bounded derived category Db(X) of a smooth projective variety X encodes essential information of its birational geometry and classical moduli spaces on it. Recently, in [7], the authors provide a criterion to induce a stabilityconditionσ ontheKuznetsovcomponents Ku(X)ofaseriesprimeFanothreefolds X from weak stability conditions on Db(X). Thus, it becomes possible to construct Bridgeland moduli spaces Mσ (Ku(X), c) of σ-stable objects of class c in Ku(X) to study birational geometry of classical moduli spaces of semistable sheaves on X. In the present note, we make an attempt in this direction. Let Yd be a prime Fano threefold of index 2 and degree d. We consider the semiorthogonal decomposition of Yd given by Db(Yd) = ⟨Ku(Yd), OYd , OYd (1)⟩ The numerical Grothendieck group N(Ku(Yd)) of Ku(Yd) is a rank two lattice spanned The numerical Grothendieck group N(Ku(Yd)) of Ku(Yd) is a rank two lattice spanned by v := [IL] = 1 −1 d H2, w := H −1 2 H2 + 1 6 −1 d H3 where IL is an ideal sheaf of a line L on Yd. Let X2g−2 be an index one prime Fano threefold of genus g and degree 2g −2. The semi-orthogonal decomposition of X2g−2 are given in [25, 26]: Db(X2g−2) = ⟨AX2g−2, OX2g−2, E∨⟩. Abstract We study Bridgeland moduli spaces of semistable objects of (−1)-classes and (−4)-classes in the Kuznetsov components on index one prime Fano threefold X4d+2 of degree 4d + 2 and index two prime Fano threefold Yd of degree d for d = 3, 4, 5. For every Serre-invariant stability condition on the Kuznetsov components, we show that the moduli spaces of stable objectsof(−1)-classeson X4d+2 andYd areisomorphic.Weshowthatmodulispacesofstable objects of (−1)-classes on X14 are realized by Fano surface C(X) of conics, moduli spaces of semistable sheaves MX(2, 1, 6) and MX(2, −1, 6) and the correspondent moduli spaces on cubic threefold Y3 are realized by moduli spaces of stable vector bundles Mb Y (2, 1, 2) and Mb Y (2, −1, 2). We show that moduli spaces of semistable objects of (−4)-classes on Yd are isomorphic to the moduli spaces of instanton sheaves Minst Y when d ̸= 1, 2, and show that there are open immersions of Minst Y into moduli spaces of semistable objects of (−4)-classes when d = 1, 2. Finally, when d = 3, 4, 5 we show that these moduli spaces are all isomorphic to Mss X (2, 0, 4). Keywords Derived categories · Bridgeland moduli spaces · Kuznetsov components Keywords Derived categories · Bridgeland moduli spaces · Kuznetsov components Mathematics Subject Classiﬁcation Primary 14F05; Secondary 14J45 · 14D20 · 14D23 2 School of Mathematics, The University of Edinburgh, JCMB Building, Kings Building, Edinburgh EH9 3FD, UK 123 Z. Liu , S. Zhang 804 1.1 Background The numerical Grothendieck group N(AX2g−2) of AX2g−2 is a rank two lattice spanned by The numerical Grothendieck group N(AX2g−2) of AX2g−2 is a rank two lattice spanned by by s := [IC] = 1 − 1 g −1 H2, t := H − g 2 + 1 L −16 −g 12 P where IC is an ideal sheaf of a conic C on X2g−2. We call u ∈N(Ku(Yd)) (or N(X2g−2)) a (−r)-class if χ(u, u) = −r, where χ(−, −) is the Euler pair. As noted in [26, Proposition 3.9], the Chern character map identiﬁes the numer- ical Grothendieck group with the lattice generated by Chern characters of some sheaves, then we will use notations of numerical class and Chern character alternatively. In [26, 28], the author established equivalences of triangulated categories Ku(Yd) ∼= AX4d+2 for some pairs (Yd, X4d+2) ∈Zd ⊂MF2 d × MF1 4d+2. Theorem 1.1 [26, 28] Let MFi d be the moduli space of index i degree d prime Fano threefold. Then for each 3 ≤d ≤5, there is a correspondence Zd ⊂MF2 d × MF1 4d+2 which 123 A note on Bridgeland moduli spaces and moduli... 805 is dominant over each factor and such that for any point (Yd, X4d+2) ∈Zd there is an equivalence is dominant over each factor and such that for any point (Yd, X4d+2) ∈Zd there is an equivalence : Ku(Yd) ∼= AX4d+2. Moreover, this equivalence induces an isometry between numerical Grothendieck groups, and maps the class of the ideal sheaves of lines v := [IL] to the class of the ideal sheaves of conics s := [IC]. Using this, [28] proved the isomorphisms of Fano surface (Yd) of lines and Fano surface C(X4d+2) of conics. In the present note, we mainly study various classical moduli spaces on these Fano threefolds from a modern point of view. We apply the techniques developed in [1, 9, 35, 40] to identify several interesting classical moduli spaces of sheaves with Bridgeland moduli spaces of stable objects in Kuznetsov components of prime Fano threefolds of index one and two. Then we use equivalence in Theorem 1.1 to study moduli spaces on index one prime Fano threefolds via the ones on index two Fano threefold. We focus on moduli space Mσ ′(AX, c′)(or Mσ (Ku(Y), c)) of stable objects of (−1)-classes and (−4)-classes in Kuznetsov component AX and Ku(Y). 1.1 Background The former ones provide moduli spaces of minimal dimension. Typical stable object of (−1)-class in Ku(Y) are given by the ideal sheaves of lines v up to auto-equivalences of Ku(Y). The latter ones can be regarded as moduli spaces of stable objects of twice of (−1)-classes. We denote by Mss σ ′(AX, c′) the moduli space of S-equivalence classes of σ ′-semistable objects. Mb Y (2, 1, 2) ∼= (Y) Mb Y (2, 1, 2) ∼= (Y) Next, we focus on moduli spaces of stable objects of (−4)-class in AX and Ku(Y). Typical examples of these moduli spaces are moduli space of instanton sheaves on del Pezzo threefold Y and compactiﬁcation of ACM sheaves on prime Fano threefolds of index one studied in [12]. Originally, instanton bundles appeared in [3] as a way to describe Yang-Mills instantons on a 4-sphere S4, which play an important role in Yang-Mills gauge theory. They provide extremelyinterestinglinksbetweenphysicsandalgebraicgeometry.Themathematicalnotion of an instanton bundle was ﬁrst introduced on P3 and generalized to Fano threefolds in [18, 27]. On Y = Y3 and Y4, the instantonic condition is automatically satisﬁed for stable bundles of rank 2 with c1 = 0, c2 = 2, c3 = 0 (for precise deﬁnitions, see Sect. 6), as proved in [17, 37]. On Y5, this is conjectured in [39, Conjecture 3.7] and we will prove this in Lemma 6.8. On the index one side, there are also some interesting moduli spaces of sheaves. As shown in [12], when X is a non-hyperelliptic prime Fano threefold, there are some arithmetically Cohen- Macaulay (ACM) bundles in the moduli space Mss X (2, 0, 4), and the sheaves in Mss X (2, 0, 4) are classiﬁed when genus g ≥7. We denote the moduli space of S-equivalence classes of instanton sheaves by Minst Y and study its relation with the moduli space and the Bridgeland moduli spaces of S-equivalence classes of semistable objects of (−4)-classes in Ku(Y). More precisely, we have: Next, we focus on moduli spaces of stable objects of (−4)-class in AX and Ku(Y). Typical examples of these moduli spaces are moduli space of instanton sheaves on del Pezzo threefold Y and compactiﬁcation of ACM sheaves on prime Fano threefolds of index one studied in [12]. Originally, instanton bundles appeared in [3] as a way to describe Yang-Mills instantons on a 4-sphere S4, which play an important role in Yang-Mills gauge theory. They provide extremelyinterestinglinksbetweenphysicsandalgebraicgeometry.Themathematicalnotion of an instanton bundle was ﬁrst introduced on P3 and generalized to Fano threefolds in [18, 1.2 Main results Let Mss X (r, c1, c2) be the moduli space of S-equivalence classes of rank r torsion-free Gieseker-semistable sheaves with Chern classes c1, c2 and ci = 0 for i ≥3, and MX(r, c1, c2) be its stable locus. We ﬁrst focus on the moduli spaces of (−1)-classes. Up to sign, there are three (−1)- classes in N(AX): s, 3s −t and 2s −t, and three (−1)-classes in N(Ku(Y)): v, v −w and 2v −w. We realize all these Bridgeland moduli spaces as classical moduli spaces. Theorem 1.2 (1) Let X be a prime Fano threefold of index 1 and degree 14. Let σ ′ be a Serre-invariant stability condition on AX. Then we have the following isomorphisms between classical moduli spaces and Bridgeland moduli spaces of stable objects: (a) (Proposition 4.4) C(X) ∼= −→Mσ ′(AX, s). (b) (Proposition 5.8) MX(2, 1, 6) ∼= −→Mσ ′(AX14, 3s −t). (c) (Proposition 5.11) MX(2, −1, 6) ∼= −→Mσ ′(AX14, 2s −t). (a) (Proposition 4.4) C(X) ∼= −→Mσ ′(AX, s). ∼ (b) (Proposition 5.8) MX(2, 1, 6) ∼= −→Mσ ′(AX14, 3s −t). ∼ (b) (Proposition 5.8) MX(2, 1, 6) ∼= −→Mσ ′(AX14, 3s −t). ∼ (c) (Proposition 5.11) MX(2, −1, 6) ∼= −→Mσ ′(AX14, 2s −t) (c) (Proposition 5.11) MX(2, −1, 6) ∼= −→Mσ ′(AX14, 2s −t). (2) Let Y be a cubic threefold. Let σ be a Serre-invariant stability condition on Ku(Y).Then we have the following isomorphisms between moduli spaces of stable sheaves and Bridge- land moduli spaces of stable objects: (2) Let Y be a cubic threefold. Let σ be a Serre-invariant stability condition on Ku(Y).Then we have the following isomorphisms between moduli spaces of stable sheaves and Bridge- land moduli spaces of stable objects: (a) (Proposition 5.15) Mb Y (2, 1, 2) ∼= −→Mσ (Ku(Y), v −w). (b) (Proposition 5.18) Mb Y (2, −1, 2) ∼= −→Mσ (Ku(Y), 2v −w). Using the equivalence in Theorem 1.1 for degree 14 prime Fano threefold X14 and associ- ated cubic threefold Y3 and the properties of Serre-invariant stability conditions on Kuznetsov components, we recover two classical results: 806 Z. Liu , S. Zhang Corollary 1.3 [23, Theorem 7.2] Let X be a prime Fano threefold of index 1 and degree 14. Then we have the following isomorphism: Corollary 1.3 [23, Theorem 7.2] Let X be a prime Fano threefold of index 1 and degree 14. 1.2 Main results Then we have the following isomorphism: MX(2, 1, 6) ∼= C(X) Corollary 1.4 [10, Theorem 1] Let Y be a cubic threefold. Then we have the following iso- morphism: Theorem 1.5 (Theorems 7.6, 7.7, 9.4) (1) Let Y := Yd be a prime Fano threefold of index 2 and degree d. When d ̸= 1, 2, we have an isomorphism between moduli space of S-equivalence classes of instanton sheaves and Bridgeland moduli space: Minst Y ∼= −→Mss σ (Ku(Y), 2v) for every Serre-invariant stability condition σ on Ku(Yd). When d = 1, 2, we have an open immersion: When d = 1, 2, we have an open immersion: Minst Y →Mss σ (Ku(Y), 2v) for every stability condition σ ∈K, where K is a certain family of stability conditions. for every stability condition σ ∈K, where K is a certain family of stability conditions. (2) L t X b i F th f ld f i d 1 d d 14 18 22 Th h for every stability condition σ ∈K, where K is a certain family of stability conditions. (2) Let X be a prime Fano threefold of index 1 and degree 14, 18 or 22. Then we have an for every stability condition σ ∈K, where K is a certain family of stability conditions. (2) Let X be a prime Fano threefold of index 1 and degree 14, 18 or 22. Then we have an isomorphism between moduli space of S-equivalence classes of semistable sheaves and d l d d l (2) Let X be a prime Fano threefold of index 1 and degree 14, 18 or 22. Then we have an isomorphism between moduli space of S-equivalence classes of semistable sheaves and Bridgeland moduli space: (2) Let X be a prime Fano threefold of index 1 and degree 14, 18 or 22. Then we have an isomorphism between moduli space of S-equivalence classes of semistable sheaves and Bridgeland moduli space: Mss X (2, 0, 4) ∼= −→Mss σ ′(AX, 2s) such that the restriction gives such that the restriction gives MX(2, 0, 4) ∼= −→Mσ ′(AX, 2s) for every Serre-invariant stability condition σ ′ on AX. 12 A note on Bridgeland moduli spaces and moduli... 807 In fact, one is able to show that there is an open immersion Minst Y2 →Mss σ (Ku(Y2), 2v) for every Serre-invariant stability condition σ on Ku(Y2). As a corollary, we have the following isomorphisms of moduli spaces of sheaves: Corollary 1.6 Let (Y, X) ∈Zd ⊂MF2 d × MF1 4d+2 and 3 ≤d ≤5. Theorem 1.5 (Theorems 7.6, 7.7, 9.4) Then we have the following isomorphism of moduli spaces Minst Y ∼= Mss X (2, 0, 4) More generally, we prove following results. Theorem 1.7 Let (Y, X) ∈Zd ⊂MF2 d × MF1 4d+2 and 3 ≤d ≤5. Let σ ′, σ be two Serre-invariant stability conditions on AX and Ku(Y) respectively. Then the equivalence in Theorem 1.1 induces the following isomorphisms between Bridgeland moduli spaces: Theorem 1.1 induces the following isomorphisms between Bridgeland moduli spaces: (1) (Corollary 4.2) Mσ (Ku(Y), a) ∼= −→Mσ ′(AX, a′) where a′, a are (−1)-classes in AX and Ku(Y) respectively. (1) (Corollary 4.2) Mσ (Ku(Y), a) ∼= −→Mσ ′(AX, a′) where a′, a are (−1)-classes in AX and Ku(Y) respectively. (2) (Corollary 8.5) When d ̸= 4, we have Mss σ (Ku(Y), b) ∼= −→Mss σ ′(AX, b′) where b′, b are two (−4)-classes in AX and Ku(Y) respectively. (2) (Corollary 8.5) When d ̸= 4, we have Mss σ (Ku(Y), b) ∼= −→Mss σ ′(AX, b′) where b′, b are two (−4)-classes in AX and Ku(Y) respectively. (3) (Theorem 8.4) When d = 4, we have Mss σ (Ku(Y), 2v) ∼= −→Mss σ ′(AX, 2s). (3) (Theorem 8.4) When d = 4, we have Mss σ (Ku(Y), 2v) ∼= −→Mss σ ′(AX, 2s). (3) (Theorem 8.4) When d = 4, we have Mss σ (Ku(Y), 2v) ∼= −→Mss σ ′(AX, 2s). 1.3 Related work The ﬁrst example of stability conditions constructed in the Kuznetsov component of a cubic threefold is given in [9]. After a direct construction of stability conditions in the Kuznetsov components of a series Fano threefolds in [7], the various Bridgeland moduli space of stable objects in the Kuznetsov components of index two and three Fano threefolds are studied in [1, 8, 11, 35, 36]. In [35], the Fano surface of lines on Yd, d ̸= 1 is identiﬁed with moduli space of stable objects with (−1)-class v in Ku(Yd), where d = 1 case is treated in [36]. In [24, 40], the authors dealt with Bridgeland moduli spaces for Gushel-Mukai threefolds, and used them to study several conjectures. In particular, the Bridgeland moduli spaces of stable objects with (−1)-classes in AX10 are realized as Cm(X) and MX(2, 1, 5), while in our note, in Theorem 1.2, we realize Bridgeland moduli space of stable objects of (−1)-classes in AX14 as C(X), MX(2, 1, 6) and MX(2, 1, −6). In our note, we identify moduli space of instanton sheaves with minimal charge on Yd, d = 3, 4, 5 with moduli space of (semi)stable objects of (−4)-class 2v in Ku(Yd). On cubic threefolds, these Bridgeland moduli spaces were studied in [29] via derived category of coherent sheaves of P2 with the action of a sheaf of Clifford algebras, and on Y4, Y5 these were studied in [37, 39] via classical stability of sheaves on curves and representations of quivers. q When the ﬁrst draft of our paper was ﬁnished, we learned that Xuqiang Qin independently proved similar results in [38] to Theorem 7.6. He studied Minst Yd for d = 3, 4, 5 and showed the isomorphism between Minst Yd and Mss σ (Ku(Yd), 2v) for a family of stability conditions K constructed in [35], while we show this for every Serre invariant stability condition deﬁned on Ku(Yd). Thus, we use slightly different techniques in proving Theorem 7.6. In [38], the author applies wall-crossing techniques developed in [8, 35], while we have to not only use wall-crossing techniques but also use weak Mukai lemma to prove stability for all Serre- invariant stability conditions. He also showed the stability of instanton bundles of minimal charge, while we prove this for all instanton sheaves in Proposition 7.2. Moreover, in [38], 123 Z. Liu , S. 1.5 Structure of the paper In Sects. 2 and 3, we recall some basic deﬁnitions and properties of Kuznetsov component and (weak)stability conditions. Fix Serre-invariant stability conditions σ and σ ′ on Ku(Y) and AX respectively. In Sect. 4, we ﬁrst show that all moduli spaces of stable objects of (−1)-classes in Ku(Y) and AX are isomorphic. Then we show that there is an isomorphism C(X) ∼= Mσ ′(AX, s) for every X := X4d+2 and d = 3, 4, 5. In Sect. 5 we focus on Y3 and X14. Using similar arguments in Sect. 4, we realize three moduli spaces of stable objects of (−1)-classes as classical moduli space. In Sect. 6 we ﬁrst recall some deﬁnitions and properties of instanton sheaves, and show that the instantonic condition is closely related to the walls of Bridgeland stability conditions. In Sect. 7, we use the results in Sect. 6 to show that for Y := Yd there are isomorphisms Minst Y →Mss σ (Ku(Y), 2v) when d ̸= 1, 2 and open immersions when d = 1, 2. In Sect. 8 we show that for every pair (Yd, X4d+2) ∈ Zd ⊂MF2 d × MF1 4d+2 and d = 3, 4, 5, the equivalence Ku(Yd) ∼= AX4d+2 gives an isomorphism Mσ (Ku(Y), 2v) ∼= Mσ ′(AX, 2s). As a corollary, when d = 3, 5 we show that all moduli spaces of semistable objects of (−4)-classes in Ku(Y) and AX are isomorphic. As an application, in Sect. 9 we show that for X := X4d+2 and d = 3, 4, 5 the projection functor gives an isomorphism Mss X (2, 0, 4) ∼= Mss σ ′(AX, 2s). 2.1 Prime Fano threefolds A complex smooth projective variety with ample anticanonical bundle is called Fano. A Fano variety is called prime if it has Picard number 1. For a prime Fano variety X, we can choose a unique ample divisor such that Pic(X) ∼= Z · H, which is called the fundamental divisor of X. The index of a prime Fano variety is the least integer i such that −K X = i · H. The degree of a prime Fano variety is d := H3. The genus g is deﬁned as 2g −2 = d. 1.4 Further questions It is an interesting question to classify instanton sheaves on a general quartic double solid Y2 and prove similar result as Theorem 7.6. On the other hand, in [40], it is shown that Ku(Y2) is not equivalent to AX10, so it is interesting to study the relation between Bridgeland moduli spaces Mσ (Ku(Y2), 2v) and Mσ ′(AX10, 2s) for Serre invariant stability conditions. 1.3 Related work Zhang 808 the author makes use of the notion of 2-Gieseker stability while we do not but apply more elementary techniques. In addition we prove Lemmas 7.11, 7.12 and Proposition 7.13 to make the theorem work for every Serre-invariant stability condition. the author makes use of the notion of 2-Gieseker stability while we do not but apply more elementary techniques. In addition we prove Lemmas 7.11, 7.12 and Proposition 7.13 to make the theorem work for every Serre-invariant stability condition. 2.2 Derived category of Fano threefolds Let Yd be a prime Fano threefold of index 2 and degree d. We consider the semiorthogonal decomposition of Yd given by Db(Yd) = ⟨Ku(Yd), OYd , OYd (1)⟩ 123 A note on Bridgeland moduli spaces and moduli... 809 The numerical Grothendieck group N(Ku(Yd)) of Ku(Yd) is a rank 2 lattice spanned by v := [IL] = 1 −1 d H2, w := H −1 2 H2 + 1 6 −1 d H3 where IL is an ideal sheaf of a line L on Yd. The Euler form is given by where IL is an ideal sheaf of a line L on Yd. The Euler form is given by −1 −1 1 −d −d (2.1) (2.1) In the case of index one, the semiorthogonal decomposition of X2g−2 of even genus 6 ≤g ≤ 12 are given in [25, 26]. In the case of index one, the semiorthogonal decomposition of X2g−2 of even genus 6 ≤g ≤ 12 are given in [25, 26]. Db(X2g−2) = ⟨Ku(X2g−2), E, OX2g−2⟩ Db(X2g−2) = ⟨Ku(X2g−2), E, OX2g−2⟩ where E is a rank 2 stable vector bundle with ch(E) = 2 −H + g−4 2 L + 10−g 12 P. However we will use another semiorthogonal decomposition in [28, Section B.2], which is given by Db(X2g−2) = ⟨AX2g−2, OX2g−2, E∨⟩ Db(X2g−2) = ⟨AX2g−2, OX2g−2, E∨⟩ The numerical Grothendieck group N(AX2g−2) of AX2g−2 is a rank 2 lattice spanned by The numerical Grothendieck group N(AX2g−2) of AX2g−2 is a rank 2 lattice spanned by s := [IC] = 1 − 1 g −1 H2, t := H − g 2 + 1 L −16 −g 12 P where IC is an ideal sheaf of a conic C on X2g−2. The Euler form is given by where IC is an ideal sheaf of a conic C on X2g−2. The Euler form is given by −1 −2 1 −g 2 1 −g (2.2) (2.2) As noted in [26, Proposition 3.9], the Chern character map identify the numerical Grothendieck group with the lattice generated by Chern characters of some sheaves. We will use notations of numerical class and Chern character alternatively. Up to sign, there are three (−1)-classes s, 3s −t and 2s −t in N(AX14), and three (−1)- classes v, 2v −w and v −w in N(Ku(Y3)). And there are three (−4)-classes, which are the twice of three (−1)-classes. μ −stable ⇒Gieseker stable ⇒Gieseker semistable ⇒μ −semistable μ −stable ⇒Gieseker stable ⇒Gieseker semistable ⇒μ −semistable We denote the moduli space of S-equivalence classes of rank r torsion-free Gieseker- semistable sheaves with Chern classes c1, c2 and c3 = 0 by Mss X (r, c1, c2). And we denote the moduli space of S-equivalence classes of rank r torsion-free Gieseker-stable sheaves with Chern classes c1, c2 and c3 = 0 by MX(r, c1, c2). 3 Review on Bridgeland stability conditions In this section, we recall the deﬁnition and some properties of (weak)stability conditions on triangulated category. 2.2 Derived category of Fano threefolds It is also easy to see that there are inﬁnitely many (0)-class, (−1)-class and (−4)-class, and no (−2)-class and (−3)-class in Ku(Y4) ∼= AX18; And there are inﬁnitely many (−1)-class and (−4)-class, and no (0)-class, (−2)-class and (−3)-class in Ku(Y5) ∼= AX22. We also need some results from [12, 28, 33]. Recall that a bundle F on a polarised smooth projective variety (X, H) of dimension n is called arithmetically Cohen–Macaulay (ACM) if Hi(F( j H)) = 0 for all 1 ≤i ≤n −1 and all j. Lemma 2.1 Let Db(X2g−2) = ⟨AX2g−2, OX, E∨⟩as above, then (1) For each g = 8, 10, 12 there is a closed immersion X2g−2 →Gr(2, g 2 + 2) such that E is the pullback of the tautological bundle on Gr(2, g 2 + 2). (1) For each g = 8, 10, 12 there is a closed immersion X2g−2 →Gr(2, g 2 + 2) such that E is the pullback of the tautological bundle on Gr(2, g 2 + 2). (2) E∨is the unique rank 2 stable vector bundle with c1 = H, c2 = g+2 2 L, c3 = 0, cal Mukai bundle. Moreover, E∨is globally generated and ACM. 123 810 Z. Liu , S. Zhang 2.3 Moduli spaces of sheaves In this subsection, we recall some deﬁnitions about (semi)stable sheaves. We refer to the book [22] for a more detailed account of notions. Let (X, H) be a smooth polarized n-dimensional projective variety. Recall that a torsion- free sheaf F is Gieseker-semistable if for any coherent subsheaf E with 0 < rk(E) < rk(F), one has p(E,t) rk(E) ≤p(F,t) rk(F) for t ≫0. The sheaf F is called Gieseker-stable if the inequality above is always strict. 1 The slope of a sheaf F with positive rank is deﬁned as μ(F) := c1(F)Hn−1 rk(F)Hn . We recall that a torsion-free coherent sheaf F is μ-semistable if for any coherent subsheaf E with 0 < rk(E) < rk(F), on has μ(E) ≤μ(F). The sheaf F is called μ-stable if the above inequality is always strict. The two notions are related as following: 3.1 (Weak)stability conditions σ-stable) if for every non-zero proper subobject B ⊂A, we have μσ (B) ≤μσ (A) (resp. μσ (B) < μσ (A)). An object F ∈T is called σ-(semi)stable if F[k] ∈A is σ-(semi)stable for some k ∈Z. (3) Every object A ∈A has a Harder-Narasimhan ﬁltration in σ-semistble objects. (3) Every object A ∈A has a Harder-Narasimhan ﬁltration in σ-semistble objects. (4) There is a quadratic form Q on ⊗R such that Q\|Ker(Z) is negative deﬁnite, and Q(A) ≥0 for all σ-semistable A ∈A. (4) There is a quadratic form Q on ⊗R such that Q\|Ker(Z) is negative deﬁnite, and Q(A) ≥0 for all σ-semistable A ∈A. A weak stability condition σ = (A, Z) on T with respect to the lattice is called a Bridgeland stability condition if Z ◦v is a stability function on A. Deﬁnition 3.4 The phase of a σ-semistable object A ∈A is Deﬁnition 3.4 The phase of a σ-semistable object A ∈A is φ(A) := 1 π arg(Z(A)) ∈(0, 1] and for A[n], we set φ(A[n]) := φ(A) + n. A slicing Pσ of T is a collection of full additive subcategories Pσ (φ) ⊂T for φ ∈R, such that the subcategory Pσ (φ) is given by the zero object and all σ-semistable objects with phase φ. We both use the notation σ = (Pσ , Z) and σ = (Aσ , Z) for a (weak)stability condition with heart Aσ := Pσ ((0, 1]), where Pσ is a slicing and Pσ ((0, 1]) is the extension closure of subcategories {Pσ (φ) \| φ ∈(0, 1]}. We denote by Stab(T ) the set of stability conditions on T . The universal covering space ˜ GL+ 2 (R) of GL+ 2 (R) has a right action on Stab(T ), deﬁned in [14, Lemma 8.2]. 2 2 Starting from a weak stability condition σ = (A, Z) on T , we can produce a new heart of a bounded t-structure, by tilting A. Let μ ∈R, we deﬁne following subcategoies of A: T μ σ := {A ∈A : all HN factors B of A have slope μσ (B) > μ} Fμ σ := {A ∈A : all HN factors B of A have slope μσ (B) ≤μ} Thus by [19], the category Aμ σ := ⟨T μ σ , Fμ σ [1]⟩is the heart of a bounded t-structure on T . 3.1 (Weak)stability conditions Let T be a triangulated category. Deﬁnition 3.1 A heart of a bounded t-structure on T is a full subcategory A ⊂T such that (1) for A, B ∈A and n < 0 we have Hom(A, B[n]) = 0, and (2) for every object F ∈T there exists a sequence of morphisms 0 = F0 φ1 −→F1 →· · · φm −→Fm = F such that cone(φi) is of form Ai[ki] for some sequence k1 > k2 > · · · > km of integers and objects Ai ∈A. We denote Ai by H−ki (F). Deﬁnition 3.2 Let A be an abelian category. A weak stability function on A is a group homomorphism Z : K(A) →C such that for every non-zero object A ∈A, we have ImZ(A) ≥0, and ImZ(A) = 0 ⇒ReZ(A) ≤0. We say Z is a stability function if ImZ(A) ≥0, and ImZ(A) = 0 ⇒ReZ(A) < 0. Fix a ﬁnite rank lattice and a surjective homomorphism v : K(A) ↠. Deﬁnition 3.3 A weak stability condition on T with respect to the lattice is a pair σ = (A, Z), where Z : →C is a group homomorphism and A is the heart of a bounded t-structure, satisfying the following conditions: 123 A note on Bridgeland moduli spaces and moduli... 811 (1) The composition K(A) = K(T ) v→ Z→C is a weak stability function on A; we denote Z(A) := Z(v(A)). We deﬁne the slope for any object A ∈A as (1) The composition K(A) = K(T ) v→ Z→C is a weak stability function on A; we denote Z(A) := Z(v(A)). We deﬁne the slope for any object A ∈A as (1) The composition K(A) = K(T ) v→ Z→C is a weak stability function on A; we denote Z(A) := Z(v(A)). We deﬁne the slope for any object A ∈A as μσ (A) := −ReZ(A) ImZ(A) μσ (A) := −ReZ(A) ImZ(A) for ImZ(A) > 0 and μσ (A) := +∞otherwise. (2) A non-zero object A ∈A is called σ-semistable (resp. σ-stable) if for every non-zero proper subobject B ⊂A, we have μσ (B) ≤μσ (A) (resp. μσ (B) < μσ (A)). An object F ∈T is called σ-(semi)stable if F[k] ∈A is σ-(semi)stable for some k ∈Z. (2) A non-zero object A ∈A is called σ-semistable (resp. 3.1 (Weak)stability conditions We say that the heart Aμ σ is obtained by tilting A with respect to the weak stability condition σ at the slope μ. 3.2 Weak stability condition on Db(X) Let X be a smooth projective variety of dimension n and H be an ample divisor on X. Following [7, Section 2], we review the construction of weak stability conditions on Db(X). For any j ∈{0, 1, 2, ..., n}, we consider the lattice j H ∼= Z j+1 generated by (Hnch0, Hn−1ch1, ..., Hn−jch j) ∈Q j+1 with surjective map v j H : K(X) → j H induced by Chern character. The pair σH := (Coh(X), Z H) where Z H : 1 H →C is given by Z H(F) := −Hn−1ch1(F) + i Hnch0(F) 123 812 Z. Liu , S. Zhang deﬁnes a weak stability condition on Db(X) with respect to the lattice 1 H (see [7, Example 2.8]). Moreover, any μH-semistable sheaf F satisﬁes the following Bogomolov–Gieseker inequality: deﬁnes a weak stability condition on Db(X) with respect to the lattice 1 H (see [7, Example 2.8]). Moreover, any μH-semistable sheaf F satisﬁes the following Bogomolov–Gieseker inequality: H(F) := (Hn−1ch1(F))2 −2Hnch0(F) · Hn−2ch2(F) ≥0. Given a parameter β ∈R, we denote by Cohβ(X) the heart of bounded t-structure obtained by tilting the weak stability condition σH at the slope μH = β. For F ∈Db(X), we set chβ(F) := e−βch(F). Explicitly, for each non-negative integer k we have chβ k (F) = k i=0 (−β)i Hi i! chk−i(F). Proposition 3.5 [7, Proposition 2.12] For any (α, β) ∈R>0 × R, the pair σα,β = (Cohβ(X), Zα,β) with Proposition 3.5 [7, Proposition 2.12] For any (α, β) ∈R>0 × R, the pair σα,β = (Cohβ(X), Zα,β) with Zα,β(F) := 1 2α2Hnchβ 0 (F) −Hn−2chβ 2 (F) + i Hn−1chβ 1 (F) deﬁnes a weak stability condition on Db(X) with respect to 2 H. The quadratic form Q can be given by the discriminant H. Moreover, these weak stability conditions vary continuously as (α, β) ∈R>0 × R varies. deﬁnes a weak stability condition on Db(X) with respect to 2 H. The quadratic form Q can be given by the discriminant H. Moreover, these weak stability conditions vary continuously as (α, β) ∈R>0 × R varies. We can visualize the weak stability conditions σα,β in the upper half plane R>0 × R. Deﬁnition 3.6 Let v be a vector in 2 H. Deﬁnition 3.6 Let v be a vector in 2 H. (1) A numerical wall for v is the set of pairs (α, β) ∈R>0 × R such that there is a vector w ∈2 H verifying the numerical relation μα,β(v) = μα,β(w). β H (2) A wall for E ∈Cohβ(X) is a numerical wall for v := ch≤2(E), where ch≤2(E) := (ch0(E), ch1(E), ch2(E)),suchthatforevery(α, β)onthewallthereisanexactsequence of semistable objects 0 →F →E →G →0 in Cohβ(X) such that μα,β(F) = μα,β(E) = μα,β(G) gives rise to the numerical wall. β (3) A wall for E ∈Cohβ(X) is called an actual wall if E becomes unstable after crossing the wall. (4) A chamber is a connected component in the complement of the union of walls in the upper half-plane. (4) A chamber is a connected component in the complement of the union of walls in the upper half-plane. An important property is that the weak stability conditions σα,β satisfy well-behaved wall-crossing: walls respect to a class v ∈2 H are locally ﬁnite. By [6, Proposition B.5], if v = ch≤2(E) with E ∈Cohβ(X), then the stability of E remains unchanged as (α, β) varies in a chamber. According to [31], a numerical wall for v ∈2 H is either a semicircle centered along the β-axis or a vertical wall parallel to the α-axis. No two walls intersect. Finally, we recall the following variant of the weak stability conditions of Proposition 3.5, which will be used frequently. Fix μ ∈R and let u be the unit vector in upper half plane with μ = −Reu Imu . We denote by Cohμ α,β(X) the heart obtained by tilting the weak stability condition σα,β = (Cohβ(X), Zα,β) at the slope μα,β = μ. Proposition 3.7 [7, Proposition 2.15] The pair σ μ α,β := (Cohμ α,β(X), Zμ α,β), where Zμ α,β := 1 u Zα,β is a weak stability condition on Db(X). 123 A note on Bridgeland moduli spaces and moduli... 813 3.3 Serre-invariant stability conditions on Kuznetsov component Hence K = V · ˜ GL+ 2 (R) = σ(α0, −1 2) · ˜ GL+ 2 (R). (2) Fix 0 < α0 < 1 2. For every (α, β) ∈V , there is a ˜g ∈ ˜ GL+ 2 (R) such that σ(α, β) = σ(α0, −1 2) · ˜g. Hence K = V · ˜ GL+ 2 (R) = σ(α0, −1 2) · ˜ GL+ 2 (R). 2 2 (3) Every stability condition in K is Serre-invariant. 2 2 (3) Every stability condition in K is Serre-invariant. (3) Every stability condition in K is Serre-invariant. (3) Every stability condition in K is Serre-invariant. Thus via the equivalences Ku(Yd) ∼= AX4d+2 for d = 3, 4, 5, since this equivalence commutes with Serre functor, a Serre-invariant stability condition on Ku(Yd) induces a Serre-invariant stability condition on AX4d+2. Thus via the equivalences Ku(Yd) ∼= AX4d+2 for d = 3, 4, 5, since this equivalence commutes with Serre functor, a Serre-invariant stability condition on Ku(Yd) induces a Serre-invariant stability condition on AX4d+2. As shown in [35, Section 5.2], there are some useful numerical properties for Serre- invariant stability condition on Ku(Y). Note that in the proof of following lemmas in [35], the only properties of Y they used is that S3 Ku(Y) ∼= [5], hence via the equivalence in Theorem [28], these properties also hold for Serre-invariant stability conditions on AX. For X = X14, we have: As shown in [35, Section 5.2], there are some useful numerical properties for Serre- invariant stability condition on Ku(Y). Note that in the proof of following lemmas in [35], the only properties of Y they used is that S3 Ku(Y) ∼= [5], hence via the equivalence in Theorem [28], these properties also hold for Serre-invariant stability conditions on AX. For X = X14, we have: Lemma 3.10 For every Serre-invariant stability condition σ on AX14, if F ∈AX14 is a σ-semistable object of phase φ(F), then the phase of SAX14 (F) satisﬁes φ(F) < φ(SAX14 (F)) < φ(F) + 2. In particular, we have Ext2(F, F) = 0. Lemma 3.11 The heart of a Serre-invariant stability condition σ on AX14 has homological dimension 2. Lemma 3.12 For every Serre-invariant stability condition σ = (A, Z) on AX14 and every non-zero object F ∈A, we have χ(F, F) ≤−1 and ext1(F, F) ≥2. 3.3 Serre-invariant stability conditions on Kuznetsov component In this subsection, we recall some results in [35], which we will use frequently in the next sections. Assume that T is a triangulated category with the Serre functor ST . Deﬁnition 3.8 A stability σ on T is called Serre-invariant if ST (σ) = σ · ˜g for some ˜g ∈ ˜ GL+ 2 (R). Recall that Ku(Y4) ∼= AX18 ∼= Db(C2) and Ku(Y5) ∼= AX22 ∼= Db(Q3). Here C2 is a smooth curve of genus 2 and Q3 is the 3-Kronecker quiver (see [28]). Stability conditions on these two categories are studied in [16, 32]. Following [35], we deﬁne Following [35], we deﬁne V := {(α, β) ∈R>0 × R \| −1 2 ≤β < 0, α < −β, or −1 < β < −1 2, α ≤1 + β} and let K be the orbit of V by ˜ GL+(R) We set Z(α β) : Z0 \| and A(α β) : V := {(α, β) ∈R>0 × R \| −1 2 ≤β < 0, α < −β, or −1 < β < −1 2, α ≤1 + β} and let K be the orbit of V by ˜ GL+ 2 (R). We set Z(α, β) := Z0 α,β\|Ku(Y), and A(α, β) := Coh0 α,β(Y) ∩Ku(Y). We deﬁne a lattice 2 H,Ku(Y) := Im(K(Ku(Y)) →K(Y) →2 H) ∼= Z2. and let K be the orbit of V by ˜ GL+ 2 (R). We set Z(α, β) := Z0 α,β\|Ku(Y), and A(α, β) := Coh0 α,β(Y) ∩Ku(Y). We deﬁne a lattice 2 H,Ku(Y) := Im(K(Ku(Y)) →K(Y) →2 H) ∼= Z2. Theorem 3.9 [7], [35, Theorem 3.3, Proposition 3.6, Corollary 5.5] Let Y be a prime Fano threefold of index 2. (1) The pair σ(α, β) := (A(α, β), Z(α, β)) is a Bridgeland stability condition on Ku(Y) with respect to 2 H,Ku(Y) ∼= Z2 for every (α, β) ∈V . (2) Fix 0 < α0 < 1 2. For every (α, β) ∈V , there is a ˜g ∈ ˜ GL+ 2 (R) such that σ(α, β) = σ(α0, −1 2) · ˜g. Hence K = V · ˜ GL+ 2 (R) = σ(α0, −1 2) · ˜ GL+ 2 (R). (2) Fix 0 < α0 < 1 2. For every (α, β) ∈V , there is a ˜g ∈ ˜ GL+ 2 (R) such that σ(α, β) = σ(α0, −1 2) · ˜g. 4 Isomorphisms between Bridgeland moduli spaces of (−1)-classes In this section, we ﬁx (Y, X) = (Yd, X4d+2) ∈Zd ⊂MF2 d × MF1 4d+2 as in [28] for d = 3, 4, 5. We denote the closed point in Mσ ′ that corresponds to E ∈AX by [E]. In this section, we ﬁx (Y, X) = (Yd, X4d+2) ∈Zd ⊂MF2 d × MF1 4d+2 as in [28] for d = 3, 4, 5. We denote the closed point in Mσ ′ that corresponds to E ∈AX by [E]. Proposition 4.1 The equivalence : Ku(Y) ∼= −→AX in Theorem 1.1 induces an isomorphism between moduli spaces Proposition 4.1 The equivalence : Ku(Y) ∼= −→AX in Theorem 1.1 induces an isomorphism between moduli spaces Mσ (Ku(Y), 1 −L) ∼= −→Mσ ′(AX, 1 −2L) such that maps [E] to [(E)] on the level of closed points. Here σ, σ ′ are Serre-invariant stabilityconditionson Ku(Y), AX respectively.Inparticular, Mσ ′(AX, 1−2L)isirreducible and smooth of dimension 2. Proof From [35, Theorem 1.1] we know that (Y) ∼= Mσ (Ku(Y), 1 −L), hence there is a universal family on Mσ (Ku(Y), 1 −L). Since is of Fourier-Mukai type, the same argument in Lemma 4.3 shows that induces a morphism such that maps [E] to [(E)]. By Lemma 3.14 we know that this morphism is a bijection between closed points. Now since is an equivalence, the induced morphism is étale. Therefore, this morphism is bijective and étale, which is an isomorphism. The last statement also follows from [35, Theorem 1.1]. ⊓⊔ Corollary 4.2 Let a be a (−1)-class in N(Ku(Y)) and a′ be a (−1)-class in N(AX). Then the Bridgeland moduli spaces Mσ (Ku(Y), a) ∼= Mσ ′(AX, a′) for any Serre invariant stability condition σ, σ ′ on Ku(Y), AX respectively. Corollary 4.2 Let a be a (−1)-class in N(Ku(Y)) and a′ be a (−1)-class in N(AX). Then the Bridgeland moduli spaces Mσ (Ku(Y), a) ∼= Mσ ′(AX, a′) for any Serre invariant stability condition σ, σ ′ on Ku(Y), AX respectively. Proof (1) When d = 3, all (−1)-classes in AX14 up to signs are [IC] = 1 −2L, [SAX (IC)] and [S2 AX (IC)], and all (−1)-classes in Ku(Y3) up to signs are [IL] = 1−L, [SKu(Y3)(IL)] and [S2 Ku(Y3)(IL)]. Now σ and σ ′ are Serre-invariant stability conditions, thus Bridgeland moduli spaces of stable objects with these (−1)-classes are all isomorphic. 3.3 Serre-invariant stability conditions on Kuznetsov component Lemma 3.13 (Weak Mukai Lemma) Let σ be a Serre-invariant stability condition on AX2g−2. Let A →F →B be a triangle in the heart Aσ such that hom(A, B) = Hom(B, A[2]) = 0. Then ext1(A, A) + ext1(B, B) ≤ext1(F, F) ext1(A, A) + ext1(B, B) ≤ext1(F, F) 123 123 123 814 Z. Liu , S. Zhang When g = 8, as in [35], Hom(B, A[2]) = 0 if σ-semistable factors of A have phases greater or equal than the phases of the σ-semistable factors of B. When g = 10 and 12, since every heart of a stability condition on A2g−2 has homological mension 1, hence automatically we have Hom(B, A[2]) = 0. When g = 10 and 12, since every heart of a stability condition on A2g−2 has homological dimension 1, hence automatically we have Hom(B, A[2]) = 0. The same argument in [35, Lemma 5.13] shows that: Lemma 3.14 Let X := X14 or X22. Let σ be a Serre-invariant stability condition on AX. Then every F ∈AX with ext1(F, F) = 2 is σ-stable. If X = X18, then every F ∈AX with ext1(F, F) = 1 or χ(F, F) = −1 and ext1(F, F) = 2 is σ-stable. eorem 3.15 [35, Theorem 1.2] Let X := X14, X18 or X22. Every non-empty m σ ′-stable objects in AX with respect to a Serre-invariant stability condition σ 4 Isomorphisms between Bridgeland moduli spaces of (−1)-classes On the other hand, the linear isometry of N(Ku(Y5)) on (−1)-classes, given by (x, y) →(−4x −5y, x + y) is induced by the rotation functor R : Ku(Y5) →Ku(Y5). It is straightforward to check its action on (x, y)(representing a (−1)-class) corresponds to the multiplication by η. This means that all (−1)-classes in N(Ku(Y5)) are given by action of rotation functor R on 1−L(up to sign). Thus desired result follows from similar arguments in Proposition 4.1. it is the Pell’s equation x2 −5y2 = 1, which is a Diophantine equation of the form x2−Ny2 = 1 with a positive non-square integer N. It is a norm equation in the sense that allsolutions of the equation is the setof allelements in the ringQ( √ 5)of the form x+ √ 5y whose norm is 1. Then by Dirichlet unit theorem, the set of the solutions is an index 2 subgroup of the unit group of the ring of algebraic integers in Q( √ 5), which is isomorphic to Z(up to sign), generated by the fundamental unit η = 1 2(3 + √ 5) by [15]. Thus all the integer solutions (x, y) are parametrised by ηi, i ∈Z, up to sign. On the other hand, the linear isometry of N(Ku(Y5)) on (−1)-classes, given by (x, y) →(−4x −5y, x + y) is induced by the rotation functor R : Ku(Y5) →Ku(Y5). It is straightforward to check its action on (x, y)(representing a (−1)-class) corresponds to the multiplication by η. This means that all (−1)-classes in N(Ku(Y5)) are given by action of rotation functor R on 1−L(up to sign). Thus desired result follows from similar arguments in Proposition 4.1. 4.1 Hilbert schemes of conics as Bridgeland moduli spaces In this subsection, we show that there is an isomorphism between the Fano surface of conics C(X) and the Bridgeland moduli space p : C(X) ∼= →Mσ ′(AX, 1 −2L). for X := X14, X18 or X22 and every Serre-invariant stability condition σ ′ on AX. y y Let σ ′ be a Serre-invariant Bridgeland stability condition on AX. First, we construct a natural morphism p : C(X) →Mσ ′(AX, 1 −2L): Let σ ′ be a Serre-invariant Bridgeland stability condition on AX. First, we construct a ural morphism p : C(X) →Mσ ′(AX, 1 −2L): 4 Isomorphisms between Bridgeland moduli spaces of (−1)-classes (2) When d = 4, there are inﬁnitely many (−1)-classes in N(Ku(Y4)) and N(AX18) respec- tively. Note that Ku(Y4) ∼= AX18 ∼= Db(C2). All (−1)-classes in Ku(Y4) are permuted by rotation functor R : E →LOY (E ⊗OY (H)) and the functor preserves the stability con- ditions on Ku(Y4) by [35, Proposition 5.7]. Then by similar arguments in Proposition 4.1, all the moduli spaces are isomorphic. (2) When d = 4, there are inﬁnitely many (−1)-classes in N(Ku(Y4)) and N(AX18) respec- tively. Note that Ku(Y4) ∼= AX18 ∼= Db(C2). All (−1)-classes in Ku(Y4) are permuted by rotation functor R : E →LOY (E ⊗OY (H)) and the functor preserves the stability con- ditions on Ku(Y4) by [35, Proposition 5.7]. Then by similar arguments in Proposition 4.1, all the moduli spaces are isomorphic. (3) d = 5. Let xv + yw ∈N(Ku(Y5)) be a (−1)-class, then the pair of integers (x, y) are solutions of equation x2+5xy+5y2 = 1. Up to a linear transform: X = x+ 5 2 y, Y = 1 2 y, (3) d = 5. Let xv + yw ∈N(Ku(Y5)) be a (−1)-class, then the pair of integers (x, y) are solutions of equation x2+5xy+5y2 = 1. Up to a linear transform: X = x+ 5 2 y, Y = 1 2 y, (3) d = 5. Let xv + yw ∈N(Ku(Y5)) be a (−1)-class, then the pair of integers (x, y) are solutions of equation x2+5xy+5y2 = 1. Up to a linear transform: X = x+ 5 2 y, Y = 1 2 y, 123 A note on Bridgeland moduli spaces and moduli... 815 it is the Pell’s equation x2 −5y2 = 1, which is a Diophantine equation of the form x2−Ny2 = 1 with a positive non-square integer N. It is a norm equation in the sense that allsolutions of the equation is the setof allelements in the ringQ( √ 5)of the form x+ √ 5y whose norm is 1. Then by Dirichlet unit theorem, the set of the solutions is an index 2 subgroup of the unit group of the ring of algebraic integers in Q( √ 5), which is isomorphic to Z(up to sign), generated by the fundamental unit η = 1 2(3 + √ 5) by [15]. Thus all the integer solutions (x, y) are parametrised by ηi, i ∈Z, up to sign. Next, we show that p is an isomorphism. Lemma 4.3 The projection functor pr induces a morphism Lemma 4.3 The projection functor pr induces a morphism p : C(X) →Mσ ′(AX, 1 −2L) where p([C]) = [pr(IC)] = [IC] on the level of closed points. 5 Bridgeland moduli spaces of (−1)-classes on X14 and Y3 In this section we ﬁx X := X14 and Y := Y3. We show that MX(2, 1, 6) ∼= Mσ ′(AX14, 3s−t) and MX(2, −1, 6) ∼= Mσ ′(AX14, 2s−t) on X. And we have Mb Y (2, 1, 2) ∼= Mσ (Ku(Y), v− w) and Mb Y (2, −1, 2) ∼= Mσ (Ku(Y), 2v −w) on Y. In this section we ﬁx X := X14 and Y := Y3. We show that MX(2, 1, 6) ∼= Mσ ′(AX14, 3s−t) and MX(2, −1, 6) ∼= Mσ ′(AX14, 2s−t) on X. And we have Mb Y (2, 1, 2) ∼= Mσ (Ku(Y), v− w) and Mb Y (2, −1, 2) ∼= Mσ (Ku(Y), 2v −w) on Y. where p([C]) = [pr(IC)] = [IC] on the level of closed points. Hence we know that dp is an isomorphism at every closed point, and therefore p is étale. Now since C(X) is projective and Mσ ′(AX, 1 −2L) is proper, p is projective. Thus p is an embedding. But Mσ ′(AX, 1 −2L) is irreducible and smooth by Proposition 4.1, hence p is actually an isomorphism. ⊓⊔ Corollary 4.5 If F ∈AX is a σ ′-stable object for some Serre-invariant stability conditions σ ′ on AX with [F] = [IC] ∈N(AX), then F ∼= IC[2k] for some conics C on X and k ∈Z. 5.1 MX(2, 1, 6) as Bridgeland moduli space As shown in [23], there are two classes of sheaves in MX(2, 1, 6): globally generated locally free sheaves and non-locally free sheaves. We ﬁrst deal with locally free sheaves. When E ∈MX(2, 1, 6) is a locally free sheaf, by [23, Section 5.2] we have an exact sequence: 0 →OX →E →IC(1) →0 where C is an elliptic sextic. p First, we determine the image of E under the projection functor pr. Recall that for a rank two locally free sheaf F, we have F∨∼= F ⊗det F∨. Lemma 5.1 Let E ∈MX(2, 1, 6) be a locally free sheaf, then we have: Lemma 5.1 Let E ∈MX(2, 1, 6) be a locally free sheaf, then we have: (1) h0(E) = 5, h1(E) = h2(E) = h3(E) = 0. (2) h0(E∨) = h1(E∨) = h2(E∨) = h3(E∨) = 0. (3) hom(E, E) = 1, ext1(E, E) = 2, ext2(E, E) = ext3(E, E) = 0. ( ) ( ) , ( ) ( ) ( ) (2) h0(E∨) = h1(E∨) = h2(E∨) = h3(E∨) = 0. (3) hom(E, E) = 1, ext1(E, E) = 2, ext2(E, E) = ext3(E, E) = 0. Proof (1) is from [23, Lemma 5.1, Proposition 5.2] and the fact χ(E) = 5. Proof (1) is from [23, Lemma 5.1, Proposition 5.2] and the fact χ(E) = 5. For (2), by Serre duality we have h1(E∨) = h2(E(−1)) = h2(E∨), hence h1(E∨) = h2(E∨) = 0 by [23, Proposition 5.2]. And h0(E∨) = 0 from stability of E, therefore by Serre duality we have h0(E∨) = h3(E(−1)) = h3(E∨) = 0. ⊓⊔ (3) is from [23, Proposition 5.10]. ⊓⊔ Lemma 5.2 Let E ∈MX(2, 1, 6) be a locally free sheaf, then we have exti(E∨, E) = 0 for all i. Lemma 5.2 Let E ∈MX(2, 1, 6) be a locally free sheaf, then we have exti(E∨, E) = 0 for all i. Proof From the stability and Serre duality, we know exti(E∨, E) = 0 for i = 0, 3. Since E is globally generated, a general section of E will vanish along an elliptic sextic C in Gr(2, 6). Then we have an exact sequence as in [23]: Proof From the stability and Serre duality, we know exti(E∨, E) = 0 for i = 0, 3. where p([C]) = [pr(IC)] = [IC] on the level of closed points. where p([C]) = [pr(IC)] = [IC] on the level of closed points. Proof From [28, Lemma B.3.3] we know that IC ∈AX for every conic C on X. Hence pr(IC) = IC. On the other hand, we know that ext1(IC, IC) = 2, hence by Lemma 3.14 IC is in the heart Aσ ′ up to a shift and σ ′-stable. Similar arguments in [9, Section 5.1] and [1, Section 5.1] shows that this shift can be chosen uniformly. Now we are going to show that there is a natural morphism p induced by functor pr. Since the projection functor pr : Db(X) →AX is of Fourier-Mukai type, we can assume K ∈Db(X × X) is the integral kernel and ψK : Db(X) pr −→AX →Db(X) be the Fourier- Mukai transform deﬁned by K. Let I be the universal ideal sheaf on C(X) × X. We deﬁne ψ′ := ψK × idC(X) = ψK⊠O C(X) : Db(X × C(X)) →Db(X × C(X)) Then ψ′(I) is a family of objects in AX parametrised by C(X), which deﬁnes a morphism p : C(X) →Mσ ′(AX, 1 −2L). Then ψ′(I) is a family of objects in AX parametrised by C(X), which deﬁnes a morphism p : C(X) →Mσ ′(AX, 1 −2L). To show p([C]) is given by pr(IC) for any closed point c = [C] ∈C(X), we denote ic : {c} × X →C(X) × X. Then we have: ψK (i∗ c (I)) ∼= pr(IC) and i∗ c (ψK⊠O C(X) (I)) ∼= ψi∗c K⊠O C(X) (IC) ∼= ψK (IC) = pr(IC) This means p([C]) is given by [pr(IC)]. ⊓⊔ Next, we show that p is an isomorphism. 123 816 Z. Liu , S. Zhang Proposition 4.4 The morphism p : C(X) →Mσ ′(AX, 1 −2L) deﬁned in Lemma 4.3 is an isomorphism. Proposition 4.4 The morphism p : C(X) →Mσ ′(AX, 1 −2L) deﬁned in Lemma 4.3 is an isomorphism. Proof It is clear that p is injective. The tangent map dp at a closed point [C] is given by dp[C] : Ext1(IC, IC) →Ext1(pr(IC), pr(IC)) dp[C] : Ext1(IC, IC) →Ext1(pr(IC), pr(IC)) Since IC is already in AX, we have pr(IC) = IC. Hence we know that dp is an isomorphism at every closed point, and therefore p is étale. Since IC is already in AX, we have pr(IC) = IC. 5.1 MX(2, 1, 6) as Bridgeland moduli space Since E is globally generated, a general section of E will vanish along an elliptic sextic C in Gr(2, 6). Then we have an exact sequence as in [23]: Then we have an exact sequence as in [23]: 0 →OX →E →IC(1) →0 123 A note on Bridgeland moduli spaces and moduli... 817 Applying Hom(E∨, −) to this sequence, and since exti(E∨, OX) = 0 for every i, we have Exti(E∨, E) = Exti(E∨, IC(1)) = Hi(X, E∨⊗IC), ∀i ≥0. Applying Hom(E∨, −) to this sequence, and since exti(E∨, OX) = 0 for every i, we have Exti(E∨, E) = Exti(E∨, IC(1)) = Hi(X, E∨⊗IC), ∀i ≥0. As in [23, Section 5.2], E∨\|C is split of type (3, 3) or unsplit. When E∨\|C is split, it is clear that h0(E∨\|C) = 6. When E∨\|C is unsplit, in this case E∨\|C is the tensor of a degree 3 line bundle N with a unique vector bundle F obtained as a non-trivial extension of OC. Thus E∨\|C is a non-trivial extension of N, which gives h0(E∨\|C) = 6. Now if the restriction map H0(E∨) →H0(E∨\|C) is not injective, then C is contained in a copy of Gr(2, 5). But this is impossible since C is of degree 6 and Gr(2, 5) has degree 5. Then the restriction map H0(E∨) →H0(E∨\|C) is an isomorphism from the injectivity. Now applying (X, E∨⊗−) to the standard exact sequence of C, we obtain Exti(E∨, E) = Hi(E∨⊗IC) = 0. ⊓⊔ ⊓⊔ Therefore by Lemma 5.2, we have pr(E) = LOX (E). From Lemma 5.1 we know that pr(E) = LOX (E) is given by ker(ev)[1], where ev is the evaluation map: 0 →ker(ev) →H0(E) ⊗OX ev −→E →0 (5.1) It is clear that ch(ker(ev)) = 3−H −L+ 2 3 P. We are going to check that pr(E) = ker(ev)[1] is σ ′-stable. 0 →ker(ev) →H0(E) ⊗OX ev −→E →0 (5.1) 2 (5.1) It is clear that ch(ker(ev)) = 3−H −L+ 2 3 P. We are going to check that pr(E) = ker(ev)[1] is σ ′-stable. Lemma 5.3 We have ext1(ker(ev), ker(ev)) = 2. Hence pr(E) = ker(ev)[1] ∈AX is σ ′- stable with respect to every Serre-invariant stability condition σ ′ on AX. Proof The second statement follows from the ﬁrst one and Lemma 3.14, hence we only need to show the ﬁrst statement. TensoringOX(−1)withthesequence5.1andtakingcohomology,wehavehi(ker(ev)(−1)) = 5 · hi(O(−1)) for all i by Lemma 5.1. 5.1 MX(2, 1, 6) as Bridgeland moduli space Zhang Lemma 5.4 Let E ∈MX(2, 1, 6) be a non-locally free sheaf, then we have: Lemma 5.4 Let E ∈MX(2, 1, 6) be a non-locally free sheaf, then we have: (1) h0(E) = 5, h1(E) = h2(E) = h3(E) = 0. (2) h0(E(−1)) = h1(E(−1)) = h2(E(−1)) = h3(E(−1)) = 0. (3) hom(E, E) = 1, ext1(E, E) = 2, ext2(E, E) = ext3(E, E) = 0. (2) h0(E(−1)) = h1(E(−1)) = h2(E(−1)) = h3(E(−1)) = 0. 1 2 3 Proof (1) and (2) both follow from Lemma 2.1 and the above exact sequence 5.2. (3) is from [23, Proposition 5.12]. ⊓⊔ Proof (1) and (2) both follow from Lemma 2.1 and the above exact sequence 5.2. (3) is from [23, Proposition 5.12]. ⊓⊔ Lemma 5.5 Let E ∈MX(2, 1, 6) be a non-locally free sheaf, then we have Exti(E∨, E) = 0 for every i. Proof By Proposition 2.1, we know ext1(T , T ) = ext2(T , T ) = ext3(T , T ) = 0 and hom(T , T ) = 1. From [23, Proposition 5.11] we have T ∨⊗OL ∼= OL ⊕OL(−1), hence we obtain hom(T , OL) = 1 and ext1(T , OL) = 0. Since Hom(T , T ) →Hom(T , OL) is induced by the surjective map T ↠OL, this map is non-trivial, hence for dimensional reason it is an isomorphism. Thus applying Hom(T , −) to the sequence 5.2 above, from the long exact sequence we obtain that Exti(T , E) = 0, ∀i ≥0. ⊓⊔ ⊓⊔ Therefore by Lemma 5.5, pr(E) = LOX (E), which is given by ker(ev)[1], where ev is the evaluation map: 0 →ker(ev) →H0(E) ⊗OX ev −→E →0. Since H0(E) ⊗E is locally free and E is torsion free, by [20, Proposition 1.1] we know that in this case ker(ev) is reﬂexive. Now we want to check pr(E) = ker(ev)[1] is σ ′-stable. This is almost the same as locally free case: Lemma 5.6 We have ext1(ker(ev), ker(ev)) = 2. Hence pr(E) = ker(ev)[1] ∈AX is σ ′- stable with respect to every Serre-invariant stability condition σ ′ on AX. Proof The second statement follows from Lemma 3.14, hence we only need to show the ﬁrst statement. Using Lemma 5.4, the same arguments in Lemma 5.3 shows that exti(ker(ev), ker(ev)) = 0 for i = 2, 3. 5.1 MX(2, 1, 6) as Bridgeland moduli space Hence we know h3(ker(ev)(−1)) = 5 and hi(ker(ev)(−1)) = 0 for i ̸= 3. Thus by Serre duality we have hom(ker(ev), H0(E) ⊗ OX) = 25, exti(ker(ev), H0(E) ⊗OX) = 0 for i ̸= 0. By Serre duality we have Exti(ker(ev), E) = Ext3−i(E(1), ker(ev)). Again by Serre duality, we have exti(E(1), H0(E) ⊗OX) = 0 when i ̸= 3 and ext3(E(1), H0(E) ⊗ OX) = 25. Also we have Exti(E(1), E) = Ext3−i(E, E), hence by Lemma 5.1 we know that hom(E(1), E) = ext1(E(1), E) = 0. Therefore applying Hom(E(1), −) to the exact sequence 5.1 and taking long exact sequence, we obtain hom(E(1), ker(ev)) = ext1(E(1), ker(ev)) = ext2(E(1), ker(ev)) = 0. Finally, applying Hom(ker(ev), −) to the sequence 5.1, we obtain a long exact sequence. From computations above, we have exti(ker(ev), ker(ev)) = 0 for i = 2, 3. Since χ(ker(ev), ker(ev)) = −1, we only need to show hom(ker(ev), ker(ev)) = 1. Note that H0(X, ker(ev)) = 0, then via the inclusion p ker(ev) →p−1 ker(ev) ⊗H0(E) we know that H0(X, p ker(ev)) = 0 for all p ≥1. And we have −1 < μ(p ker(ev)) = pμ(ker(ev)) < 0 for every 1 ≤p ≤2. Therefore ker(ev) is a stable bundle by Hoppe’s criterion [21] (see also [2, Theorem 1.2]). Hence we obtain that hom(ker(ev), ker(ev)) = 1 and ext1(ker(ev), ker(ev)) = 2. ⊓⊔ ⊓⊔ When E ∈MX(2, 1, 6) is not locally free, by [23, Proposition 5.11], there is an exact sequence: When E ∈MX(2, 1, 6) is not locally free, by [23, Proposition 5.11], there is an exact sequence: 0 →E →T →OL →0 (5.2) (5.2) where L is a line on X. By [13, Proposition 3.5], we know T is a rank 2 globally generated stable vector bundle with c1(T ) = H, c2(T ) = 5L. Thus from Proposition 2.1, we have T ∼= E∨. where L is a line on X. By [13, Proposition 3.5], we know T is a rank 2 globally generated stable vector bundle with c1(T ) = H, c2(T ) = 5L. Thus from Proposition 2.1, we have T ∼= E∨. 818 Z. Liu , S. 5.1 MX(2, 1, 6) as Bridgeland moduli space Since χ(ker(ev), ker(ev)) = −1, we only need to show hom(ker(ev), ker(ev)) = 1. Note that ker(ev) is reﬂexive, hence is determined by the complement of any closed subset of codimension ≥2 (see for example, [20]). And since the non-locally free locus of ker(ev) has codimension ≥3, without loss of generality we can assume ker(ev) is locally free. Hence the same argument in Lemma 5.3 shows that hom(ker(ev), ker(ev)) = 1. ⊓⊔ Lemma 5.7 The projection functor induces a morphism Lemma 5.7 The projection functor induces a morphism q : MX(2, 1, 6) →Mσ ′(AX, 3s −t) for every Serre-invariant stability condition σ ′ on AX, such that q([E]) = [pr(E)]. Proof Since χ(E, E) = −1, by [22, Theorem 4.6.5] we know MX(2, 1, 6) is a ﬁne moduli space. Thus using Lemmas 5.3 and 5.6, this lemma follows from the same argument in Lemma 4.3. ⊓⊔ Proposition 5.8 The morphism q : MX(2, 1, 6) →Mσ ′(AX, 3s −t) deﬁned in Lemma 5.7 is an isomorphism. Proposition 5.8 The morphism q : MX(2, 1, 6) →Mσ ′(AX, 3s −t) deﬁned in Lemma 5.7 is an isomorphism. 123 A note on Bridgeland moduli spaces and moduli... 819 Proof It is clear that q is injective. Let [E] ∈MX(2, 1, 6) be a closed point, then T[E]MX(2, 1, 6) = Ext1(E, E). And we know T[q(E)]Mσ ′ = Ext1(ker(ev), ker(ev)) = Ext1(ker(ev)[1], ker(ev)[1]) = Ext1(pr(E), pr(E)). By deﬁnition of projection, we have an exact triangle H0(E) ⊗OX ev −→E →pr(E) H0(E) ⊗OX ev −→E →pr(E) ying Hom(E, −) to this triangle and using Lemma 5.4, we obtain a long exact sequence: Applying Hom(E, −) to this triangle and using Lemma 5.4, we obtain a long exact sequence: 0 →Hom(E, E) →Hom(E, pr(E)) →0 →Ext1(E, E) d[E]q −−−→Ext1(E, pr(E)) = Ext1(pr(E), pr(E)) →0 0 →Hom(E, E) →Hom(E, pr(E)) →0 →Ext1(E, E) d[E]q −−−→Ext1(E, pr(E)) = Ext1(pr(E), pr(E)) →0 The last equality is from the adjunction of pr and i, where i is the embedding AX →Db(X). Thus the tangent map d[E]q : Ext1(E, E) →Ext1(E, pr(E)) = Ext1(pr(E), pr(E)) is an isomorphism. The last equality is from the adjunction of pr and i, where i is the embedding AX →Db(X). Thus the tangent map d[E]q : Ext1(E, E) →Ext1(E, pr(E)) = Ext1(pr(E), pr(E)) is an isomorphism. Finally, we know that MX(2, 1, 6) and Mσ ′(AX, 3s −t) are both proper, hence q is also proper. And since q is injective and étale, it is an embedding. By Proposition 4.1 and Corollary 4.2, we know that Mσ ′(AX, 3s −t) is irreducible and smooth, therefore q is actually an isomorphism. ⊓⊔ ⊓⊔ 5.2 MX(2, −1, 6) as Bridgeland moduli space Zhang Proposition 5.11 The projection functor pr induces an isomorphism p′ : MX(2, −1, 6) ∼= −→Mσ ′(AX, 2s −t) Proposition 5.15 The projection functor pr induces an isomorphism Proposition 5.15 The projection functor pr induces an isomorphism Proposition 5.15 The projection functor pr induces an isomorphism Mb Y (2, 1, 2) ∼= −→Mσ (Ku(Y), v −w) Proof Using Lemmas 5.13 and 5.14, a similar argument in Lemma 5.7 shows that pr induces a morphism Mb Y (2, 1, 2) →Mσ (Ku(Y), v −w) such that maps [E] to [pr(E)]. And as in Proposition 5.8, we know that this morphism is étale, injective and proper, hence is an embedding. Since Mσ (Ku(Y), v −w) is irreducible and smooth by Corollary 4.2, this is an isomorphism. ⊓⊔ 5.2 MX(2, −1, 6) as Bridgeland moduli space Lemma 5.9 Let E ∈MX(2, −1, 6), then we have: (1) Hi(E) = 0, ∀i ≥0. (2) Exti(E∨, E) = 0, ∀i ≥0. (2) Exti(E∨, E) = 0, ∀i ≥0. Proof (1): Since E(1) ∈MX(2, 1, 6), this follows from Lemmas 5.1 and 5.4. 3 (2): When E is locally free, we have Exti(E∨, E) = Ext3−i(E∨, E∨). Then the statem follows from Lemma 5.2. (2): When E is locally free, we have Exti(E∨, E) = Ext3−i(E∨, E∨). Then the statement follows from Lemma 5.2. When E is not locally free, by Maruyama’s restriction theorem we can choose a sufﬁciently general linear section S, such that E(1)\|S and E∨\|S are both μ-semistable. Since these two sheaves correspond to primitive vectors, E(1)\|S and E∨\|S are actually μ-stable. From Lemma 5.5 we know exti(E∨, E(1)) = 0 for all i. Since E∨\|S and E(1)\|S are stable and not isomorphic to each other, we have Hom(E∨, E(1)\|S) = 0 and Ext2(E∨, E(1)\|S) = Ext2(E∨\|S, E(1)\|S) = Hom(E(1)\|S, E∨\|S) = 0. Now since χ(E∨, E(1)\|S) = 0, we have Ext1(E∨, E(1)\|S) = 0. Thus applying Hom(E∨, −) to the short exact sequence 0 →E → E(1) →E(1)\|S →0, we obtain Exti(E∨, E) = 0, ∀i ≥0. ⊓⊔ Therefore we know that E ∈AX, which means pr(E) = E. Lemma 5.10 Let E ∈ MX(2, −1, 6), then hom(E, E) = 1, ext1(E, E) = 2 and exti(E, E) = 0 for i ≥2. Hence E is σ ′-stable for every Serre-invariant stability con- dition σ ′ on AX. Lemma 5.10 Let E ∈ MX(2, −1, 6), then hom(E, E) = 1, ext1(E, E) = 2 and exti(E, E) = 0 for i ≥2. Hence E is σ ′-stable for every Serre-invariant stability con- dition σ ′ on AX. Proof The second statement follows from Lemma 3.14 and the ﬁrst one. For the ﬁrst state- ment, we have Exti(E, E) = Exti(E(−1), E(−1)), hence the statement follows from Lemmas 5.1 and 5.4. ⊓⊔ Proof The second statement follows from Lemma 3.14 and the ﬁrst one. For the ﬁrst state- ment, we have Exti(E, E) = Exti(E(−1), E(−1)), hence the statement follows from Lemmas 5.1 and 5.4. ⊓⊔ Now using Lemmas 5.9, 5.10 and the same argument in the proof of Proposition 5.8, we obtain the following proposition. 12 820 Z. Liu , S. 5.3 Mb Y(2, 1, 2) as Bridgeland moduli space Recall that a vector bundle is called normalised if h0(E(−1)) = 0 and h0(E) ̸= 0. We denote by Mb Y (2, 1, 2) the moduli space of (semi)stable bundle of rank 2 and Chern class c1 = 1, c2 = 2, c3 = 0 on Y. Lemma 5.12 Every E ∈Mb Y (2, 1, 2) is normalised. Proof Since E is stable, we have h0(E(−1)) = hom(O(1), E) = 0. A similar argument in [23, Lemma 5.1] shows that h2(E) = h3(E) = 0, hence h0(E) ̸= 0 follows from χ(E) = 3. ⊓⊔ Proof Since E is stable, we have h0(E(−1)) = hom(O(1), E) = 0. A similar argument in [23, Lemma 5.1] shows that h2(E) = h3(E) = 0, hence h0(E) ̸= 0 follows from χ(E) = 3. ⊓⊔ Thus by [10, Lemma 1], every E ∈Mb Y (2, 1, 2) is ACM, i.e. Hi(E( j)) = 0 for all 1 ≤i ≤2 and all j. Lemma 5.13 For every E ∈Mb Y (2, 1, 2), we have h∗(E(−1)) = 0 and h0(E) = 3, hi(E) = 0 for i ̸= 0. Proof Since E is ACM, we have hi(E(−1)) = hi(E) = 0 for i ̸= 0, 3. And from stability we have h0(E(−1)) = 0. By Serre duality and stability of E, we have h3(E) = h0(E∨(−2)) = h0(E(−3)) = 0. Since χ(E(−1)) = 0 and χ(E) = 3, we know that h3(E(−1)) = 0 and h0(E) = 3. ⊓⊔ Therefore by Lemma 5.13, pr(E) is given by cone(ev), where ev : H0(E) ⊗OY →E is the evaluation map. Lemma 5.14 We have ext1(cone(ev), cone(ev)) = 2. Hence pr(E) = cone(ev) is σ-stable for every Serre-invariant stability condition σ on Ku(Y). Proof Using [10, Lemma 3], a similar argument in Lemma 5.3 shows that ext2(cone(ev), cone(ev)) = ext3(cone(ev), cone(ev)) = 0.Thusthestatementfollowsfromhom(cone(ev), cone(ev)) = 1. ⊓⊔ Proof Using [10, Lemma 3], a similar argument in Lemma 5.3 shows that ext2(cone(ev), cone(ev)) = ext3(cone(ev), cone(ev)) = 0.Thusthestatementfollowsfromhom(cone(ev), cone(ev)) = 1. ⊓⊔ 5.4 Mb Y(2, −1, 2) as Bridgeland moduli space We denote by Mb Y (2, −1, 2) the moduli space of (semi)stable bundle of rank 2 and Chern class c1 = −1, c2 = 2, c3 = 0 on Y. 123 A note on Bridgeland moduli spaces and moduli... 821 Lemma 5.16 For every E ∈Mb Y (2, −1, 2), we have E ∈Ku(Y). Proof Since E(1) ∈Mb Y (2, 1, 2), by Lemma 5.13 we have h∗(E) = 0. And by Serre dual- ity we have hi(E(−1)) = h3−i(E∨(−1)) = h3−i(E), thus from Lemma 5.13 we know h∗(E(−1)) = 0. ⊓⊔ Proof Since E(1) ∈Mb Y (2, 1, 2), by Lemma 5.13 we have h∗(E) = 0. And by Serre dual- ity we have hi(E(−1)) = h3−i(E∨(−1)) = h3−i(E), thus from Lemma 5.13 we know h∗(E(−1)) = 0. ⊓⊔ Lemma 5.17 For every E ∈Mb Y (2, −1, 2), we have hom(E, E) = 1, ext1(E, E) = 2, ext2(E, E) = ext3(E, E) = 0. Hence pr(E) = E is σ-stable with respect to every Serre-invariant stability condition σ on Ku(Y). Lemma 5.17 For every E ∈Mb Y (2, −1, 2), we have hom(E, E) = 1, ext1(E, E) = 2, ext2(E, E) = ext3(E, E) = 0. Hence pr(E) = E is σ-stable with respect to every Serre-invariant stability condition σ on Ku(Y). Proof Since E(1) ∈Mb Y (2, 1, 2), this is from hom(E, E) = 1 and [10, Lemma 3]. Proposition 5.18 The projection functor pr induces an isomorphism Mb Y (2, −1, 2) ∼= −→Mσ (Ku(Y), 2v −w) Proof Using Lemmas 5.16 and 5.17, a similar argument in Proposition 5.11 shows that pr induces a morphism Mb Y (2, −1, 2) →Mσ (Ku(Y), 2v−w) such that maps [E] to [pr(E)] = [E]. And as in Proposition 5.11, we know that this morphism is étale, injective and proper, hence is an embedding. Since Mσ (Ku(Y), 2v −w) is irreducible and smooth by Corollary 4.2, this is an isomorphism. ⊓⊔ ⊓⊔ 6 Instanton sheaves and wall-crossing Let Y := Yd be a prime Fano threefold of index 2 and degree d. The notion of mathematical instanton bundle was ﬁrst introduced on P3 and generalized to Y in [18, 27]. Deﬁnition 6.1 Let Y be a prime Fano threefold of index 2. An instanton bundle of charge n on Y is a Gieseker-stable vector bundle E of rank 2 with c1(E) = 0, c2(E) = n, and satisﬁes the instantonic condition: H1(Y, E(−1)) = 0 As shown in [17, 37], every Gieseker-semistable sheaf with (r, c1, c2, c3) = (2, 0, 2, 0) on Y3 and Y4 automatically satisﬁes H1(E(−1)) = 0. Therefore, we can give a more general deﬁnition: Deﬁnition 6.2 Let Y be a prime Fano threefold of index 2. An instanton sheaf on Y is a Gieseker-semistable sheaf E of rank 2 with c1(E) = 0, c2(E) = 2 c3(E) = 0, and satisﬁes the instantonic condition: H1(Y, E(−1)) = 0 6.1 Wall-crossing Proof If there is a wall, then it is given by a short exact sequence 0 →A →E →B →0 0 →A →E →B →0 such that: (1) μα,−1 2 (A) = μα,−1 2 (B) = μα,−1 2 (E); (2) H(A) ≥0, H(B) ≥0; (3) H(A) ≤ H(E), H(B) ≤ H(E) (4) ch−1 2 (A) + ch−1 2 (B) = ch−1 2 (E); (1) μα,−1 2 (A) = μα,−1 2 (B) = μα,−1 2 (E); (3) H(A) ≤ H(E), H(B) ≤ H(E) (4) ch−1 2 (A) + ch−1 2 (B) = ch−1 2 (E); For (4) we can assume For (4) we can assume 2, H, d −8 4d H2 = a, b 2 H, c 8d H2 + 2 −a, 2 −b 2 H, 2d −16 −c 8d H2 for some a, b, c ∈Z such that 2\|b −a (this is from ch −1 2 1 −a 2 H = ch1 = c1 ∈Z · H). Since A, B ∈Coh−1 2 (Y), we have Im(Z(A)) ≥0 and Im(Z(B)) ≥0. Thus b ≥0 and 2 −b ≥0, which implies b = 0 or b = 1 or b = 2. d 8 2 8 3 2 p Since μα,−1 2 (E) = d−8 4d −α2, H(E) = 8 d (H3)2, the previous conditions can be written as: as: (1) 1 b c 4d −α2a = d−8 4d −α2 = 1 2−b 2d−16−c 4d −α2(2 −a) ; (2) b2 4 −ac 4d ≥0, (2−b)2 4 + (2−a)(c+16−2d) 4d ≥0; (3) b2 4 −ac 4d ≤8 d , (2−b)2 4 + (2−a)(c+16−2d) 4d ≤8 d Now from d−8 4d −α2 < ∞for all α > 0, we know that b ̸= 0 or 2. Then b = 1, and therefore c −d + 8 = 4d(a −1)α2 by (1). Since 2\|b −a, we have a is odd. By (2) and (3) we have d −32 ≤ac ≤d and d −32 ≤(a −2)(c + 16 −2d) ≤d. 1 Now note that ch −1 2 1 (A) = 1 2 H = ch1(A) + 1 2aH, thus ch1(A) = c1(A) = 1−a 2 H. 6.1 Wall-crossing In this subsection, we show that when d ̸= 2, the instantonic condition is equivalent to the non-existence of the maximal semicircle wall with respect to σα,β. Lemma 6.3 Let E ∈Coh−1 2 (Y) be a σα,−1 2 -semistable object for some α > 0 with ch≤2(E) = (1, 0, −1 d H2). Then E is σα,−1 2 -semistable for every α > 0 and ch(E) = 1 −L + x P for x ∈Z≤0 when d ̸= 1, and x ∈Z≤1 when d = 1. 123 822 Z. Liu , S. Zhang Z. Liu , S. Zhang Proof Assume ch(E) = 1 −L + x P for x ∈Z. From the wall-crossing argument in [35, Proposition 4.1] we know that E is σα,−1 2 -semistable for every α > 0. Hence by [30], [6, Conjecture 4.1] holds for E and α = 0. Therefore when d ̸= 1, we have x < 1, which means x ≤0. When d = 1, we have x ≤3 2, which means x ≤1 ⊓⊔ Proof Assume ch(E) = 1 −L + x P for x ∈Z. From the wall-crossing argument in [35, Proposition 4.1] we know that E is σα,−1 2 -semistable for every α > 0. Hence by [30], [6, Conjecture 4.1] holds for E and α = 0. Therefore when d ̸= 1, we have x < 1, which means x ≤0. When d = 1, we have x ≤3 2, which means x ≤1 ⊓⊔ Next we rule out all possible walls of [E] = 2 −2L on β = −1 2. Next we rule out all possible walls of [E] = 2 −2L on β = −1 2. Proposition 6.4 When d ̸= 5, the only possible walls for ch(E) = 2 −2L on β = −1 2 are realized by extensions of ideal sheaves of lines. 1 When d = 5, there are two possible walls on β = −1 2, realized by extensions of ideal sheaves of lines and object O(−1)[1]. A note on Bridg After solv when d ̸= 5, When (a, where ch −1 2 ≤2 ( Lemma 6.3 w α > 0. Henc sheaves, hen Whend = or ch −1 2 ≤2 (B) = that A ∼= O( From a sim Lemma 6.5 L wall intersec d+2 2d )2 + α2 The follow walls: Lemma 6.6 L (1) E satisﬁe (2) The wall When d = 1, Proof Since tonic conditi is equivalent (1) ⇒(2 the wall by d (2) ⇒(1) (2) The wall given by O(−1)[1] with respect to σα,β is not an actual wall for E. When d = 1, we have (1) implies (2). When d = 1, we have (1) implies (2). When d = 1, we have (1) implies (2). Proof Since χ(E(−1)) = 0 and h0(E(−1)) = h3(E(−1)) = 0 from stability, the instan- tonic condition is equivalent to H2(E(−1)) = 0. By Serre duality, the instantonic condition is equivalent to H2(E(−1)) = Hom(O(1), E[2]) = Hom(E, O(−1)[1]) = 0. Proof Since χ(E(−1)) = 0 and h0(E(−1)) = h3(E(−1)) = 0 from stability, the instan- tonic condition is equivalent to H2(E(−1)) = 0. By Serre duality, the instantonic condition is equivalent to H2(E(−1)) = Hom(O(1), E[2]) = Hom(E, O(−1)[1]) = 0. (1) ⇒(2): Since we have Hom(O(−1)[1], E) = 0, these lead to the non-existence of the wall by deﬁnition. y (2) ⇒(1): The instantonic condition is equivalent to H2(E(−1)) = Hom(E, O(−1)[1]) = 0. By [8, Proposition 4.8] we know E is σα,β-semistable for α ≫0 and β < 0. Now by Lemma 6.5, we know that O(−1)[1] gives the biggest semicircle wall. So if O(−1)[1] is not an actual wall, then after crossing this wall, E remains σα,β-semistable. But when d = 3, 4, 5, under this wall we have E ∈Cohβ(Y) and O(−1)[1] ∈Cohβ(Y), and μα,β(E) > μα,β(O(−1)[1]), which gives Hom(E, O(−1)[1]) = 0. ⊓⊔ Corollary 6.7 Let E be an instanton sheaf. Then E ∈Cohβ(Y) is σα,β-semistable for every α > 0 and β = −1 2, −1. Corollary 6.7 Let E be an instanton sheaf. Then E ∈Cohβ(Y) is σα,β-semistable for every α > 0 and β = −1 2, −1. Proof This immediately follows from Proposition 6.4, Lemmas 6.5 and 6.6. Proof This immediately follows from Proposition 6.4, Lemmas 6.5 and 6.6. A note on Bridg After solv when d ̸= 5, When (a, where ch −1 2 ≤2 ( Lemma 6.3 w α > 0. Henc sheaves, hen Whend = or ch −1 2 ≤2 (B) = that A ∼= O( From a sim Lemma 6.5 L wall intersec d+2 2d )2 + α2 The follow walls: Lemma 6.6 L (1) E satisﬁe (2) The wall When d = 1, Proof Since tonic conditi is equivalent (1) ⇒(2 the wall by d (2) ⇒(1) A note on Bridgeland moduli spaces and moduli... 823 After solving this, we ﬁnd that the only possible solutions are (a, c, α) = (1, d −8, R>0) when d ̸= 5, and (a, c, α) = (1. −3, R>0), (−1, −5, 1 √ 20), (3, −1, 1 √ 20) when d = 5. When (a c) = (1 d 8) the destabilizing sequence is given by √ 20 √ 20 When (a, c) = (1, d −8), the destabilizing sequence is given by 0 →A →E →B →0 where ch −1 2 ≤2 (A) = ch −1 2 ≤2 (B) = (1, 1 2 H, d−8 8d H2). Since ch3(A)+ch3(B) = ch3(E) = 0, by Lemma 6.3 we know actually ch(A) = ch(B) = 1 −1 d H2 and σα,−1 2 -semistable for every α > 0. Hence from [6, Lemma 2.7] and Pic(Y) ∼= Z, we know that A and B are μ-semistable sheaves, hence both isomorphic to ideal sheaves of lines. 1 Whend = 5and(a, c) = (−1, −5), (3, −1),wehaveeitherch −1 2 ≤2 (A) = (−1, 1 2 H, −1 8 H2) or ch −1 2 ≤2 (B) = (−1, 1 2 H, −1 8 H2). Thus by a standard result in [7, Proposition 2.14] we know that A ∼= O(−1)[1] or B ∼= O(−1)[1]. ⊓⊔ From a similar computation in Proposition 6.4, we have: Lemma 6.5 Let d ̸= 2 and E ∈Coh−1(Y) with ch(E) = 2−2L. Then there is no semicircle wall intersect with β = −1. Therefore, the maximal possible semicircle wall for E is (β − d+2 2d )2 + α2 = ( d−2 2d )2 realized by O(−1)[1]. The following lemma gives another description of instantonic condition as existence of walls: Lemma 6.6 Let d ̸= 1, 2 and E ∈Mss Y (2, 0, 2). Then the following are equivalent: (1) E satisﬁes instantonic condition. Lemma 6.6 Let d ̸= 1, 2 and E ∈Mss Y (2, 0, 2). Then the following are equivalent: (1) E satisﬁes instantonic condition. Lemma 6.6 Let d ̸= 1, 2 and E ∈Mss Y (2, 0, 2). Then the following are equivalent: (1) E satisﬁes instantonic condition. (1) E satisﬁes instantonic condition. (1) E satisﬁes instantonic condition. ﬁ (2) The wall given by O(−1)[1] with respect to σα,β is not an actual wall for E. 6.1 Wall-crossing And ch −1 2 2 (A) = c 8d H2 = ch2 + 1 2ch1H + 1 8ch0H2 = c2 1−2c2 2 + 1−a 4 H2 + 1 8aH2, hence ( c 8d −(1−a)2 8 −2−a 8 )H2 = −c2 ∈Z·H.Insummary,wehavefollowingsystemofinequalities: (1) d = 1, 2, 3, 4, 5 and a, c ∈Z, a odd and α > 0; 2 (1) d = 1, 2, 3, 4, 5 and a, c ∈Z, a odd and α > 0; 2 (2) c −d + 8 = 4d(a −1)α2; (3) d −32 ≤ac ≤d; (4) d −32 ≤(a −2)(c + 16 −2d) ≤d; (5) c 8 −d(1−a)2 8 −d(2−a) 8 ∈Z. 123 123 Proof This immediately follows from Proposition 6.4, Lemmas 6.5 and 6.6. ⊓⊔ We denote the moduli space of instanton sheaves by Minst Y . Let Y = Y3, Y4 or Y5. We collect some properties and classiﬁcations of instanton sheaves from [17, 27, 37, 39]. Recall that on a smooth conic C ∼= P1, the theta-characteristic is given by θ ∼= OP1(−1). Lemma 6.8 Let Y = Y3, Y4 or Y5. Let E ∈Mss Y (2, 0, 2). 123 Z. Liu , S. Zhang 824 (1) E is an instanton sheaf. (2) If E is Gieseker-stable, then E is either locally free or deﬁned by 0 →E →H0(θ(1)) ⊗OY →θ(1) →0 where θ is a theta-characteristic of a smooth conic C ⊂Y3. If E is strictly Gieseker- semistable, then E is an extension of two ideal sheaves of lines. i (3) For every E ∈Minst Y , we have E ∈Ku(Y). (4) Minst Y is irreducible, projective and smooth of dimension 5. Proof (1): When Y = Y3, this is proved in the proof of [17, Theorem 2.4]. When Y = Y4, this is from [37, Theorem 1.2]. When Y = Y5, if E is not an instanton sheaf, then O(−H)[1] gives an actual wall on β = −1 2 by Lemma 6.6, and we have an exact sequence in Coh−1 2 (Y) 0 →F →E →O(−H)[1] →0 where F ∈Coh−1 2 (Y) is σα,−1 2 -semistable for some α > 0. In this case we have ch≤2(F) = 1 where F ∈Coh−1 2 (Y) is σα,−1 2 -semistable for some α > 0. In this case we have ch≤2(F) = (3 −H 1 H2) which contradicts [30 Proposition 3 2] (3, −H, 1 10 H2), which contradicts [30, Proposition 3.2]. 10 p (2): This is from [17, Theorem 3.5], [37, Theorem 1.4] and [39, Theorem 1.2]. 10 (2): This is from [17, Theorem 3.5], [37, Theorem 1.4] and [39, Theorem 1.2]. (3): This is from the classiﬁcation of such sheaves and the same argument as in [37, mma 4.1]. (3): This is from the classiﬁcation of such sheaves and the same argument as in [37, Lemma 4.1]. (4): This is from [17, Theorem 4.6], [37, Theorem 5.4] and [39, Theorem 5.6]. (4): This is from [17, Theorem 4.6], [37, Theorem 5.4] and [39, Theorem 5.6]. 7 Moduli of instanton sheaves on Yd as Bridgeland moduli space Let Y := Yd. In this section, we are going to show that the projection functor pr induces an isomorphism Minst Y ∼= −→Mss σ (Ku(Y), 2 −2L) for d ̸= 1, 2 and an open immersion Minst Y →Mss σ (Ku(Y), 2 −2L) for d = 1, 2. (1) E is an instanton sheaf. 7.2 Bridgeland (semi)stable objects are (semi)stable sheaves In this subsection we assume 3 ≤d ≤5. We show that for every σ(α, −1 2)-(semi)stable object F ∈A(α, −1 2) with [F] = 2L −2, we have F[−1] is Gieseker-(semi)stable sheaf. Our argument is as follows: 2 Our argument is as follows: First, using Proposition 6.4, a similar argument in [7, Lemma 6.11] shows that when starting at (α0, β0) = ( d−2 2d , −d+2 2d ) and β0 approaching to −1 2, the only wall we will meet is the wall C realized by O(−1)⊕a[2] for some a ∈Z>0. Then following the argument in [35, Proposition 4.6], if C is not an actual wall, we can show that F[−1] ∈Coh−1 2 (Y) is σα,−1 2 -semistable for every α > 0. Then again by [6, Lemma 2.7], F[−1] is a μ-semistable sheaf. Therefore, by Lemma 7.4, Lemma 6.8 and the standard slope-comparison argument, F[−1] is actually an instanton sheaf. When C is an actual wall, we also follow the argument in [35, Proposition 4.6] and show that crossing this wall will lead to a contradiction. Lemma 7.3 Let (α0, β0) = ( d−2 2d , −d+2 2d ) and F ∈Coh0 α0,β0(Y) be a σ 0 α0,β0-semistable object with ch(F) = 2L −2. Then when β0 approaching β = −1 2, the only wall of F with respect to σ 0 α,β can be only realized by O(−1)⊕a[2] for some a ∈Z>0. Proof We consider the location of possible walls for F. As shown in Lemma 6.5, there is no semicircle wall intersecting with β = −1. Thus the point (α0, β0) already lies on the maximal numerical wall C, and we only need to show that this wall is realized by O(−1)⊕a[2] for some a ∈Z>0. Proof We consider the location of possible walls for F. As shown in Lemma 6.5, there is no semicircle wall intersecting with β = −1. Thus the point (α0, β0) already lies on the maximal numerical wall C, and we only need to show that this wall is realized by O(−1)⊕a[2] for some a ∈Z>0. We assume that C is given by a sequence 0 →A →F →B →0 such that when (α, β) lies on the wall, then Z0 α,β(A) and Z0 α,β(B) lie on the open line segment connecting 0 and Z0 α,β(F). By continuity, this still holds at the end point (α, β) = (0, −1). 7.1 In Kuznetsov component Thus taking α = 1 2 −ϵ for 0 < ϵ ≪1 gives μα,−3 2 (E) > μα,−3 2 (O(−2)[1]), which implies hom(E, O(−2)[1]) = hom(O(−2)[1], E(−2)[3]) = h2(E) = 0. ⊓⊔ ⊓⊔ Proposition 7.2 Let E ∈Minst Y be an instanton sheaf. Then we have E[1] ∈A(α, −1 2) is σ(α, −1 2)-semistable for every 0 < α < 1 2. If d ̸= 1 and E is Gieseker-stable, then E[1] is σ(α, −1 2)-stable for every 0 < α < 1 2. Proof This is from Lemma 7.1 and Corollary 6.7. When d ̸= 1 and E[1] is strictly σ(α, −1 2)- semistable, by Lemmas 7.11, 7.12 and [35, Theorem 1.1] we know that E is an extension of ideal sheaf of lines, hence is strictly Gieseker-semistable. ⊓⊔ 7.1 In Kuznetsov component First, we show that every instanton sheaf E is in the Kuznetsov component Ku(Y). When d = 3, 4 and 5, this is shown in [17, 37, 39] by using the classiﬁcations of instanton sheaves. We give another proof, which does not need classiﬁcation results on Y1 and Y2, and also works for d = 3, 4, 5. Lemma 7.1 For every E ∈Minst Y , we have H∗(E) = H∗(E(−1)) = 0. Thus E ∈Ku(Y). Lemma 7.1 For every E ∈Minst Y , we have H∗(E) = H∗(E(−1)) = 0. Thus E ∈Ku(Y) Proof When d = 3, 4, 5, this is from Lemma 6.8. Assume d = 1, 2. From the stability and Serre duality, we have h0(E) = h0(E(−1)) = h3(E) = h3(E(−1)) = 0. By the instantonic condition and χ(E(−1))=0, we have h1(E(−1)) = 0. Since χ(E) = 0, we only need to show h2(E) = 0. Assume d = 1, 2. From the stability and Serre duality, we have h0(E) = h0(E(−1)) = h3(E) = h3(E(−1)) = 0. By the instantonic condition and χ(E(−1))=0, we have h1(E(−1)) = 0. Since χ(E) = 0, we only need to show h2(E) = 0. When d = 2, from Corollary 6.7 we know that E ∈Coh−1(Y) is σα,−1-semistable for every α > 0. By [7, Proposition 2.14] we have that O(−2)[1] ∈Coh−1(Y) is σα,−1-stable for every α > 0. Thus taking 0 < α ≪1 gives μα,−1(E) > μα,−1(O(−2)[1]), which implies hom(E, O(−2)[1]) = hom(O(−2)[1], E(−2)[3]) = h2(E) = 0. 3 When d = 1, from Lemma 6.4, 6.6 and Corollary 6.7, we know that E ∈Coh−3 2 (Y) is σα,−3 2 -semistable for α ≫0, and remain semistable when α = 1 2 −ϵ for 0 < ϵ ≪1. By 123 A note on Bridgeland moduli spaces and moduli... 825 [7, Proposition 2.14] we have that O(−2)[1] ∈Coh−3 2 (Y) is σα,−3 2 -stable for every α > 0. Thus taking α = 1 2 −ϵ for 0 < ϵ ≪1 gives μα,−3 2 (E) > μα,−3 2 (O(−2)[1]), which implies hom(E, O(−2)[1]) = hom(O(−2)[1], E(−2)[3]) = h2(E) = 0. ⊓⊔ [7, Proposition 2.14] we have that O(−2)[1] ∈Coh−3 2 (Y) is σα,−3 2 -stable for every α > 0. ⊓⊔ Proposition 7.5 Assume d ̸= 1, 2. For every object F ∈A(α, −1 2) with [F] = 2L −2 and σ(α, −1 2)-(semi)stable for some 0 < α < 1 2, we have F[−1] is a (semi)stable instanton sheaf. Proof We argue as [35, Proposition 4.6]. By [35, Proposition 3.6], without loss of generality we can assume F is in the heart Coh0 α,β(Y) for (α, β) ∈V . ,β From d ≥3 we can ﬁnd a point (α0, β0) = ( d−2 2d , −d+2 2d ) ∈V such that F ∈Coh0 α0,β0(Y) and μ0 α0,β0 = +∞. By Lemmas 6.5 and 7.3, this is the top point of the semicircle wall, denoted by C, realized by O(−1)⊕a[2]. Assume that C is not an actual wall for F. Thus F is σ 0 α0,β0-semistable and remains semistable when β0 approaches −1 2. By the deﬁnition of Coh0 α,−1 2 (Y), there is an exact triangle A[1] →F →B such that A (resp. B) ∈Coh−1 2 (Y) with σα,−1 2 -semistable factors having slope μα,−1 2 ≤0 (resp. μα,−1 2 > 0). Since F is σ 0 α,−1 2 -semistable, we have that Zα,−1 2 (B) = 0. Hence either B is supported on a point or B = 0, and therefore ch(A) = (2, 0, −2 d H2, mP) where m ≥0 is the length of B. Moreover, A[1] is σ 0 α,−1 2 -semistable and, since A ∈Coh−1 2 (Y), we have that A is σα,−1 2 -semistable. such that A (resp. B) ∈Coh−1 2 (Y) with σα,−1 2 -semistable factors having slope μα,−1 2 ≤0 (resp. μα,−1 2 > 0). Since F is σ 0 α,−1 2 -semistable, we have that Zα,−1 2 (B) = 0. Hence either B is supported on a point or B = 0, and therefore ch(A) = (2, 0, −2 d H2, mP) where m ≥0 is the length of B. Moreover, A[1] is σ 0 α,−1 2 -semistable and, since A ∈Coh−1 2 (Y), we have that A is σα,−1 2 -semistable. 2 But as we showed in Proposition 6.4, A is actually σα,−1 2 -semistable for every α > 0 when d = 3, 4 and σα,−1 2 -semistable for α > 1 √ 20 when d = 5. Hence A is a μ-semistable sheaf by [6, Lemma 2.7]. 7.2 Bridgeland (semi)stable objects are (semi)stable sheaves Assume ch−1 ≤2(A) = (a, bH, c 2d H2) for a, b, c ∈Z. Since μ0 α0,β0(F) = +∞, we have Re(Z0 α0,β0(A)) ≤0 and Re(Z0 α0,β0(A)) ≤0. Thus we have b = −2, −1, 0. First we assume b = −1, then we have μ0,−1(A) = −c 2d . Since ch−1 ≤2(O(−1)) = (1, 0, 0), we have μ0 0,−1(O(−1)) = +∞. Therefore, μ0 0,−1(A) = μ0 0,−1(O(−1)) implies c = 0. Hence we have ch−1 ≤2(A) = (a, −H, 0) and ch−1 ≤2(B) = (−2 −a, −H, ( 2 d −1)H2). But this is impossible since we must have μ0 0,−1(B) = +∞. 123 826 Z. Liu , S. Zhang Thus we have b = 0 or 2, and in these cases either ch≤2(A) = (a, −aH, a 2 H2) or ch≤2(B) = (a, −aH, a 2 H2). By a standard argument, we conclude that A ∼= O(−1)⊕a[2] or B ∼= O(−1)⊕a[2]. ⊓⊔ Lemma 7.4 Let A be a μ-semistable sheaf with ch(A) = 2−2L, such that σα,−1 2 -semistable for α ≫0, then A is Gieseker-semistable. Proof If A is not Gieseker-semistable, let G ⊂A be the destabilizing sheaf. Then rk(G) = 1, χ(G(n)) > χ(A(n))/2 when n ≫0 and G is Gieseker-semistable. Assume that ch(G) = 1 + aH + b 2 L + c 2 P where a, b, c ∈Z. 2 2 We know that χ(G(n)) = (1 + d+3 3 a + b+c 2 ) + ( d+3 3 + da + b 2)n + (a + 1) d 2 n2 + d 6 n3 and χ(A(n))/2 = d 3 n + d 2 n2 + d 6 n3. From χ(G(n)) > χ(A(n))/2 for n ≫0, we have: (1) a > 0; (2) a = 0, −2 < b; (3) a = 0, b = −2, c > 0. By the μ-semistability of A, (1) is impossible. For (2) we have μα,−1 2 (G) > μα,−1 2 (A), which contradicts the σα,−1 2 -semistability of A. Hence the only possible case is ch(G) = 1−L+ c 2 P and c > 0. Since χ(G) ∈Z, we can assume that ch(G) = 1 −L + x P for x ∈Z+. By the μ-semistability of A, (1) is impossible. For (2) we have μα,−1 2 (G) > μα,−1 2 (A), which contradicts the σα,−1 2 -semistability of A. 7.2 Bridgeland (semi)stable objects are (semi)stable sheaves Hence the only possible case is ch(G) = 1−L+ c 2 P and c > 0. Since χ(G) ∈Z, we can assume that ch(G) = 1 −L + x P for x ∈Z+. Since 1 −L + x P is a primitive class, G is actually μ-stable. Then by [6, Lemma 2.7], G is σα,−1 2 -stable for α ≫0. But by Lemma 6.3 this contradicts x ∈Z+. Hence we conclude that A is Gieseker-semistable. ⊓⊔ Since 1 −L + x P is a primitive class, G is actually μ-stable. Then by [6, Lemma 2.7], G is σα,−1 2 -stable for α ≫0. But by Lemma 6.3 this contradicts x ∈Z+. Hence we conclude that A is Gieseker-semistable. ⊓⊔ ⊓⊔ By [30], after taking α →0, [6, Conjecture 4.1] holds for A and α = 0, β = −1 2. And when d = 5 we take α = 1 √ 20, β = −1 2. 123 A note on Bridgeland moduli spaces and moduli... 827 Thus we have m ≤1 when d = 3, 4, and m = 0 when d = 5. Assume m = 1, then we apply Hom(OY , −) to A[1] →F →B and obtain hom(OY , A[2]) = 1 and hom(OY , A[i]) = 0 for i ̸= 2. By Serre duality we have hom(A, O(−2)[1]) = 1. But using Lemma 6.5, a similar slope-comparison argument in Lemma 7.1 shows that hom(A, O(−2)[1]) = 0, which makes a contradiction. Thus we conclude that F = A[1], hence F[−1] is σα,−1 2 -semistable for every α > 0. By Lemma 7.4, F[−1] = A is actually a Gieseker-semistable sheaf. If F is σ(α, −1 2)- stable, it can not be an extension of ideal sheaves of lines up to some shifts. Hence F[−1] is Gieseker-stable in this case. When d = 3, 4, by Lemma 6.8 we know that F[−1] is an instanton sheaf. When d = 5, from Lemma 6.5 we know that the only wall intersecting with β = −1 2 is realized by O(−1)[2], and this is not an actual wall for F by assumption. Since μα,−1 2 (O(−1)[1]) = α2 −1 4 and μα,−1 2 (F[−1]) = −3 20 −α2, if we take α < 1 √ 20, we obtain F, O(−1)[2] ∈Coh0 α,−1 2 (Y) and μ0 α,−1 2 (F) > μ0 α,−1 2 (O(−1)[2]), which implies Hom(F[−1], O(−1)[1]) = 0. Hence F[−1] is an instanton sheaf. 1 Now we assume that C is an actual wall. Then F becomes unstable when β0 →−1 2, and F is strictly σ 0 α0,β0-semistable and there is a sequence in 0 →P →F →Q →0 Coh0 α0,β0(Y), where P, Q are σ 0 α0,β0-semistable with the same slope +∞. From Lemma 7.3 we know that P ∼= O(−1)⊕a[2]. Coh0 α0,β0(Y), where P, Q are σ 0 α0,β0-semistable with the same slope +∞. From Lemma 7.3 we know that P ∼= O(−1)⊕a[2]. Therefore, we have χ(P, Q) < 0. Since P, Q are in the same heart, we know that Hom(P, Q[i]) = 0 for i ≤0. ⊓⊔ And by Serre duality we have Hom(P, Q[i]) = Hom(Q, O(−3H)⊕a[5 −i]) = 0 for i ≥3, since O(−3H)[2] ∈Coh0 α0,β0(Y) is σ 0 α0,β0- stable with slope μ0 α0,β0(Q) = +∞> μ0 α0,β0(O(−3H)[2]). So Hom(P, Q[1]) ̸= 0 and we can deﬁne an object G as the non-trivial extension 0 →Q →G →P →0 in Coh0 α0,β0(Y). Now G is σ 0 α,β-semistable when β →−1 2. The argument in previous cases shows that G[−1] is an instanton sheaf. Thus we have Hom(G[−1], O(−1)[1]) = Hom(G, O(−1)[2]) = 0 and makes a contradiction. ⊓⊔ in Coh0 α0,β0(Y). Now G is σ 0 α,β-semistable when β →−1 2. The argument in previous cases shows that G[−1] is an instanton sheaf. Thus we have Hom(G[−1], O(−1)[1]) = Hom(G, O(−1)[2]) = 0 and makes a contradiction. ⊓⊔ ⊓⊔ Now we construct isomorphisms between moduli spaces: Theorem 7.6 Let Y = Y3, Y4, Y5. The projection functor pr induces an isomorphism Minst Y ∼= −→Mss σ (Ku(Y), 2L −2) for every Serre-invariant stability condition σ on Ku(Y). for every Serre-invariant stability condition σ on Ku(Y). Proof By Proposition 7.13, we can assume σ = σ(α, −1 2) for 0 < α < 1 2. Using Proposition 7.2 and the GIT construction of Mss Y (2, 0, 2), a similar argument in [37, Sec- tion 5] and [39, Section 5] shows that the projection functor pr induces a morphism Minst Y →Mss σ (Ku(Y), 2L −2). Y By Proposition 7.2 and Proposition 7.5, we know that this morphism is bijective on closed points. Since the functor pr is the identity on E, we know that this morphism is étale. Thus the projection functor induces a bijective étale morphism, which is an isomorphism. ⊓⊔ 828 Z. Liu , S. Zhang Theorem 7.7 Let Y = Y1, Y2. The projection functor pr induces an open immersion Theorem 7.7 Let Y = Y1, Y2. The projection functor pr induces an open immersion Minst Y →Mss σ (Ku(Y), 2L −2) for every Serre-invariant stability condition σ ∈K. Proof By [35, Proposition 3.6], we can assume σ = σ(α, −1 2). Using Proposition 7.2 and the GIT construction of Mss Y (2, 0, 2), a similar argument in [37, Section 5] and [39, Section 5] shows that the projection functor pr induces a morphism Minst Y →Mss σ (Ku(Y), 2L −2). Y By Proposition 7.2, we know that this is injective on closed points. Since the functor pr is the identity on E, we know that this morphism is étale. Thus the projection functor induces an injective étale morphism, which is an open immersion. ⊓⊔ 7.3 All Serre-invariant stability conditions 829 The table of dimension of page 2 is: The table of dimension of page 2 is: on of page 2 is: dim E p,q 2 = 0 0 0 a b c 2 4 0 d e f dim E p,q 2 = 0 0 0 a b c 2 4 0 d e f From the convergence of spectral sequence, we know c = 0, b = 0. And from χ(N, M) = −1 we know a −b + c = −1, which implies a = −1, thus makes a contradiction. p Next we assume d = 4, 5. In both cases the heart of a stability condition on Ku(Y) has homological dimension 1, hence E p,q 2 = E p,q ∞. If we take q = 0, we obtain 5 = ext1(F, F) ≥ i ext1(Hi(F), Hi(F)) ≥r If r = 1 then we are done. Otherwise if r ≥2, then this is also impossible since 1 = hom(F, F) = dim E0,0 2 + dim E1,−1 2 ≥dim E0,0 2 = 2 from the convergence of spectral sequence, which makes a contradiction. ⊓⊔ From some elementary computations, we have: Lemma 7.9 Let A, B ∈Ku(Y). Assume ch(A) = a1v + b1w and ch(B) = a2v + b2w for ai, bi ∈Z, then μα,−1 2 (A) = μα,−1 2 (B) if and only if one of the following holds: (1) a1 = a2 = 0 or b1 = b2 = 0 (2) a1, a2, b1, b2 ̸= 0, and a1 b1 = a2 b2 . Lemma 7.9 Let A, B ∈Ku(Y). Assume ch(A) = a1v + b1w and ch(B) = a2v + b2w for ai, bi ∈Z, then μα,−1 2 (A) = μα,−1 2 (B) if and only if one of the following holds: (1) a1 = a2 = 0 or b1 = b2 = 0 (2) a1, a2, b1, b2 ̸= 0, and a1 b1 = a2 b2 . Proposition 7.10 Let F ∈Ku(Y) be an object with ch(F) = 2 −2L and hom(F, F) = 1, ext1(F, F) = 5, ext2(F, F) = ext3(F, F) = 0, then F is σ-stable for every Serre- invariant stability condition on Ku(Y). In particular, every E ∈MY (2, 0, 2) is σ-stable. Proof By Lemma 7.8, without loss of generality we can assume that F ∈Aσ . 7.3 All Serre-invariant stability conditions In this subsection, we assume Y := Yd for d = 3, 4, 5. We can consider all Serre-invariant stability conditions, not only for σ ∈K. Lemma 7.8 Let F ∈Ku(Y) be an object with [F] = 2v = 2 −2L and hom(F, F) = 1, ext1(F, F) = 5, ext2(F, F) = ext3(F, F) = 0. Then F is in the heart of every Serre- invariant stability on Ku(Y) up to some shifts. Proof As in [9, Lemma 4.5], we consider the spectral sequence for objects in Ku(Y) whose second page is given by E p,q 2 = i Hom p(Hi(F), Hi+q(F)) ⇒Hom p+q(F, F) where the cohomology is taken with respect to the heart. 1 1 where the cohomology is taken with respect to the heart. 1 1 where the cohomology is taken with respect to the heart. 1 1 First we assume d = 3. Then E1,q 2 = E1,q ∞by Lemma 3.11. Thus ext1(∗, ∗) ≥2 for ∗∈Ku(Y). If we take q = 0, we obtain 5 = ext1(F, F) ≥ i ext1(Hi(F), Hi(F)) ≥2r where r is the number of non-zero cohomology objects of F. where r is the number of non-zero cohomology objects of F. If r = 1. then F is already in the heart up to some shifts. Otherwise if r = 2, we denote these two cohomology objects by M, N. Thus we have ext1(M, M) = ext1(N, N) = 2 or ext1(M, M) = 2, ext1(N, N) = 3 or ext1(M, M) = 3, ext1(N, N) = 2. But χ(M, M) ≤ −1 and χ(N, N) ≤−1, hence if ext1(M, M) = 2, ext1(N, N) = 3, then χ(M, M) = −1 and −2 ≤χ(N, N) ≤−1. Since there is no (-2)-class in Ku(Y3), we have χ(M, M) = χ(N, N) = −1. In other two cases we also have χ(M, M) = χ(N, N) = −1, i.e. M and N are always (-1)-classes. Now since ch(F) = 2 −2L = 2v, by classiﬁcations of (−1)-classes we know ch(M) = ch(N) = 1 −L = v. Thus the second page is: E p,q 2 = 0 0 0 Hom(N, M) Ext1(N, M) Ext2(N, M) Hom(M, M) ⊕Hom(N, N) Ext1(M, M) ⊕Ext1(N, N) 0 Hom(M, N) Ext1(M, N) Ext2(M, N) 123 123 A note on Bridgeland moduli spaces and moduli... 7.3 All Serre-invariant stability conditions If F is not σ-semistable, then there is an exact triangle If F is not σ-semistable, then there is an exact triangle A →F →B such that A, B are both σ-semistable with φ(A) > φ(B). Hence we have Hom(A, B) = 0 and ext2(B, A) = ext2(A, A) = ext2(B, B) = 0. such that A, B are both σ-semistable with φ(A) > φ(B). Hence we have Hom(A, B) = 0 and ext2(B, A) = ext2(A, A) = ext2(B, B) = 0. First we assume d = 3. By Lemma 3.13 we have ext1(A, A) + ext1(B, B) ≤ext1(F, F) = 5 ext1(A, A) + ext1(B, B) ≤ext1(F, F) = 5 By Lemma 3.12, the only possible cases are ext1(A, A) = ext1(B, B) = 2 or ext1(A, A) = 2, ext1(B, B) = 3 or ext1(A, A) = 3, ext1(B, B) = 2. But χ(A, A) ≤−1 and χ(B, B) ≤ −1, hence if ext1(A, A) = 2, ext1(B, B) = 3, then −1 ≤χ(A, A) ≤−1 and −2 ≤ χ(B, B) ≤−1. Since there is no (-2)-class in Ku(Y3), we have χ(A, A) = χ(B, B) = −1. In other two cases we also have χ(A, A) = χ(B, B) = −1, i.e. A and B are always (-1)- classes. Now since ch(F) = 2−2L = 2v, by classiﬁcation of (−1)-classes we know ch(A) = ch(B) = 1 −L = v. But in this case we have φ(A) = φ(B), which is a contradiction. When d = 4, 5, the heart Aσ of Ku(Y) has homological dimension 1, then it is easy to make contradictions by the spectral sequence in [40, Proposition 4.16] and Mukai Lemma. First we assume d = 5, then the only possible cases are ext1(A, A) = ext1(B, B) = 2 or ext1(A, A) = 2, ext1(B, B) = 3 or ext1(A, A) = 3, ext1(B, B) = 2. 123 830 Z. Liu , S. 7.3 All Serre-invariant stability conditions Zhang From the triangle B[−1] →A →F we have a spectral sequence which degenerates at E3 converging to Ext∗(F, F) with E1-page being E p,q 1 = ⎧ ⎪⎪⎪⎪⎨ ⎪⎪⎪⎪⎩ Extq(A, B[−1]) = Extq−1(A, B), p = −1 Extq(A, A) ⊕Extq(B, B), p = 0 Extq(B[−1], A) = Extq+1(B, A), p = 1 0, p /∈[−1, 1] When ext1(A, A) + ext1(B, B) = 4, the dimension of E1 page looks like: When ext1(A, A) + ext1(B, B) = 4, the dimension of E1 page looks like: dim E p,q 1 = 0 0 0 0 0 0 0 0 0 c 0 0 0 0 4 0 0 0 2 b 0 0 0 a dim E p,q 1 = 0 0 0 0 0 0 0 0 0 c 0 0 0 0 4 0 0 0 2 b 0 0 0 a The dimension of E2 page looks like: dim E p,q 2 = 0 0 0 0 0 0 0 0 0 c 0 0 0 0 4 0 0 0 x y 0 0 0 a dim E p,q 1 = 0 0 0 0 0 0 0 0 0 c 0 0 0 0 4 0 0 0 2 b 0 0 0 a The dimension of E2 page looks like: The dimension of E2 page looks like: dim E p,q 2 = 0 0 0 0 0 0 0 0 0 c 0 0 0 0 4 0 0 0 x y 0 0 0 a Hence we have x + a = 1, y + c = 1, x −y = 2 −b and all variables are non-negative. Whenever x = 0 or x = 1, we have χ(A, B) = −1, χ(B, A) = −1 or χ(A, B) = 0, χ(B, A) = −2. Assume ch(A) = a1v + b1w and ch(B) = a2v + b2w, we have a1 + a2 = 2, b1 + b2 = 0 and ai, bi ∈Z. When χ(A, B) = 0, χ(B, A) = −2, we have −a2(a1 + b1) + b2((1 −d)a1 −db1) = 0 and −a1(a2 + b2) + b1((1 −d)a2 −db2) = −2, which has no solution under our assumptions. 7.3 All Serre-invariant stability conditions When χ(A, B) = −1, χ(B, A) = −1, we have −a2(a1+b1)+b2((1−d)a1−db1) = −1 and −a1(a2+b2)+b1((1−d)a2−db2) = −1, which has the only solution a1 = a2 = 1, b1 = b2 = 0. 1 1 nce we have x + a = 1, y + c = 1, x −y = 2 −b and all variables are non-negative. Hence we have x + a = 1, y + c = 1, x −y = 2 −b and all variables are non-negative. Whenever x = 0 or x = 1, we have χ(A, B) = −1, χ(B, A) = −1 or χ(A, B) = 0, χ(B, A) = −2. Assume ch(A) = a1v + b1w and ch(B) = a2v + b2w, we have a1 + a2 = 2, b1 + b2 = 0 and ai, bi ∈Z. When χ(A, B) = 0, χ(B, A) = −2, we have −a2(a1 + b1) + b2((1 −d)a1 −db1) = 0 and −a1(a2 + b2) + b1((1 −d)a2 −db2) = −2, Hence we have x + a = 1, y + c = 1, x −y = 2 −b and all variables are non-negative. Whenever x = 0 or x = 1, we have χ(A, B) = −1, χ(B, A) = −1 or χ(A, B) = 0 χ(B A) = −2 Assume ch(A) = a1v + b1w and ch(B) = a2v + b2w we have a1 + Hence we have x + a = 1, y + c = 1, x −y = 2 −b and all variables are non-negative. Whenever x = 0 or x = 1, we have χ(A, B) = −1, χ(B, A) = −1 or χ(A, B) = 0, χ(B, A) = −2. Assume ch(A) = a1v + b1w and ch(B) = a2v + b2w, we have a1 + a2 = 2, b1 + b2 = 0 and ai, bi ∈Z. When χ(A, B) = 0, χ(B, A) = −2, we have −a2(a1 + b1) + b2((1 −d)a1 −db1) = 0 and −a1(a2 + b2) + b1((1 −d)a2 −db2) = −2, which has no solution under our assumptions. When χ(A, B) = −1, χ(B, A) = −1, we have −a2(a1+b1)+b2((1−d)a1−db1) = −1 and −a1(a2+b2)+b1((1−d)a2−db2) = −1, which has the only solution a1 = a2 = 1, b1 = b2 = 0. 7.3 All Serre-invariant stability conditions Since ch(A) + ch(B) = ch(F) = 2v, we know that only possible case is ch(A) = ch(B) = v. When d = 4, since there is only one orbit in Stab(Ku(Y4)), we can assume σ = σ(α, −1 2). Then by Lemma 7.9 we know that only possible case is ch(A) = ch(B) = v. But as shown in [35, Proposition 4.6], this implies that A ∼= IL[2k] and B ∼= IL′[2k] for some lines L, L′ on Y and k ∈Z. If all stable factors of F are isomorphic to S, then χ(F, F) = n2χ(S, S) = −4, which implies n = 2. This means F is also an extension of S ∼= IL[2k]. But both cases contradict our assumptions by [17, Lemma 4.3]. ⊓⊔ ⊓⊔ By [35, Remark 5.14], for every pair of Serre-invariant stability conditions σ1 = (P1, Z1) and σ2 = (P2, Z2) on Ku(Y) we have Z1 = T ◦Z2 for some T = (ti j)1≤i, j≤2 ∈GL+ 2 (R). Thus an elementary computation shows that: By [35, Remark 5.14], for every pair of Serre-invariant stability conditions σ1 = (P1, Z1) and σ2 = (P2, Z2) on Ku(Y) we have Z1 = T ◦Z2 for some T = (ti j)1≤i, j≤2 ∈GL+ 2 (R). Thus an elementary computation shows that: Lemma 7.11 Let σ1 = (P1, Z1) and σ2 = (P2, Z2) be two Serre-invariant stability condi- tions on Ku(Y). Let E, E′ ∈Ku(Y) be any two objects, then μ1(E) = μ1(E′) if and only if μ2(E) = μ2(E′); And μ1(E) > μ1(E′) if and only if μ2(E) > μ2(E′). Lemma 7.12 Let σ1 = (P1, Z1) be a Serre-invariant stability condition on Ku(Y) and E ∈ P1(φ1(E)) be a σ1-stable object with [E] = av for a ∈Z. Then for any σ1-stable object F ∈P1(φ1(E)) we have [F] = bv for b ∈Z and ab > 0. Proof When F ∼= E, this is clear. Assume E and F are not isomorphic. Then by Lemmas 7.11 and 7.9 we know [F] = bv for b ∈Z. It is clear that ab ̸= 0. If ab < 0, then χ(F, E) = hom(F, E) −ext1(F, E) = −ab > 0. This means hom(F, E) > 0. But this is impossible since E and F are both stable and not isomorphic. 7.3 All Serre-invariant stability conditions When ext1(A, A) + ext1(B, B) = 5, since there is no (−2)-class, the dimension of E1 page looks like: dim E p,q 1 = 0 0 0 0 0 0 0 0 0 c 0 0 0 0 5 0 0 0 3 b 0 0 0 a The dimension of E2 page looks like: dim E p,q 2 = 0 0 0 0 0 0 0 0 0 c 0 0 0 0 5 0 0 0 x y 0 0 0 a 123 dim E p,q 1 = 0 0 0 0 0 0 0 0 0 c 0 0 0 0 5 0 0 0 3 b 0 0 0 a The dimension of E2 page looks like: dim E p,q 2 = 0 0 0 0 0 0 0 0 0 c 0 0 0 0 5 0 0 0 x y 0 0 0 a 123 123 A note on Bridgeland moduli spaces and moduli... 831 Hence we have c = y = 0, x+a = 1, x = 3−b and all variables are non-negative. Whenever x = 0 or x = 1, we have χ(A, B) = 0, χ(B, A) = −2. Assume ch(A) = a1v + b1w and ch(B) = a2v + b2w, we have a1 + a2 = 2, b1 + b2 = 0 and ai, bi ∈Z. Since χ(A, B) = 0, χ(B, A) = −2, we have −a2(a1 + b1) + b2((1 −d)a1 −db1) = 0 and −a1(a2 + b2) + b1((1 −d)a2 −db2) = −2, which has no solution under our assumptions. Next we assume d = 4. In this case we must have χ(A, A) + χ(B, B) = −4, hence ext1(A, A) + ext1(B, B) = 4 + hom1(A, A) + hom1(B, B) ≥6, which contradicts the Mukai lemma. Now we are going to show that F is σ-stable. By [35, Proposition 3.6] we can assume σ = σ(α, −1 2). Assume that F is strictly σ-semistable. If F has more than one non-isomorphic stable factors, then there is an exact sequence 0 →A →F →B →0 where φ(A) = φ(B) = φ(F) and A, B are σ-semistable with Hom(A, B)=0. When d = 3, 5, by Mukai Lemma we know χ(A, A) = χ(B, B) = −1. 7.3 All Serre-invariant stability conditions ⊓⊔ ⊓⊔ Proposition 7.13 Let σ1 = (P1, Z1) and σ2 = (P2, Z2) = σ(α, −1 2) be two Serre-invariant stability conditions on Ku(Y). Let E ∈Ku(Y) be an object with [E] = ±2v. Then E ∈ P1((0, 1]) is σ1-(semi)stable if and only if E[m] ∈P2((0, 1]) is σ2-(semi)stable for m = m(σ1) ∈Z. Proof Without loss of generality, we can assume [E] = −2v. As shown in [35], if F ∈Ku(Y) is a σ1-stable object with [F] = −v, then F is isomorphic to an ideal sheaf of line up to some shifts, and this shifts only depend on σ1; And if F ∈Ku(Y) is a σ2-stable object with [F] = −v, then F ∼= IL[1] where L is a line on Y. We can assume IL[1 −m] ∈P1((0, 1]), where m = m(σ1) ∈Z only depends on σ1. Thus if F ∈Ku(Y) is an object with [F] = −v, then F ∈P1((0, 1]) is σ1-stable if and only if F[m] ∈P2((0, 1]) is σ2-stable. 123 123 832 Z. Liu , S. Zhang By Proposition 7.10, E ∈P1((0, 1]) is σ1-stable if and only if E is σ2-stable up to some shifts. To show E[m] is in the heart of σ2, we can do as in [1, Section 5.1]: Let F = IL[1 −m]. Since Hom(F, E[1]) = Ext1(F, E) ̸= 0, we have φ2(F) < φ2(E) + 1. Since Hom(E, F[1]) = Ext1(E, F) ̸= 0, we have φ2(E) < φ2(F) + 1. Thus −1 < φ2(E)−φ2(F) < 1. But by Lemma 7.11 we know μ2(E) = μ2(F), thus φ2(E)−φ2(F) ∈Z, which shows φ2(E) = φ2(F). Thus since F[m] ∈P2((0, 1]), we have E[m] ∈P2((0, 1]). Now we assume E ∈P1((0, 1]) is strictly σ1-semistable, let E1, ..., En be the JH-factors under σ1 of E. Then Ei are σ1-stable with φ1(E1) = ... = φ1(En). Since ch(Ei) = ch(E) = −2v, by Lemmas 7.9, 7.11 and 7.12 we have n = 2 and ch(E1) = ch(E2) = −v. This means E1 ∼= IL1[1 −m] and E2 ∼= IL2[1 −m], where L1, L2 are two lines on Y. Thus we know that E[m] is an extension of IL1[1] and IL2[1], hence is σ2-semistable. 1 2 Conversely, if E is σ2-stable, then by Proposition 7.10 and the same argument above shows that E[−m] is σ1-stable. 7.3 All Serre-invariant stability conditions If E is strictly σ2-semistable, then E is an extension of two ideal sheaves of lines up to shift by 1. Thus we also have E[−m] is σ1-semistable. ⊓⊔ 8 Isomorphisms between Bridgeland moduli spaces of (−4)-classes Proof From Theorem 7.6 we know Minst Y ∼= Mss σ (Ku(Y), 2L −2). Since the equivalences in [28] map the numerical class 2(1 −L) to 2(1 −2L), hence using Corollary 8.3, a similar argument in Theorem 7.6 shows that induces a morphism s. This is a proper morphism since moduli spaces on both sides are proper. From Corollary 8.3 we know that s is bijective at closed points and Mσ (Ku(Y), 2−2L) = s−1(Mσ ′(AX, 2 −4L)). Since s is induced by equivalence, it is étale. Now we know s is a bijective étale proper morphism, which is an isomorphism. ⊓⊔ From Corollary 8.3 we know that s is bijective at closed points and Mσ (Ku(Y), 2−2L) = s−1(Mσ ′(AX, 2 −4L)). Since s is induced by equivalence, it is étale. Now we know s is a bijective étale proper morphism, which is an isomorphism. ⊓⊔ Corollary 8.5 Let b be any (−4)-class in N(Ku(Yd)) and b′ be any (−4)-class in N(AX4d+2). Then the Bridgeland moduli spaces Mss σ (Ku(Yd), b) ∼= Mss σ ′(AX4d+2, b′) for any Serre invariant stability condition σ, σ ′ and for each d = 3, 5. Proof When d = 3, 5, note that (x0, y0) is an integer solution of x2 + dxy + dy2 = 4 if and only if (x0/2, y0/2) is an integer solution of x2 + dxy + dy2 = 1, Thus the result follows from a similar argument in Corollary 4.2. Indeed, if d = 3, then there are three (−4)- classes in N(Ku(Y3)): 2[IL], 2[SKu(Y3)(IL)], 2[S2 Ku(Y3)(IL)] up to sign. Similarly, there are three (−4)-classes in N(AX14): 2[IC], 2[SKu(Y3)(IC)], 2[S2 Ku(Y3)(IC)] up to sign, where C is a conic on X14. Now σ and σ ′ are Serre-invariant stability conditions, thus Bridgeland moduli spaces of stable objects with these (−4)-classes are all isomorphic. If d = 5, then we have an equation x2 + 5xy + 5y2 = 1, then by exactly the same argument in Corollary 4.2, the solutions of the equation x2 + 5xy + 5y2 = 1 and hence x2 + 5xy + 5y2 = 4 are parametrised by Z, generated by the fundamental unit, up to sign. Then all the moduli space of stable objects of (−4)-classes are isomorphic since stability condition σ are invariant under rotation functor. ⊓⊔ 8 Isomorphisms between Bridgeland moduli spaces of (−4)-classes Let X = X4d+2 for d = 3, 4, 5. In this section we are going to show that Mss σ (Ku(Y), 2 − 2L) ∼= Mss σ ′(AX, 2 −4L) and this isomorphism restricts to the isomorphism of stable locus. σ Almost the same as Lemma 7.8 and Proposition 7.10, but using Corollary 4.5 instead of [35, Proposition 4.6], we have: Lemma 8.1 Let F ∈AX be an object with [F] = 2s = 2 −4L and hom(F, F) = 1, ext1(F, F) = 5, ext2(F, F) = ext3(F, F) = 0. Then up to some shifts F is in the heart of every Serre-invariant stability on AX. Proposition 8.2 Let F ∈AX be an object with ch(F) = 2 −4L and hom(F, F) = 1, ext1(F, F) = 5, ext2(F, F) = ext3(F, F) = 0, then F is σ ′-stable with respect to every Serre-invariant stability condition σ ′ on AX. Using the equivalence in [28], Proposition 7.13 holds for AX4d+2 and d = 3, 4, 5 if we replace σ(α, −1 2) by (σ(α, −1 2)). In summary, we have: Corollary 8.3 Let (Y, X) ∈Zd ⊂MF2 d × MF1 4d+2 for d = 3, 4, 5. Let E be an object in Ku(Y) with [E] = 2 −2L. Let σ and σ ′ be two Serre-invariant stability conditions on Ku(Y) and AX respectively. Then E ∈Pσ ((0, 1]) is σ-(semi)stable if and only if (E)[m] ∈ Pσ ′((0, 1]) is σ ′-(semi)stable for m = m(σ, σ ′) ∈Z. Theorem 8.4 Let (Y, X) ∈Zd ⊂MF2 d × MF1 4d+2 for d = 3, 4, 5. Let σ and σ ′ be Serre-invariant stability conditions on Ku(Y) and AX respectively. Then the equivalence : Ku(Y) ∼= AX in Theorem 1.1 induces an isomorphism between moduli spaces: s : Mss σ (Ku(Y), 2 −2L) ∼= −→Mss σ ′(AX, 2 −4L) such that the restriction Mσ (Ku(Y), 2 −2L) ∼= −→Mσ ′(AX, 2 −4L) is also an isomorphism. A note on Bridgeland moduli spaces and moduli... 833 Proof From Theorem 7.6 we know Minst Y ∼= Mss σ (Ku(Y), 2L −2). Since the equivalences in [28] map the numerical class 2(1 −L) to 2(1 −2L), hence using Corollary 8.3, a similar argument in Theorem 7.6 shows that induces a morphism s. This is a proper morphism since moduli spaces on both sides are proper. 9 Moduli space Mss X (2, 0, 4) on X4d+2 as Bridgeland moduli space In this section, we are going to show that there is an isomorphism between Mss X (2, 0, 4) and Mss σ ′(AX, 2 −4L) for X = X14, X18 or X22. σ ( ) As shown in [12], we have the following classiﬁcation of sheaves in Mss X (2, 0, 4): (1) E is strictly Gieseker-semistable if and only if E is an extension of ideal sheaves of conics. (2) E is Gieseker-stable and locally free. (2) E is Gieseker-stable and locally free. (3) E is Gieseker-stable but not locally free, and ﬁt into an exact sequence 0 →IC →E →IL →0 where C is a cubic and L is a line. In this case χ(IC) = 0, ch(IC) = 1 −3L + 1 2 P. where C is a cubic and L is a line. In this case χ(IC) = 0, ch(IC) = 1 −3L + 1 2 P. (4) E is Gieseker stable but not locally free and ﬁt into an exact sequence where C is a cubic and L is a line. In this case χ(IC) = 0, ch(IC) = 1 −3L + 1 2 P. (4) E is Gieseker-stable but not locally free, and ﬁt into an exact sequence E is Gieseker-stable but not locally free, and ﬁt into an exact sequence 0 →IC →E →Ix →0 where C is a quartic and x ∈C is a point. In this case χ(IC) = 0, ch(IC) = 1 −4L + P. (5) E i Gi k bl b l ll f d ﬁi where C is a quartic and x ∈C is a point. In this case χ(IC) = 0, ch(IC) = 1 −4L + P. (5) E is Gieseker-stable but not locally free, and ﬁt into an exact sequence where C is a quartic and x ∈C is a point. In this case χ(IC) = 0, ch(IC) = 1 −4L + P. (5) E is Gieseker-stable but not locally free, and ﬁt into an exact sequence where C is a quartic and x ∈C is a point. In this case χ(IC) = 0, ch(IC) = 1 −4L + P. (5) E is Gieseker-stable but not locally free, and ﬁt into an exact sequence (5) E is Gieseker-stable but not locally free, and ﬁt into an exact sequence 0 →IC →E →OX →0 where C is of degree 4 with χ(OC) = 2. 9 Moduli space Mss X (2, 0, 4) on X4d+2 as Bridgeland moduli space Since χ(E∨, IC) = 2, we only need to show that ext1(E∨, IC) = 0, that is h0(E\|C) = 0. If E is in case (4), from stability we have hom(E∨, IC) = ext3(E∨, IC) = hom(E∨, Ix) = ext3(E∨, Ix) = 0. From the long exact sequence we have ext1(E∨, Ix) = 2. Since χ(E∨, IC) = 2, we only need to show that ext1(E∨, IC) = 0, that is h0(E\|C) = 0. If E is in case (5), we have ext∗(E∨, OX) = 0 and hom(E∨, IC) = ext3(E∨, IC) = 0. Since χ(E∨, IC) = 0, we only need to show that ext1(E∨, IC) = 0, that is h0(E\|C) = 0. Now for all these three cases, a similar argument in [28, Lemma B.3.3] shows that these cohomology groups are vanishing as we want. If E is in case (5), we have ext∗(E∨, OX) = 0 and hom(E∨, IC) = ext3(E∨, IC) = 0. Since χ(E∨, IC) = 0, we only need to show that ext1(E∨, IC) = 0, that is h0(E\|C) = 0. Now for all these three cases, a similar argument in [28, Lemma B.3.3] shows that these cohomology groups are vanishing as we want. omology groups are vanishing as we want. When E is locally free, from stability and Serre duality we have hom(E∨, E) = 0 and ext3(E∨, E) = 0. 1 When E is locally free, from stability and Serre duality we have hom(E∨, E) = 0 and ext3(E∨, E) = 0. Since χ(E∨, E) = 0, we only need to show ext1(E∨, E) = hom(E∨, E[1]) = 0. To this end, 1 Since χ(E∨, E) = 0, we only need to show ext1(E∨, E) = hom(E∨, E[1]) = 0. To this end, by [6, Lemma 2.7 (ii)] we know that E∨and E[1] are in Coh 1 4 (X) and are σα, 1 4 -stable for α ≫0. But μα, 1 4 (E∨) > μα, 1 4 (E[1]) when α ≫0, which gives hom(E∨, E[1]) = ext1(E∨, E) = 0. ⊓⊔ by [6, Lemma 2.7 (ii)] we know that E∨and E[1] are in Coh 1 4 (X) and are σα, 1 4 -stable for α ≫0. But μα, 1 4 (E∨) > μα, 1 4 (E[1]) when α ≫0, which gives hom(E∨, E[1]) = ext1(E∨, E) = 0. 9 Moduli space Mss X (2, 0, 4) on X4d+2 as Bridgeland moduli space In this case χ(IC) = −1, ch(IC) = 1 −4L. When E is strictly Gieseker-semistable, it is clear from the classiﬁcation above that E ∈ AX and is semistable with respect to every Serre-invariant stability condition. When E is Gieseker-stable, this is also true. 123 834 Z. Liu , S. Zhang Lemma 9.1 Let E ∈MX(2, 0, 4), then hi(E) = 0 and exti(E∨, E) = 0 for every i. Thus E ∈AX. Lemma 9.1 Let E ∈MX(2, 0, 4), then hi(E) = 0 and exti(E∨, E) = 0 for every i. Thus E ∈AX. Proof By [12, Lemma 4.3] we know h2(E) = 0. From the stability and Serre duality, we have h3(E) = ext3(OX, E) = hom(E, OX(−1)) = 0 and h0(E) = hom(OX, E) = 0. Since χ(E) = 0, we know h1(E) = 0. Thus hi(E) = 0 for every i. y To prove exti(E∨, E) = 0, we assume X = X14 for simplicity, but other cases are almost the same. First we assume that E is in case (3). From stability we have hom(E∨, IC) = ext3(E∨, IC) = hom(E∨, IL) = ext3(E∨, IL) = 0. From E\|L = OL ⊕OL(−1) we know ext1(E∨, IL) = 1 and ext2(E∨, IL) = 0. Since χ(E∨, IC) = 1, we only need to show that ext1(E∨, IC) = 0, that is h0(E\|C) = 0. First we assume that E is in case (3). From stability we have hom(E∨, IC) = ext3(E∨, IC) = hom(E∨, IL) = ext3(E∨, IL) = 0. From E\|L = OL ⊕OL(−1) we know ext1(E∨, IL) = 1 and ext2(E∨, IL) = 0. Since χ(E∨, IC) = 1, we only need to show that ext1(E∨, IC) = 0, that is h0(E\|C) = 0. ∨ 3 ∨ ∨ If E is in case (4), from stability we have hom(E∨, IC) = ext3(E∨, IC) = hom(E∨, Ix) = ext3(E∨, Ix) = 0. From the long exact sequence we have ext1(E∨, Ix) = 2. Since χ(E∨, IC) = 2, we only need to show that ext1(E∨, IC) = 0, that is h0(E\|C) = 0. If E is in case (4), from stability we have hom(E∨, IC) = ext3(E∨, IC) = hom(E∨, Ix) = ext3(E∨, Ix) = 0. From the long exact sequence we have ext1(E∨, Ix) = 2. for every Serre-invariant stability condition σ ′ on AX. Proof A similar argument in Theorem 7.6 shows that the projection functor pr induces a morphism s′ : Mss X (2, 0, 4) →Mss σ ′(AX, 2 −4L). We know that Mss X (2, 0, 4) is projective and Mss σ ′(AX, 2 −4L) is proper, hence s′ is also projective. By Proposition 9.3, we have s′−1(Mσ ′(AX, 2 −4L)) = MX(2, 0, 4) and s′ is injective. From the fact that the functor pr is the identity on E ∈Mss X (2, 0, 4), we know s′ is étale. Thus s′ is an embedding. Since Mss σ ′(AX, 2 −4L) is irreducible and smooth by Lemma 6.8, Theorems 7.6 and 8.4, s′ is an isomorphism. ⊓⊔ ⊓⊔ Acknowledgements This note originated from a seminar talk given by the second author in Tianyuan Mathe- matical Center in Southwest China(TMCSC), Sichuan University. It is part of the Undergraduate Mathematical Research Project of the ﬁrst author mentored by the second author. We would like to thank professor Xiaojun Chen for the invitation and TMCSC for their hospitality. We thank Daniele Faenzi, Li Lai, Girivaru Ravin- dra, Junyan Xu, and Song Yang for answering us several questions. We also thank Arend Bayer, Augustinas Jacovskis for useful conversations on several related topics. The ﬁrst author would like to thank Jiahui Gao, Songtao Ma, Rui Xiong, and Jiajin Zhang for useful discussion. The second author thanks Tingyu Sun for support. We also would like to thank the referee for the careful reading of the manuscript, and for providing detailed and interesting comments. In addition, we would like to thank Laura Pertusi and Xuqiang Qin for informing us the very recent preprint [38] and giving useful comments on the earlier draft of our paper. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. and the restriction gives an isomorphism MX(2, 0, 4) ∼= −→Mσ ′(AX, 2 −4L) for every Serre-invariant stability condition σ ′ on AX. for every Serre-invariant stability condition σ ′ on AX. 9 Moduli space Mss X (2, 0, 4) on X4d+2 as Bridgeland moduli space Thus the only possible case is ch≤2(A) = ch≤2(B) = (1, 0, −2L), which does not effect the semi-stability of E.Therefore,thereisnowallfor E withrespecttoσα,β onβ = −ϵ whereϵ > 0 sufﬁciently small. Thus E is σα,β-semistable for every α > 0 and β < 0 with −β sufﬁciently small. Hence E[1] ∈Coh0 α,β(X) is σ 0 α,β-semistable, and E[1] ∈A(α, β) is σ(α, β)-semistable for such (α, β). ⊓⊔ a = 0, 1, 2, and μα,β(A) = μα,β(E) = μα,β(B) < +∞implies a ̸= 0, 2. Thus the only possible case is ch≤2(A) = ch≤2(B) = (1, 0, −2L), which does not effect the semi-stability of E.Therefore,thereisnowallfor E withrespecttoσα,β onβ = −ϵ whereϵ > 0 sufﬁciently small. Thus E is σα,β-semistable for every α > 0 and β < 0 with −β sufﬁciently small. Hence E[1] ∈Coh0 α,β(X) is σ 0 α,β-semistable, and E[1] ∈A(α, β) is σ(α, β)-semistable for such (α, β). ⊓⊔ ⊓⊔ Proposition 9.3 Let E ∈Mss X (2, 0, 4). Then E ∈AX and if E is Gieseker-(semi)stable, then E is σ ′-(semi)stable. Proposition 9.3 Let E ∈Mss X (2, 0, 4). Then E ∈AX and if E is Gieseker-(semi)stable, then E is σ ′-(semi)stable. Proof First we assume that E is strictly Gieseker-semistable. Then from the classiﬁcation we know that E is an extension of ideal sheaves of conics. Thus the statement follows from Lemma 4.3. When E is Gieseker-stable, the result follows from Lemmas 9.1, 9.2 and Proposition 8.2. ⊓⊔ ⊓⊔ eorem 9.4 Let X = X14, X18 or X22. Then the projection functor induces an isomorphism s′ : Mss X (2, 0, 4) ∼= −→Mss σ ′(AX, 2 −4L) s′ : Mss X (2, 0, 4) ∼= −→Mss σ ′(AX, 2 −4L) and the restriction gives an isomorphism 9 Moduli space Mss X (2, 0, 4) on X4d+2 as Bridgeland moduli space ⊓⊔ Now we are going to show that E ∈MX(2, 0, 4) is stable with respect to every Serre- invariant stability condition σ ′ on AX. Lemma 9.2 Let E ∈MX(2, 0, 4), then hom(E, E) = 1, ext1(E, E) = 5, ext2(E, E) = ext3(E, E) = 0. Lemma 9.2 Let E ∈MX(2, 0, 4), then hom(E, E) = 1, ext1(E, E) = 5, ext2(E, E) = ext3(E, E) = 0. Proof It is clear χ(E, E) = −4. And ext3(E, E) = hom(E, E(−1)) = 0 from stability. Hence we only need to show ext2(E, E) = 0. Proof It is clear χ(E, E) = −4. And ext3(E, E) = hom(E, E(−1)) = 0 from stability. Hence we only need to show ext2(E, E) = 0. By [7, Section 6], σ(α, β) = σ 0 α,β\|AX is a stability condition on AX for every β < 0 and 0 < α with −β, α both sufﬁciently small. When d = 4, 5, the heart A(α, β) has homological dimension 1. When d = 3, from [34] we know that these stability conditions are Serre-invariant. Thus by Lemma 3.10, to show ext2(E, E) = 0, we only need to show E ∈AX is σ(α, β)-semistable. To this end, from [8, Proposition 4.8] we know that E is σα,β-semistable for all β < 0 and α ≫0. Thus by locally-ﬁniteness of walls, to show E is σα,β-semistable for every α > 0 and β < 0 with −β sufﬁciently small, we only need to show that there is no semicircle wall tangent with line β = 0. If there is a such semicircle wall C, then it is given by a sequence 0 →A →E →B →0 in Cohβ(X) for (α, β) ∈C, such that μα,β(A) = μα,β(E) = μα,β(B). Assume ch≤2(A) = (a, bH, c d H2) for a, b, c ∈Z. By continuity, we have μ0,0(A) = μ0,0(E) = μ0,0(B). Thus we have b = 0. But as in Proposition 6.4, chβ 1 (A) ≥0 and chβ 1 (B) ≥0 for β < 0 implies 123 A note on Bridgeland moduli spaces and moduli... 835 a = 0, 1, 2, and μα,β(A) = μα,β(E) = μα,β(B) < +∞implies a ̸= 0, 2. for every Serre-invariant stability condition σ ′ on AX. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. 123 Z. Liu , S. Zhang 836 References 1. Altavilla, M., Petkovic, M., Rota, F.: Moduli spaces on the Kuznetsov component of Fano threefolds of index 2 (2021). arXiv:1908.10986 ( ) 2. Ancona, V., Ottaviani, G.: Stability of apecial instanton bundles on P2n+1. Trans. Am. Math. Soc. 341(2), 677–693 (1994) 3. 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https://openalex.org/W4251787421	http://www.vliz.be/imisdocs/publications/344389.pdf	English	null	Impact of small-scale disturbances on geochemical conditions, biogeochemical processes and element fluxes in surface sediments of the eastern Clarion-Clipperton Zone, Pacific Ocean	null	2,019	cc-by	14,734	Impact of small-scale disturbances on geochemical conditions, biogeochemical processes and element ﬂuxes in surface sediments of the eastern Clarion–Clipperton Zone, Paciﬁc Ocean Jessica B. Volz1, Laura Haffert2, Matthias Haeckel2, Andrea Koschinsky3, and Sabine Kasten1,4 1Alfred Wegener Institute Helmholtz Centre for Polar and Marine Research, 27570 Bremerhaven, Germany 2GEOMAR Helmholtz Centre for Ocean Research Kiel, 24148 Kiel, Germany 3Department of Physics and Earth Sciences, Jacobs University Bremen, 28759 Bremen, Germany 4Faculty of Geosciences, University of Bremen, Klagenfurter Strasse, 28359 Bremen, Germany Correspondence: Jessica B. Volz (jessica.volz@awi.de) Received: 12 August 2019 – Discussion started: 20 August 2019 Revised: 18 December 2019 – Accepted: 14 January 2020 – Published: 28 February 2020 iments was removed during various small-scale disturbance experiments in the different exploration contract areas. Tran- sient transport-reaction modeling for the Polish and German contract areas reveals that the removal of the surface sedi- ments is associated with the loss of the reactive labile to- tal organic carbon (TOC) fraction. As a result, oxygen con- sumption rates decrease signiﬁcantly after the removal of the surface sediments, and, consequently, oxygen penetrates up to 10-fold deeper into the sediments, inhibiting denitriﬁca- tion and Mn(IV) reduction. Our model results show that the return to steady-state geochemical conditions after the dis- turbance is controlled by diffusion until the reactive labile TOC fraction in the surface sediments is partly re-established and the biogeochemical processes commence. While the re- establishment of bioturbation is essential, steady-state geo- chemical conditions are ultimately controlled by the deliv- ery rate of organic matter to the seaﬂoor. Hence, under cur- rent depositional conditions, new steady-state geochemical conditions in the sediments of the CCZ are reached only on a millennium scale even for these small-scale disturbances simulating deep-sea mining activities. Abstract. The thriving interest in harvesting deep-sea min- eral resources, such as polymetallic nodules, calls for en- vironmental impact studies and, ultimately, for regulations for environmental protection. Industrial-scale deep-sea min- ing of polymetallic nodules most likely has severe conse- quences for the natural environment. However, the effects of mining activities on deep-sea ecosystems, sediment geo- chemistry and element ﬂuxes are still poorly understood. Pre- dicting the environmental impact is challenging due to the scarcity of environmental baseline studies as well as the lack of mining trials with industrial mining equipment in the deep sea. Thus, currently we have to rely on small-scale distur- bances simulating deep-sea mining activities as a ﬁrst-order approximation to study the expected impacts on the abyssal environment. Here, we investigate surface sediments in disturbance tracks of seven small-scale benthic impact experiments, which have been performed in four European contract areas for the exploration of polymetallic nodules in the Clarion– Clipperton Zone (CCZ) in the NE Paciﬁc. These small-scale disturbance experiments were performed 1 d to 37 years prior to our sampling program in the German, Polish, Belgian and French contract areas using different disturbance devices. Biogeosciences, 17, 1113–1131, 2020 https://doi.org/10.5194/bg-17-1113-2020 © Author(s) 2020. This work is distributed under the Creative Commons Attribution 4.0 License. Biogeosciences, 17, 1113–1131, 2020 https://doi.org/10.5194/bg-17-1113-2020 © Author(s) 2020. This work is distributed under the Creative Commons Attribution 4.0 License. Impact of small-scale disturbances on geochemical conditions, biogeochemical processes and element ﬂuxes in surface sediments of the eastern Clarion–Clipperton Zone, Paciﬁc Ocean Jessica B. Volz1, Laura Haffert2, Matthias Haeckel2, Andrea Koschinsky3, and Sabine Kasten1,4 1Alfred Wegener Institute Helmholtz Centre for Polar and Marine Research, 27570 Bremerhaven, Germany 2GEOMAR Helmholtz Centre for Ocean Research Kiel, 24148 Kiel, Germany 3Department of Physics and Earth Sciences, Jacobs University Bremen, 28759 Bremen, Germany 4Faculty of Geosciences, University of Bremen, Klagenfurter Strasse, 28359 Bremen, Germany We show that the depth distribution of solid-phase Mn in the up- per 20 cm of the sediments in the CCZ provides a reliable tool for the determination of the disturbance depth, which has been proposed in a previous study from the SE Paciﬁc (Paul et al., 2018). We found that the upper 5–15 cm of the sed- Introduction The accelerating global demand for metals and rare-earth el- ements drives the economic interest in deep-sea mining (e.g., Glasby, 2000; Hoagland et al., 2010; Wedding et al., 2015). J. B. Volz et al.: Impact of small-scale disturbances Based on the observation that bulk solid-phase Mn contents decrease over depth in the surface sediments of the Distur- bance and Recolonization Experiment (DISCOL) area in the Peru Basin, SE Paciﬁc, Paul et al. (2018) have suggested that the depth distribution of solid-phase Mn and associated met- als (e.g., Mo, Ni, Co, Cu) could be used to trace the sediment removal by disturbances. In addition, other solid-phase prop- erties such as total organic carbon (TOC) contents, porosity and radioisotopes may be suitable for the determination of the disturbance depth. Although a considerable number of environmental impact studies have been conducted in different nodule ﬁelds, the prediction of environmental consequences of potential future deep-sea mining is still difﬁcult (e.g., Ramirez-Llodra et al., 2011; Jones et al., 2017; Gollner et al., 2017; Cuvelier et al., 2018). In case of the CCZ, the evaluation of the environmen- tal impact of deep-sea mining activities is challenging due to the fact that baseline data on the natural spatial heterogeneity and temporal variability in depositional conditions, benthic communities and the biogeochemical processes in the sedi- ments are scarce (e.g., Mewes et al., 2014, 2016; Vanreusel et al., 2016; Mogollón et al., 2016; Juan et al., 2018; Volz et al., 2018, 2020; Menendez et al., 2018; Hauquier et al., 2019). In addition, there is no clear consensus on the most appro- priate mining techniques for the commercial exploitation of nodules, and technical challenges due to the inaccessibility of nodules at great water depths between 4000 and 5000 m have limited the deployment of deep-sea mining systems un- til today (e.g., Chung, 2010; Jones et al., 2017). The most reactive TOC compounds, found in the biotur- bated uppermost sediment layer, are the main drivers for early diagenetic processes (e.g., Froelich et al., 1979; Berner, 1981) and are expected to be removed during mining ac- tivities (König et al., 2001). Thus, strong biogeochemical implications can be expected in the sediments after deep- sea mining activities. König et al. (2001) have applied nu- merical modeling to study the consequences of the removal of the upper 10 cm of the sediments in the DISCOL area. They showed that the degradation of TOC during aerobic respiration, denitriﬁcation and Mn(IV) reduction may be decreased for centuries, increasing the oxygen penetration depth (OPD). J. B. Volz et al.: Impact of small-scale disturbances J. B. Volz et al.: Impact of small-scale disturbances 1114 Seaﬂoor minerals of interest include (1) polymetallic nod- ules (e.g., Mero, 1965), (2) massive sulﬁde deposits (e.g., Scott, 1987) and (3) cobalt-rich crusts (e.g., Halkyard, 1985). As the seaﬂoor within the Clarion–Clipperton Zone (CCZ) in the NE Paciﬁc holds one of the most extensive deposits of polymetallic nodules with considerable base metal quan- tities, commercial exploitation of seaﬂoor mineral deposits may focus on the CCZ (e.g., Mero, 1965; Halbach et al., 1988; Rühlemann et al., 2011; Hein et al., 2013; Kuhn et al., 2017a). The exploration and, ultimately, industrial exploita- tion of polymetallic nodules demand international regula- tions for the protection of the environment (e.g., Halfar and Fujita, 2002; Glover and Smith, 2003; Davies et al., 2007; van Dover, 2011; Ramirez-Llodra et al., 2011; Boetius and Haeckel, 2018). The International Seabed Authority (ISA) is responsible for regulating the exploration and exploita- tion of marine mineral resources as well as for protecting and conserving the marine environment beyond the exclusive economic zones of littoral states from harmful effects (ISA, 2010). The ISA has granted temporal contracts for the explo- ration of polymetallic nodules in the CCZ, engaging all con- tract holders to explore resources, test mining equipment and assess the environmental impacts of deep-sea mining activi- ties (ISA, 2010; Lodge et al., 2014; Madureira et al., 2016). ments; (2) the re-deposition of material from the suspended sediment plume; and (3) potentially also the compaction of the surface sediments due to weight of the nodule collector (Thiel and Forschungsverband Tiefsee-Umweltschutz, 2001; Oebius et al., 2001; König et al., 2001; Grupe et al., 2001; Radziejewska, 2002; Khripounoff et al., 2006; Cronan et al., 2010; Paul et al., 2018; Gillard et al., 2019). The wide range of estimates for the disturbance depth may be associated with (1) various devices used for the deep-sea disturbance exper- iments (Brockett and Richards, 1994; Oebius et al., 2001; Jones et al., 2017), (2) distinct sediment properties in differ- ent nodule ﬁelds of the Paciﬁc Ocean (e.g., Cronan et al., 2010; Hauquier et al., 2019) and (3) different approaches for the determination of the disturbance depth (e.g., Oebius et al., 2001; Grupe et al., 2001; Khripounoff et al., 2006). Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. www.biogeosciences.net/17/1113/2020/ 2.1.1 Small-scale disturbances Since the 1970s, several comprehensive environmental im- pact studies of deep-sea mining simulations have been car- ried out in the CCZ, including the Benthic impact experi- ment (BIE; e.g., Trueblood and Ozturgut, 1997; Radziejew- ska, 2002) and the Japan deep-sea impact experiment (JET; Fukushima, 1995). In addition, numerous small-scale seaﬂoor disturbances have been carried out in the CCZ in the past 40 years using various tools such as epibenthic sledge (EBS) and dredges (e.g., Vanreusel et al., 2016; Jones et al., 2017). The EBS is towed along the seabed for the collec- tion of benthic organisms (and nodules), thereby also remov- ing the upper few centimeters of the sediments (e.g., Brenke, 2005). In 2015, some of these disturbances that were up to 37 years old were revisited as part of the BMBF–EU JPI Oceans pilot action “Ecological aspects of deep-sea min- ing (MiningImpact)” project in order to evaluate the long- term consequences of such small-scale disturbances on the abyssal benthic ecosystem (Table 1; Fig. 2; Martínez Arbizu and Haeckel, 2015). For comparison, DISCOL, which was conducted in a nodule ﬁeld in the Peru Basin (PB) in 1989, was revisited as part of MiningImpact (Boetius, 2015; Grein- ert, 2015). In the framework of DISCOL, a seaﬂoor area of ∼11 km2 was disturbed with a plow harrow. The impact of the DISCOL experiment was studied 0.5, 3 and 7 years after the disturbance had been set (e.g., Thiel and Forschungsver- band Tiefsee-Umweltschutz, 2001). Furthermore, new small- scale disturbance tracks were created during SO239 in the BGR-RA and in the GSR area “B6” using an EBS in or- der to add also initial temporal datasets (Table 1; Fig. 2; Martínez Arbizu and Haeckel, 2015). The EBS weighed about 400 kg and created a disturbance track of about 1.5 m width (Brenke, 2005). The fresh EBS disturbance tracks in the BGR-RA and GSR areas were revisited 1 d after their creation. Eight months prior to the cruise SO239, towed g The different investigated European contract areas within the CCZ include the BGR, IOM, GSR and IFREMER areas. Comprehensive pore-water and solid-phase analyses on the MUC and GC sediment cores from undisturbed sites have been conducted in previous baseline studies and are pre- sented elsewhere (Volz et al., 2018, 2020). J. B. Volz et al.: Impact of small-scale disturbances zonation, sedimentation rates, ﬂuxes of particulate organic carbon (POC) to the seaﬂoor and bioturbation depths, we summarized these key parameters, which are originally pre- sented elsewhere, in Table 2 (Volz et al., 2018). Steady-state transport-reaction models have shown that aerobic respira- tion is the dominant biogeochemical process at all investi- gated sites, consuming more than 90 % of the organic matter delivered to the seaﬂoor (Mogollón et al., 2016; Volz et al., 2018). Below the OPD at more than 0.5 m depth, Mn(IV) and nitrate reduction succeed in the suboxic zone, where oxygen and sulﬁde are absent (e.g., Mewes et al., 2014; Mogollón et al., 2016; Kuhn et al., 2017b; Volz et al., 2018). At several sites investigated in this study, including the BGR “reference area” (BGR-RA) and IOM sites, decreasing Mn2+ concen- trations at depth are probably associated with the oxidation of Mn2+ by upwardly diffusing oxygen circulating through the underlying basaltic crust (Mewes et al., 2016; Kuhn et al., 2017b; Volz et al., 2018). bances on redox zonation and element ﬂuxes and (2) deter- mine how much time is needed for the re-establishment of a new steady-state geochemical system in the sediments after the disturbances. Our work includes pore-water and solid- phase analyses as well as the application of a transient one- dimensional transport-reaction model. 2 Material and methods As part of the European JPI Oceans pilot action “Ecological aspects of deep-sea mining (MiningImpact)”, multiple-corer (MUC) and gravity-corer (GC) sediment cores were taken during the RV SONNE cruise SO239 in March–April 2015 from undisturbed sites in various European contract areas for the exploration of polymetallic nodules (Fig. 1; Table 1; Martínez Arbizu and Haeckel, 2015). These undisturbed ref- erence sites were chosen in close proximity (< 5 km) to small-scale disturbance experiments for the simulation of deep-sea mining, which were created up to 37 years ago and revisited during cruise SO239 (Table 1; see Sect. 2.1.1.; Martínez Arbizu and Haeckel, 2015). The sampling of sedi- ments in the disturbance tracks of these experiments was con- ducted by video-guided push coring (PC) between 1 d and 37 years after the initial disturbances using the ROV Kiel 6000 (Table 1; Fig. 2; Martínez Arbizu and Haeckel, 2015). 2.1.1 Small-scale disturbances These analyses in- clude the determination of pore-water oxygen; NO− 3 , Mn2+ and NH+ 4 concentrations; and TOC contents for MUC and GC sediment cores (Volz et al., 2018) as well as solid-phase bulk Mn contents for the MUC sediment cores (Volz et al., 2020). In the framework of this study, we used these pre- viously published pore-water and solid-phase data as undis- turbed reference data for geochemical conditions and sedi- ment composition (Table 1). On this basis, here we inves- tigate seven small-scale disturbances for the simulation of deep-sea mining (Table 1; see Sect. 2.1.1.; Martínez Arbizu and Haeckel, 2015). J. B. Volz et al.: Impact of small-scale disturbances p Here, we investigate the impact of various small-scale dis- turbances on geochemical conditions, biogeochemical pro- cesses and element ﬂuxes in surface sediments of the CCZ. These small-scale disturbance tracks were created up to 37 years ago in four different European contract areas for the exploration of polymetallic nodules, including the German BGR (Bundesanstalt für Geowissenschaften und Rohstoffe) area, the Belgian GSR (Global Sea Mineral Resources NV) area, the French IFREMER (Institut Français de Recherche pour l’Exploitation de la Mer) area and the Polish IOM (In- teroceanmetal) area. In order to determine the disturbance depths of the different small-scale disturbances in the differ- ent European contract areas, we correlate the depth distri- butions of solid-phase Mn and TOC between disturbed sites and undisturbed reference sites using the Pearson product– moment correlation coefﬁcient. On this basis, we (1) assess the short- and long-term consequences of small-scale distur- The physical removal of nodules as hard-substrate habitats has severe consequences for the nodule-associated sessile fauna as well as the mobile fauna (Bluhm, 2001; Smith et al., 2008; Purser et al., 2016; Vanreusel et al., 2016). With slow nodule growth rates of a few millimeters per million years (e.g., Halbach et al., 1988; Kuhn et al., 2017a), the deep-sea fauna may not recover for millions of years (Vanreusel et al., 2016; Jones et al., 2017; Gollner et al., 2017; Stratmann et al., 2018). In addition to the removal of deep-sea fauna as well as seaﬂoor habitats, the exploitation of nodules is associated with (1) the removal, mixing and re-suspension of the upper 4 cm to more than several tens of centimeters of the sedi- Biogeosciences, 17, 1113–1131, 2020 www.biogeosciences.net/17/1113/2020/ 1115 2.1 Site description The CCZ is deﬁned by two transform faults, the Clarion Frac- ture Zone in the north and the Clipperton Fracture Zone in the south, and covers an area of about 6×106 km2 (Fig. 1; e.g., Halbach et al., 1988). The sediments at the investigated sites (Table 1) are dominated by clayey siliceous oozes, with Mn nodules varying in size (1–10 cm) and spatial density (0– 30 kg m−2) at the sediment surface (Berger, 1974; Kuhn et al., 2012; Mewes et al., 2014; Volz et al., 2018). In order to characterize the investigated sediments with respect to redox Biogeosciences, 17, 1113–1131, 2020 www.biogeosciences.net/17/1113/2020/ www.biogeosciences.net/17/1113/2020/ 1116 J. B. Volz et al.: Impact of small-scale disturbances J. B. Volz et al.: Impact of small-scale disturbances Figure 1. Sampling sites (black circles; black star) in various European contract areas for the exploration of manganese nodules within the Clarion–Clipperton Zone (CCZ). Investigated stations are located in the German BGR area (blue), eastern European IOM area (yellow), Belgian GSR area (green) and French IFREMER area (red). The two stations within the German BGR area are located in the “prospective area” (BGR-PA; black star) and in the “reference area” (BGR-RA; black circle). The contract areas granted and governed by the International Seabed Authority (ISA; white areas) are surrounded by nine areas of particular environmental interest (APEIs), which are excluded from any mining activities (green shaded squares). Geographical data provided by the ISA. Figure 1. Sampling sites (black circles; black star) in various European contract areas for the exploration of manganese nodules within the Clarion–Clipperton Zone (CCZ). Investigated stations are located in the German BGR area (blue), eastern European IOM area (yellow), Belgian GSR area (green) and French IFREMER area (red). The two stations within the German BGR area are located in the “prospective area” (BGR-PA; black star) and in the “reference area” (BGR-RA; black circle). The contract areas granted and governed by the International Seabed Authority (ISA; white areas) are surrounded by nine areas of particular environmental interest (APEIs), which are excluded from any mining activities (green shaded squares). Geographical data provided by the ISA. 1. MUC and PC cores investigated in this study, including information on geographic position, water depth, type and age bances (years: yr; months: mth; days: d). 2.2 Sediment sampling and solid-phase analyses Total organic carbon (TOC) contents were determined using an Eltra CS-2000 element analyzer. Approximately 100 mg of freeze-dried, homogenized sediment was transferred into a ceramic cup and decalciﬁed with 0.5 mL of 10 % HCl at 250 ◦C for 2 h before analysis. Based on an in-house refer- ence material, precision of the analysis was better than 3.7 % (n = 83). ROV-operated push cores were sampled at intervals of 1 cm for solid-phase analyses. Bulk sediment data and TOC con- tents have been corrected after Kuhn (2013) for the interfer- ence of the pore-water salt matrix with the sediment com- position (Volz et al., 2018). The mass percentage of the pore water was determined gravimetrically before and af- ter freeze-drying of the wet sediment samples. The salt- corrected sediment composition c′ was calculated from the measured solid-phase composition c using the mass percent- age of H2O of the wet sediment (w), which contains 96.5 % J. B. Volz et al.: Impact of small-scale disturbances During the BIONOD cruises aboard RV L’Atalante in 2012, the same EBS setup as used during cruise SO239 was deployed in the BGR “prospective area” (BGR-PA) and in the IFREMER area (Table 1; Rühlemann et al., 2013; Menot et al., 2013; Martínez Arbizu and Haeckel, 2015). In 1995, the deep-sea sediment re-suspension system (DSSRS) was used during the IOM-BIE (Benthic Impact Ex- periment) disturbance in the IOM area (Table 1; e.g., Kotlin- ski and Stoyanova, 1998). The DSSRS weighed 3.2 t under normal atmospheric pressure and was designed to dredge the seaﬂoor while producing a re-suspended particle plume about 5 m above the seaﬂoor (Brockett and Richards, 1994; Sharma, 2001). Based on the dimensions of the DSSRS de- vice, the disturbance track created during the IOM-BIE dis- turbance experiment is about 2.5 m wide (Fig. 2; Brockett and Richards, 1994). In 1978, the Ocean Mineral Company (OMCO) created disturbance tracks in the French IFREMER area by towed dredge sampling (Table 1; e.g., Spickermann, 2012). (1) (1) 2.2.1 Total acid digestions Total acid digestions were performed in the microwave sys- tem MARS Xpress (CEM) after the protocols by Kretschmer et al. (2010) and Nöthen and Kasten (2011). Approximately 50 mg of freeze-dried, homogenized bulk sediment was di- gested in an acid mixture of 65 % sub-boiling distilled HNO3 (3 mL), 30 % sub-boiling distilled HCl (2 mL) and 40 % Suprapur® HF (hydroﬂuoric acid; 0.5 mL) at ∼230 ◦C. Di- gested solutions were fumed off to dryness; the residue was re-dissolved under pressure in 1 M HNO3 (5 mL) at ∼200 ◦C and then ﬁlled up to 50 mL with 1 M HNO3. Total bulk Mn and Al contents were determined using inductively coupled plasma optical emission spectrometry (ICP-OES; IRIS In- trepid ICP-OES Spectrometer, Thermo Elemental). Based on the standard reference material NIST 2702, accuracy and precision of the analysis were 3.7 % and 3.5 % for Mn, re- spectively (n = 67). www.biogeosciences.net/17/1113/2020/ Area Site Coring Disturbance device and Disturbance Latitude Longitude Water depth SO239- device type age (N) (W) (m) BGR-PA 39 MUC – – 11◦50.64′ 117◦03.44′ 4132.0 BGR-PA 41 PC EBS1 3 yr 11◦50.92′ 117◦03.77′ 4099.2 BGR-RA 62 GC – – 11◦49.12′ 117◦33.22′ 4312.2 BGR-RA 64 PC EBS2 1 d 11◦48.27′ 117◦30.18′ 4332 BGR-RA 66 MUC – – 11◦49.13′ 117◦33.13′ 4314.8 IOM 84 MUC – – 11◦04.73′ 119◦39.48′ 4430.8 IOM 87 GC – – 11◦04.54′ 119◦39.83′ 4436 IOM 101 PC IOM-BIE3 20 yr 11◦04.38′ 119◦39.38′ 4387.4 GSR 121 MUC – – 13◦51.25′ 123◦15.3′ 4517.7 GSR 131 PC EBS2 1 d 13◦52.38′ 123◦15.1′ 4477.6 GSR 141 PC Dredge4 8 mth 13◦51.95′ 123◦15.33′ 4477 IFREMER 157 PC Dredge5 37 yr 14◦02.06′ 130◦07.23′ 4944.5 IFREMER 161 PC EBS1 3 yr 14◦02.20′ 130◦05.87′ 4999.1 IFREMER 175 MUC – – 14◦02.45′ 130◦05.11′ 5005.5 1 Epibenthic sledge (EBS) during BIONOD cruises in 2012 aboard L’Atalante (Brenke, 2005; Rühlemann et al., 2013; Menot et al., 2013). 2 Epibenthic sledge (EBS) during RV SONNE cruise SO239 in 2015 (Brenke, 2005; Martínez Arbizu and Haeckel, 2015). 3 Benthic impact experiment (BIE); disturbance created with the deep-sea sediment re-suspension system (DSSRS; e.g., Brocket and Richards, 1994; Kotlinski and Stoyanova, 1998). 4 Towed dredge sampling during GSR cruise in 2014 aboard MV Mt. Mitchell (Jones et al., 2017). 5 Towed dredge sampling by the Ocean Minerals Company (OMCO) in 1978 aboard Hughes Glomar Explorer (Morgan et al., 1993; Spickermann, 2012). es investigated in this study, including information on geographic position, water depth, type and age of th ths: mth; days: d). 1 Epibenthic sledge (EBS) during BIONOD cruises in 2012 aboard L’Atalante (Brenke, 2005; Rühlemann et al., 2013; Menot et al., 2013). 2 Epibenthic sledge (EBS) during RV SONNE cruise SO239 in 2015 (Brenke, 2005; Martínez Arbizu and Haeckel, 2015). 3 Benthic impact experiment (BIE); disturbance created with the deep-sea sediment re-suspension system (DSSRS; e.g., Brocket and Richards, 1994; Kotlinski and Stoyanova, 1998). 4 Towed dredge sampling during GSR cruise in 2014 aboard MV Mt. Mitchell (Jones et al., 2017). 5 Towed dredge sampling by the Ocean Minerals Company (OMCO) in 1978 aboard Hughes Glomar Explorer (Morgan et al., 1993; Spickermann, 2012). www.biogeosciences.net/17/1113/2020/ Biogeosciences, 17, 1113–1131, 2020 www.biogeosciences.net/17/1113/2020/ 1117 J. B. Volz et al.: Impact of small-scale disturbances Table 2. Information of sedimentation rate (Sed. rate), ﬂux of particulate organic carbon (POC) to the seaﬂoor, bioturbation depth (Bioturb. depth), and oxygen penetration depth (OPD) based on GC cores from the investigated sites and determined in the study by Volz et al. (2018). Information for the BGR-PA area is taken from an adjacent site (A5-2-SN; 11◦57.22′ N, 117◦0.42′ W) studied by Mewes et al. (2014) and Mogollón et al. (2016). Area Sed. rate POC ﬂux Bioturb. depth OPD (cm kyr−1) (mg Corg m−2 d−1) (cm) (m) BGR-PA ∼0.53a ∼6.9a ∼5a ∼2a,b BGR-RA 0.65 1.99 7 0.5 IOM 1.15 1.54 13 3 GSR 0.21 1.51 8 > 7.4 IFRE-1 0.64 1.47 7 4.5 IFRE-2 0.48 1.5 8 3.8 APEI3 0.2 1.07 6 > 5.7 a Mogollón et al. (2016). b Mewes et al. (2014). H2O (Eq. 1): c′ = c · 100 100 − 100 · (w· 100 96.5 )−w 100−w . (1) 2 2 1 T t l id di ti dredge sampling was performed in the GSR area by the Belgian contractor (Martínez Arbizu and Haeckel, 2015; Jones et al., 2017). During the BIONOD cruises aboard RV L’Atalante in 2012, the same EBS setup as used during cruise SO239 was deployed in the BGR “prospective area” (BGR-PA) and in the IFREMER area (Table 1; Rühlemann et al., 2013; Menot et al., 2013; Martínez Arbizu and Haeckel, 2015). In 1995, the deep-sea sediment re-suspension system (DSSRS) was used during the IOM-BIE (Benthic Impact Ex- periment) disturbance in the IOM area (Table 1; e.g., Kotlin- ski and Stoyanova, 1998). The DSSRS weighed 3.2 t under normal atmospheric pressure and was designed to dredge the seaﬂoor while producing a re-suspended particle plume about 5 m above the seaﬂoor (Brockett and Richards, 1994; Sharma, 2001). Based on the dimensions of the DSSRS de- vice, the disturbance track created during the IOM-BIE dis- turbance experiment is about 2.5 m wide (Fig. 2; Brockett and Richards, 1994). In 1978, the Ocean Mineral Company (OMCO) created disturbance tracks in the French IFREMER area by towed dredge sampling (Table 1; e.g., Spickermann, 2012). dredge sampling was performed in the GSR area by the Belgian contractor (Martínez Arbizu and Haeckel, 2015; Jones et al., 2017). 2.3 Pearson correlation coefﬁcient In order to determine the disturbance depths, solid-phase bulk Mn contents were correlated between disturbed sedi- ments and undisturbed reference sediments using the Pearson Biogeosciences, 17, 1113–1131, 2020 www.biogeosciences.net/17/1113/2020/ www.biogeosciences.net/17/1113/2020/ Biogeosciences, 17, 1113–1131, 2020 1118 J. B. Volz et al.: Impact of small-scale disturbances J. B. Volz et al.: Impact of small-scale disturbances J p Figure 2. Examples of undisturbed reference sediments in the German BGR-PA area and the French IFREMER area and pictures of small- scale disturbances for the simulation of deep-sea mining within the CCZ, which are investigated in the framework of this study (years: yr; months: mth; days: d). Copyright: ROV KIEL 6000 Team, GEOMAR Helmholtz Centre for Ocean Research Kiel, Germany. Figure 2. Examples of undisturbed reference sediments in the German BGR-PA area and the French IFREMER area and pictur cale disturbances for the simulation of deep-sea mining within the CCZ, which are investigated in the framework of this study months: mth; days: d) Copyright: ROV KIEL 6000 Team GEOMAR Helmholtz Centre for Ocean Research Kiel Germany Figure 2. Examples of undisturbed reference sediments in the German BGR-PA area and the French IFREMER area and pictures of small- scale disturbances for the simulation of deep-sea mining within the CCZ, which are investigated in the framework of this study (years: yr; months: mth; days: d). Copyright: ROV KIEL 6000 Team, GEOMAR Helmholtz Centre for Ocean Research Kiel, Germany. product–moment correlation coefﬁcient r (Eq. 1; Table 1; Pearson, 1895). The Pearson correlation coefﬁcient is a sta- tistical measure of the linear relationship between two arrays of variables, with solid-phase bulk Mn contents from undisturbed reference sediments were taken from Volz et al. (2020). The high- est positive linear correlations of solid-phase Mn contents (rMn ∼1) between the disturbed sites and the respective undisturbed reference sites (Table 1) were used to determine the depths of the disturbances. In a second step, the same cor- relation was applied to the TOC contents (rTOC) in order to verify the depth of disturbance. While the TOC contents in the disturbed sediments were determined in the framework of this study, TOC contents from undisturbed reference sed- iments were taken from Volz et al. (2018). y = 1 n Pn i=1yi. www.biogeosciences.net/17/1113/2020/ rxy = Pn i=1 (xi −x)(yi −y) qPn i=1(xi −x)2Pn i=1(yi −y)2 , (2) (2) (2) where n is the sample size, x and y are individual sample points, and x and y are the sample means x = 1 n Pn i=1xi and where n is the sample size, x and y are individual sample points, and x and y are the sample means x = 1 n Pn i=1xi and y = 1 n Pn i=1yi. While the solid phase bulk Mn contents of the disturbed y = 1 n Pn i=1yi. y = 1 n Pn i=1yi. n While the solid-phase bulk Mn contents of the disturbed sediments were determined in the framework of this study, www.biogeosciences.net/17/1113/2020/ Biogeosciences, 17, 1113–1131, 2020 2.4 Geochemical model setup and reaction network Assuming steady-state compaction, the model applies an exponential function that is parameterized according to the available porosity data at each station (e.g., Berner, 1980; Supplement Fig. S1): A transient one-dimensional transport-reaction model (Eq. 3; e.g., Boudreau, 1997; Haeckel et al., 2001) was used (1) to assess the impact of small-scale disturbances on biogeo- chemical processes, geochemical conditions and element ﬂuxes in sediments of the CCZ and (2) to estimate the time required to establish a new steady-state geochemical sys- tem after a small-scale disturbance. We applied a transient transport-reaction model for the sites in the BGR-RA and IOM areas (Table 1). These sites were chosen due to distinc- tively different sedimentation rates and OPD (Table 2). We adapted the code of the steady-state transport-reaction model, which was originally presented by Volz et al. (2018), and used pore-water oxygen, NO− 3 , Mn2+ and NH+ 4 data as well as TOC contents of GC sediment cores from the same study as undisturbed reference data (Tables 1, 2). Thus, the model parameters and baseline input data used for the transient transport-reaction model are the same as presented in the study by Volz et al. (2018). The transient transport-reaction model consists of four aqueous species (O2, NO− 3 , Mn2+, NH+ 4 ), four solid species (TOC1, TOC2, TOC3, MnO2) and six reactions (R1–R6; Supplement Table S1), with ϕi = ϕ∞(ϕ0 −ϕ∞)exp(−βz), (6) (6) where ϕ∞is the porosity at the “inﬁnite depth”, at which point compaction is completed, ϕ0 is the porosity at the sediment water interface (z = 0), and β is the porosity- attenuation coefﬁcient. Organic matter was treated in three reactive fractions (3G- model) with ﬁrst-order kinetics. The rate expressions for the reactions (R1–R6) include inhibition terms, which are listed together with the rate constants (Table S3). Based on the Pearson correlation coefﬁcient rMn, we re- moved the upper 7 cm of sediments in the transport-reaction model for the IOM-BIE site and the upper 10 cm of sedi- ments in the transport-reaction model for the BGR-RA site. Due to the lack of data on the re-establishment of bioturba- tion, i.e., the recovery of the bioturbation “pump” after small- scale disturbance experiments, we tested the effect of dif- ferent bioturbation scenarios in the transport-reaction model. 3.1 Characterization of disturbed sites Bi = B0 exp zmix −zi zatt . 1 + exp zmix −zi zatt , (4) αi = α0 exp zmix −zi zatt . 1 + exp zmix −zi zatt , (5) Most of the small-scale disturbances investigated in the framework of this study were created with an EBS (Table 1; Fig. 2). Based on the visual impact inspection of the EBS disturbance tracks in the CCZ, the sediments were mostly pushed aside by the EBS and piled up next to the left and right side of the tracks (Fig. 2). In particular, the freshly cre- ated 1 d old EBS tracks in the BGR-RA and GSR areas indi- cate that the sediments were mostly scraped off and accumu- lated next to the freshly exposed sediment surfaces (Fig. 2). Small sediment lumps occur on top of the exposed sediment surfaces on the EBS tracks, which indicates that some sedi- where α0 and B0 are constants indicating the maximum bioir- rigation and bioturbation intensity at the sediment–water in- terface (SWI), the depth where the bioturbation and bioir- rigation intensity is halved is denoted by zmix, and the at- tenuation of the biogenically induced mixing with depth is controlled by zatt. Biogeosciences, 17, 1113–1131, 2020 Biogeosciences, 17, 1113–1131, 2020 2.4 Geochemical model setup and reaction network For the different post-disturbance bioturbation scenarios, we assumed that bioturbation is inhibited immediately after the disturbance with a linear increase to undisturbed reference bioturbation coefﬁcients (Volz et al., 2018). Based on the work by Miljutin et al. (2011) and Vanreusel et al. (2016), we assumed that bioturbation should be fully re-established after 100, 200 and 500 years. As the modeling results for the dif- ferent time spans were almost identical, we only present here the model that assumes that bioturbation is at pre-disturbance intensity 100 years after the impact (Volz et al., 2018; Ta- ble S2). We applied the transient transport-reaction model under the assumption that the sedimentation rates as well as the POC ﬂuxes to the seaﬂoor remain constant over time (Table 2). The model was coded in MATLAB, with a dis- cretization and reaction setup closely following the steady- state model (Volz et al., 2018). ∂(θiCi,j) ∂t = ∂Di,jθi ∂Ci,j/∂z ∂z −∂ωiθiCi,j ∂z + αiθi Ci,j −C0,j + θi X Ri,j, (3) (3) where z is sediment depth, and subscripts i and j represent depth and species dependence, respectively; aqueous or solid species concentration is denoted by C (Table S2), and D is, in the case of solutes, the effective diffusive mixing coefﬁcient, which has been corrected for tortuosity (Dm,i,j; Boudreau, 1997). In the case of solids, D represents the bioturbation coefﬁcient (Bi; Eq. 4), ϑ is the volume fraction represent- ing the porosity ϕ for the aqueous phase and 1 −ϕ for the solid phase, the velocity of either the aqueous (v) or the solid phase (w) is denoted by the symbol ω, αi is the bioirrigation coefﬁcient (0 for solid species; Eq. 5), and PRi,j is the sum of the reactions affecting the given species. The bioturbation and bioirrigation proﬁles, i.e., biologi- cally induced mixing of sediment and pore water, respec- tively, are represented by a modiﬁed logistic function: Biogeosciences, 17, 1113–1131, 2020 1119 3.2 Sediment porosity and solid-phase composition The sediment porosity shows little lateral variability and ranges between 0.65 and 0.8 throughout the upper 25 cm of the sediments at all investigated disturbed sites (Fig. 3). At the disturbed IOM-BIE site, sediment porosity is about 5 % higher in the upper 4 cm of the sediments than below. Bulk Mn contents in the upper 25 cm of the sediments at the dis- turbed sites are between 0.1 and 0.9 wt % (Fig. 3). Solid- phase Mn contents decrease with depth at all investigated sites. Total organic carbon (TOC) contents in the upper 25 cm of the sediments at the disturbed sites are within 0.2 wt % and 0.5 wt % (Fig. 3). The TOC contents slightly decrease with depth at all investigated sites. 3.4 Transport-reaction modeling The removal of the surface sediments in the transient transport-reaction model for the BGR-RA and IOM-BIE sites is associated with the loss of the reactive labile or- ganic matter (Figs. 5 and 6). About 10 kyr after the removal of the upper 10 cm of the sediments in the model for the BGR-RA site, oxygen penetrates about 10-fold deeper into the disturbed sediments than in undisturbed sediments (Ta- ble 2; Fig. 5; Volz et al., 2018). At the IOM-BIE site, oxy- gen reaches the maximum OPD at about 100 years after the removal of the upper 7 cm of the sediments. At this site, the oxygen front migrates only ∼1 m deeper than the cor- responding OPD in undisturbed sediments (Table 2; Fig. 5; Volz et al., 2018). As a consequence of deeper OPDs at both sites, the oxic–suboxic redox boundary is located at greater depth, with a signiﬁcant consumption of pore-water Mn2+ in the path of the oxygen front. The NH+ 4 concen- trations are also being diminished, reaching minima within 100–1000 years and 1–10 years after the disturbance experi- ments in the BGR-RA and IOM areas, respectively. The trend for the NO− 3 is more complicated, with lower concentrations during the downward migration of the OPD and augmented concentrations once oxygen concentrations reach their max- imum (Figs. 5 and 6). J. B. Volz et al.: Impact of small-scale disturbances Table 3. Calculated Pearson correlation coefﬁcients rMn and rTOC for the determination of the disturbance depth of various small-scale dis- turbances investigated in the framework of this study (compare Table 1). For both correlations, the highest positive linear Pearson coefﬁcient for solid-phase Mn contents (rMn ∼1) between the disturbed sites and the respective undisturbed reference sites was used. Table 3. Calculated Pearson correlation coefﬁcients rMn and rTOC for the determination of the disturbance depth of various small-scale dis- turbances investigated in the framework of this study (compare Table 1). For both correlations, the highest positive linear Pearson coefﬁcient for solid-phase Mn contents (rMn ∼1) between the disturbed sites and the respective undisturbed reference sites was used. Exploration Disturbance device and Disturbed Reference rMn Disturbance depth rTOC area type site SO239- site SO239- (cm) BGR-PA EBS 41 39 0.86 5 – BGR-RA EBS 64 66 0.82 15 −0.4 IOM IOM-BIE 101 87 0.97 7 0.77 GSR EBS 131 121 0.72 6 0.88 GSR Dredge 141 121 0.88 6 0.91 IFREMER Dredge 157 175 0.74 10 0.73 IFREMER EBS 161 175 0.93 7 0.74 ment slid off from the adjacent ﬂanks of the sediment accu- mulation after the disturbances (Fig. 2). However, the mostly smooth sediment surfaces of the EBS tracks suggest that sed- iment mixing during the EBS disturbance experiments may be mostly negligible (Fig. 2; Table 1). In the 8-month-old dredge track in the GSR area, small furrows occur at the dis- turbed sediment surface, most likely caused by the shape of the dredge (Fig. 2). in the disturbed sediments correlates well with the porosity in the respective undisturbed reference sediments (Fig. 4), sedi- ment compaction due to the weight of the disturbance device may be negligible during the small-scale disturbances inves- tigated in the framework of this study. www.biogeosciences.net/17/1113/2020/ 1120 J. B. Volz et al.: Impact of small-scale disturbances J. B. Volz et al.: Impact of small-scale disturbances J. B. Volz et al.: Impact of small scale disturbances 1121 Figure 3. Solid-phase Mn and TOC contents for all disturbed sites investigated in the framework of this study (years: yr; months: mth; days: d). Figure 3. Solid-phase Mn and TOC contents for all disturbed sites investigated in the framework of this study (years: yr; months: mth; days: d). Figure 3. Solid-phase Mn and TOC contents for all disturbed sites investigated in the framework of this study d). 4 Discussion ment, which was conducted in the DISCOL area in the Peru Basin in 2015 (Greinert, 2015). The freshly created EBS track in the DISCOL area was revisited 5 weeks after the disturbance experiment, where the surface sed- iment was mostly removed and deeper sediment layers were exposed without visible sediment mixing (Boetius, 2015; Paul et al., 2018). In a study on the geochemi- cal regeneration in disturbed sediments of the DISCOL area, Paul et al. (2018) have shown that the bulk Mn-rich top sediment layer, which has been observed in undis- turbed sediments, is removed in the 5-week old EBS disturbance track. Thus, an important prerequisite for this method is met, and the authors have proposed that the depth distribution of solid-phase Mn may be suit- able for the evaluation of the impact as well as for the 4.1 Depths of small-scale disturbance experiments Our work demonstrates that the depth distribution of solid- phase Mn provides a reliable tool for the determination of the disturbance depths in the sediments of the CCZ (Fig. 4; Table 3). The success of the correlation of solid-phase Mn contents between disturbed and undisturbed reference sedi- ments beneﬁts from several factors. 1. Sediment mixing during the small-scale disturbance ex- periments is negligible: the visual impact assessment of the investigated disturbance tracks in the CCZ suggests that sediment mixing during the small-scale disturbance experiments was insigniﬁcant (Fig. 2). This observation is in agreement with a recent EBS disturbance experi- Biogeosciences, 17, 1113–1131, 2020 Biogeosciences, 17, 1113–1131, 2020 www.biogeosciences.net/17/1113/2020/ www.biogeosciences.net/17/1113/2020/ 3.3 Pearson correlation coefﬁcient and disturbance depths The Pearson correlation coefﬁcient rMn for the correlation of solid-phase Mn contents between the disturbed sites and the respective reference sites ranges between 0.72 and 0.97 (Ta- ble 3). Based on rMn, 5–15 cm of sediment was removed by various disturbance experiments in the different contract ar- eas (Fig. 4). Applying these rMn-derived disturbance depths for the correlation of the TOC depth distributions between disturbed sites and respective adjacent reference sites gives Pearson correlation coefﬁcients rTOC within 0.73 and 0.91 (Table 3; Fig. 4), which may support the estimates for the dis- turbance depth based on rMn. At the BGR-RA site, the corre- lation of TOC contents between the disturbed site and the ref- erence site shows negative values. As the sediment porosity Naturally, the solute ﬂuxes across the SWI are strongly affected after the surface sediment removal (Fig. 7). The transient transport-reaction model suggests that the oxygen ﬂuxes into the sediments are lowered by a factor of 3–6 af- ter 10–100 years at the IOM-BIE and BGR-RA sites, respec- tively. This trend is mirrored by the decreased release of NH+ 4 and NO− 3 into the bottom water. Biogeosciences, 17, 1113–1131, 2020 www.biogeosciences.net/17/1113/2020/ Biogeosciences, 17, 1113–1131, 2020 1121 www.biogeosciences.net/17/1113/2020/ 1122 www.biogeosciences.net/17/1113/2020/ J. B. Volz et al.: Impact of small-scale disturbances J. B. Volz et al.: Impact of small-scale disturbances p Figure 4. Correlation of solid-phase Mn and TOC contents between the disturbed sites and the respective undisturbed reference sediments (grey shaded proﬁles) using the disturbance depths determined with the Pearson correlation coefﬁcient (compare Table 3). For the undisturbed reference sediments, solid-phase Mn contents are taken from Volz et al. (2020) and TOC contents are taken from Volz et al. (2018; years: yr; months: mth; days: d). Figure 4. Correlation of solid-phase Mn and TOC contents between the disturbed sites and the respective undisturbed reference sediments (grey shaded proﬁles) using the disturbance depths determined with the Pearson correlation coefﬁcient (compare Table 3). For the undisturbed reference sediments, solid-phase Mn contents are taken from Volz et al. (2020) and TOC contents are taken from Volz et al. (2018; years: yr; months: mth; days: d). monitoring of the recovery of small-scale disturbance experiments. monitoring of the recovery of small-scale disturbance experiments. tinctly decreasing contents below this level (Fig. 4; Volz et al., 2020). Similar bulk solid-phase Mn distribution patterns have been reported for other sites within the CCZ (e.g., Khripounoff et al., 2006; Mewes et al., 2014; Widmann et al., 2015). Volz et al. (2020) have suggested that the widely observed solid-phase Mn enrichments in CCZ surface sediments formed in association with a more compressed oxic zone, which may have prevailed as a result of lower bottom-water oxygen concentrations during the last glacial period than today. Strong indica- tion for lower glacial bottom-water oxygen concentra- tions throughout the eastern Paciﬁc Ocean have been provided by a number of independent proxies (e.g., An- 2. The solid-phase Mn maxima in the surface sediments appear to be a regional phenomenon across the CCZ area as has been observed throughout the different ex- ploration areas studied in the framework of this study (Volz et al., 2020): the investigated disturbed sediments as well as the undisturbed reference sediments in the CCZ show decreasing solid-phase Mn contents with depth in the upper 20–30 cm of the sediments (Figs. 3; 4; Volz et al., 2020). In the undisturbed reference sedi- ments, solid-phase Mn contents show maxima of up to 1 wt % in the upper 10 cm of the sediments, with dis- www.biogeosciences.net/17/1113/2020/ Biogeosciences, 17, 1113–1131, 2020 J. B. Volz et al.: Impact of small-scale disturbances J. B. Volz et al.: Impact of small-scale disturbances 1123 results of the transient transport-reaction model showing depth proﬁles of TOC, O2, NO− 3 , Mn2+ and NH+ 4 for (a) the EBS e German BGR-RA area and (b) the IOM-BIE disturbance in the eastern European IOM area. The model is adapted after ransport-reaction model presented in Volz et al. (2018) and shows the response of the geochemical system in the sediments nditions are disturbed by the removal of the upper 10 cm (BGR-RA; average disturbance depth of BGR-PA and BGR-RA; 7 cm (IOM; see Table 3) of the sediments while maintaining the same boundary conditions but with reduced bioturbation years after the disturbance. Figure 5. Model results of the transient transport-reaction model showing depth proﬁles of TOC, O2, NO− 3 , Mn2+ and NH+ 4 for (a) the EBS disturbance in the German BGR-RA area and (b) the IOM-BIE disturbance in the eastern European IOM area. monitoring of the recovery of small-scale disturbance experiments. The model is adapted after the steady-state transport-reaction model presented in Volz et al. (2018) and shows the response of the geochemical system in the sediments if steady-state conditions are disturbed by the removal of the upper 10 cm (BGR-RA; average disturbance depth of BGR-PA and BGR-RA; see Table 3) and 7 cm (IOM; see Table 3) of the sediments while maintaining the same boundary conditions but with reduced bioturbation over the ﬁrst 100 years after the disturbance. Biogeosciences, 17, 1113–1131, 2020 www.biogeosciences.net/17/1113/2020/ www.biogeosciences.net/17/1113/2020/ 1124 J. B. Volz et al.: Impact of small-scale disturbances The development of a re-suspended particle plume during the disturbance experiments highly depends on various factors, such as sediment properties, seaﬂoor topography, bottom- water currents and the disturbance device (e.g., Gillard et al., 2019). Although local and regional variations in these factors have been reported for the CCZ, they are not well constrained (e.g., Mewes et al., 2014; Aleynik et al., 2017; Volz et al., 2018; Gillard et al., 2019; Hauquier et al., 2019). As the dis- turbance tracks investigated in the framework of this study are relatively small, with a maximum width of 2.5 m (Fig. 2; Brockett and Richards, 1994; Brenke, 2005), re-suspended particles may (1) only partly deposit on the disturbance track and (2) mostly be transported laterally by currents and de- posit on top of undisturbed sediments in the proximity of the disturbance tracks (e.g., Fukushima, 1995; Aleynik et al., 2017; Gillard et al., 2019). This is in accordance with the close correlation of the sediment porosity between the dis- turbed and undisturbed reference sites, which indicates that the deposition of re-settling particles with higher porosity at the sediment surface in the disturbance tracks is insigniﬁcant at all sites, except for the IOM-BIE site (Fig. 4). The poros- ity data further show that sediment compaction, potentially caused by the weight of the disturbance device (Cuvelier et al., 2018; Hauquier et al., 2019), is insigniﬁcant at all dis- turbed sites. Figure 7. Pore-water ﬂuxes of oxygen (O2), nitrate (NO− 3 ) and am- monia (NH+ 4 ) at the sediment–water interface obtained by the ap- plication of the transient transport-reaction model. Oxygen ﬂuxes into the sediment and ﬂuxes of nitrate and ammonia towards the sediment surface are shown as a function of time after the EBS and IOM-BIE disturbances in the German BGR-RA area (blue) and in the eastern European IOM area (black), respectively. derson et al., 2019, and references therein). As a conse- quence of the condensed oxic zone, upwardly diffusing pore-water Mn2+ may have precipitated as authigenic Mn(IV) at a shallow oxic–suboxic redox boundary in the upper few centimeters of the sediments. After the last glacial period, the authigenic Mn(IV) peak was con- tinuously mixed into subsequently deposited sediments by bioturbation, causing the observed broad solid-phase Mn(IV) enrichment in the surface sediments (Fig. 4; Volz et al., 2020). 3. J. B. Volz et al.: Impact of small-scale disturbances J. B. Volz et al.: Impact of small-scale disturbances J. B. Volz et al.: Impact of small-scale disturbances Figure 7. Pore-water ﬂuxes of oxygen (O2), nitrate (NO− 3 ) and am- monia (NH+ 4 ) at the sediment–water interface obtained by the ap- plication of the transient transport-reaction model. Oxygen ﬂuxes into the sediment and ﬂuxes of nitrate and ammonia towards the sediment surface are shown as a function of time after the EBS and IOM-BIE disturbances in the German BGR-RA area (blue) and in the eastern European IOM area (black), respectively. other estimates for small-scale disturbances by similar gear in the CCZ and in the DISCOL area, which suggest that the upper 4–20 cm of the sediments was removed (e.g., Thiel and Forschungsverband Tiefsee-Umweltschutz, 2001; Oebius et al., 2001; König et al., 2001; Grupe et al., 2001; Radziejew- ska, 2002; Khripounoff et al., 2006; Paul et al., 2018). How- ever, as the disturbed sites investigated in this study and the respective undisturbed reference sites are located up to 5 km apart from each other, the correlation of solid-phase Mn may be inﬂuenced by some spatial heterogeneities in solid-phase Mn contents (Table 1; Mewes et al., 2014). Furthermore, it should be noted that for the correlation of solid-phase Mn contents between the disturbed and undisturbed reference sites, we did not consider that (1) particles may have re- settled on the freshly exposed sediment surfaces from re- suspended particle plumes (e.g., Jankowski and Zielke, 2001; Thiel and Forschungsverband Tiefsee-Umweltschutz, 2001; Radziejewska, 2002; Gillard et al., 2019), (2) sediment slid off from adjacent ﬂanks of the sediment accumulation after the disturbances (Fig. 2), and (3) sediments were deposited after the small-scale disturbances at sedimentation rates be- tween 0.2 and 1.2 cm kyr−1 (Table 2; Volz et al., 2018). How- ever, only in the case of the IOM-BIE disturbance, the vi- sual impact assessment suggested that the disturbance sur- face was concealed, here by re-settling sediments (Fig. 2). The development of a re-suspended particle plume during the disturbance experiments highly depends on various factors, such as sediment properties, seaﬂoor topography, bottom- water currents and the disturbance device (e.g., Gillard et al., 2019). Although local and regional variations in these factors have been reported for the CCZ, they are not well constrained (e.g., Mewes et al., 2014; Aleynik et al., 2017; Volz et al., 2018; Gillard et al., 2019; Hauquier et al., 2019). J. B. Volz et al.: Impact of small-scale disturbances J. B. Volz et al.: Impact of small-scale disturbances d model results of the transient transport-reaction model (Fig. 5) showing depth proﬁles of TOC, O2, NO− 3 , Mn2+ and r 1 m of the sediments with the ﬁt of the simulated proﬁles with the analytical data for undisturbed sediments at current emical conditions and for the new steady-state geochemical system after the disturbance (dark blue proﬁles) for (a) EBS German BGR-RA area and (b) the IOM-BIE disturbance in the eastern European IOM area. Figure 6. Detailed model results of the transient transport-reaction model (Fig. 5) showing depth proﬁles of TOC, O2, NO− 3 , Mn2+ and NH+ 4 for the upper 1 m of the sediments with the ﬁt of the simulated proﬁles with the analytical data for undisturbed sediments at current steady-state geochemical conditions and for the new steady-state geochemical system after the disturbance (dark blue proﬁles) for (a) EBS disturbance in the German BGR-RA area and (b) the IOM-BIE disturbance in the eastern European IOM area. Biogeosciences, 17, 1113–1131, 2020 www.biogeosciences.net/17/1113/2020/ 1125 J. B. Volz et al.: Impact of small-scale disturbances As the dis- turbance tracks investigated in the framework of this study are relatively small, with a maximum width of 2.5 m (Fig. 2; Brockett and Richards, 1994; Brenke, 2005), re-suspended particles may (1) only partly deposit on the disturbance track and (2) mostly be transported laterally by currents and de- posit on top of undisturbed sediments in the proximity of the disturbance tracks (e.g., Fukushima, 1995; Aleynik et al., 2017; Gillard et al., 2019). This is in accordance with the close correlation of the sediment porosity between the dis- turbed and undisturbed reference sites, which indicates that the deposition of re-settling particles with higher porosity at the sediment surface in the disturbance tracks is insigniﬁcant at all sites, except for the IOM-BIE site (Fig. 4). The poros- ity data further show that sediment compaction, potentially caused by the weight of the disturbance device (Cuvelier et al., 2018; Hauquier et al., 2019), is insigniﬁcant at all dis- other estimates for small-scale disturbances by similar gear in the CCZ and in the DISCOL area, which suggest that the upper 4–20 cm of the sediments was removed (e.g., Thiel and Forschungsverband Tiefsee-Umweltschutz, 2001; Oebius et al., 2001; König et al., 2001; Grupe et al., 2001; Radziejew- ska, 2002; Khripounoff et al., 2006; Paul et al., 2018). How- ever, as the disturbed sites investigated in this study and the respective undisturbed reference sites are located up to 5 km apart from each other, the correlation of solid-phase Mn may be inﬂuenced by some spatial heterogeneities in solid-phase Mn contents (Table 1; Mewes et al., 2014). Furthermore, it should be noted that for the correlation of solid-phase Mn contents between the disturbed and undisturbed reference sites, we did not consider that (1) particles may have re- settled on the freshly exposed sediment surfaces from re- suspended particle plumes (e.g., Jankowski and Zielke, 2001; Thiel and Forschungsverband Tiefsee-Umweltschutz, 2001; Radziejewska, 2002; Gillard et al., 2019), (2) sediment slid off from adjacent ﬂanks of the sediment accumulation after the disturbances (Fig. 2), and (3) sediments were deposited after the small-scale disturbances at sedimentation rates be- tween 0.2 and 1.2 cm kyr−1 (Table 2; Volz et al., 2018). How- ever, only in the case of the IOM-BIE disturbance, the vi- sual impact assessment suggested that the disturbance sur- face was concealed, here by re-settling sediments (Fig. 2). 4.2 Impact of small-scale disturbances on the geochemical system The geochemical conditions found at the study sites in the CCZ are the result of a balanced interplay of key factors, such as the input of fresh, labile TOC, sedimentation rate and bioturbation intensity (e.g., Froelich et al., 1979; Berner, 1981; Zonneveld et al., 2010; Mogollón et al., 2016; Volz et al., 2018). Together they characterize the upper reactive layer, which in turn plays a crucial role in the location of the OPD in the sediments of the CCZ (e.g., Mewes et al., 2014; Mogollón et al., 2016; Volz et al., 2018). Oxygen is con- sumed via aerobic respiration during the degradation of or- ganic matter, while bioturbation transports fresh, labile TOC into deeper sediments (e.g., Haeckel et al., 2001; König et al., 2001). The presence of labile TOC throughout the biotur- bated zone signiﬁcantly enhances the consumption of oxy- gen with depth, where oxygen is not as easily replenished by seawater oxygen. Thus, the availability of labile TOC in the bioturbated layer controls the amount of oxygen that passes through the reactive layer into deeper sediments (e.g., König et al., 2001). Below the highly reactive layer, refrac- tory organic matter degradation and secondary redox reac- tions – such as oxidation of Mn2+ – control the consump- tion of oxygen (Table S1; Mogollón et al., 2016; Volz et al., 2018). The oxygen proﬁle, more precisely the position of the OPD, in turn, strongly inﬂuences the distribution of other so- lutes. Below the OPD, denitriﬁcation and Mn(IV) reduction commence, albeit at much lower rates, consuming pore-water NO− 3 and releasing Mn2+ (Mogollón et al., 2016; Volz et al., 2018). The study sites in the CCZ provide an excellent ex- ample for how slight differences in key environmental factors can profoundly change the overall solute proﬁles, with OPDs ranging between 0.5 m (BGR-RA) and > 7.4 m (GSR), as outlined by Volz et al. (2018). Figure 8. Conceptual model for time-dependent pore-water ﬂuxes of oxygen (O2), nitrate (NO− 3 ) and ammonia (NH+ 4 ) at the sediment–water interface after the removal of the upper 7–10 cm of the sediments. The re-establishment of bioturbation, the maxi- mum oxygen penetration depth (OPD) and the re-establishment of the surface sediment layer dominated by the reactive labile organic matter fraction are indicated as a function of time after the sediment removal (years: yr). J. B. Volz et al.: Impact of small-scale disturbances J. B. Volz et al.: Impact of small-scale disturbances Figure 8. Conceptual model for time-dependent pore-water ﬂuxes of oxygen (O2), nitrate (NO− 3 ) and ammonia (NH+ 4 ) at the sediment–water interface after the removal of the upper 7–10 cm of the sediments. The re-establishment of bioturbation, the maxi- mum oxygen penetration depth (OPD) and the re-establishment of the surface sediment layer dominated by the reactive labile organic matter fraction are indicated as a function of time after the sediment removal (years: yr). 4.2 Impact of small-scale disturbances on the geochemical system main driver shaping solute proﬁles towards a new geo- chemical steady-state system in the absence of the re- active layer (Figs. 5 and 6). This entails the downward migration of the OPD, as oxygen is no longer effectively consumed in the upper sediment layer. The presence of oxygen outcompetes denitriﬁcation and Mn(IV) reduc- tion and induces NH+ 4 and Mn2+ oxidation instead, thus minimizing pore-water NH+ 4 and Mn2+ concentrations (Figs. 5 and 6). At the same time, NO− 3 , produced dur- ing nitriﬁcation in the presence of oxygen (e.g., Froelich et al., 1979; Berner, 1981; Haeckel et al., 2001; Mogol- lón et al., 2016; Volz et al., 2018), is accordingly re- duced during denitriﬁcation, and NO− 3 concentrations are lowered during this ﬁrst phase. The removal of the upper 5–15 cm of the sediment re- sults, on one hand, in an almost complete loss of the labile TOC fraction (Fig. 4), as this fraction is restricted to the upper 20 cm of the sediment in the CCZ (e.g., Müller and Mangini, 1980; Emerson, 1985; Müller et al., 1988; Mewes et al., 2014; Mogollón et al., 2016; Volz et al., 2018). On the other hand, studies on faunal diversity and density in small- scale disturbances in the sediments of the CCZ and in the DISCOL area show that most of the biota is lost immediately after the disturbance experiment (Borowski and Thiel, 1998; 2001; Bluhm et al., 2001; Thiel et al., 2001; Vanreusel et al., 2016; Jones et al., 2017; Gollner et al., 2017). Thus, a dras- tic decline or standstill of bioturbation can be expected in the surface sediments. 2. The second phase is characterized by the increasing in- ﬂuence of reactive ﬂuxes across the seaﬂoor. It takes ap- proximately 1000 years before any signiﬁcant build-up of an upper labile TOC layer is re-established (Fig. 6), at which point solute proﬁles slowly shift towards their pre-disturbance shape (Fig. 7). Interestingly, during the transition time, when oxygen is still present at depth but aerobic respiration in the upper sediments has already began to pick up, NO− 3 concentrations are strongly ele- vated in the BGR sediments (Figs. 5 and 6). This is due to the fact that NO− 3 is not consumed during denitriﬁ- cation or the Mn–anammox reaction in the presence of oxygen (Mogollón et al., 2016; Volz et al., 2018). 2. 1. Since the labile TOC fraction and bioturbating fauna are mostly removed, downward diffusion of oxygen is the J. B. Volz et al.: Impact of small-scale disturbances Lastly, the OPD at all sites is located at sediment depths greater than 0.5 m, and, thus, diagenetic precipitation of Mn(IV) in the surface sediments (e.g., Gingele and Kas- ten, 1994) since the last glacial period can be ruled out (Table 2; Mewes et al., 2014; Volz et al., 2020). Based on the depth distribution of solid-phase Mn, our work suggests that between 5 and 15 cm of the surface sedi- ments was removed and pushed aside by the different small- scale disturbance experiments in the CCZ (Table 3; Fig. 4). This range of disturbance depths is in good agreement with Biogeosciences, 17, 1113–1131, 2020 Biogeosciences, 17, 1113–1131, 2020 www.biogeosciences.net/17/1113/2020/ 1126 J. B. Volz et al.: Impact of small-scale disturbances 5 Conclusion Through correlation of solid-phase Mn contents of disturbed and undisturbed reference sedi- ments, we (1) propose that the depth distribution of solid- phase Mn in the sediments of the CCZ provides a reliable tool for the estimation of the disturbance depth and (2) show that 5–15 cm of the sediments was removed during the small- scale disturbance experiments investigated in this study. As the small-scale disturbances are associated with the removal of the surface sediments characterized by reactive labile or- ganic matter, the disturbance depth ultimately determines the impact on the geochemical system in the sediments. The ap- plication of a transient transport-reaction model reveals that the removal of the upper 7–10 cm of the surface sediments is associated with a meter-scale downward extension of the oxic zone and the shutdown of denitriﬁcation and Mn(IV) re- duction. As a consequence of lower respiration rates after the disturbance experiments, the geochemical system in the sed- iments is controlled by downward oxygen diffusion. While the re-establishment of bioturbation within centuries after the disturbance is important for the development of steady-state geochemical conditions in the disturbed sediments, the rate at which geochemical steady-state conditions are reached ul- timately depends on the delivery rate of organic matter to the seaﬂoor. Assuming the accumulation of labile organic matter to proceed at current Holocene sedimentation rates in the dis- turbed sediments, biogeochemical reactions resume in the re- active surface sediment layer, and, thus, the new steady-state geochemical system in the disturbed sediments in the CCZ is reached on a millennial timescale after the disturbance of the surface sediments. It should be noted that while bioturbation has a pivotal in- ﬂuence on the undisturbed steady-state proﬁle, it only plays a secondary role in re-establishing the steady-state geochem- ical system at the disturbed sites in the CCZ. Studies sug- gest that faunal abundances fully recover within centuries af- ter the disturbance even though the benthic community may be different than prior to the disturbance (e.g., Miljutin et al., 2011; Vanreusel et al., 2016). Due to the extremely slow build-up of the reactive layer with labile TOC, the bioturba- tion “pump” is active again before any signiﬁcant amount of labile TOC is present about 1–100 kyr after the distur- bance. Thus, full recovery is mainly controlled by the re- establishment of the upper reactive layer, i.e., the delivery rate of labile TOC to the seaﬂoor. 5 Conclusion considered. The release of nutrients complements the close link between sediment geochemistry and the food web struc- ture (e.g., Smith et al., 1979; Dunlop et al., 2016; Stratmann et al., 2018) and further emphasizes their interdependencies. Figure 7 depicts ﬂuxes of oxygen, NO− 3 and NH+ 4 across the seaﬂoor. As expected, with the reactive layer being mostly absent, ﬂuxes across the seaﬂoor are severely reduced, which particularly affects the oxygen uptake of the sediments as well as the release of NO− 3 and NH+ 4 into the bottom wa- ter. At about 100 to 1000 years after the disturbance, concur- rent with the build-up of an upper sediment layer containing signiﬁcant amounts of labile organic matter, ﬂuxes begin to increase again, albeit much slower than the rate of the de- crease in ﬂuxes subsequently after the disturbances (Fig. 7; note the logarithmic scale). considered. The release of nutrients complements the close link between sediment geochemistry and the food web struc- ture (e.g., Smith et al., 1979; Dunlop et al., 2016; Stratmann et al., 2018) and further emphasizes their interdependencies. Figure 7 depicts ﬂuxes of oxygen, NO− 3 and NH+ 4 across the seaﬂoor. As expected, with the reactive layer being mostly absent, ﬂuxes across the seaﬂoor are severely reduced, which particularly affects the oxygen uptake of the sediments as well as the release of NO− 3 and NH+ 4 into the bottom wa- ter. At about 100 to 1000 years after the disturbance, concur- rent with the build-up of an upper sediment layer containing signiﬁcant amounts of labile organic matter, ﬂuxes begin to increase again, albeit much slower than the rate of the de- crease in ﬂuxes subsequently after the disturbances (Fig. 7; note the logarithmic scale). We studied surface sediments from seven small-scale dis- turbance experiments for the simulation of deep-sea mining, which were performed between 1 d and 37 years prior to our sampling in the NE Paciﬁc Ocean. These small-scale distur- bance tracks were created using various disturbance devices in different European contract areas for the exploration of polymetallic nodules within the eastern part of the Clarion– Clipperton Zone (CCZ). 4.2 Impact of small-scale disturbances on the geochemical system The second phase is characterized by the increasing in- ﬂuence of reactive ﬂuxes across the seaﬂoor. It takes ap- proximately 1000 years before any signiﬁcant build-up of an upper labile TOC layer is re-established (Fig. 6), at which point solute proﬁles slowly shift towards their pre-disturbance shape (Fig. 7). Interestingly, during the transition time, when oxygen is still present at depth but aerobic respiration in the upper sediments has already began to pick up, NO− 3 concentrations are strongly ele- vated in the BGR sediments (Figs. 5 and 6). This is due to the fact that NO− 3 is not consumed during denitriﬁ- cation or the Mn–anammox reaction in the presence of oxygen (Mogollón et al., 2016; Volz et al., 2018). Based on the results of the transient transport-reaction model, geochemical recovery after small-scale sediment dis- turbances can be divided into two main phases (Fig. 8). With the importance of bioturbation and the mining- related removal of associated fauna in mind, solute and in particular nutrient ﬂuxes across the seaﬂoor should also be 1. 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Bluhm H.: Re-establishment of an abyssal megabenthic community after experimental physical disturbance of the seaﬂoor, Deep-Sea Res. Pt. II, 48, 3841–3868, 2001. Special issue statement. This article is part of the special issue “Assessing environmental impacts of deep-sea mining – revisiting decade-old benthic disturbances in Paciﬁc nodule areas”. It is not associated with a conference. Boetius, A.: RV SONNE Fahrtbericht/Cruise Report SO242-2: JPI OCEANS Ecological Aspects of Deep-Sea Mining, DISCOL Revisited, Guayaquil-Guayaquil (Equador), 28 August–1 Octo- ber 2015, GEOMAR Helmholtz-Zentrum für Ozeanforschung, Kiel, 2015. Boetius, A. and Haeckel, M.: Mind the seaﬂoor, Science, 359, 34– 36, https://doi.org/10.1126/science.aap7301, 2018. Acknowledgements. We thank captain Lutz Mallon, the crew and the scientiﬁc party of RV SONNE cruise SO239 for the technical and scientiﬁc support. Thanks to Jennifer Ciomber, Benjamin Löf- ﬂer and Vincent Ozegowski for their participation in sampling and analysis onboard. For analytical support in the home laboratory and during data evaluation we are grateful to Ingrid Stimac, Olaf Kreft, Dennis Köhler and Ingrid Dohrmann (all at AWI). J. B. Volz et al.: Impact of small-scale disturbances with ongoing natural environmental changes (e.g., bottom- water warming, acidiﬁcation, changes in the POC ﬂux to the seaﬂoor), it is difﬁcult to assess whether the surface sediment removal may trigger a tipping point for deep-sea ecosys- tems. This study also provides valuable data for further in- vestigations on the environmental impact of deep-sea mining, such as during the launched JPI Oceans follow-up project MiningImpact 2. Financial support. This research was funded by the Bundesmin- isterium für Bildung und Forschung (grant no. 03F0707A+G) as part of the JPI Oceans pilot action “Ecological aspects of deep-sea mining (MininigImpact). Further ﬁnancial support was provided by the Alfred Wegener Institute Helmholtz Centre for Polar and Marine Research. The article processing charges for this open-access The article processing charges for this open-access publication were covered by a Research publication were covered by a Research Centre of the Helmholtz Association. Centre of the Helmholtz Association. Data availability. The data are available via the data man- agement portal OSIS-Kiel and the WDC database PAN- GAEA, including the solid-phase bulk sediment Mn and TOC contents (https://doi.org/10.1594/PANGAEA.904560; Volz et al., 2019a) as well as the porosity data (https://doi.org/10.1594/PANGAEA.904578; Volz et al., 2019b). Data availability. The data are available via the data man- agement portal OSIS-Kiel and the WDC database PAN- GAEA, including the solid-phase bulk sediment Mn and TOC contents (https://doi.org/10.1594/PANGAEA.904560; Volz et al., 2019a) as well as the porosity data (https://doi.org/10.1594/PANGAEA.904578; Volz et al., 2019b). Review statement. This paper was edited by Jack Middelburg and reviewed by two anonymous referees. Review statement. This paper was edited by Jack Middelburg and reviewed by two anonymous referees. 5 Conclusion The transport-reaction model reveals that under current de- positional conditions, the new steady-state geochemical sys- tem is established after 1–10 kyr at the IOM-BIE site, while the re-establishment of steady-state geochemical conditions at the BGR-RA site takes 10–100 kyr (Figs. 5 and 6). Shorter recovery times at the IOM site compared to the BGR-RA site are related to higher sedimentation rates (1.15 instead of 0.65 cm kyr−1) and shallower impact on the sediment (7 cm instead of 10 cm sediment removal). Accordingly, the maxi- mum OPD is reached after 100 years and 10 kyr at the IOM and BGR-RA site, respectively (Figs. 5 and 6), while the reactive layer is clearly established sooner at the IOM site compared to the BGR-RA site (Fig. 7). Thus, the disturbance depth clearly has a strong inﬂuence on the recovery pro- cess of the geochemical system of the sediments, highlight- ing the importance of low-impact mining equipment. Con- sidering that in the CCZ areas of about 8500 km2 could be commercially mined in 20 years per individual mining op- eration (Madureira et al., 2016), this impact assessment of small-scale disturbance experiments may only represent a ﬁrst approach for the prediction of the environmental impact of large-scale deep-sea mining activities. Our study represents the ﬁrst study on the impact of small- scale disturbance experiments on the sedimentary geochem- ical system in the prospective areas for polymetallic nodule mining in the CCZ. Our ﬁndings on the evaluation of the disturbance depths using solid-phase Mn contents as well as the quantiﬁcation of the development of a new geochemi- cal steady-state system in the sediments advance our knowl- edge about the potential long-term consequences of deep-sea mining activities. We propose that mining techniques poten- tially used for the potential commercial exploitation of nod- ules in the CCZ may remove less than 10 cm of the sur- face sediments in order to minimize the impact on the geo- chemical system in the sediments. The depth distribution of solid-phase Mn may be used for environmental monitoring purposes during future mining activities in the CCZ. 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https://openalex.org/W3081615416	https://www.mdpi.com/2304-8158/9/9/1208/pdf?version=1598943695	English	null	Impact of Bottle Aging on the Composition and Sensory Properties of Flavored Chardonnay and Shiraz Wines	Foods	2,020	cc-by	9,381	Received: 13 August 2020; Accepted: 27 August 2020; Published: 1 September 2020 Abstract: Natural ﬂavorings could potentially be used to enhance the intensity of wine aroma and ﬂavor; albeit since ﬂavor additives are not legally permitted winemaking aids, ﬂavored wines would need to be labeled as wine products. In this study, changes in the composition and sensory proﬁles of ﬂavored Chardonnay (n = 2) and Shiraz (n = 2) wines were compared at bottling, and then again after 12 months of bottle aging. Flavorings and ﬂavored wines were also analyzed by gas chromatography-mass spectrometry (GC-MS) to determine the key constituents responsible for changes to aroma and ﬂavor proﬁles. However, many of the volatile compounds identiﬁed in ﬂavor additives were not detected at appreciably higher concentrations in ﬂavored wines, which was attributed to the very small quantities of ﬂavorings that were added to base wines. The sensory proﬁles of control and ﬂavored wines were determined by descriptive analysis, and the addition of ﬂavorings to base wines signiﬁcantly inﬂuenced the perception of some sensory attributes. Flavored Chardonnay wines exhibited enhanced fruit aromas and ﬂavors, while fruit and developed attributes were enhanced in ﬂavored Shiraz wines. Diﬀerences in sensory proﬁles were less apparent in Chardonnay wines following bottle aging, but depending on the ﬂavorings added, ﬂavored Shiraz wines could still be discriminated from their corresponding control wines after bottle aging. Results from this study demonstrate the potential for ﬂavor additives to be used to enhance desirable attributes and/or mitigate wine sensory deﬁciencies. Keywords: bottle aging; descriptive analysis; ﬂavor additives; GC-MS; wine; wine product Impact of Bottle Aging on the Composition and Sensory Properties of Flavored Chardonnay and Shiraz Wines Yaelle Saltman, Julie A. Culbert, Trent E. Johnson , Renata Ristic , Kerry L. Wilkinson * an Susan E. P. Bastian School of Agriculture, Food and Wine, Waite Campus, The University of Adelaide, PMB 1, Glen Osmond, SA 5064, Australia; yaelle.saltman@adelaide.edu.au (Y.S.); julie.culbert@adelaide.edu.au (J.A.C.); trent.johnson@adelaide.edu.au (T.E.J.); renata.ristic@adelaide.edu.au (R.R.); sue.bastian@adelaide.edu.au (S.E.P.B.) * Correspondence: kerry.wilkinson@adelaide.edu.au; Tel.: +61-8-8313-7360 foods foods foods www.mdpi.com/journal/foods 1. Introduction Aroma and ﬂavor intensity are important indicators of wine quality, and can be attributed to the presence of volatile compounds derived from grapes, primary and secondary fermentation, maturation and/or aging [1,2]. Whereas, ﬂavor additives can be used to moderate aroma and ﬂavor intensity in food and beverage production [3], regulations governing the use of additives and processing aids in most wine-producing countries prevent their use during winemaking. In Australia, the addition of ﬂavorings to wine renders it a ‘wine product’, according to the Australian and New Zealand Food Standard Code [4]. However, research suggests a signiﬁcant proportion of consumers (up to 50%) do not understand the meaning of this term, and they may, therefore, be misled by ‘wine product’ labeling [5]. Whilst it is unlikely that ﬂavor additives would ever be used in the production of premium quality wines, they might oﬀer winemakers the ability to moderate the sensory properties of commodity wines or address wines with sensory deﬁciencies. For example, ﬂavorings could be Foods 2020, 9, 1208; doi:10.3390/foods9091208 www.mdpi.com/journal/foods www.mdpi.com/journal/foods Foods 2020, 9, 1208 2 of 14 used to: (i) Enhance the aroma and ﬂavor intensity of wines aﬀected by adverse weather conditions; (ii) mask undesirable ‘green’ (unripe) characters; or (iii) introduce oak-related characters to wine, without the investment in time or capital associated with traditional barrel maturation. Recent studies have, therefore, explored consumer attitudes towards the addition of ﬂavorings to wine [6] and the potential for ﬂavor additives to inﬂuence the sensory proﬁles and consumer acceptance of wine [7]. An online survey (with 1031 participants) concerning attitudes towards the use of additives in food and wine production found Australian wine consumers were generally more accepting of the addition of natural ﬂavorings to wine, than many of the (legally permitted) additives currently used in winemaking (e.g., tartaric acid and sulfur dioxide) [6]. A subsequent study evaluated consumer liking of control (unﬂavored) and ﬂavored Chardonnay and Shiraz wines, and identiﬁed consumer segments who accepted, and in some cases preferred, the ﬂavored wines [7]. The ﬂavorings added to wines were chosen according to ﬁndings from previous studies on wine consumers’ ﬂavor preferences [8,9], with ﬂavor combinations optimized via bench-top trials, then reﬁned using feedback obtained from consumers via focus panels [7]. The composition of wine evolves with time, as a consequence of chemical transformations that occur after bottling [10]. These compositional changes can be either desirable or undesirable. 1. Introduction For example, hydrolysis of esters during aging of wine can result in the loss of varietal expression, i.e., a decrease in the intensity of fruity, ﬂoral characters [11,12], while the formation of phenylacetaldehyde and methional, due to oxidative eﬀects can give rise to over-ripe fruit or cooked vegetable notes [13]. In other cases, pleasant toasty, biscuit, honey, nutty, and/or toﬀee characters may arise with the aging of white wine [14–16]. Similarly, temporal changes in the volatile compounds present in red wines can result in primary fruit characters giving way to caramel, savory, truﬄe, leather, chocolate, cedar, and/or coﬀee developed notes [17,18]. Aging of red wine is also associated with modiﬁcations in the wine color and mouthfeel properties, due to reactions of polyphenolic compounds [19]. Whereas, some wine styles beneﬁt from extended aging in the bottle, many inexpensive (commodity) wines are intended to be consumed soon after bottling, when varietal expression is at its peak. The stability of ﬂavor additives in the acidic wine medium has not yet been investigated, but the sensory perception of ﬂavorings would be expected to diminish with time. As such, this study sought to determine the impact of bottle aging on the composition and sensory proﬁles of ﬂavored wines, using a combination of gas chromatography-mass spectrometry (GC-MS) and descriptive sensory analysis. 2.1. Flavorings and Reagents Flavor additives were sourced from the Product Makers Pty. Ltd. (Melbourne, VIC, Australia; chocolate1039, cinnamon1525, custard1989, orange1883, raspberry228 and vanilla1729) and FlavorSense Corporation (San Rafael, CA, USA; apricotWW3, berry8819, butter10-1206, honey, oak and passion fruit77116). Analytical grade reagents, solvents, and standards used in GC-MS analysis were purchased from Sigma Aldrich (Castle Hill, NSW, Australia), CDN Isotopes (Pointe-Claire, QC, Canada) and Chem-Supply (Gillman, SA, Australia). 2.4.2. GC-MS Instrumentation Samples were analyzed by gas chromatography-mass spectrometry (GC-MS), with a 7890A GC coupled to a 5975C inert XL mass selective detector (Agilent Technologies, Santa Clara, CA, USA) and equipped with a Gerstel MPS2 Multipurpose autosampler (Gerstel, Mülheim an der Ruhr, Germany). Instrument control and data analysis were performed with Agilent ChemStation software (E.02.02.1431, Agilent Technologies, Santa Clara, CA, USA) and Gerstel MASter software (version 1.3, Lasersan Australiasia Pty. Ltd., Robina, QLD, Australia). Samples were incubated with agitation for 10 min at 50 ◦C, prior to headspace solid-phase micro-extraction (HS-SPME) for 30 min at 50 ◦C (with agitation) using a Supelco 50/30 µm divinylbenzene/carboxen/polydimethylsiloxane 1 cm SPME ﬁber. The SPME ﬁber was desorbed in the GC inlet containing an ultra-inert glass SPME liner (straight taper with 0.75 mm i.d.), operating in splitless mode at a temperature of 240 ◦C. The SPME ﬁber remained in the inlet for 10 min, but with a purge, ﬂow to split vent of 20 mL/min after 3 min. Separation of volatile compounds was achieved using an Agilent J&W DB-WAX capillary column (60 m × 0.25 mm i.d. × 0.25 µm) with ultrapure helium (Coregas, Cavan, SA, Australia) as the carrier gas at a constant ﬂow rate of 1.5 mL/min. The oven program was as follows: 40 ◦C (held for 5 min), increased to 210 ◦C at 2 ◦C/min (held for 5 min), and then to 240 ◦C at 5 ◦C/min (held for 10 min), giving a total runtime of 111 min. The MS was operated using positive ion electron impact at 70 eV in either full scan mode (m/z 35–350) or select ion monitoring (SIM), with MS source and quad temperatures of 230 ◦C and 150 ◦C, respectively. The MS transfer line was held at 240 ◦C. 2.2. Preparation and Aging of Wines The preparation of ﬂavored wines has previously been reported in full [7]. Brieﬂy, four inexpensive commercial wines (retailing at ≤AUD$10 per 750 mL bottle), two 2011 Chardonnay wines from the Riverland and South Eastern Australia (hereafter CH1 and CH2) and two 2011 Shiraz wines, both from South Eastern Australia (hereafter SH1 and SH2), were sourced from Australian wineries and spiked with diﬀerent combinations of ﬂavorings (Table S1) to generate two ﬂavored versions of each wine (on a 20 L scale). Flavor combinations were selected and optimized based on consumer surveys, bench-top trials, and focus panels described previously [6,7]. Control and ﬂavored wines were then bottled Foods 2020, 9, 1208 3 of 14 (in 375 mL dark green colored glass bottles) under metal screwcap closures, with minimal ullage (<1–2 mL) and carbon dioxide blanketing, and cellared (in an upright position), in darkness at 15 ◦C. 2.4.1. Sample Preparation For analysis of ﬂavorings, ﬂavor additives (2–3 drops, approx. 0.1 g) were added to 20 mL screw-cap autosampler vials (Sigma Aldrich), together with Milli-Q water (5 mL) and sodium chloride (2.0 g). Vials were sealed and thoroughly mixed with a vortex mixer prior to GC-MS analysis. For analysis of ﬂavored wines, wine (0.5 mL) was placed in a 20 mL screw-cap autosampler vial containing sodium chloride (2.0 g) and Milli-Q water (4.5 mL) and 2-octanol (10 µL, 50 mg/L in ethanol) added as an internal standard. Vials were sealed and thoroughly mixed using a vortex mixer prior to GC-MS analysis. Flavorings were analyzed ﬁve weeks after bottling (i.e., at t = 0), whereas wines were analyzed ﬁve weeks after bottling, then again following 12 months of bottle aging (i.e., at t = 0 and t = 1). 2.4. Volatile Composition of Flavorings and Wines 2.4. Volatile Composition of Flavorings and Wines 2.3. Basic Wine Composition The pH, titratable acidity (TA, as g/L of tartaric acid) alcohol (% v/v), residual sugar (as g/L of glucose and fructose) and volatile acidity (VA, as g/L of acetic acid) of wines were measured (in duplicate) according to published methodology [20]. Results from chemical analyses performed ﬁve weeks after bottling (hereafter ‘t = 0’) were reported previously [7]; in the current study, analyses were repeated following 12 months bottle aging of wines (hereafter ‘t = 1’). 2.5. Sensory Analysis of Wines The sensory proﬁles of control and ﬂavored wines were determined by descriptive analysis (DA). The sensory analyses performed ﬁve weeks after bottling (i.e., at ‘t = 0’) have previously been described in full [7]; in the current study, analyses were repeated following 12 months bottle aging of wines (i.e., at ‘t = 1’). A DA panel comprising twelve panelists (seven females and ﬁve males, aged between 22 and 60 years), all of whom had previous DA experience and six of whom participated in DA at t = 0, was assembled. Panelists underwent ﬁve training sessions (1 × 2 h session per week, held over ﬁve consecutive weeks). During training sessions, the panel evaluated the aroma, ﬂavor, taste, and mouthfeel attributes of wines, according to standard DA protocol [21], and were introduced to the tasting booths in which formal evaluations would be held (i.e., under controlled ventilation, light conditions, and temperature, being 22–23 ◦C). The panel generated twelve aroma, seven ﬂavor, and ﬁve taste and mouthfeel descriptors for Chardonnay wines and eleven aroma, eight ﬂavor, and ﬁve taste and mouthfeel descriptors for Shiraz wines (Table S3); these included the same aroma, ﬂavor, taste, and mouthfeel attributes used for DA of wines at t = 0, but several additional descriptors were generated for the bottle-aged wines. Reference standards were developed during early training sessions and were freshly prepared (in covered, opaque black glasses) for use at subsequent training sessions and throughout formal evaluations. Examples of taste and mouthfeel attributes (from low to high) were also provided and comprised creaminess (low-fat milk to full cream milk), acidity (base wine spiked with 0.5 to 2 g/L tartaric acid), bitterness (base wine spiked with 5 to 20 mg/L quinine sulfate), and astringency (felt material to sandpaper). The aftertaste was deﬁned as the length of time for which fruit and/or phenolic attributes were perceived after expectoration. During training, panelists practiced rating the intensity of each descriptor, and their performance was evaluated using SENPAQ (version 5.01, Qi Statistics, Reading, UK). Further training was provided to panelists for any attributes with the signiﬁcant judge by sample interactions. Panel performance was considered to be satisfactory once interactions were minimized, after which, formal evaluations commenced. Four formal evaluation sessions were held (two each for Chardonnay and Shiraz wines), with nine wines presented per session, such that three replicates of each wine were assessed. 2.4.2. GC-MS Instrumentation SIM parameters were as follows: Group 1 (Start time 0.00 min) m/z 43.1, 70.1, 71.1, 86.0, 88.1, 101.1 and 116.1; Group 2 (start time 18.01 min) m/z 68.1, 79.1, 93.0 and 136.1; Group 3 (start time 30.00 min) m/z 39.1, 41.1, 55.1, 57.1, 67.1, 70.1, 71.1, 82.1, 83.1, 84.1, 89.1, 93.1, 95.0, 96.0, 105.0, 106.0, 121.1, 129.1 and 136.1; Group 4 (start time 48.00 min) m/z 59.1, 65.1, 69.1, 91.1, 93.1, 104.1, 121.1, 123.1, 136.1, 138.1, 156.1, 163.0, 164.1 and 192.1; Group 5 (start time of 62.00 min) m/z 43.1, 55.1, 57.1, 65.1, 77.1, 85.0, 91.1, 92.1, 93.1, 103.1, 104.1, 121.1, 122.1, 128.1, 131.1, 132.1, 135.1, 136.1, 147.1, 176.1, 177.1, and 192.1. Ions in groups 1 and 2 had a dwell time of 100 ms, while those in groups 3, 4, and 5 had a dwell time of 50 ms. Compound identiﬁcation was achieved using the National Institute of Standards and Technology (NIST) 05 Mass Spectral library database 4 of 14 Foods 2020, 9, 1208 and by comparing retention times and mass spectra with those of reference standards (Table S2), when available. Compound peak areas were corrected relative to 2-octanol. and by comparing retention times and mass spectra with those of reference standards (Table S2), when available. Compound peak areas were corrected relative to 2-octanol. 2.6. Data Analysis Sensory data were analyzed using a mixed model analysis of variance (ANOVA) with wine sample and replicate as ﬁxed factors and panelists as random factors, including two-way interactions (Tables S4–S7). Fisher’s least signiﬁcant diﬀerence (LSD) was applied as post-hoc comparison p < 0.05. Data analyses were performed with XLSTAT (version 2020.1., Addinsoft, New York, NY, USA). 2.5. Sensory Analysis of Wines Wines (30 mL) were assigned random three-digit codes and served in XL5 (ISO standard) 215 mL wine glasses covered with plastic lids, using a randomized presentation order, with wines presented in brackets of four or ﬁve samples. Chardonnay wines were served at 14–16 ◦C and Shiraz wines were served at 22–24 ◦C. Panelists evaluated wines and recorded the intensity of each sensory attribute using FIZZ data acquisition software (Version 2.47b, Biosystèms, Couternon, France) on 15 cm unstructured line scales with anchor points of ‘low’ and ‘high’ placed at 0% and 100% on the scale, respectively. Between samples, panelists cleansed their palate with ﬁltered water and unsalted crackers during a one min break. Panelists were required to have ﬁve min breaks after each bracket. DA panelists gave informed consent before participating in the study, which was approved by the Human Research Ethics Committee of The University of Adelaide (Project No. H-174-2011). 2.7. Ethical Statement DA panelists gave informed consent before participating in the study, which was approved by the Human Research Ethics Committee of The University of Adelaide (Project No. H-174-2011). 5 of 14 Foods 2020, 9, 1208 3. Results and Discussion 3.1. Inﬂuence of Flavoring and Aging on Basic Wine Composition Foods 2020, 9, x FOR PEER REVIEW The pH, TA, alcohol, residual sugar, and VA of control and ﬂavored wines were measured after 12 months of bottle aging (t = 1), to investigate compositional diﬀerences amongst wines attributable to the addition of natural ﬂavorings (Table S8). As expected, compositional diﬀerences were observed between the four wines, but no signiﬁcant diﬀerences were observed between control wines and their corresponding ﬂavored wines. Nor were there signiﬁcant diﬀerences between wines after bottling and bottle aging (i.e., at t = 0 and t = 1, data not shown). Neither the addition of natural ﬂavorings nor bottle aging signiﬁcantly inﬂuenced the basic wine parameters that were measured. 3.1. Influence of Flavoring and Aging on Basic Wine Composition The pH, TA, alcohol, residual sugar, and VA of control and flavored wines were measured after 12 months of bottle aging (t = 1), to investigate compositional differences amongst wines attributable to the addition of natural flavorings (Table S8). As expected, compositional differences were observed between the four wines, but no significant differences were observed between control wines and their corresponding flavored wines. Nor were there significant differences between wines after bottling and bottle aging (i.e., at t = 0 and t = 1, data not shown). Neither the addition of natural flavorings nor bottle aging significantly influenced the basic wine parameters that were measured 3.2. Volatile Composition of Flavor Additives and Flavored Wines g g g y p 3 2 Volatile Composition of Flavor Additives and Flavored Wines The composition of ﬂavor additives was analyzed by GC-MS in an attempt to identify the key volatile compounds responsible for their characteristic aromas and ﬂavors. The complexity of ﬂavorings varied considerably, with some ﬂavor additives comprising relatively few volatile compounds, e.g., the raspberry ﬂavor additive (Figure 1a), while others contained an array of constituents; around 20, in the case of the passion fruit ﬂavoring (Figure 1b). The most abundant ﬂavoring constituents were isoprenoids, furans, esters, alcohols and volatile phenols (Table 1), all of which have previously been identiﬁed as constituents of grapes and/or wine [22–25]. 3.2. Volatile Composition of Flavor Additives and Flavored Wines The composition of flavor additives was analyzed by GC-MS in an attempt to identify the key volatile compounds responsible for their characteristic aromas and flavors. The complexity of flavorings varied considerably, with some flavor additives comprising relatively few volatile compounds, e.g., the raspberry flavor additive (Figure 1a), while others contained an array of constituents; around 20, in the case of the passion fruit flavoring (Figure 1b). The most abundant flavoring constituents were isoprenoids, furans, esters, alcohols and volatile phenols (Table 1), all of which have previously been identified as constituents of grapes and/or wine [22–25]. (a) (b) Figure 1. Chromatograms of (a) raspberry and (b) passion fruit flavor additives. 0 10,000,000 20,000,000 30,000,000 0 10 20 30 40 50 60 70 80 90 100 110 Abundance Time (min) Ethyl butanoate Ethyl 2-methylbutyrate Ethyl 3-methylbutyrate Linalool β-Ionone 0 10,000,000 20,000,000 30,000,000 0 10 20 30 40 50 60 70 80 90 100 110 Abundance Time (min) Ethyl butanoate unidentified unidentified unidentified D-Limonene Ethyl hexanoate (Z)-3-Hexen-1-ol acetate (Z)-3-Hexen-1-ol 3-Hexenyl butyrate Linalool α-Terpineol Phenyl isovalerate Figure 1. Chromatograms of (a) raspberry and (b) passion fruit ﬂavor additives. 0 10,000,000 20,000,000 30,000,000 0 10 20 30 40 50 60 70 80 90 100 110 Abundance Time (min) Ethyl butanoate Ethyl 2-methylbutyrate Ethyl 3-methylbutyrate Linalool β-Ionone (a) (b) 0 10,000,000 20,000,000 30,000,000 0 10 20 30 40 50 60 70 80 90 100 110 Abundance Time (min) Ethyl butanoate unidentified unidentified unidentified D-Limonene Ethyl hexanoate (Z)-3-Hexen-1-ol acetate (Z)-3-Hexen-1-ol 3-Hexenyl butyrate Linalool α-Terpineol Phenyl isovalerate (b) Figure 1. Chromatograms of (a) raspberry and (b) passion fruit flavor additives. Figure 1. Chromatograms of (a) raspberry and (b) passion fruit ﬂavor additives. 6 of 14 Foods 2020, 9, 1208 Table 1. Volatiles identiﬁed as abundant constituents of natural ﬂavor additives by GC-MS analysis. 3.2. Volatile Composition of Flavor Additives and Flavored Wines g g g y p 3 2 Volatile Composition of Flavor Additives and Flavored Wines Table 1. Volatiles identiﬁed as abundant constituents of natural ﬂavor additives by GC-MS analys Flavor Additives Volatile Compounds apricot linalool, hexyl butanoate berry linalool, α-terpineol, α-ionone butter ethyl butanoate chocolate 2-ethyl-1-hexanol, ethyl butanoate cinnamon cinnamaldehyde, ethyl cinnamate, benzaldehyde custard ethyl butanoate honey 2-phenylethyl acetate, ethyl acetate oak 2-phenylethyl alcohol, furfural orange linalool, ethyl butanoate, limonene passion fruit 2-phenethyl isovalerate, cis-3-hexenyl butyrate raspberry β-ionone, linalool vanilla vanillin, 2-ethyl-1-hexanol Control and ﬂavored wines were also analyzed by GC-MS (at both t = 0 and t = 1), to determine compositional changes attributable to the addition of ﬂavorings and/or bottle aging. However, ﬂavorings were added to wines in such small quantities, i.e., as 1% solutions prepared from ≤3.0 g/L standards of ﬂavor additives (Table S1), that many of the volatile compounds identiﬁed as constituents of ﬂavor additives were either not detected in ﬂavored wines or were present at similar concentrations to those of corresponding control wines (data not shown); irrespective of whether samples were analyzed using full scan mode or following the development of SIM methods to improve selectivity and sensitivity. However, there were some notable exceptions (Table 2). Similar levels of cis-3-hexenyl butyrate were found in CH1 and CH1 + PF at t = 0, but almost 30-fold higher concentrations were observed in CH1 + PF, than in CH1, at t = 1. Comparable results were obtained following the addition of passion fruit ﬂavoring to CH2; approximately 50-fold higher cis-3-hexenyl butyrate concentrations were found in CH2 + PF, than in CH2 at t = 1. Although similar levels of linalool were found in CH1 and CH1 + PF at t = 0, CH1+PF contained approximately double the linalool content of CH1 at t = 1. The linalool and limonene concentrations of SH1 + C and SH1 + R were similarly found to increase (relative to SH1) following bottle aging. Signiﬁcant quantities of 2-ethyl hexanol were detected in all control and ﬂavored SH1 wines; levels were higher in SH1 + C than SH1 at t = 0, but lower in SH1 + C (than SH1) at t = 1. The addition of berry ﬂavoring to SH2 resulted in signiﬁcantly higher concentrations of linalool and α- and β-ionone in SH2 + B (approximately 55%, 1900% and 360% higher levels, respectively, at t = 0). 3.2. Volatile Composition of Flavor Additives and Flavored Wines g g g y p 3 2 Volatile Composition of Flavor Additives and Flavored Wines Linalool levels remained similar for control and ﬂavored SH2 wines following bottle aging, but the α- and β-ionone content of SH2 + B increased (by an additional 50–100%) during bottle aging. Approximately two-fold higher concentrations of phenethyl acetate were found in CH2 + H than in CH2. In some instances, compositional diﬀerences between control and ﬂavored wines were directly attributable to the addition of ﬂavor additives, but changes observed after bottle aging likely reﬂect chemical transformations of wine and/or ﬂavor constituents [10]. The detection of volatile compounds derived from ﬂavor additives could be improved through various method development strategies, for example, through the extraction of larger volumes of ﬂavored wine, diﬀerent sampling methods, and/or the use of more speciﬁc standards (i.e., isotopically labeled internal standards). In this study, the impact of ﬂavor addition and bottle aging was instead assessed via sensory analysis. However, it should be acknowledged that challenges associated with detecting ﬂavor constituents in wine could have implications for policing the use of ﬂavor additives by industry; i.e., where their use is legally prohibited, ﬂavorings can seemingly impact wine aroma and ﬂavor at concentrations that cannot be readily detected. 7 of 14 Foods 2020, 9, 1208 Table 2. Peak areas for selected volatile constituents of control and ﬂavored Chardonnay (CH1 and CH2) and Shiraz (SH1 and SH2) wines, after bottling (t = 0) and then after 12 months of bottle aging (t = 1) (t = 1). 3.2. Volatile Composition of Flavor Additives and Flavored Wines g g g y p 3 2 Volatile Composition of Flavor Additives and Flavored Wines Flavor Target Compound Wine Composition berry linalool SH2 (t = 0): 77,936 SH2 + B (t = 0): 120,887 SH2 (t = 1): 74,909 SH2 + B (t = 1): 110,252 α-ionone SH2 (t = 0): 1797 SH2 + B (t = 0): 35,769 SH2 (t = 1): 1842 SH2 + B (t = 1): 53,500 β-ionone SH2 (t = 0): 2686 SH2 + B (t = 0): 12,429 SH2 (t = 1): 2416 SH2 + B (t = 1): 27,276 chocolate 2-ethyl-1-hexanol SH1 (t = 0): 693,470 SH1 + C (t = 0): 880,936 SH1 (t = 1): 903,129 SH1 + C (t = 1): 783,384 linalool SH1 (t = 0): 65,153 SH1 + C (t = 0): 67,702 SH1 (t = 1): 66,725 SH1 + C (t = 1): 430,975 limonene SH1 (t = 0): 14,347 SH1 + C (t = 0): 18,966 SH1 (t = 1): 18,458 SH1 + C (t = 1): 50,955 honey 2-phenylethyl acetate CH2 (t = 0): 327,217 CH2 + H (t = 0): 734,763 CH2 (t = 1): 325,031 CH2 + H (t = 1): 511,441 passion fruit cis-3-hexenyl butyrate CH1 (t = 0): 4309 CH1 + PF (t = 0): 3232 CH1 (t = 1): 3272 CH1 + PF (t = 1): 87,350 CH2 (t = 0): 3531 CH2 + PF (t = 0): 2990 CH2 (t = 1): 3400 CH2 + PF (t = 1): 161,211 linalool CH1 (t = 0): 46,231 CH1 + PF (t = 0): 43,152 CH1 (t = 1): 45,623 CH1 + PF (t = 1): 95,449 raspberry linalool SH1 (t = 0): 65,153 SH1 + R (t = 0): 62,545 SH1 (t = 1): 66,725 SH1 + R (t = 1): 282,633 limonene SH1 (t = 0): 14,347 SH1 + R (t = 0): 19,719 SH1 (t = 1): 18,458 SH1 + R (t = 1): 41,333 Peak areas were corrected against the internal standard (i.e., 2-octanol). 3.3. Sensory Proﬁles of Control and Flavored Wines Results from descriptive analysis of control and ﬂavored Chardonnay and Shiraz wines performed at t = 0 and t = 1 were compared to determine the impact of ﬂavor addition and bottle aging on wine sensory proﬁles. As previously reported, the addition of ﬂavorings inﬂuenced the sensory proﬁles of Chardonnay and Shiraz wines [7], but bottle aging also inﬂuenced wine aroma, ﬂavor, and/or mouthfeel attributes. The addition of apricot and passion fruit ﬂavorings to CH1 enhanced the intensity of selected fruit and/or ﬂoral characters, and diminished the perception of astringency (Table 3). However, after 12 months of bottle aging, diﬀerences between control and ﬂavored wines were less apparent; only melon aroma and caramel-lolly ﬂavor were found to be signiﬁcantly higher in ﬂavored CH1 wines at t = 1. The intensity of other sensory attributes increased for CH1, CH1 + A, and CH1 + PF alike. This likely reﬂects the development of some complexity, due to aging, i.e., increases in the intensity of vanilla, butter, mixed spice, caramel, and oak characters, as well as the occurrence of dried fruit, toast and green notes (Table 3). However, it should be acknowledged that this could also reﬂect diﬀerences in the composition and/or performance of the DA panels between t = 0 and t = 1. LSD values were higher at t = 1 compared to t = 0 (data not shown), despite each panel undergoing training. Increased LSD values might also reﬂect the DA panel’s broader use of the intensity scales at t = 1. Foods 2020, 9, 1208 8 of 14 Table 3. Mean intensity ratings for aroma, ﬂavor, taste, and mouthfeel attributes of control and ﬂavored Chardonnay 1 (CH1) wines after bottling (t = 0) and then after 12 months of bottle aging (t = 1). Chardonnay 1 (CH1) wines after bottling (t = 0) and then after 12 months of bottle aging (t = 1). 3.3. Sensory Proﬁles of Control and Flavored Wines Values are mean scores from 4 replicates per treatment, determined by 11 judges at t = 0 and from 3 replicates per treatment, determined by 12 judges at t = 1. Mean values followed by a diﬀerent letter within a row (by treatment for each time point) are signiﬁcantly diﬀerent (p ≤0.05, one way ANOVA, Fisher’s LSD post hoc); ns = not signiﬁcant. a Attributes associated with aged wines only. Less favorable results were achieved following the addition of ﬂavor additives to Chardonnay 2 (Table 4). The honey ﬂavoring had little impact on wine aroma or ﬂavor; the honey aroma was not signiﬁcantly enhanced, and only the perception of oak ﬂavor increased. The addition of passion fruit ﬂavoring surprisingly resulted in less intense fruit characters (including passion fruit aroma), while bitterness, acidity, and astringency were perceived to be more prominent. This may reﬂect cross-modal interactions [26], as previous studies have shown certain aromas can enhance taste perceptions without directly imparting taste properties [27–29]. Certainly, the enhanced acidity perceived in CH2+PF at t = 0 was not indicative of any diﬀerences in pH or TA (data not shown). p y p Bottle aging of CH2 gave similar outcomes to those observed for CH1; i.e., more intense vanilla, butter, orange blossom, mixed spice, caramel-lolly, and oak characters, together with dried fruit, toast and green vegetable notes (Table 4). Interestingly, the bitterness, acidity, and astringency observed in CH2 + PF at t = 0 were no longer prominent after bottle aging. At t = 1, control and ﬂavored CH2 wines had quite similar sensory proﬁles, albeit CH2 + H exhibited more intense honey and oak notes, than CH2 (at t = 1). Again, the emergence of additional attributes, dried fruit and toast, in particular (Table 4), was consistent with the developed notes associated with bottle age in white wine. The addition of ﬂavorings to SH1 signiﬁcantly increased the perception of confectionary and chocolate-vanilla characters, and diminished the earthy aroma of SH1 + R, but there were few statistically signiﬁcant sensory diﬀerences between SH1 and SH1 + C (Table 5). This was surprising given the chocolate ﬂavoring was intended to enhance chocolate notes, i.e., to mimic oak characters. In the case of SH1 + R, sensory diﬀerences were attributed to the butter and custard ﬂavor additives present in the raspberry ﬂavoring. 3.3. Sensory Proﬁles of Control and Flavored Wines Attributes CH1 t = 0 CH1 + A t = 0 CH1 + PF t = 0 p CH1 t = 1 CH1 + A t = 1 CH1 + PF t = 1 p Aroma passion fruit 7.4 7.4 8.9 ns 10.2 11.8 10.4 ns tropical fruit 8.6 9.8 9.7 ns 9.5 9.4 9.0 ns stone fruit 7.3 b 9.1 a 9.6 a 0.017 8.5 10.3 9.2 ns citrus 3.6 b 6.3 a 5.8 a 0.001 6.7 6.3 5.9 ns green 2.3 1.9 2.0 ns 2.9 1.5 1.0 ns honey 5.1 5.3 4.1 ns 7.7 9.6 8.7 ns vanilla 4.4 4.4 4.5 ns 7.5 10.5 8.1 ns butter 3.8 a 3.2 a 2.1 b 0.002 8.0 9.3 8.6 ns orange blossom 3.1 b 5.7 a 4.0 b 0.010 7.5 8.3 8.1 ns dried stone fruit a - - - - 8.5 10.6 10.3 ns Melon a - - - - 4.0 b 5.3 ab 6.5 a 0.009 Toast a - - - - 6.4 6.9 5.8 ns Flavor passion fruit 9.5 9.2 9.2 ns 11.1 10.6 9.6 ns stone fruit 9.0 9.5 9.6 ns 10.2 9.4 10.1 ns mixed spice 4.6 b 5.9 a 3.6 c <0.001 8.8 8.2 8.8 ns caramel-lolly 4.3 b 5.3 a 3.7 b 0.005 6.8 b 10.0 a 9.5 a 0.017 oak 3.3 b 4.8 a 3.3 b 0.013 8.7 9.9 9.1 ns dried stone fruit a - - - - 10.0 12.5 11.7 ns green vegetable a - - - - 3.1 1.9 2.7 ns Taste and mouthfeel bitterness 5.5 5.6 5.2 ns 6.4 6.9 6.0 ns acidity 8.0 7.9 8.3 ns 9.0 9.7 8.9 ns astringency 6.1 a 3.9 c 4.9 b <0.0001 8.4 7.1 7.2 ns creaminess 5.8 a 3.8 b 4.0 b <0.0001 6.3 b 9.5 a 7.5 ab 0.044 aftertaste 10.9 a 6.4 b 7.1 b <0.0001 10.0 10.8 10.1 ns Values are mean scores from 4 replicates per treatment, determined by 11 judges at t = 0 and from 3 replicates per treatment, determined by 12 judges at t = 1. Mean values followed by a diﬀerent letter within a row (by treatment for each time point) are signiﬁcantly diﬀerent (p ≤0.05, one way ANOVA, Fisher’s LSD post hoc); ns = not signiﬁcant. a Attributes associated with aged wines only. 3.3. Sensory Proﬁles of Control and Flavored Wines This combination of ﬂavors (i.e., butter, orange, custard, and raspberry) seemingly maintained its inﬂuence on wine aroma and ﬂavor during bottle aging, and the intensity of 9 of 14 Foods 2020, 9, 1208 confectionary and chocolate-vanilla characters were still signiﬁcantly diﬀerent from SH1 at t = 1. Again, additional attributes were observed at t = 1, i.e., plum, licorice and dried herb aromas, and cherry and green vegetable ﬂavors, possibly due to aging (Table 5). The sensory proﬁles of SH1 and SH1 + C were still similar. These results suggest the raspberry ﬂavoring that was added to SH1 had greater persistence than any of the ﬂavorings added to the Chardonnay base wines. Table 4. Mean intensity ratings for aroma, ﬂavor, taste, and mouthfeel attributes of control and ﬂavored Chardonnay 2 (CH2) wines after bottling (t = 0) and then after 12 months of bottle aging (t = 1). 3.3. Sensory Proﬁles of Control and Flavored Wines Attributes CH2 t = 0 CH2 + H t = 0 CH2 + PF t = 0 p CH2 t = 1 CH2 + H t = 1 CH2 + PF t = 1 p Aroma passion fruit 10.0 a 9.5 a 7.6 b <0.001 11.7 10.4 11.3 ns tropical fruit 10.5 10.5 10.1 ns 10.2 8.8 10.8 ns stone fruit 6.5 ab 8.2 a 6.0 b ns 8.0 b 8.9 ab 10.8 a 0.016 citrus 6.2 a 3.3 b 2.6 b <0.0001 7.7 7.1 8.7 ns green 1.7 a 1.0 b 1.0 b 0.012 3.3 2.1 3.0 ns honey 5.1 a 5.2 a 3.2 b 0.029 6.1 b 9.5 a 7.3 b 0.010 vanilla 5.5 4.1 5.3 ns 7.0 8.9 6.5 ns butter 3.5 a 2.9 a 1.7 b <0.001 8.1 8.0 7.6 ns orange blossom 2.4 2.1 2.7 ns 7.3 8.1 9.3 ns dried stone fruit a - - - - 8.4 10.2 8.3 ns Melon a - - - - 5.6 5.6 6.1 ns Toast a - - - - 5.2 ab 6.6 a 4.5 b 0.026 Flavor passion fruit 9.8 10.8 9.5 ns 12.0 a 10.1 b 12.1 a 0.036 stone fruit 7.7 8.8 7.1 ns 10.0 9.6 11.6 ns mixed spice 2.5 b 3.3 b 2.4 b ns 5.7 6.3 7.0 ns caramel-lolly 3.1 3.5 3.2 ns 6.2 9.0 7.0 ns oak 2.3 b 3.7 a 2.9 b 0.008 6.6 b 9.3 a 6.0 b 0.004 dried stone fruit a - - - - 9.6 11.0 9.9 ns green vegetable a - - - - 3.5 2.0 4.1 ns Taste and mouthfeel bitterness 3.0 c 5.3 b 9.7 a <0.0001 6.1 4.8 4.9 ns acidity 7.2 b 6.7 b 11.2 a <0.0001 10.0 9.8 11.3 ns astringency 5.4 b 5.0 b 9.3 a <0.0001 6.6 6.6 7.4 ns creaminess 7.3 ab 6.4 b 8.5 a 0.006 7.2 7.8 6.3 ns aftertaste 7.9 b 9.6 ab 10.7 a <0.001 8.9 b 11.2 a 10.0 ab 0.0003 Values are mean scores from 4 replicates per treatment, determined by 11 judges at t = 0 and from 3 replicates per treatment, determined by 12 judges at t = 1. Mean values followed by a diﬀerent letter within a row (by treatment for each time point) are signiﬁcantly diﬀerent (p ≤0.05, one way ANOVA, Fisher’s LSD post hoc); ns = not signiﬁcant. a Attributes associated with aged wines only. 3.3. Sensory Proﬁles of Control and Flavored Wines These results demonstrate that ﬂavorings could be used to inﬂuence the sensory proﬁles of wine, but the optimization of the concentration and composition of ﬂavorings would be needed to achieve lasting sensory outcomes. Table 5. Mean intensity ratings for aroma, ﬂavor, taste, and mouthfeel attributes of control and ﬂavored Shiraz 1 (SH1) wines after bottling (t = 0) and then after 12 months of bottle aging (t = 1). 3.3. Sensory Proﬁles of Control and Flavored Wines Table 4. Mean intensity ratings for aroma, ﬂavor, taste, and mouthfeel attributes of control and ﬂavored Chardonnay 2 (CH2) wines after bottling (t = 0) and then after 12 months of bottle aging (t = 1). Values are mean scores from 4 replicates per treatment, determined by 11 judges at t = 0 and from 3 replicates per treatment, determined by 12 judges at t = 1. Mean values followed by a diﬀerent letter within a row (by treatment for each time point) are signiﬁcantly diﬀerent (p ≤0.05, one way ANOVA, Fisher’s LSD post hoc); ns = not signiﬁcant. a Attributes associated with aged wines only. The addition of berry ﬂavoring, which comprised berry, custard, and butter ﬂavor additives, to SH2 enhanced the perception of confectionary ﬂavor and diminished the perceived intensity of oak ﬂavor at t = 0 (Table 6). After bottle aging, the intensity of red berry and confectionary aromas and chocolate-vanilla ﬂavor were still signiﬁcantly higher than in SH2. In contrast, the raspberry ﬂavoring did not signiﬁcantly inﬂuence wine aroma or ﬂavor, at either t = 0 or t = 1. This suggests a higher dose of ﬂavor additives might have been needed to modify wine sensory properties. A key aim of this study was to determine the impact of bottle aging on the sensory proﬁles of ﬂavored Chardonnay and Shiraz wines, and the persistence of sensory qualities imparted by ﬂavorings. In some, but not all cases, the addition of ﬂavor additives did modify the perception of wine aroma and/or ﬂavor. Where ﬂavorings had limited impact on wine sensory proﬁles, the addition of more concentrated ﬂavorings, or ﬂavorings comprised of diﬀerent combinations of ﬂavor additives, might achieve more apparent sensory outcomes. Regardless, bottle aging seemingly inﬂuenced ﬂavored Chardonnay and Shiraz wines diﬀerently. The diﬀerences observed between the sensory proﬁles of control and ﬂavored Chardonnay wines after bottling (i.e., at t = 0) were not as apparent after 12 months bottle aging (i.e., at t = 1); which may have reﬂected the development of secondary vanilla, butter, spice, caramel and/or honey characters in both control and ﬂavored wines. In contrast, 10 of 14 Foods 2020, 9, 1208 sensory diﬀerences observed between some control and ﬂavored Shiraz wines, SH1+R in particular, persisted during bottle aging, such that the sensory impact of ﬂavor additives on wine aroma and/or ﬂavor was still apparent. 3.3. Sensory Proﬁles of Control and Flavored Wines Attributes SH1 t = 0 SH1 + C t = 0 SH1 + R t = 0 p SH1 t = 1 SH1 + C t = 1 SH1 + R t = 1 p Aroma red berry 5.2 6.4 6.1 ns 4.9 6.2 5.0 ns dark berry 7.7 8.1 7.6 ns 6.9 6.4 7.1 ns confectionary 5.4 b 5.8 b 8.3 a <0.001 3.6 b 4.6 b 6.5 a <0.0001 chocolate-vanilla 5.4 b 5.3 b 8.4 a <0.0001 4.7 b 4.8 b 7.8 a <0.0001 mixed spice 6.4 6.1 5.5 ns 4.4 4.8 4.9 ns earthy 2.2 b 3.0 a 1.7 b 0.003 4.8 3.5 4.2 ns green 2.1 2.8 1.6 ns 4.6 a 3.4 ab 2.5 b 0.006 black pepper 4.8 5.2 4.6 ns 5.7 4.8 4.3 ns Plum a - - - - 5.2 4.8 4.9 ns Licorice a - - - - 4.1 4.0 4.0 ns dried herbs a - - - - 3.5 3.0 3.5 ns Flavor red berry 6.7 6.4 7.0 ns 6.5 6.3 6.7 ns dark berry 9.2 8.7 9.3 ns 7.7 7.1 7.2 ns confectionary 5.2 ab 4.8 b 6.6 a 0.043 4.4 b 5.1 b 6.5 a 0.003 mixed spice 6.5 6.0 6.3 ns 5.2 ab 4.7 b 6.0 a 0.021 chocolate-vanilla 4.2 b 3.9 b 6.4 a <0.0001 5.0 b 5.2 b 7.1 a <0.001 oak 7.4 7.0 7.9 ns 5.8 b 5.6 b 6.8 a 0.020 Cherry a - - - - 6.4 a 4.8 b 6.8 a 0.013 green vegetable a - - - - 4.2 3.4 2.8 ns Taste and mouthfeel bitterness 6.5 6.1 5.2 0.006 5.9 6.3 4.8 0.045 acidity 7.4 7.9 7.5 ns 6.0 6.0 6.2 ns astringency 7.9 7.8 8.4 ns 7.8 8.3 7.5 ns alcohol 8.4 7.7 7.7 ns 7.3 7.4 6.6 0.040 length 10.6 10.4 10.9 ns 7.0 7.1 7.5 ns Values are mean scores from 4 replicates per treatment, determined by 12 judges at t = 0 and from 3 replicates per treatment, determined by 12 judges at t = 1. Mean values followed by a diﬀerent letter within a row (by treatment for each time point) are signiﬁcantly diﬀerent (p ≤0.05, one way ANOVA, Fisher’s LSD post hoc); ns = not signiﬁcant. a Attributes associated with aged wines only. Table 5. 3.3. Sensory Proﬁles of Control and Flavored Wines Mean intensity ratings for aroma, ﬂavor, taste, and mouthfeel attributes of control and ﬂavored Shiraz 1 (SH1) wines after bottling (t = 0) and then after 12 months of bottle aging (t = 1). Values are mean scores from 4 replicates per treatment, determined by 12 judges at t = 0 and from 3 replicates per treatment, determined by 12 judges at t = 1. Mean values followed by a diﬀerent letter within a row (by treatment for each time point) are signiﬁcantly diﬀerent (p ≤0.05, one way ANOVA, Fisher’s LSD post hoc); ns = not signiﬁcant. a Attributes associated with aged wines only. Although ﬂavor additives are routinely used in many food and beverage industries, they are not legally permitted winemaking aids [4], so their use in wine is currently prohibited. It is unlikely that ﬂavorings would ever be used in the production of premium quality wines, for which winemakers and consumers alike value traditional approaches to winemaking. However, this study demonstrates the potential for ﬂavorings to be used to mitigate sensory deﬁciencies in lower quality and/or commodity wines, if the regulations governing winemaking additives were reviewed. For example, ﬂavorings could be used to enhance the aroma and/or ﬂavor of wines which lack intensity (e.g., due to adverse weather conditions, such as unusually cool seasons, or prolonged drought or heat), or to mask the presence of undesirable characters (e.g., green or earthy notes) or even mild faults or taints (e.g., reductive characters or mustiness). Given the current (global) trend towards producing wines of lower alcohol content [30], there might also be opportunities for ﬂavor additives to be used in conjunction with various alcohol adjustment strategies, which can also aﬀect wine aroma, ﬂavor, and body. Additionally, ﬂavorings could be used to introduce oak characters to wine (e.g., vanilla, coconut, 11 of 14 Foods 2020, 9, 1208 or spice notes), without the investment in time or capital associated with traditional barrel maturation. Findings could also be applied in the production of wine products, i.e., wine made with the addition of ﬂavorings, legally deﬁned in Australia as ‘food containing no less than 700 mL/L of wine which has been formulated, processed, modiﬁed or mixed with other foods’ [4]. Table 6. 3.3. Sensory Proﬁles of Control and Flavored Wines Mean intensity ratings for aroma, ﬂavor, taste, and mouthfeel attributes of control and ﬂavored Shiraz 2 (SH2) wines after bottling (t = 0) and then after 12 months of bottle aging (t = 1). Attributes SH2 t = 0 SH2 + B t = 0 SH2 + R t = 0 p SH2 t = 1 SH2 + B t = 1 SH2 + R t = 1 p Aroma red berry 6.3 7.0 6.8 ns 5.4 b 7.7 a 6.2 b <0.001 dark berry 7.9 7.7 8.4 ns 6.0 6.6 7.1 ns confectionary 6.4 8.0 6.7 ns 4.1 b 6.4 a 5.0 b 0.002 chocolate-vanilla 4.6 5.3 4.9 ns 3.9 5.2 4.4 ns mixed spice 6.0 5.5 5.6 ns 3.6 3.7 4.9 ns earthy 2.3 2.7 2.1 ns 4.7 a 3.0 b 4.2 a 0.013 green 2.4 2.6 2.8 ns 4.5 4.0 3.6 ns black pepper 5.2 4.7 4.8 ns 4.3 5.3 4.5 ns Plum a - - - - 5.4 4.8 6.0 ns Licorice a - - - - 4.6 5.0 4.1 ns dried herbs a - - - - 3.1 2.9 2.9 ns Flavor red berry 7.5 8.7 7.8 ns 7.3 8.2 7.7 ns dark berry 9.2 9.8 9.7 ns 7.4 7.6 7.4 ns confectionary 7.5 ab 8.8 a 6.7 b 0.035 6.6 7.1 7.1 ns mixed spice 5.4 5.4 5.5 ns 4.1 5.2 5.3 ns chocolate-vanilla 4.8 5.5 4.4 ns 4.3 b 5.8 a 4.9 ab 0.016 oak 8.2 a 6.2 b 7.6 a 0.002 4.9 4.9 4.9 ns Cherry a - - - - 6.7 6.9 4.9 ns green vegetable a - - - - 3.7 3.7 2.9 ns Taste and mouthfeel bitterness 5.3 4.6 5.0 ns 5.3 a 4.4 b 4.4 b 0.050 acidity 6.8 7.3 7.3 ns 6.2 5.8 6.4 ns astringency 7.9 7.9 7.9 ns 7.0 6.0 6.6 ns alcohol 7.1 b 8.1 a 7.9 a 0.040 6.7 6.4 6.9 ns length 9.8 10.3 10.1 ns 7.1 7.5 7.4 ns Values are mean scores from 4 replicates per treatment, determined by 12 judges at t = 0 and from 3 replicates per treatment, determined by 12 judges at t = 1. Mean values followed by a diﬀerent letter within a row (by treatment for each time point) are signiﬁcantly diﬀerent (p ≤0.05, one way ANOVA, Fisher’s LSD post hoc); ns = not signiﬁcant. a Attributes associated with aged wines only. Table 6. 4. Conclusions The natural ﬂavorings used in this study were found to contain volatile compounds previously identiﬁed in grapes and/or wine, but their addition to base wines did not always signiﬁcantly impact wine composition; i.e., many of the volatile compounds identiﬁed as constituents of ﬂavor additives were not detected at appreciably higher concentrations in ﬂavored wines, which likely reﬂects the extremely small quantities of ﬂavorings added to base wines. However, the addition of ﬂavorings signiﬁcantly modiﬁed the sensory proﬁles of wines, with ﬂavored wines, CH1 + A, CH1 + PF, CH2 + PF, SH1 + R and SH2 + B in particular, exhibiting enhanced fruit and/or developed aromas and ﬂavors, as a consequence of the use of ﬂavor additives. In the case of Chardonnay wines, the variation in sensory properties resulting from the addition of ﬂavorings diminished with time. However, the sensory impact arising from the addition of selected ﬂavorings to Shiraz persisted after 12 months of bottle aging. This demonstrates the potential for ﬂavor additives to be used to enhance desirable sensory attributes and/or mitigate sensory deﬁciencies. Supplementary Materials: The following are available online at http://www.mdpi.com/2304-8158/9/9/1208/s1, Table S1: Flavorings added to Chardonnay and Shiraz wines. Table S2: Aroma descriptors and GC-MS method characteristics (retention times and ions) of key constituents of ﬂavor additives. Table S3: Attributes and standards used in the descriptive analysis of Chardonnay and Shiraz wines. Table S4: Analysis of variance p values for sensory attributes of Chardonnay 1 (CH1) wines after bottling (at t = 0) and after 12 months bottle aging (at t = 1). Table S5: Analysis of variance p values for sensory attributes of Chardonnay 2 (CH2) wines after bottling (at t = 0) and after 12 months bottle aging (at t = 1). Table S6: Analysis of variance p values for sensory attributes of Shiraz 1 (SH1) wines after bottling (at t = 0) and after 12 months bottle aging (at t = 1). Table S7: Analysis of variance p values for sensory attributes of Shiraz 2 (SH2) wines after bottling (at t = 0) and after 12 months bottle aging (at t = 1). Table S8: pH, titratable acidity (TA), alcohol, residual sugar, and volatile acidity (VA) of control and ﬂavored Chardonnay and Shiraz wines after 12 months bottle aging (t = 1). Author Contributions: All authors were involved in conceptualization; formal analysis, Y.S., J.A.C., T.E.J. 3.3. Sensory Proﬁles of Control and Flavored Wines Mean intensity ratings for aroma, ﬂavor, taste, and mouthfeel attributes of control and ﬂavored Shiraz 2 (SH2) wines after bottling (t = 0) and then after 12 months of bottle aging (t = 1). Values are mean scores from 4 replicates per treatment, determined by 12 judges at t = 0 and from 3 replicates per treatment, determined by 12 judges at t = 1. Mean values followed by a diﬀerent letter within a row (by treatment for each time point) are signiﬁcantly diﬀerent (p ≤0.05, one way ANOVA, Fisher’s LSD post hoc); ns = not signiﬁcant. a Attributes associated with aged wines only. As indicated above, diﬀerences in the composition and/or performance of the DA panel between t = 0 and t = 1 are acknowledged as an inherent limitation of the study. The diﬀerences observed in the sensory proﬁles of control and ﬂavored wines between time points may, in part, have been attributable to the DA panel. Nonetheless, signiﬁcant diﬀerences were still observed between the sensory properties of some control and ﬂavored wines at each time point. The DA panels identiﬁed several new attributes in bottle-aged wines, some of which were consistent with descriptors associated with bottle aging, i.e., dried fruit and toast for white wine and licorice and plum for red wine. Most importantly, there was no evidence to suggest that any chemical transformation of natural ﬂavorings that might have occurred resulted in the formation of oﬀ-odors during bottle aging; i.e., at t = 1, the ﬂavor additives had not negatively impacted wine sensory proﬁles. Foods 2020, 9, 1208 12 of 14 12 of 14 4. Conclusions and R.R.; investigation, Y.S.; resources, S.E.P.B.; writing—original draft preparation, Y.S. and K.L.W.; writing—review and editing, J.A.C., T.E.J., R.R. and S.E.P.B.; supervision, K.L.W. and S.E.P.B.; project administration, S.E.P.B.; funding acquisition, Y.S., K.L.W. and S.E.P.B. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Wine Australia and Y.S. held a Wine Australia PhD scholarship (GWR Ph1102). ding: This research was funded by Wine Australia and Y.S. held a Wine Australia PhD scholarship R Ph1102). Acknowledgments: The authors gratefully acknowledge: the industry partners who contributed wine; FlavorSense and The Product Makers for the provision of ﬂavor additives; Laura Fassier for assistance with wine analysis; and the University of Adelaide staﬀand students involved in DA. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. References 1. Fischer, U. Wine aroma. In Flavours and Fragrances; Berger, R.G., Ed.; Springer: Berlin/Heidelberg, Germany, 2007; pp. 241–267. 2. Rapp, A.; Mandery, H. 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W4391822516.txt	https://www.qeios.com/read/038K9H.2/pdf	en		Behaviour change intervention tailoring	Definitions	2,024	cc-by	279	Qeios · Definition, February 14, 2024 Open Peer Review on Qeios Behaviour change intervention tailoring Behaviour Change Intervention Ontology (BCIO) Source Behaviour Change Intervention Ontology (BCIO) Definition: A BCI attribute in which the content or delivery of a BCI for some member of the BCI population varies according to their characteristics or setting. Informal definition: An attribute of a behaviour change intervention (BCI) or a BCI component whereby its content or delivery is varied according to characteristics of members of the target population or setting. Comment: Tailoring can be static (i.e., conducted before intervention delivery in a case case), or dynamic (i.e., conducted one or more times after the intervention has started in a given case based on population or setting characteristics present at that time. Population and setting characteristics include the historical factors such as prior exposure to the intervention. This definition was imported from the Behaviour Change Intervention Ontology (seehttps://bciosearch.org/). Comments and suggestions for improvements are welcome using the Qeios review system. Definitions imported from the Behaviour Change Intervention Ontology (BCIO) are what are known as ‘ontological definitions’. See this article in Qeios for an explanation https://www.qeios.com/read/YGIF9B. Ontological definitions can sometimes be hard to read. In those cases we also include an informal definition. Definitions also often require elaboration to make it clear how they should be used and what they include. In those cases we include a comment. Qeios ID: 038K9H.2 · https://doi.org/10.32388/038K9H.2 1/2 Qeios · Definition, February 14, 2024 Also, definitions sometimes require an explanation as to how they came about to help users understand how they relate to alternative definitions. In those cases we include a curator note. Qeios ID: 038K9H.2 · https://doi.org/10.32388/038K9H.2 2/2
https://openalex.org/W4392971183	https://healthcare.renaciptamandiri.org/index.php/ojs/article/download/2/2	Indonesian	null	Mewujudkan Generasi Emas dengan Optimalisasi Bounding Attachment Melalui Pijat Bayi oleh Ibu & Kader Posyandu di Kelurahan Sepanjang Jaya Kecamatan Rawalumbu, Bekasi	Deleted Journal	2,023	cc-by-sa	2,850	Abstrak Pertumbuhan dan perkembangan anak merupakan hal yang sangat penting dan salah satu aspek yang harus diperhatikan secara serius sejak usia dini. Upaya yang dapat dilakukan dalam mengoptimalkan di samping nutrisi yang diberikan oleh ibu dan salah satu cara lain perlu adanya rangsangan stimulasi pijat bayi. Metode kegiatan pengabdian masyarakat dilakukan dengan cara melakukan pendidikan kesehatan dan praktik mengajarkan pijat bayi untuk optimalisasi bounding attachment ibu dan bayi serta keterampilan seputar pijat bayi dan memberikan edukasi mengenai manfaat pijat bayi. Bertempat di Kelurahan Sepanjang Jaya Kecamatan Rawalumbu dan waktu pelaksanaan bulan Desember 2022. Hasil kegiatan terjadi peningkatan Pengetahuan Ibu sebelum dan sesudah Pendidikan Kesehatan dan Mempraktekkan Pijat Bayi Optimalisasi Bounding Attachment. Pengetahuan definisi, tujuan dan manfaat pijat bayi hasil pretest sebanyak 60% peserta menyatakan jawaban benar, hasil posttest sebanyak 100% peserta menyatakan benar. Pengetahuan tentang indikasi dan kontraindikasi pijat hasil pretest sebanyak 36,7% peserta menyatakan jawaban benar, hasil posttest sebanyak 96,5% peserta menyatakan benar. Pengetahuan persiapan yang dilakukan pijat bayi hasil pretest sebanyak 53,3% peserta menyatakan jawaban benar, kemudian hasil postest sebanyak 97% peserta menyatakan benar. Pengetahuan prosedur dan tata cara pijat bayi hasil 33,3% peserta menyatakan jawaban benar, kemudian hasil posttest 100% peserta menyatakan benar. Pijat bayi sebagai aplikasi stimulasi sentuhan, karena dalam pijat bayi terdapat unsur sentuhan berupa kasih sayang, perhatian, suara atau bicara, pandangan mata, gerakan, dan pijat. Kata Kunci: Asuhan, komplementer, pijat bayi, optimalisasi, bounding attachment ORIGINAL ARTICLE HEALTH CARE: JOURNAL OF COMMUNITY SERVICE / VOL 1 NO. 1 FEBRUARY 2024 ORIGINAL ARTICLE HEALTH CARE: JOURNAL OF COMMUNITY SERVICE / VOL 1 NO. 1 FEBRUARY 2024 HEALTH CARE: JOURNAL OF COMMUNITY SERVICE / VOL 1 NO. 1 FEBRUARY 2024 ORIGINAL ARTICLE \| Health Care: Journal of Community Service 5 Mewujudkan Generasi Emas dengan Optimalisasi Bounding Attachment Melalui Pijat Bayi oleh Ibu & Kader Posyandu di Kelurahan Sepanjang Jaya Kecamatan Rawalumbu, Bekasi Wiwit Desi Intarti1, Evi Nur Akhiriyanti2, Lenny Irmawaty3, Puri Kresna Wati4, Wiwit Desi Intarti1, Evi Nur Akhiriyanti2, Lenny Irmawaty3, Puri Kresna Wati4, Farida Simanjutak5, Renince Siregar6, Hainun Nisa7, Tetty Rina Aritonang8, Marni Br. Karo9, Riyen Sari Manullang10, Friska Junita11, Rupdi Lumban Siantar12, Dewi Rostianingsih13, Maslan Pangaribuan14, Siti Munawaroh15 1-15 STIKES Medistra Indonesia, Bekasi, Indonesia e-mail: wiwit.desti1982@gmail.com1 DOI : 10.62354/healthcare.v1i1.2 1-15 STIKES Medistra Indonesia, Bekasi, Indonesia e-mail: wiwit.desti1982@gmail.com1 DOI : 10.62354/healthcare.v1i1.2 Abstract The growth and development of children is very important and an aspect that must be taken seriously from an early age. Efforts that can be made to optimize the nutrition provided by the mother and one other way include the stimulation of baby massage. The method of community service activities is carried out by conducting health education and practicing teaching baby massage to optimize the bonding attachment of mother and baby as well as skills regarding baby massage and providing education about the benefits of baby massage. Located in the Lulu Jaya Subdistrict, Rawalumbu District and the implementation time is December 2022. The results of the activity are an increase in Mother's Knowledge before and after Health Education and Practicing Baby Massage Optimizing Bounding Attachment. Knowledge of the definition, purpose and benefits of baby massage, 60% of participants stated the correct answer in the pretest, and 100% of participants stated \| Health Care: Journal of Community Service 5 ISSN 3046-6075 the correct answer in the posttest. Knowledge about indications and contraindications for massage, 36.7% of participants stated the correct answer in the pretest, and 96.5% of participants stated the correct answer in the posttest. Knowledge of preparations for baby massage, 53.3% of participants stated the correct answer in the pretest, then 97% of participants stated the correct answer in the posttest. Knowledge of procedures and procedures for baby massage resulted in 33.3% of participants stating the correct answer, then posttest results 100% of participants stated correctly. Baby massage is an application of tactile stimulation, because in baby massage there are elements of touch in the form of affection, attention, sound or speech, eye gaze, movement and massage. Keyword: Complementary care, baby massage, optimization, bonding attachment 2. METODE Kegiatan pengabdian masyarakat dilakukan dengan cara melakukan pendidikan kesehatan dan praktik pijat bayi untuk optimalisasi bounding attachment ibu dan bayi serta keterampilan seputar pijat bayi dan memberikan edukasi mengenai manfaat pijat bayi, kegiatan dilaksanakan oleh Mahasiswa STIKes Medistra Indonesia dan didampingi oleh dosen pengampu dengan memberikan materi yang mudah dimengerti dan menggunakan bahasa yang mudah dipahami oleh sasaran. Mengajarkan cara melakukan pijat bayi dengan sentuhan yang mempererat bounding attachment bertempat di Kelurahan Sepanjang Jaya Kecamatan Rawalumbu dan waktu pelaksanaan bulan Desember 2022. 1. PENDAHULUAN Upaya kesehatan anak diatur dalam Permenkes Nomor 25 Tahun 2014 bahwa setiap anak berhak atas kelangsungan hidup, tumbuh dan berkembang serta berhak atas perlindungan dari kekerasan dan diskriminasi, sehingga perlu dilakukan upaya kesehatan anak secara terpadu, menyeluruh, dan berkesinambungan. Upaya kesehatan anak dimulai sejak bayi dari kesehatan bayi terlihat dari penurunan angka kesakitan dan kematian dari tahun ke tahun. Angka Kematian Bayi (AKB) atau Infant Mortality Rate (IMR) merupakan indikator yang sangat sensitif terhadap upaya pelayanan kesehatan terutama yang berhubungan dengan bayi baru lahir perinatal dan neonatal. Dari kematian bayi sebesar 3,26/1.000 kelahiran hidup, 82 % terjadi pada saat neonatal (0-28 hari), 17,39 % post neonatal (29 hari -11 bulan) (Gefrianti, 2019). Dari hasil penelitian yang ada, angka kematian bayi ini tidak berdiri sendiri, melainkan terkait dengan faktor- faktor lain terutama gizi. Bayi atau anak balita yang kekurangan gizi sangat rentan terhadap penyakit-penyakit infeksi, termasuk diare dan infeksi saluran akut, utamanya pneumonia (Notoatmodjo, 2010) dalam (Khairunnisa, 2021). Berat badan adalah salah satu indikator antropometri untuk menilai tumbuh pada bayi atau anak (Marlina & Lestari, 2019) dalam (Khairunnisa, 2021). World Health Organization (WHO) 2017 menyatakan angka kejadian berat badan bayi di dunia masih di bawah standar yaitu lebih dari 5% dengan prevalensi underweight di asia tenggara 26,9%, sedangkan prevalensi underweight di dunia secara global sebesar 14% (Carolin, 2020) dalam (Khairunnisa, 2021). Pertumbuhan dan perkembangan anak merupakan hal yang sangat penting dan salah satu aspek yang harus diperhatikan secara serius sejak usia dini. Salah satu penyebab permasalahan berat badan adalah nafsu makan anak yang turun. Upaya yang dapat dilakukan dalam mengoptimalkan berat badan pada bayi di samping nutrisi yang diberikan oleh ibu dan salah satu cara lain perlu adanya rangsangan stimulus atau yang biasa dikenal dengan pijat bayi (Roesli, 2016) dalam (Khairunnisa, 2021). \| Health Care: Journal of Community Service 6 ISSN 3046-6075 \| Health Care: Journal of Community Service 7 3. HASIL DAN PEMBAHASAN Pendidikan kesehatan dalam bentuk ceramah dan demonstrasi. Untuk mengukur pengetahuan peserta tentang pijat bayi pemateri juga melakukan pretest dan post test. Hasil rekapitulasi pretest sebelum dilakukan penyampaian materi diinterpretasikan dalam tabel sebagai berikut: Tabel 1. Rekapitulasi Pengetahuan Ibu Sebelum dan Sesudah Pendidikan Kesehatan dan Mempraktekkan Pijat Bayi Optimalisasi Bounding Attachment Tabel 1. Rekapitulasi Pengetahuan Ibu Sebelum dan Sesudah Pendidikan Kesehatan dan Mempraktekkan Pijat Bayi Optimalisasi Bounding Attachment abe e ap tu as e geta ua bu Sebe u da Sesuda e d d a ese ata dan Mempraktekkan Pijat Bayi Optimalisasi Bounding Attachment Pertanyaan Prosentase Hasil Jawaban Benar (%) Sebelum Sesudah Definisi, tujuan, manfaat 60 100 Indikasi dan kontraindikasi 36,7 96,5 Persiapan yang dilakukan 53,3 97 Prosedur dan tata cara 33,3 100 Sumber: Data Olahan Sendiri (Desember, 2022) Berdasarkan Tabel 1 menyatakan bahwa terjadi peningkatan pengetahuan saat sebelum dan sesudah pendidikan kesehatan serta demonstrasi pijat bayi, hal ini terlihat dari jawaban benar pada peserta pengabdian masyarakat pada saat menjawab pertanyaan sebelum dan sesudah. Pengetahuan tentang definisi, tujuan dan manfaat pijat bayi hasil pretest sebanyak 60% peserta menyatakan jawaban benar, kemudian hasil posttest sebanyak 100% peserta menyatakan benar. Pengetahuan tentang indikasi dan kontraindikasi pijat hasil pretest sebanyak 36,7% peserta menyatakan jawaban benar, kemudian hasil posttest sebanyak 96,5% peserta menyatakan benar. Pengetahuan tentang persiapan apa saja yang dilakukan pijat bayi hasil pretest sebanyak 53,3% peserta menyatakan jawaban benar, kemudian hasil postest sebanyak 97% peserta menyatakan benar. Pengetahuan tentang prosedur dan tata cara pijat bayi hasil 33,3% peserta kegiatan pengabdian masyarakat menyatakan jawaban benar, kemudian hasil posttest sebanyak 100% peserta menyatakan benar. \| Health Care: Journal of Community Service 7 ISSN 3046-6075 Pijat bayi dapat digolongkan sebagai aplikasi stimulasi sentuhan, karena dalam pijat bayi terdapat unsur sentuhan berupa kasih sayang, perhatian, suara atau bicara, pandangan mata, gerakan, dan pijat. Stimulasi ini akan merangsang perkembangan struktur maupun fungsi sel - sel otak (Soedjatmiko, 2006) dalam (Triana, 2017). Pijat bayi merupakan terapi sentuh kontak langsung dengan tubuh yang dapat memberikan rasa aman dan nyaman pada bayi. Sentuhan dan pelukan dari seorang ibu adalah kebutuhan dasar bayi. Jika pijat bayi dilakukan secara teratur akan meningkatkan hormon katekolamin (epinefrin dan norepinefrin) yang dapat memicu stimulasi tumbuh kembang karena dapat meningkatkan nafsu makan, meningkatkan berat badan, dan merangsang perkembangan struktur maupun fungsi otak (Riksani, 2018) dalam (Sinaga & Laowo, 2020). \| Health Care: Journal of Community Service 8 3. HASIL DAN PEMBAHASAN Masa tumbuh kembang bayi merupakan masa keemasan sekaligus masa kritis perkembangan seseorang yaitu pada usia 0-12 bulan, masa ini dibutuhkan bounding attachment antara ibu dan bayi sejak dini. Dikatakan masa keemasan karena masa bayi berlangsung sangat singkat dan tidak dapat diulang kembali. Dikatakan masa kritis karena pada masa ini bayi sangat peka terhadap lingkungan dan membutuhkan asupan gizi serta stimulasi yang baik untuk pertumbuhan dan perkembangannya. Kenaikan berat badan anak pada tahun pertama kehidupan apabila anak mendapat gizi yang baik yaitu dari bayi lahir sampai 6 bulan pertama pertambahan berat badan setiap minggu 140-200 gram. Berat badan bayi menjadi 2 kali lipat berat badan lahir pada akhir 6 bulan pertama. Sedangkan pada umur 6-12 bulan pertambahan berat badan setiap minggu berkisar antara 85-400 gram. Berat badan akan meningkat sebesar 3 kali berat badan lahir pada akhir tahun pertama (Wong, Hockenberry, Wilson, Perry, & Lowdermilk; Hidayat, 2018) dalam (Irva et al, 2014). Pertumbuhan adalah perubahan fisik dan peningkatan ukuran bagian tubuh dari seorang individu yang masing-masing berbeda, sedangkan perkembangan adalah bertambah sempurnanya kemampuan, keterampilan, dan fungsi tubuh yang lebih kompleks dalam motorik kasar, motorik halus, bicara dan bahasa, serta sosialisasi dan kemandirian yang dimiliki individu untuk beradaptasi dengan lingkungan. Stimulasi pijat bayi merupakan salah satu cara untuk meningkatkan perkembangan pada bayi, agar bayi dapat berkembang dengan baik (Nugrohowati & Nurhidayati, 2015) dalam (Susanti, 2019). Hasil peningkatan pengetahuan tentang pijat bayi sejalan dengan teori yang disampaikan oleh Roesli (2016) dalam (Khairunnisa, 2021) yang mengemukakan tentang manfaat dari pemijatan bayi yaitu merangsang nafsu makan bayi. Dengan dilakukan pemijatan bayi merangsang aktivitas nervus vagus, di mana saraf ini (saraf otak ke-10) yang akan menyebabkan peningkatan kadar enzim penyerapan gastrin dan insulin, sehingga penyerapan makanan akan lebih baik serta meningkatkan peristaltik usus dan pengosongan lambung meningkat yang dapat merangsang nafsu makan bayi Salah satu mekanisme dasar pijat bayi adalah aktivitas Nervus Vagus meningkatkan volume ASI yaitu penyerapan makanan menjadi lebih baik karena \| Health Care: Journal of Community Service 8 ISSN 3046-6075 peningkatan Aktivitas Nervus Vagus menyebabkan bayi cepat lapar sehingga akan lebih sering menyusu pada ibunya. Seperti diketahui, ASI akan semakin banyak diproduksi jika semakin banyak diminta. 3. HASIL DAN PEMBAHASAN selain itu, ibu yang memijat bayinya akan merasa lebih tenang dan hal ini berdampak positif pada peningkatan volume ASI (Julianti, 2017) dalam (Harahap, 2019), Sesuai dengan penelitian yang dilakukan Yuliana, Suharto, dan Handayani (2013) dalam Triana (2017), kepada bayi usia 3-5 bulan didapatkan peningkatan berat badan bayi yang dipijat selama 4 minggu yang dilakukan secara rutin lebih tinggi dibandingkan berat badan bayi yang tidak dipijat. Hal ini terjadi karena hormon stress pada bayi menurun, maka bayi dapat menghisap ASI lebih banyak, sehingga produksi ASI meningkat dan berat badan akan meningkat. Beberapa penelitian telah menunjukan bahwa 31-49% pijat bayi pada prematur dapat meningkatkan 31- 49% berat badan yang diikuti juga dengan peningkatan besar lingkar kepala dan tinggi badan. Hasil penelitian yang dilakukan Astriana & Suryani (2017), pada bayi cukup bulan usia 1-6 bulan dipijat 15 menit, dalam dua kali seminggu, akan mengalami kenaikan berat badan lebih baik dari pada bayi yang tidak dipijat. Namun hasil ini tidak sejalan dengan penelitian Fauziyah & Wijayanti (2018) dalam Khairunnisa (2021), yang menyatakan bahwa tidak terdapat pengaruh pemberian pijat bayi terhadap perubahan berat badan bayi. 4. SIMPULAN Pengabdian Masyarakat dengan tema “Mewujudkan Generasi Emas dengan Optimalisasi Bounding Attachment melalui Pijat Bayi oleh Ibu dan Kader Posyandu di Wilayah Kelurahan Sepanjang Jaya, Kota Bekasi” berhasil meningkatkan pengetahuan Ibu dan Kader tentang Optimalisasi Bounding Attachment melalui Pijat Bayi. \| Health Care: Journal of Community Service 9 ISSN 3046-6075 ISSN 3046-6075 ISSN 3046-6075 \| Health Care: Journal of Community Service 9 Daftar Pustaka Fida, M. 2012. Pengantar Ilmu Kesehatan Anak. Jakarta : D Medika Gelenia, 2014. Home Baby Spa. Jakarta: Prevarication Gefrianti, S. (2019) Profil Kesehatan Jawa Barat. Bandung. Hanifah, L. dan Febriani, M., 2014. Gambaran Pengetahuan Ibu Tentang Perkembangan Bayi Harahap, N.R. (2019) ‘Pijat Bayi Meningkatkan Berat Badan Bayi Usia 0-6 Bulan’, Jurnal Kesehatan Prima, 13(2), p. 99. Available at: https://doi.org/10.32807/jkp.v13i2.226. Hutasuhut, A.S. (2018) ‘Pengaruh Pijat Bayi terhadap Pertumbuhan (Berat Badan) Bayi Usia 1-3 Bulan di Wilayah Kerja Puskesmas Pematang Pasir Kota Tanjung Balai Tahun 2018’, Journal of Chemical Information and Modeling, 53(9), pp. 1689–1699 Irva, T.S., Hasanah, O. and Worfest, R. (2014) ‘Pengaruh Terapi Pijat terhadap Peningkatan Berat Badan Bayi’, JOM PSIK, 1(2), pp. 1–9. Julianti, 2017. Rahasia Baby Spa. Jakarta: Writerpreneur Club Kartini. 2014. Pijat Bayi. Diakses pada tanggal 25 Januari 2018 Kaisa, A. (2020) Hubungan SPA Bayi dengan Perubahan Berat Badan pada Bayi di Ummi Humairah Makassar. Sekolah Tinggi Ilmu Kesehatan Panakkukang Makassar. Kementerian Kesehatan RI. 2009. Pedoman pelaksanaan stimulasi, deteksi dan intervensi dini tumbuh kembang anak di tingkat pelayanan kesehatan dasar. Jakarta: Kementerian Kesehatan RI Keputusan Menteri Kesehatan Republik Indonesia Nomor 369/MENKES/SK/III/ 2007. Standar Profesi Bidan. Jakarta: Kepmenkes RI Khairunnisa (2021) Pengaruh Pemberian Stimulasi Pijat Bayi terhadap Berat Badan pada Bayi. Politeknik Kesehatan Kemenkes Bengkulu. Latif, H.H.A. (2017) ‘Hubungan Pijat Bayi Terhadap Kenaikan Berat Badan Pada Bayi Berat Lahir Rendah (Bblr)Di Rskia Ummi Khasanah Yogyakarta’, Journal of Chemical Information and Modeling, pp. 26–54. Lilis Suryani, I. (2017) ‘Pengaruh Pijat Bayi Terhadap Peningkatan Berat Badan Pada Bayi Di Bps Masnoni Teluk Betung Utara Kota Bandar Lampung’, The Journal of Holistic Healthcare), 11(2), pp. 72–76. Marni (2019) ‘Pengaruh Pijat Bayi terhadap Peningkatan Berat Badan pada Bayi’, Jurnal Kebidanan Indonesia, 10(1), pp. 12–18. Marni (2019) ‘Pengaruh Pijat Bayi terhadap Peningkatan Berat Badan pada Bayi’, Jurnal Kebidanan Indonesia, 10(1), pp. 12–18. Maryunani, Anik. 2011. Ilmu Kesehatan Anak Dalam Kebidanan. Jakarta: CV. Trans Info Media Maryunani, Anik. 2011. Ilmu Kesehatan Anak Dalam Kebidanan. Jakarta: CV. Trans Info Media Media Muslihatun, Wafi Nur. 2011. Asuhan Neonatus Bayi dan Balita. Yogyakarta: Fitramaya Notoatmodjo, Soekidjo. 2012. Metodologi Penelitian Kesehatan. Jakarta: Rineka Cipta Muslihatun, Wafi Nur. 2011. Asuhan Neonatus Bayi dan Balita. Yogyakarta: Fitramaya Notoatmodjo, Soekidjo. 2012. Metodologi Penelitian Kesehatan. Jakarta: Rineka Cipta Noviyanti, I.S. (2017) Perbandingan Kualitas Tidur Bayi yang dilakukan Pijat (Baby Massage) dan Bayi yang tidak dilakukan Pijat (Baby Massage) (Studi di Noviyanti, I.S. (2017) Perbandingan Kualitas Tidur Bayi yang dilakukan Pijat (Baby Massage) dan Bayi yang tidak dilakukan Pijat (Baby Massage) (Studi di \| Health Care: Journal of Community Service 10 ISSN 3046-6075 Mojo Baby Spa Kota Malang dan di Posyandu Bina Sejahtera 04 Desa Wonorejo Lawang). Universitas Muhammadiyah Malang. Prasetyono, D. . (2013). Buku Pintar Pijat Bayi. Buku Biru. ———. 2016. Pengaruh Pijat Bayi Dengan Kualitas Tidur Bayi Usia 3-6 Bulan Di Desa Bandung Kecamatan Diwek Kabupaten Jombang.Jurnal Ilmiah Kebidanan 3(1): 77–83. https://jurnal.uns.ac.id/placentum/article/viewFile/22834/16948. Prasetyono, D.S. 2017. Buku Pintar Pijat Bayi. Yogyakarta: Buku Biru Prasetyono, D.S. 2017. Buku Pintar Pijat Bayi. Yogyakarta: Buku Biru Profil Kesehatan Sumatera Utara. 2012. Tumbuh Kembang Bayi dan Balita. Medan: Dinas kesehatan Sumatera Utara y , j y gy Profil Kesehatan Sumatera Utara. 2012. Tumbuh Kembang Bayi dan Balita. 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(2020) Efektivitas Baby Massage terhadap Peningkatan Berat Badan pada Bayi Umur 5-6 Bulan. Sekolah Tinggi Ilmu Kesehatan Insan Cendekia Medika Jombang. Sinaga, A. and Laowo, N. (2020) ‘Pengaruh Pijat Bayi terhadap Kualitas Tidur Bayi Usia 0- 6 Bulan di BPM Pera Kecamatan Medan Tuntungan Tahun 2019’, Excellent Midwifery Journal, 3(1), pp. 27–31. Susila, Ida. 2017. Pengaruh Teknik Pijat Bayi Terhadap Kenaikan Berat Badan Bayi Usia 0-7 Bulan. 9(2): 14–19. Susanti, J. \| Health Care: Journal of Community Service 11 Media (2019) Efektivitas Pijat Bayi terhadap Peningkatan Motorik pada Bayi Usia 6- 12 Bulan di Klinik Hanna Kasih Medan. Institut Kesehatan Helvetia Medan. Susanto, R. (2019) ‘Modul Metodologi Penelitian (KIP 311)’. Jakarta: Universitas Esa Unggul, pp. 1–22. Triana, M. (2017) Pengaruh Pemberian Pijat Bayi Terhadap Perubahan Berat Badan Bayi Usia 1 - 4 Bulan Di Puskesmas Tamalanrea Makassar. Universitas Hasanuddin \| Health Care: Journal of Community Service 11 ISSN 3046-6075
https://openalex.org/W2109918527	https://ccsenet.org/journal/index.php/mas/article/download/476/426	English	null	Kinematics and Dynamics of a Master Maipulator	Modern applied science	2,009	cc-by	4,641	1. Introduction The first telerobotic system was developed by Raymond C. Goertz in 1940s to let an operator handle radioactive materials behind a shielded wall, at National Argonne Laboratory in the US. In the past decades, teleoperation has found applications in many areas including space technologies, underwater explorations and assistance, surgery and rehabilitations, nuclear/toxic material handling and waste disposal, military/firefighting operation, mining. Recently, the applications of teleoperation systems have been extended to training, education, entertainment, and virtual reality areas as well. As can be seen in figure 1, a teleoperation system generally has five components: operator, master, control system, slave, and environment. The main function of the system can be explained as follows: a control command is sent through the control system to the slave to make the remote manipulator perform a task as desired; to prevent damage, to reduce task completion time and to enhance performance, contact interaction from the remote site has to be transmitted to the operator. As shown in figure 1, throughout an interaction, the master mechanism must perform the dual task of position measurement and force display. The kinematics especially the forward kinematics is of great importance for the control of the telerobotic system. While, when dealing with the dynamics of robotic manipulators, frictional effects is often neglected. This paper mainly discusses the forward kinematics using Denavit-Hartenberg method, and derives the dynamic equations incorporating frictional effects in the joints. Kinematics and Dynamics of a Master Maipulator Yechu Hu College of Mechanical and Electronic Engineering Tianjin Polytechnic University Tianjin 300160, China E-mail: hyc820815@yahoo.cn Modern App Modern App Abstract I analyze the kinematics and Dynamics of Phantom Premium 1.5 made and sold by SenSable Technologies Inc., which is widely-used as the master manipulator in telerobotic systems. The forward kinematics is studied using Denavit-Hartenberg method, the manipulator Jacobian is also presented. The dynamic equations incorporating frictional effects of Premium 1.5 are derived. Keywords: Master manipulator, Kinematics analysis, Dynamic model, Frictional effects 2. Kinematics analysis Figure 2 is the photo and structural sketch of a typical master manipulator (PHANToM Premium 1.5), which can provide the operator with 3 DOF motion and three dimensional force feedbacks. As shown in figure 2(b), Premium 1.5 consists of three rotational joints, zero configuration of Premium 1.5 is shown in figure 2(b), spatial frame and tool frame are superposed in zero configuration. Figure 3 depicts the side and top views of the configuration of Premium 1.5. And, the reference frames are shown in figure 4, the D-H parameter is listed in table 1, where ai represents the distance from Zi to Zi+1 measured along Xi, ai represents the distance from Zi to Zi+1 measured along Xi, αi represents the angle from Zi to Zi+1 measured about Xi, αi represents the angle from Zi to Zi+1 measured about Xi, di represents the distance from Xi-1 to Xi along Zi, di represents the distance from Xi-1 to Xi along Zi, resents the distance from Xi-1 to Xi along Zi, θi represents the angle from Xi-1 to Xi measured about Zi. Vol. 3, No. 3 Modern Applied Science And the final transformation matrix can be written in equation 2 with some mediated matrixes from 0 wT to 3 t T omitted. And the final transformation matrix can be written in equation 2 with some mediated matrixes from 0 wT to 3 t T omitted. 0 1 2 3 0 1 2 3 ( ) ( ) 1 0 s w t t T T T T T T θ θ ⎡ ⎤ = = ⎢ ⎥ ⎣ ⎦ R p (2) 0 1 2 3 0 1 2 3 ( ) ( ) 1 0 s w t t T T T T T T θ θ ⎡ ⎤ = = ⎢ ⎥ ⎣ ⎦ R p (2) Where (2) Where Where Where 1 1 3 1 3 3 3 1 1 3 1 3 cos sin sin sin cos ( ) 0 cos sin sin cos sin cos cos θ θ θ θ θ θ θ θ θ θ θ θ − ⎛ ⎞ ⎜ ⎟ = ⎜ ⎟ ⎜ ⎟ − − ⎝ ⎠ R θ (3) 1 1 2 2 3 2 1 2 2 3 1 1 1 2 2 3 sin ( cos sin ) ( ) sin cos cos ( cos sin ) l l l l l l l l θ θ θ θ θ θ θ θ + ⎛ ⎞ ⎜ ⎟ = + − ⎜ ⎟ ⎜ ⎟ −+ + ⎝ ⎠ p θ (4) 1 1 3 1 3 3 3 1 1 3 1 3 cos sin sin sin cos ( ) 0 cos sin sin cos sin cos cos θ θ θ θ θ θ θ θ θ θ θ θ − ⎛ ⎞ ⎜ ⎟ = ⎜ ⎟ ⎜ ⎟ − − ⎝ ⎠ R θ (3) 1 1 2 2 3 2 1 2 2 3 1 1 1 2 2 3 sin ( cos sin ) ( ) sin cos cos ( cos sin ) l l l l l l l l θ θ θ θ θ θ θ θ + ⎛ ⎞ ⎜ ⎟ = + − ⎜ ⎟ ⎜ ⎟ −+ + ⎝ ⎠ p θ (4) (3) (4) Except the forward kinematics of the manipulator, the velocity relationship between the rotational joints and the end effector is often concerned. θi represents the angle from Xi-1 to Xi measured about Zi. The general transformation matrix 1 comes directly from the D-H parameters (Craig, 2005 & Murray, 1998). The general transformation matrix 1 comes directly from the D-H parameters (Craig, 2005 & Murray, 1 1 1 1 1 1 1 1 1 1 cos sin 0 sin cos cos cos sin sin sin sin cos sin cos cos 0 0 0 1 i i i i i i i i i i i i i i i i i i i a d T d θ θ θ α θ α α α θ α θ α α α − − − − − − − − − − ⎡ ⎤ ⎢ ⎥ − − ⎢ ⎥ = ⎢ ⎥ ⎢ ⎥ ⎣ ⎦ (1) 1 1 1 1 1 1 1 1 1 1 cos sin 0 sin cos cos cos sin sin sin sin cos sin cos cos 0 0 0 1 i i i i i i i i i i i i i i i i i i i a d T d θ θ θ α θ α α α θ α θ α α α − − − − − − − − − − ⎡ ⎤ ⎢ ⎥ − − ⎢ ⎥ = ⎢ ⎥ ⎢ ⎥ ⎣ ⎦ (1) (1) 155 Vol. 3, No. 3 Vol. 3, No. 3 1 1 1 2 1 2 1 2 1 2 1 1 2 1 1 2 1 2 1 2 1 1 sin sin sin ( sin ) 0 cos (cos cos ) 0 cos sin cos ( sin ) ( ) 0 0 cos 1 0 0 0 0 sin s l l l l l l l l l θ θ θ θ θ θ θ θ θ θ θ θ θ − + ⎛ ⎞ ⎜ ⎟ − ⎜ ⎟ ⎜ ⎟ − + = ⎜ ⎟ − ⎜ ⎟ ⎜ ⎟ ⎜ ⎟ ⎜ ⎟ ⎝ ⎠ J θ (5) 1 2 2 3 1 2 3 1 2 3 2 3 3 cos sin 0 0 0 cos( ) 0 0 sin( ) ( ) 0 0 1 cos 0 0 sin 0 0 b l l l l l θ θ θ θ θ θ θ θ + ⎛ ⎞ ⎜ ⎟ − ⎜ ⎟ ⎜ ⎟ − − = ⎜ ⎟ − ⎜ ⎟ ⎜ ⎟ ⎜ ⎟ ⎜ ⎟ ⎝ ⎠ J θ (6) er Manipulator 1 1 1 2 1 2 1 2 1 2 1 1 2 1 1 2 1 2 1 2 1 1 sin sin sin ( sin ) 0 cos (cos cos ) 0 cos sin cos ( sin ) ( ) 0 0 cos 1 0 0 0 0 sin s l l l l l l l l l θ θ θ θ θ θ θ θ θ θ θ θ θ − + ⎛ ⎞ ⎜ ⎟ − ⎜ ⎟ ⎜ ⎟ − + = ⎜ ⎟ − ⎜ ⎟ ⎜ ⎟ ⎜ ⎟ ⎜ ⎟ ⎝ ⎠ J θ (5) 1 2 2 3 1 2 3 1 2 3 2 3 3 cos sin 0 0 0 cos( ) 0 0 sin( ) ( ) 0 0 1 cos 0 0 sin 0 0 b l l l l l θ θ θ θ θ θ θ θ + ⎛ ⎞ ⎜ ⎟ − ⎜ ⎟ ⎜ ⎟ − − = ⎜ ⎟ − ⎜ ⎟ ⎜ ⎟ ⎜ ⎟ ⎜ ⎟ ⎝ ⎠ J θ (6) 3 D i M d l f th M t M i l t (5) (6) 3. Dynamic Model of the Master Manipulator M. C. Vol. 3, No. 3 So, the spatial and body manipulator Jacobian is written here, moreover, they can be used to describe the relationship between the end point wrench and the joint torque. Except the forward kinematics of the manipulator, the velocity relationship between the rotational joints and the end effector is often concerned. So, the spatial and body manipulator Jacobian is written here, moreover, they can be used to describe the relationship between the end point wrench and the joint torque. Vol. 3, No. 3 Cavusoglu (Cavusoglu, 2001) identified the mechanical structure of Premium 1.5 into seven segments A through G shown on figure 5. Note that the spatial frame used in dynamic calculations is centered at the intersection point of three axises of rotational joints. M. C. Cavusoglu (Cavusoglu, 2001) identified the mechanical structure of Premium 1.5 into seven segments A through G shown on figure 5. Note that the spatial frame used in dynamic calculations is centered at the intersection point of three axises of rotational joints. And he calculated the kinetic and potential energy of each segment in the spatial frame, then wrote the dynamic equation of the system using Lagrange method. And he calculated the kinetic and potential energy of each segment in the spatial frame, then wrote the dynamic equation of the system using Lagrange method. ( ) ( , ) ( ) M C N && & & + + = θ θ θ θ θ θ τ (7) (7) In equation 7, ( ) M && θ θ is inertial force, ( , ) C & & θ θ θ is Coriolis and centrifugal force, ( ) N θ is gravitational force, τ is the vector of joint torque, each element of the matrix of M, C, and N is listed in Cavusoglu’s paper (Cavusoglu, 2001). Note that the inertial force, Coriolis and centrifugal force and gravitational force are highly non-linear, making it difficult for dynamic parameter identification and controller algorithm design (McJunkin, 2007). 4. Inclusion of Frictional Effects Including the friction effect of the device and combing equation 8 with 12, I could write f f Including the friction effect of the device and combing equation 8 with 12, I could write ( ) , ,& && = τ θ θ θ π Y (15) ( ) , ,& && = τ θ θ θ π Y (15) ( ) , ,& && = τ θ θ θ π Y ( ) , ,& && = τ θ θ θ π Y (15) where d f M ⎡ ⎤ ⎢ ⎥ ⎣ ⎦ Y = Y Y (16) T d f M ⎡ ⎤ = ⎢ ⎥ ⎣ ⎦ π π π (17) (15) where d f M ⎡ ⎤ ⎢ ⎥ ⎣ ⎦ Y = Y Y (16) T d f M ⎡ ⎤ = ⎢ ⎥ ⎣ ⎦ π π π (17) (16) T d f M ⎡ ⎤ = ⎢ ⎥ ⎣ ⎦ π π π (17) (17) Equation 15 is named the dynamic model of Premium 1.5 with joint friction. Each of the element of the parameter vector is identified as shown in table 2. Vol. 3, No. 3 3, No. 3 Vol. 3, No. 3 Modern Applied Science 11 1 1 4 1 2 a b cd m gl m gl m gl π = + − (10k) 12 2 3 5 1 2 a b ef m gl m gl m gl π = + − (10l) (10k) 12 2 3 5 1 2 a b ef m gl m gl m gl π = + − (10l) (10l) The physical and geometric parameters Iaxx, Iayy, Iazz, Ibxx, Ibyy, Ibzz, Icdxx, Icdyy, Icdzz, Iefxx, Iefyy, Iefzz, Igyy, ma, mb, mcd, mef, l1, l2, l3, l4, l5 can be found in the literature (Cavusoglu, 2001), while g is the acceleration gravity. The physical and geometric parameters Iaxx, Iayy, Iazz, Ibxx, Ibyy, Ibzz, Icdxx, Icdyy, Icdzz, Iefxx, Iefyy, Iefzz, Igyy, ma, mb, mcd, mef, l1, l2, l3, l4, l5 can be found in the literature (Cavusoglu, 2001), while g is the acceleration gravity. 5. Conclusion In telerobotic systems, the kinematics especially the forward kinematics is of great importance for the control of teleoperaton, frictional effects is often neglected in dynamic equations. This paper mainly discusses the forward kinematics using D-H method, the manipulator Jacobian is also presented. And I derive the dynamic equations incorporating frictional effects in the joints. Vol. 3, No. 3 So, equation 7 could be linearly parameterized as follows: quation 7 could be linearly parameterized as follows: ( ) , , d d d & && = τ θ θ θ π Y (8) ( ) , , d d d & && = τ θ θ θ π Y (8) where Y is the regressor matrix and π is the vector 12 dynamic parameters defined as: where Y is the regressor matrix and π is the vector 12 dynamic parameters defined as: 156 March, 2009 Modern Applied Science 12 3 1 3 1 3 13 3 1 3 1 3 15 2 1 2 1 2 16 2 1 2 1 2 17 1 18 2 1 2 1 2 19 3 1 3 1 3 (1 cos2 ) 2sin 2 (1 cos2 ) 2sin 2 (1 cos2 ) 2sin 2 (1 cos2 ) 2sin 2 (1 cos2 ) 2sin 2 (1 cos2 ) 2sin 2 y y y y y y y && & & && & & && & & && & & && && & & && & & θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ = + − = − + = + − = − + = = + − = − + 10 2 3 1 2 3 1 2 2 3 1 3 2cos sin 2sin sin 2cos cos y && & & & & θ θ θ θ θ θ θ θ θ θ θ = − + (9a) 12 3 1 3 1 3 13 3 1 3 1 3 15 2 1 2 1 2 16 2 1 2 1 2 17 1 18 2 1 2 1 2 19 3 1 3 1 3 (1 cos2 ) 2sin 2 (1 cos2 ) 2sin 2 (1 cos2 ) 2sin 2 (1 cos2 ) 2sin 2 (1 cos2 ) 2sin 2 (1 cos2 ) 2sin 2 y y y y y y y && & & && & & && & & && & & && && & & && & & θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ = + − = − + = + − = − + = = + − = − + 10 2 3 1 2 3 1 2 2 3 1 3 2cos sin 2sin sin 2cos cos y && & & & & θ θ θ θ θ θ θ θ θ θ θ = − + (9a) (9a) 18 2 1 2 1 2 19 3 1 3 1 3 (1 cos2 ) 2sin 2 (1 cos2 ) 2sin 2 y y && & & && & & θ θ θ θ θ θ θ θ θ θ = + − = − + 24 2 2 25 2 1 2 26 2 1 2 28 2 2 1 2 210 2 3 3 2 3 1 2 2 3 3 211 2 sin 2 sin 2 2 sin 2 sin( ) sin sin cos( ) cos y y y y y y && & & && & && & & θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ = = = − = + = − − + + − = 31 3 2 32 3 1 2 33 3 1 2 39 3 3 1 2 310 2 3 2 2 3 1 2 2 3 2 312 3 sin 2 sin 2 2 sin 2 sin( ) cos cos cos( ) sin y y y y y y && & & && & && & & θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ = = = − = − = − − − + − = 24 2 2 25 2 1 2 26 2 1 2 28 2 2 1 2 210 2 3 3 2 3 1 2 2 3 3 211 2 sin 2 sin 2 2 sin 2 sin( ) sin sin cos( ) cos y y y y y y && & & && & && & & θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ = = = − = + = − − + + − = 31 3 2 32 3 1 2 33 3 1 2 39 3 3 1 2 310 2 3 2 2 3 1 2 2 3 2 312 3 sin 2 sin 2 2 sin 2 sin( ) cos cos cos( ) sin y y y y y y && & & && & && & & θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ θ = = = − = − = − − − + − = (9b) (9c) 312 3 sin y θ = and 1 axx efxx I I π = + ( ) 2 1 2 ayy efyy I I π = + ( ) 3 1 2 azz efzz I I π = + 4 bxx cdxx I I π = + ( ) 5 1 2 byy cdyy I I π = + ( ) 6 1 2 bzz cdzz I I π = + 7 gyy I π = 2 2 8 1 1 1 1 2 8 a b m l m l π = + 2 2 9 2 3 1 1 8 2 a b m l m l π = + 10 1 2 1 3 1 1 2 2 a b m l l m l l π = + 1 axx efxx I I π = + ( ) 2 1 2 ayy efyy I I π = + ( ) 3 1 2 azz efzz I I π = + 4 bxx cdxx I I π = + ( ) 5 1 2 byy cdyy I I π = + ( ) 6 1 2 bzz cdzz I I π = + 7 gyy I π = 2 2 8 1 1 1 1 2 8 a b m l m l π = + 2 2 9 2 3 1 1 8 2 a b m l m l π = + 10 1 2 1 3 1 1 2 2 a b m l l m l l π = + (10a) (10j) 157 Vol. 4. Inclusion of Frictional Effects Friction effect in the joints of Premium 1.5 is neglected in Equation 7 and 8, as a matter of fact, the friction effect should not be ignored. Here I use Coulomb friction plus viscous damping (Armstrong, 1994 & Olsson, 1998) to include friction effect in the dynamic model of the device (Tahmasebi, 2005). 4. Inclusion of Frictional Effects sgn( ) f fc fv & & = + τ π θ π θ (11) sgn( ) f fc fv & & = + τ π θ π θ (11) sgn( ) f fc fv & & = + τ π θ π θ (11) or 1 1 1 1 1 2 2 2 2 2 3 3 3 3 3 sgn( ) sgn( ) sgn( ) f fc fv f f fc fv f f f fc fv & & & & & & τ τ θ τ θ τ τ θ τ θ τ τ θ τ θ ⎡ ⎤ ⎡ ⎤ + ⎢ ⎥ ⎢ ⎥ ⎢ ⎥ ⎢ ⎥ = = + = ⎢ ⎥ ⎢ ⎥ ⎢ ⎥ ⎢ ⎥ + ⎢ ⎥ ⎢ ⎥ ⎣ ⎦ ⎣ ⎦ τ π Y (12) 1 1 1 1 1 2 2 2 2 2 3 3 3 3 3 sgn( ) sgn( ) sgn( ) f fc fv f f fc fv f f f fc fv & & & & & & τ τ θ τ θ τ τ θ τ θ τ τ θ τ θ ⎡ ⎤ ⎡ ⎤ + ⎢ ⎥ ⎢ ⎥ ⎢ ⎥ ⎢ ⎥ = = + = ⎢ ⎥ ⎢ ⎥ ⎢ ⎥ ⎢ ⎥ + ⎢ ⎥ ⎢ ⎥ ⎣ ⎦ ⎣ ⎦ τ π Y (12) where 1 1 2 2 3 3 sgn( ) 0 0 0 0 0 sgn( ) 0 0 0 0 0 sgn( ) 0 0 f & & & & & & θ θ θ θ θ θ ⎛ ⎞⎟ ⎜ ⎟ ⎜ ⎟ ⎜ ⎟ = ⎜ ⎟ ⎜ ⎟ ⎜ ⎟⎟ ⎜ ⎟ ⎜⎝ ⎠ Y (13) 1 2 3 1 2 3 T f f f f f f f π π π π π π ⎡ ⎤ = ⎢ ⎥ ⎣ ⎦ π (14) 1 1 1 1 1 2 2 2 2 2 3 3 3 3 3 sgn( ) sgn( ) sgn( ) f fc fv f f fc fv f f f fc fv & & & & & & τ τ θ τ θ τ τ θ τ θ τ τ θ τ θ ⎡ ⎤ ⎡ ⎤ + ⎢ ⎥ ⎢ ⎥ ⎢ ⎥ ⎢ ⎥ = = + = ⎢ ⎥ ⎢ ⎥ ⎢ ⎥ ⎢ ⎥ + ⎢ ⎥ ⎢ ⎥ ⎣ ⎦ ⎣ ⎦ τ π Y (12) (12) where 1 1 2 2 3 3 sgn( ) 0 0 0 0 0 sgn( ) 0 0 0 0 0 sgn( ) 0 0 f & & & & & & θ θ θ θ θ θ ⎛ ⎞⎟ ⎜ ⎟ ⎜ ⎟ ⎜ ⎟ = ⎜ ⎟ ⎜ ⎟ ⎜ ⎟⎟ ⎜ ⎟ ⎜⎝ ⎠ Y (13) (13) 1 2 3 1 2 3 T f fc fc fc fv fv fv π π π π π π ⎡ ⎤ = ⎢ ⎥ ⎣ ⎦ π (14) (14) Yf is the regressor matrix, πf is 6 dimensional parameter vector. References Armstrong Brian, Dupont Pierre, Canudas de Wit C. ( 1994). A Survey of Models, Analysis Tools and Compensation Methods for the Control of Machines with Friction[J]. Automatica, 30(7): 1083-1138. Armstrong Brian, Dupont Pierre, Canudas de Wit C. ( 1994). A Survey of Models, Analysis Tools a Methods for the Control of Machines with Friction[J]. Automatica, 30(7): 1083-1138. Cavusoglu M C, Feygin D. ( 2001). Kinematics and Dynamics of Phantom(TM) Model 1.5 Haptic Interface. [R]. Berkeley: University of California at Berkeley, Electronics Research Laboratory Memo M01/15. Cavusoglu M C, Feygin D. ( 2001). Kinematics and Dynamics of Phantom(TM) Model 1.5 Haptic Interface. [R]. Berkeley: University of California at Berkeley, Electronics Research Laboratory Memo M01/15. Craig J J. Introduction to Robotics. (2005). Mechanics and Control (3rd Editon)[M]. Upper Saddle River: Addison-Wesley. Craig J J. Introduction to Robotics. (2005). Mechanics and Control (3rd Editon)[M]. Upper Saddle River: Addison-Wesley. Haptic Device. [A]. Proceedings of the 2005 IEEE Conference on Control Applications[C]. Toronto, Canada: IEEE, 1251-1256. Haptic Device. [A]. Proceedings of the 2005 IEEE Conference on Control Applications[C]. Toronto, Canada: IEEE, 1251-1256. McJunkin S T. (2007). Transparency Improvement for Haptic Interfaces. [D]. Houston: Rice University McJunkin S T. (2007). Transparency Improvement for Haptic Interfaces. [D]. Houston: Rice University. 158 March, 2009 Modern Applied Science Olsson H, Astrom K J, Canudas de Wit C, et al. (1998). Friction Models and Friction Compensation[J]. European Journal of Control, 4(3): 176-195. Tahmasebi A M, Taati B, Mobasser F, et al. (2005). Dynamic Parameter Identification and Analysis of a PHANToM(TM) Olsson H, Astrom K J, Canudas de Wit C, et al. (1998). Friction Models and Friction Compensation[J]. European Journal of Control, 4(3): 176-195. Tahmasebi A M, Taati B, Mobasser F, et al. (2005). Dynamic Parameter Identification and Analysis of a PHANToM(TM) Journal of Control, 4(3): 176-195. Tahmasebi A M, Taati B, Mobasser F, et al. (2005). Dynamic Parameter Identification and Analysis of a PHANToM(TM) Table 1. D-H parameter of Premium 1.5 Frame αi-1 ai-1 θi di 0 -π/2 0 -π/2 0 1 0 -l1 θ1 l2 2 π/2 0 θ2 0 3 0 l1 -π/2-θ2+θ3 0 tool -π/2 l2 π/2 0 Table 2. Dynamic Parameter of Premium 1.5(×10-4) π1 π2 π3 π4 π5 π6 7.5784 0.3154 3.3572 12.0490 5.5095 0.2981 π7 Π8 π9 π10 π11 π12 11.8700 6.1644 0.8572 4.5693 -160 -739 π13 π14 π15 π16 π17 π18 707 251 248 -57 -35 -5 Ope. Mas. Con. Sys. Sla. References Tip Force Position Command Env. θ1 θ3 θ2 l1 l2 z x z x y y S T Figure 1. Diagram of the master-slave teleoperation system Figure 2. Photo and structural sketch of Premium1.5 x z3 zt xt π/2+θ2-θ3 Table 1. D-H parameter of Premium 1.5 θ1 θ3 θ2 l1 l2 z x z x y y S T Figure 2. Photo and structural sketch of Premium1.5 θ1 θ3 θ2 l1 l2 z x z x y y S T Figure 2. Photo and structural sketch of Premium1.5 Figure 2. Photo and structural sketch of Premium1.5 Figure 1. Diagram of the master-slave teleoperation system zs, x0 xs z0 z1 x1 z2 x3 z3 zt xt l1 l2 l1 l2 x2 θ2 π/2+θ2-θ3 θ3 θ2 z x x z y S T θ2 θ3 y θ1 z x y S z x T zs, x0 xs z0 z1 x1 z2 x3 z3 zt xt l1 l2 l1 l2 x2 θ2 π/2+θ2-θ3 Figure 3. Configuration of Premium 1.5 Figure 4. Reference frames of Premium1.5 θ3 θ2 z x x z y S T θ2 θ3 y θ1 z x y S z x T Figure 3. Configuration of Premium 1.5 Figure 3. Configuration of Premium 1.5 Figure 4. Reference frames of Premium1.5 159 Vol. 3, No. 3 Vol. 3, No. 3 Modern Applied Science θ1 θ3 θ2 y x z S A C B D E F G l1 l2 l3 Figure 5. Segments of Premium 1.5 θ1 θ3 θ2 y x z S A C B D E F G l1 l2 l3 Figure 5. Segments of Premium 1.5 Figure 5. Segments of Premium 1.5 160
https://openalex.org/W2885134534	https://gfzpublic.gfz-potsdam.de/pubman/item/item_3635895_3/component/file_3641891/3635895.pdf	English	null	Towards risk-based flood management in highly productive paddy rice cultivation – concept development and application to the Mekong Delta	Natural hazards and earth system sciences	2,018	cc-by	16,509	Nguyen Van Khanh Triet1,2, Nguyen Viet Dung1, Bruno Merz1,3, and Heiko Apel1 1GFZ German Research Centre for Geosciences, Section 5.4 Hydrology, Potsdam, 14473, Germany 2SIWRR Southern Institute of Water Resources Research, Ho Chi Minh City, Vietnam 3Institute of Earth and Environmental Science, University of Potsdam, Potsdam, 14476, Germany Correspondence: Nguyen Van Khanh Triet (triet@gfz-potsdam.de) Correspondence: Nguyen Van Khanh Triet (triet@gfz-potsdam.de) Correspondence: Nguyen Van Khanh Triet (triet@gfz-potsdam.de) Received: 25 July 2018 – Discussion started: 6 August 2018 Accepted: 14 October 2018 – Published: 5 November 2018 Abstract. Flooding is an imminent natural hazard threaten- ing most river deltas, e.g. the Mekong Delta. An appropri- ate ﬂood management is thus required for a sustainable de- velopment of the often densely populated regions. Recently, the traditional event-based hazard control shifted towards a risk management approach in many regions, driven by inten- sive research leading to new legal regulation on ﬂood man- agement. However, a large-scale ﬂood risk assessment does not exist for the Mekong Delta. Particularly, ﬂood risk to paddy rice cultivation, the most important economic activ- ity in the delta, has not been performed yet. Therefore, the present study was developed to provide the very ﬁrst insight into delta-scale ﬂood damages and risks to rice cultivation. The ﬂood hazard was quantiﬁed by probabilistic ﬂood haz- ard maps of the whole delta using a bivariate extreme value statistics, synthetic ﬂood hydrographs, and a large-scale hy- draulic model. The ﬂood risk to paddy rice was then quan- tiﬁed considering cropping calendars, rice phenology, and harvest times based on a time series of enhanced vegeta- tion index (EVI) derived from MODIS satellite data, and a published rice ﬂood damage function. The proposed concept provided ﬂood risk maps to paddy rice for the Mekong Delta in terms of expected annual damage. The presented concept can be used as a blueprint for regions facing similar prob- lems due to its generic approach. Furthermore, the changes in ﬂood risk to paddy rice caused by changes in land use currently under discussion in the Mekong Delta were esti- mated. Two land-use scenarios either intensifying or reduc- ing rice cropping were considered, and the changes in risk were presented in spatially explicit ﬂood risk maps. The ba- sic risk maps could serve as guidance for the authorities to develop spatially explicit ﬂood management and mitigation plans for the delta. The land-use change risk maps could fur- ther be used for adaptive risk management plans and as a basis for a cost–beneﬁt of the discussed land-use change sce- narios. Additionally, the damage and risks maps may sup- port the recently initiated agricultural insurance programme in Vietnam. Nat. Hazards Earth Syst. Sci., 18, 2859–2876, 2018 https://doi.org/10.5194/nhess-18-2859-2018 © Author(s) 2018. This work is distributed under the Creative Commons Attribution 4.0 License. 1 Introduction Characterized by low topography, the Mekong Delta (MD) is subjected to ﬂooding caused by high river discharge, tidal backwater effects, and storm surges. Floods in the Mekong Delta are annual events, mainly triggered by the Asian mon- soons, but also by tropical cyclones (typhoons). On the pos- itive side, ﬂoods bring various beneﬁts to the MD with an estimated annual value of USD 8–10 billion (MRC, 2012). These beneﬁts include provision of sediment to counter delta subsidence, increase in wild ﬁsh catch and enhancement of soil fertility through deposited sediment (Manh et al., 2014). On the other hand, extreme ﬂoods can result in extensive damages as recorded during the ﬂoods in 2011 and 2000. For example, the 2000 ﬂood, considered as a 20-year ﬂood (Le et al., 2007), resulted in over 450 fatalities and economic losses of USD 250 million (MRC, 2012). Recent studies sug- gest that the frequency of such extreme events is likely to in- crease (Delgado et al., 2010; Hirabayashi et al., 2013). For instance, the 100-year ﬂood in the Mekong basin in the 20th Towards risk-based ﬂood management in highly productive paddy rice cultivation – concept development and application to the Mekong Delta Nguyen Van Khanh Triet1,2, Nguyen Viet Dung1, Bruno Merz1,3, and Heiko Apel1 1GFZ German Research Centre for Geosciences, Section 5.4 Hydrology, Potsdam, 14473, Germany 2SIWRR Southern Institute of Water Resources Research, Ho Chi Minh City, Vietnam 3Institute of Earth and Environmental Science, University of Potsdam, Potsdam, 14476, Germany Nguyen Van Khanh Triet1,2, Nguyen Viet Dung1, Bruno Merz1,3, and Heiko Apel1 1GFZ German Research Centre for Geosciences, Section 5.4 Hydrology, Potsdam, 14473, Germany 2SIWRR Southern Institute of Water Resources Research, Ho Chi Minh City, Vietnam 3Institute of Earth and Environmental Science, University of Potsdam, Potsdam, 14476, Germany N. V. K. Triet et al.: Towards risk-based ﬂood management in highly productive paddy rice cultivation yield reduction as consequence of physical contact with ﬂoodwater. The methodological nov- elty is the detailed consideration of cropping calendar and plant phenology in combination with synthetic probabilis- tic ﬂood hydrographs mapping different ﬂood regimes of the Mekong. By this, the important aspect of plant phenology and temporal occurrence of ﬂood peaks is introduced into the probabilistic ﬂood risk assessment approach. The majority of the literature on ﬂood hazard assessments for the VMD focusses on changes in delta inundation haz- ards driven by upstream infrastructure development (e.g. hy- dropower dams), local ﬂood control (e.g. dyke systems), cli- mate change impacts, and sea level rise by hydrodynamic modelling (Le et al., 2007; Van, 2009; Dinh et al., 2012; Van et al., 2012; Toan, 2014; Triet et al., 2017; Dang et al., 2018). A comprehensive ﬂood hazard analysis for the whole MD can only be found in the study of Dung et al. (2015). They developed different copula-based bivariate statistical models to quantify the probability of joint occurrence of peak dis- charge and ﬂood volume of the Mekong River at Kratie, com- monly deﬁned as the upstream entrance of the MD. Apel et al. (2016) presented a detailed probabilistic ﬂuvial–pluvial ﬂood hazard assessment for the city of Can Tho in the centre of VMD using the results of Dung et al. (2015) as a boundary condition for a ﬂuvial 2-D urban ﬂood model. The ﬂood hazard is quantiﬁed following the methodology of Dung et al. (2015). To obtain spatially explicit ﬂood haz- ard maps, a large-scale hydraulic inundation model is driven by synthetic ﬂood discharge time series, which are associ- ated with probabilities of occurrences. Based on these haz- ard maps, crop damages are estimated using published dam- age functions, and explicitly considering the temporal occur- rence of high water levels, the cropping calendar, and plant phenology. Finally, the consequences of two land-use devel- opment scenarios proposed in the Mekong Delta Plan were estimated in terms of crop damages by ﬂoods. Studies on ﬂood damage are rare for the VMD. Damage assessments require rather extensive data sets, such as land- use, cropping systems and crop timing, asset values, damage functions for the different land-use types, and damage data to calibrate and validate the damage models. Consequently, ﬂood damage and risk assessments have been conducted on the scale of districts or provinces only. For example, Chinh et al. N. V. K. Triet et al.: Towards risk-based ﬂood management in highly productive paddy rice cultivation N. V. K. Triet et al.: Towards risk-based ﬂood management in highly productive paddy rice cultivation 2860 century is projected to occur every 10–20 years in the 21st century due to impacts of climate change (Hirabayashi et al., 2013). Therefore, assessing hazards and risks induced by ex- treme ﬂoods is a crucial task for developing ﬂood manage- ment strategies and climate change adaptation measures. tions were developed using observed maximum water levels at two gauging stations, and statistical damage data for the period of 2000–2007. These depth-damage functions were then applied to estimate ﬂood losses for the period 1910– 2006. Since this model requires only the water level as in- put, it can provide a quick assessment of ﬂood damage to rice crops. Applying these damage functions to the current situation is, however, not recommended due to the massive changes in land use and cropping system over the last two decades. For example, Le et al. (2018) calculated an annual rate of change in land use in the VMD of 14.9 % during 2001–2012. Hence, to our best knowledge, large-scale eco- nomic assessments of ﬂood damage to agriculture crops for the whole delta and appropriate damage models are missing. g g p Traditionally, ﬂood management in the Vietnamese Mekong Delta (VMD) has focussed on engineering solutions aiming at ﬂood control. Structural ﬂood defence measures, such as sluice gates and dyke lines, were implemented across the whole delta. The water level of the ﬂood in 2000 was commonly chosen as the design ﬂood event. Flood risk as- sessments, taking into account not only ﬂood probabilities and water levels, but also ﬂood losses, were not undertaken to support ﬂood management. Recently, non-structural mea- sures (e.g. shifting of cropping calendar) have gained more interest. This alteration is in agreement with the global trend of moving from ﬂood hazard control toward ﬂood risk man- agement (Merz et al., 2010). By deﬁnition, risk assessment is the evaluation of the frequency and magnitude of ﬂoods, or ﬂood hazard, and their consequences. Hence, damage assess- ment is an essential task for the transition from traditional hazard control to ﬂood risk management. Against this background, we provide the ﬁrst large-scale ﬂood risk assessment for the agricultural sector covering the whole VMD. Our assessment is focussed on paddy rice, the predominant land-use type in the delta. We limit our calcu- lation to direct losses, i.e. N. V. K. Triet et al.: Towards risk-based ﬂood management in highly productive paddy rice cultivation (2017) developed a model to estimate ﬂood losses us- ing surveyed damage data of the ﬂood in 2011 and assessed ﬂood risk for an urban district of Can Tho city. Similarly, publications on agro-economic ﬂood damage are practically non-existent for the VMD. The literature search on damage to agriculture in the VMD resulted in a single publication, a report by the Mekong River Commission (MRC, 2009). The MRC developed a loss model for paddy rice in two provinces in the VMD: An Giang and Dong Thap. Two damage func- Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. 2.1 Study area The VMD covers an area of approximately 40 500 km2 in the south of Vietnam, where the Mekong River discharges into the South China Sea through a number of estuary branches. The landscape is dominated by ﬂat ﬂoodplains formed by deposited river sediments. Floodplain sedimentation is esti- mated to approximately 9.5 mmyr−1 on average (Manh et al., 2013). The properties of the deposits and the associated nutrients in combination with the tropical climate form ideal conditions for high agricultural productivity. Ofﬁcial statis- tics indicate that over 64 % of the delta (2.6 million ha) is used for agriculture, with rice as the dominant crop (three- quarters of the total cultivation land), followed by orchard www.nat-hazards-earth-syst-sci.net/18/2859/2018/ Nat. Hazards Earth Syst. Sci., 18, 2859–2876, 2018 N. V. K. Triet et al.: Towards risk-based ﬂood management in highly productive paddy rice cultivation et et al.: Towards risk-based ﬂood management in highly productive paddy rice cultivation 2861 Province border River/canal Shallow inundation region Deep inundation region Vietnamese Mekong Delta ! Tidal gauge ! Hydrological gauge Tha La - Tra Su dams Xuan To weir Country border Nu Da Lancang Hong Yalong Tuo Za Chi Zi Dadu Yom Mun Han Ou Ayeyarwady Ma Wu Jialing Jinsha Ba Zuo Ping Kong You Salween Chang Jiang Mekong Yuan N’Mai Chindwin Tongtian Dam Yu Nanpan Kaladan Duliu Luhit Mytinge Balak Min Hongshui Mae Klong Yarlung Krong Dihang Rong Liu Srepok Sreng Ngun Zayü Chao Phraya Irrawaddy Delta Tonle Sap 0 250 500 125 km 0 m 5000 m 105°0’0"E 105°0’0" E 100°0’0"E 100°0’0" E 95°0’0"E 95°0’0" E 30°0’0" N 30°0’0" N 25°0’0" N 25°0’0" N 20°0’0" N 20°0’0" N 15°0’0" N 15°0’0" N 10°0’0" N 10°0’0" N Lower Mekong Basin Upper Mekong Basin Tibetan Plateau Gulf of Thailand Andaman Sea South China Sea ¬ ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 2.1 Study area 9 8 7 6 3 4 5 2 1 17 19 18 16 12 11 10 15 14 13 106°0’0" E 106°0’0" E 105°0’0" E 105°0’0" E 11°0’0" N 11°0’0" N 10°0’0" N 10°0’0" N 9°0’0" N 9°0’0" N 0 50 100 25 km South China Sea Thailand Gulf of Bassac River Tra Vinh Tien Giang Kien Giang Ca Mau Can Tho Soc Trang Bac Lieu Ben Tre Vinh Long Long An An Giang Thap Dong Hau Giang Mekong River Long Xuyen Quadrangle (LXQ) Plain of Reeds (PoR) ¬ Kratie (a) (b) Figure 1. Geographical location of the Mekong Basin (a). The Vietnamese Mekong Delta and its ﬂood-prone area (b). Deep-inundation region (above 1.5 m) marked in yellow, shallow-inundation region (below 1.5 m) presented in green. Red dots are locations of tidal gauges. Blue dots are locations of water level gauges. The numbers above blue and red dots present station codes. ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 9 8 7 6 3 4 5 2 1 17 19 18 16 12 11 10 15 14 13 106°0’0" E 106°0’0" E 105°0’0" E 105°0’0" E 11°0’0" N 11°0’0" N 10°0’0" N 10°0’0" N 9°0’0" N 9°0’0" N 0 50 100 25 km South China Sea Thailand Gulf of Bassac River Tra Vinh Tien Giang Kien Giang Ca Mau Can Tho Soc Trang Bac Lieu Ben Tre Vinh Long Long An An Giang Thap Dong Hau Giang Mekong River Long Xuyen Quadrangle (LXQ) Plain of Reeds (PoR) ¬ (b) 20°0’0" N Figure 1. Geographical location of the Mekong Basin (a). The Vietnamese Mekong Delta and its ﬂood-prone area (b). Deep-inundation region (above 1.5 m) marked in yellow, shallow-inundation region (below 1.5 m) presented in green. Red dots are locations of tidal gauges. Blue dots are locations of water level gauges. The numbers above blue and red dots present station codes. farms and sugar cane (GSO, 2015). Covering only 12 % of the total land area of Vietnam, the delta contributes 52 % to the national food production and over 80 % to the Vietnamese rice export (GSO, 2015). mally falls from mid-July to mid-August. The second, often higher peak, arrives from September to October. Floodwater from Cambodia enters the VMD via three main routes. The mainstream branches of the Mekong, i.e. Nat. Hazards Earth Syst. Sci., 18, 2859–2876, 2018 2.1 Study area Mekong and Bassac rivers, convey 90 % of the total ﬂood volume. The remaining 10 % are the transboundary overland ﬂow from Cambodian lowlands to the PoR east of the Mekong River, and the LXQ west of the Bassac River (Hung et al., 2012; Tri, 2012). Be- sides inundation caused by high river discharge, tidal ﬂoods occur in the vicinity of rivers/canals in the coastal areas, char- acterized by short but repeated durations following the high tides of the South China Sea and the Gulf of Thailand (Apel et al., 2016). From July to December, high discharge of the Mekong River triggered by the Asian monsoons cause a large-scale inundation in the delta. Our study area, referred to as the delta ﬂood-prone region in Vietnam, comprises 2 million hectares of nine provinces. The area is commonly divided into two ecological regions on the basis of inundation depth, named “deep inundation” (above 1.5 m) and “shallow inundation” (below 1.5 m). The deep-inundation areas (marked in yellow in Fig. 1) encompass the two most important ﬂoodplains in the delta, i.e. the Plain of Reeds (PoR), and the Long Xuyen Quadrangle (LXQ). Flooding in the VMD is characterized by slowly rising and receding rates, with a mean value of 5–10 cmday−1. The ﬂood hydrograph at Tan Chau and Chau Doc (blue points 1 and 2 in Fig. 1) usually has two peaks. The ﬁrst peak nor- 2.2 Data dykes can be, however, completely cut off from ﬂoodwater. The design level of the dykes was chosen to withstand water levels observed during the historical ﬂood in 2000 (Triet et al., 2017). The inundation of those areas is thus controlled by the operation of sluice gates included in the dyke lines. 2.2.1 Topography data, tidal levels and operation schemes of ﬂood control structures Tidal level data are used as downstream boundary condition of the ﬂood propagation model. Hourly tidal level records at 10 gauge stations were collected, covering the entire ﬂood season of the year 2011 from 1 June to 30 Novem- ber. The locations of these tidal gauges are given in Fig. 1. These data were provided by the Southern Regional Hydro- Meteorology Centre of Vietnam (SHRMC). To generate in- undation maps, a high-resolution (5 × 5 m) lidar-based digi- tal elevation model (DEM) for the whole VMD was acquired from the Ministry of Environment and Natural Resources of Vietnam (MONRE). The lidar data were collected and pro- cessed during 2009–2010. Operation schemes of the ﬂood control structures in 2011 were collected from the Depart- ments of Agriculture and Rural Development (DARD) of the delta provinces. Traditionally, farmers were only able to grow a single rice crop per year during the wet season. This crop was known as rainfed crop. Today, the majority of rainfed crops have been replaced by irrigated rice, except for small areas af- fected by saline water intrusion or poor soil quality (acid sul- phate soil). Farmers are able to grow two or even three crops per year. One crop is planted in the dry season in November– December and harvested in February–March (called winter– spring crop or Ðông Xuân). During the wet season, farm- ers plant one or two crops. The ﬁrst crop (summer–autumn crop or Hè Thu) is planted in April–early June and har- vested by July–early August. The second crop (autumn– winter crop or Thu Ðông) depends on how farmlands are protected against ﬂoodwater. Farmlands with full protection (located in the deep-inundation region) plant in August and harvest in November–December. In the shallow-inundation region, farmers harvest in late August–early September, be- fore the arrival of the main ﬂood peak. www.nat-hazards-earth-syst-sci.net/18/2859/2018/ www.nat-hazards-earth-syst-sci.net/18/2859/2018/ Nat. Hazards Earth Syst. Sci., 18, 2859–2876, 2018 N. V. K. Triet et al.: Towards risk-based ﬂood management in highly productive paddy rice cultivation N. V. K. Triet et al.: Towards risk-based ﬂood management in highly productive paddy rice cultivation 2862 Rice growth stage: Seedling Vegetation phase (transplanting, tiller and panicle formation) Flowering Maturity & harvest Planting calendar deep inundation region Planting calendar shallow inundation region Summer–autumn crop Summer–autumn crop Winter–spring crop Winter–spring crop Summer–autumn crop Summer–autumn crop Winter–spring crop Winter–spring crop Autumn–winter crop Autumn–winter crop J F M A M J J A S O N D N D EVI value 0 0.2 0.4 0.6 0.8 1.0 (b) Peak EVI Peak EVI Peak EVI Triple rice cropping ﬁeld Month in year 0 0.2 0.4 0.6 0.8 1.0 EVI value (a) Peak EVI Peak EVI J F M A M J J A S O N D N D Double rice cropping ﬁeld Month in year Figure 2. Time series of the smoothed enhanced vegetation index (EVI) for double (a) and triple (b) rice cropping ﬁelds in the Vietnamese Mekong Delta. The blue lines denote paddy ﬁelds in the shallow-inundation zone. Red lines represent paddy ﬁelds in the deep-inundation zone. The cropping season ends 40 days after the EVI peak (Kotera et al., 2016). J F M A M J J A S O N D N D EVI value 0 0.2 0.4 0.6 0.8 1.0 (b) Peak EVI Peak EVI Peak EVI Triple rice cropping ﬁeld Month in year 0 0.2 0.4 0.6 0.8 1.0 EVI value (a) Peak EVI Peak EVI J F M A M J J A S O N D N D Double rice cropping ﬁeld Month in year Double rice cropping ﬁeld Figure 2. Time series of the smoothed enhanced vegetation index (EVI) for double (a) and triple (b) rice cropping ﬁelds in the Vietnamese Mekong Delta. The blue lines denote paddy ﬁelds in the shallow-inundation zone. Red lines represent paddy ﬁelds in the deep-inundation zone. The cropping season ends 40 days after the EVI peak (Kotera et al., 2016). 2.2.2 Rice cropping system and planting calendar The rice cropping system in the VMD is strongly related to water availability, soil fertility and irrigation/drainage facil- ities (e.g. ﬂood control structures). Rice ﬁelds are generally encircled by dyke systems to protect them against the regular ﬂood pulse of the Mekong. These dyke systems can be clas- siﬁed as low dykes and high dykes. Low dykes protect the summer–autumn crop against the early ﬂood peak from mid- July to mid-August. They are regularly overtopped during the later stages of the ﬂood period. Farmlands protected by high The rice cropping system and planting calendar in the VMD have been well studied using optical and radar satel- lite data (e.g. Bouvet et al., 2009; Bouvet and Le Toan, 2011; Nguyen et al., 2015). Figure 2 illustrates the planting calen- dar in 2011 for four paddy ﬁelds positioned in the deep ver- sus shallow-inundation region for double- and triple-season rice ﬁelds. There is a shift of 1–1.5 months in the planting calendar between the shallow- and deep-inundation regions. The enhanced vegetation index (EVI) time series used to con- www.nat-hazards-earth-syst-sci.net/18/2859/2018/ Nat. Hazards Earth Syst. Sci., 18, 2859–2876, 2018 et et al.: Towards risk-based ﬂood management in highly productive paddy rice cultivation N. V. K. Triet et al.: Towards risk-based ﬂood management in highly productive paddy rice cultivation 2863 ¬ (a) (b) (c) Plantation area Summer-Autumn crop (SAC) Plantation area Autumn-Winter crop (AWC) Rice pixels Non-rice pixels Rice pixels Non-rice pixels Land-use VMD 2014 Unclassiﬁed Water bodies Aquaculture Aquaculture/mangroves Single-season rice Double-season rice Triple-season rice Orchards/plantations Sugar cane Eucalyptus Mangrove forests Urban and built-up lands 1 2 3 4 5 6 7 8 9 10 11 12 25 Km 50 0 100 Study area Figure 3. Panel (a) presents the land-use map of the Vietnamese Mekong Delta in 2014. Panels (b) and (c) show plantation areas of the summer–autumn crop (SAC) and the autumn–winter crop (AWC). (b) (c) Plantation area Summer-Autumn crop (SAC) Plantation area Autumn-Winter crop (AWC) Rice pixels Non-rice pixels Rice pixels Non-rice pixels (b) (c) Figure 3. Panel (a) presents the land-use map of the Vietnamese Mekong Delta in 2014. Panels (b) and (c) show plantation areas of the summer–autumn crop (SAC) and the autumn–winter crop (AWC). Figure 3. Panel (a) presents the land-use map of the Vietnamese Mekong Delta in 2014. Panels (b) and (c summer–autumn crop (SAC) and the autumn–winter crop (AWC). 2.2.2 Rice cropping system and planting calendar where G is the gain factor (G = 2.5). C1 and C2 are the coef- ﬁcients of the aerosol resistance term, which uses the 500 m blue band of MODIS to correct aerosol inﬂuences on the red band (C1 = 6.0 and C2 = 7.5). L is the canopy background adjustment (L = 1) (Huete et al., 2002). struct this plot were provided by Akihiko Kotera (personal communication, 1 March 2017). An EVI value of 0 indicates no vegetation cover, whereas a value of 1 means complete vegetation cover. The methodology used to derive the data set has been presented in Kotera et al. (2016) and applied to assess economic ﬂood damage to rice crop in the Chao Phraya delta in Thailand from 2000 to 2011. Land-use classiﬁcation was performed on the basis of the EVI time series from 2001 to 2011 (for a detailed descrip- tion see Leinenkugel et al. (2013). Within the 12 classes of the VMD land-use raster, three classes indicate rice cultiva- tion areas: single-season rice, double-season rice, and triple- season rice (values 5–7 in Fig. 3a). 3 Methodology that were tested, the Gumbel–Hougaard copula was selected as most suitable, with log-normal distributions describing the marginals of Qmax and FV . The outcomes of the mentioned study is the very ﬁrst publication on ﬂood frequency analy- sis for the MD, considering both peak discharge and ﬂood volume. We refer readers to the original paper for a detailed description. For the large-scale ﬂood risk assessment, the temporal rela- tionship between the inundation hazard and the rice planting calendar was taken into consideration. The next sections de- scribe the procedure that derives the area of rice crops ex- posed to ﬂoods, ﬂood damage (D) from a given extreme event, and the expected annual damage (EAD). These risk in- dicators were estimated for the current situation and for two land-use development scenarios, namely the reduction or ex- pansion of the triple-season rice area as given in the Mekong Delta Plan of the Vietnamese government. The methodology is outlined in Fig. 4a. Four pairs of peak discharge (Qmax) and volume at Kratie (FV ), corresponding to T10, T20, T50 and T100 ﬂoods were selected from the bivariate copula model. The most proba- ble pairs were selected from the Qmax/FV pairs with equal joint probabilities corresponding to the return periods speci- ﬁed above. A full probabilistic analysis using a large number of Qmax/FV pairs with equal joint probabilities was not per- formed due to the high computational demand of the large- scale hydraulic model (on average, 2–3 h are required for one simulation of the whole ﬂood season June–November on a PC installed with Intel i7-CPU 3.0 GHz, 16 GB RAM). The selected Qmax and FV values range from 56 500 to 66 000 m3 s−1 and from 459 to 525 km3, from the different return periods. N. V. K. Triet et al.: Towards risk-based ﬂood management in highly productive paddy rice cultivation N. V. K. Triet et al.: Towards risk-based ﬂood management in highly productive paddy rice cultivation 2864 20 40 60 80 Percentage of yield reduction Inundation duration (in days) 2 1 3 5 4 6 8 7 9 Inundation depth < 0.5 m Inundation depth 0.5–1.0 m Inundation depth >1.0 m (c) 0.2 0.4 0.6 0.8 1.0 Percentage of peak discharge Jun 01 Jul 01 Aug 01 Sep 01 Oct 01 Nov 01 Dec 01 Shape 1 p = 21 % Shape 2 p = 22 % Shape 3 p = 34 % Shape 4 p = 23 % (b) Flood risk maps rice crop in the VMD Damage function Exposed areas Cropping calendar Inundated areas Land-use map Tidal level in 2011 Lidar-based DEM Quasi-2-D flood routing model Q /F for T flood max V yr Flow series (Q) at Kratie (a) Figure 4. (a) Procedure for estimating ﬂood risk to rice production in the Vietnamese Mekong Delta. (b) The four normalized discharge hydrographs at Kratie, together with their probability of occurrence (Dung et al., 2015) used for the derivation of synthetic ﬂood events as the upper boundary of the hydraulic model. (c) Stage-damage curve for paddy rice (Dutta et al., 2003). (a) 20 40 60 80 Percentage of yield reduction Inundation duration (in days) 2 1 3 5 4 6 8 7 9 Inundation depth < 0.5 m Inundation depth 0.5–1.0 m Inundation depth >1.0 m (c) (c) Flood risk maps rice crop in the VMD Figure 4. (a) Procedure for estimating ﬂood risk to rice production in the Vietnamese Mekong Delta. (b) The four normalized discharge hydrographs at Kratie, together with their probability of occurrence (Dung et al., 2015) used for the derivation of synthetic ﬂood events as the upper boundary of the hydraulic model. (c) Stage-damage curve for paddy rice (Dutta et al., 2003). 2.2.3 Land-use data The land-use map of 2014, with a resolution of 250 × 250 m, covering all 13 provinces in the VMD was pro- vided by the German Aerospace Centre (DLR). The VMD land use is part of the product MEKONG LC2010, cover- ing the entire Mekong Basin at a spatial resolution of 500 m. MEKONG LC2010 was developed within the German– Vietnamese project “Water-related Information System for a Sustainable Development of the Mekong Delta” (WISDOM, http://www.wisdom.eoc.dlr.de/, last access: 20 July 2018). Land cover data were derived using the Moderate Resolution Imaging Spectroradiometer (MODIS) instrument aboard the Terra and Aqua satellites. Different MODIS products were combined to provide cloud free composites. The enhanced vegetation index (EVI) was calculated following Eq. (1). We reclassiﬁed the original product to two raster images presenting the summer–autumn crop, hereafter referred as SAC, and the autumn–winter crop, hereafter referred to as AWC, since their growth stages partially or fully fall in the ﬂood season. The SAC image was created by merging all pix- els with double-season and triple-season rice. The other land- use classes (e.g. orchards, sugar cane) were considered non- rice pixels (presented in Fig. 3b). To produce the AWC im- age, which is only grown in triple-season cropping schemes, only pixels with original values of 7 were considered (see Fig. 3c). EVI = G × NIR −RED NIR + C1 · RED −C2 · BLUE + L, (1) (1) Nat. Hazards Earth Syst. Sci., 18, 2859–2876, 2018 www.nat-hazards-earth-syst-sci.net/18/2859/2018/ www.nat-hazards-earth-syst-sci.net/18/2859/2018/ 3.2 Transformation of discharge to water levels To transform the discharge series into spatially distributed inundation water levels and associated timing in the VMD, a quasi-2-D, large-scale hydraulic model was used. The model domain covers the entire MD, including the VMD and Cam- bodia lowlands and the Tonle Sap Lake. It uses Kratie as the upper boundary condition and the tidal level monitoring gauges along the South China Sea and the Gulf of Thailand as downstream boundaries. All these procedures were performed using Python scripts (Python version 2.7) and the Python-supported module of Ar- cGIS (ArcGIS 10.4). The model was initially developed by Dung et al. (2011), using the 1-D river model modelling package MIKE11-HD developed by Danish Hydraulic Institute (DHI). The hydro- dynamic module (HD) provides the full dynamic solution of the 1-D Saint-Venant equations. The solution is based on an implicit ﬁnite difference scheme developed by Abbott and Ionescu (1967). Floodplain inundation (2-D ﬂow) was pre- sented by the 1-D model through wide cross sections for the Cambodian part of the model domain, which is appro- priate for the comparatively low anthropogenic impacts on the channel network, and natural inundation dynamics of in this part of the MD. In the Vietnamese part of the delta, ﬂood compartments were represented by virtual canals and con- trol structures presenting the dykes. The original model was later reﬁned and updated by Manh et al. (2014) and Triet et al. (2017) (see Fig. 5). The model was calibrated and val- idated with gauged data and maximum inundation extents derived from satellite data for a number of ﬂood events, in- cluding the moderate ﬂood of 2009, the low ﬂood of 2010, and the extreme ﬂoods in 2011 and 2000. For a detailed de- scription of the 2-D model see the original paper by Dung et al. (2011). N. V. K. Triet et al.: Towards risk-based ﬂood management in highly productive paddy rice cultivation 2865 N. V. K. Triet et al.: Towards risk-based ﬂood management in highly productive paddy rice cultivation 3.3 Transformation of water levels to ﬂood hazard indicators and, consequently, different possible damages. These shapes were adapted from Dung et al. (2015) (see Fig. 4b). The shape shp3 has the highest possibility of occurrence (p = 0.34). The ﬂood in 2000 closely followed this shape, with a minor alteration as the ﬁrst peak arrived some days ear- lier. The other three hydrograph shapes have equal probabil- ities of occurrence (p = 0.21–0.23). The shape shp2 has an early ﬂood peak, while shp1 shows a late peak. The disas- trous ﬂood in 2011 resembled shp1. The hydraulic simulations provided discharge and water level time series at the model calculation nodes. Inverse distance weighting (IDW) was applied to interpolate between nodes for complete spatial coverage. Inundation extent and depth were then obtained by intersecting the water levels with the lidar-based DEM. Based on the rice cropping system and cropping calendar, three gridded inundation maps were produced for each sim- ulation scenario. The ﬁrst map, labelled “July”, was gener- ated using the maximum water level of 61 days from the beginning of simulation from 1 June to 31 July. It was ap- plied to calculate damage to the summer–autumn crop (SAC) in both shallow- and deep-submergence regions. The second map (August) was based on the maximum water level from 1 June to 15 September in order to calculate damage to the autumn–winter crop (AWC) in the shallow-submergence re- gion. For the AWC in the deep-submergence region, we used the map deﬁned by the maximum annual submergence (an- nual) within 1 September to 30 November. The inundation grid cells of these maps were classiﬁed into three inundation depth classes to assign a stage-damage curve (Fig. 4c) to each grid cell. Exposed areas of rice crops were calculated by in- tersecting the inundation maps with the two land-use maps presenting the SAC and the AWC, as summarized in Table 1. This procedure results in 16 synthetic discharge time series at Kratie. They serve as the upper boundary condition for the ﬂood propagation model. In each simulation, the lower boundaries (i.e. tidal levels), dyke scenarios and operation schemes of ﬂood control structures were ﬁxed as recorded in 2011, i.e. the most recent damaging ﬂood. The scenarios are denoted using the return period and the hydrograph shape. For example, scenario T100shp3 corresponds to the 100-year return period of Qmax/FV and the hydrograph shp3. 3.1 Determining event hydrographs corresponding to the T -year ﬂood Synthetic ﬂood events were estimated for station Kratie (Fig. 1, left panel) with 10-, 20-, 50-, and 100-year return periods, referred to as T10, T20, T50, and T100. This station is commonly deﬁned as the upstream entrance of the MD and is used as upper boundary of the hydraulic inundation model of the MD. The estimation of ﬂood events is based on Dung et al. (2015). The authors developed and tested different bi- variate copula-based statistical models on extreme values, us- ing annual maximum discharge Qmax and ﬂood volume FV . Both variables are important for the characterization of the long-lasting annual ﬂoods in the MD. From different models Damages to agriculture crops are highly dependent on the time of occurrence of ﬂooding (Penning-Rowsell et al., 2003; Förster et al., 2008; Klaus et al., 2016). To account for the timing, each of the four Qmax/FV pairs were scaled to syn- thetic ﬂood hydrographs covering the whole ﬂood season from 1 June to 30 November using four typical hydrograph shapes (shpi, i = 1–4), representing different ﬂood patterns www.nat-hazards-earth-syst-sci.net/18/2859/2018/ Nat. Hazards Earth Syst. Sci., 18, 2859–2876, 2018 3.4 Calculation of ﬂood damage (D) and expected annual damage (EAD) Triet et al.: Towards risk-based ﬂood management in highly productive paddy rice cultivation N. V. K. Triet et al.: Towards risk-based ﬂood management in highly productive paddy rice cultivation 2866 0 50 100 150 25 km Miss sim.wet / obs.wet + sim.dry / obs.dry False 11 % 15 % 74 % Hit Sim. dry / obs. wet Sim. wet / obs. wet Sim. dry / obs. dry Sim. wet / obs. dry (b) TONLE SAP LAKE Kratie Wide cross section Real channels Cross sections extracted from DEM Virtual channels Control structures to present dykes (a) Study area (c) Sim.dry / obs.wet Sim.wet / obs.dry ¬ Figure 5. (a) Schematization of the quasi-2-D ﬂood propagation model for the Mekong Delta and the concept of simulation of compartmented ﬂoodplains in the VMD. Comparison of observed inundation extent derived from satellite data and simulated maximum inundation extent for the ﬂood event in 2011 for the whole delta (b), and evaluation of inundation simulation (c) adapted from Triet et al. (2017). TONLE SAP LAKE Kratie Wide cross section Real channels Cross sections extracted from DEM Virtual channels Control structures to present dykes (a) 0 50 100 150 25 km Miss sim.wet / obs.wet + sim.dry / obs.dry False 11 % 15 % 74 % Hit Sim. dry / obs. wet Sim. wet / obs. wet Sim. dry / obs. dry Sim. wet / obs. dry (b) Study area (c) Sim.dry / obs.wet Sim.wet / obs.dry ¬ (b) Sim. dry / obs. wet Sim. wet / obs. wet Sim. dry / obs. dry Sim. wet / obs. dry Miss sim.wet / obs.wet + sim.dry / obs.dry False 11 % 15 % 74 % Hit (c) Sim.dry / obs.wet Sim.wet / obs.dry Cross sections extracted from DEM Real channels (a) (c) Figure 5. (a) Schematization of the quasi-2-D ﬂood propagation model for the Mekong Delta and the concept of simulation of compartmented ﬂoodplains in the VMD. Comparison of observed inundation extent derived from satellite data and simulated maximum inundation extent for the ﬂood event in 2011 for the whole delta (b), and evaluation of inundation simulation (c) adapted from Triet et al. (2017). Table 1. Inundation maps for estimating damage to rice crops in the Vietnamese Mekong Delta. 3.4 Calculation of ﬂood damage (D) and expected annual damage (EAD) Flood damage (D) was calculated on a pixel basis following equation (Eq. 2) and aggregated to damage per province and to the whole study area. (2) D = Y × MP × (A1 · RD1 + A2 · RD2), (2) where D is the total monetary damage (in USD). Y and MP are the average rice yield and market prices taken from of- ﬁcial statistical data for 2011: Y = 5.0 ton per hectare, and MP = USD 280 per ton. A1 and A2 are the total exposed ar- eas classiﬁed as partial and full losses. RD1 and RD2 are the relative damage factors speciﬁed on the basis of the damage curves adopted from Dutta et al. (2003) (see Fig. 4c). The percentage of damage (i.e. in terms of yield reduction) de- pends on the duration of contact with ﬂoodwater (in days) and inundation depth, which was classiﬁed into two groups, i.e. below 0.5 m and above 0.5 m. Because ﬂooding in the VMD is characterized with a long duration of submergence of 2–5 months (Toan, 2014), loss factor RD2 was set to 1 for the areas where inundation depth is above 0.5 m (A2), while the partial loss factor RD1 was set to 0.5, which is the maxi- where D is the total monetary damage (in USD). Y and MP are the average rice yield and market prices taken from of- ﬁcial statistical data for 2011: Y = 5.0 ton per hectare, and MP = USD 280 per ton. A1 and A2 are the total exposed ar- eas classiﬁed as partial and full losses. RD1 and RD2 are the relative damage factors speciﬁed on the basis of the damage curves adopted from Dutta et al. (2003) (see Fig. 4c). The percentage of damage (i.e. in terms of yield reduction) de- pends on the duration of contact with ﬂoodwater (in days) and inundation depth, which was classiﬁed into two groups, i.e. below 0.5 m and above 0.5 m. Because ﬂooding in the VMD is characterized with a long duration of submergence of 2–5 months (Toan, 2014), loss factor RD2 was set to 1 for the areas where inundation depth is above 0.5 m (A2), while the partial loss factor RD1 was set to 0.5, which is the maxi- www.nat-hazards-earth-syst-sci.net/18/2859/2018/ Nat. Hazards Earth Syst. Sci., 18, 2859–2876, 2018 N. V. K. www.nat-hazards-earth-syst-sci.net/18/2859/2018/ Nat. Hazards Earth Syst. Sci., 18, 2859–2876, 2018 www.nat-hazards-earth-syst-sci.net/18/2859/2018/ 3.4 Calculation of ﬂood damage (D) and expected annual damage (EAD) Inundation region Cropping system Rice crop Planting calendar Input inundated raster raster name period Shallow double-season rice Summer–autumn (SAC) mid-April–mid-July July 1 Jun–31 Jul (below 1.5 m) triple-season rice Summer–autumn (SAC) March–May/early June July 1 Jun–31 Jul Autumn–winter (AWC) mid-June–mid September August 1 Jun–15 Sep Deep double-season rice Summer–autumn (SAC) May–July July 1 Jun–31 Jul (above 1.5 m) triple-season rice Summer–autumn (SAC) May–July July 1 Jun–31 Jul Autumn–winter (AWC) September–November Annual 1 Sep–30 Nov Table 1. Inundation maps for estimating damage to rice crops in the Vietnamese Mekong Delta. mum damage of this class in the damage functions, for areas with inundation depth below 0.5 m (A1). mum damage of this class in the damage functions, for areas with inundation depth below 0.5 m (A1). We calculated the EADs for each of the four hydrograph shapes following Apel et al. (2016). EAD is deﬁned as the product of probability of exceedance of a given ﬂood event 3.5 Estimation of risk variation as a result of two land-use scenarios In the ﬁnal step, we investigated how ﬂood risk will change in two land-use scenarios. Triet et al. (2017) proved that the construction of high-dyke areas in the northern delta provinces An Giang and Dong Thap increased the ﬂood haz- ard in the centre of the delta. Thus, the ﬁrst scenario con- siders the opening of the sluice gates in the high-dyke areas in these two provinces to introduce ﬂoodwater to the paddy ﬁelds during the main ﬂood period, September–October. In response to this change in the ﬂood management the farming system also changes: farmlands with triple-season cropping are converted to double-season cropping, i.e. no cultivation of the AWC. The second scenario considers an expansion of high dykes, i.e. an increase in the height of the existing low dykes, in these two provinces to enlarge the area with triple rice crop production. This scenario follows the development scenario, the so-called “food production scenario”, proposed in the Mekong Delta Plan (Deltares, 2013). Dyke height was increased using information of dyke elevation from neigh- bouring compartments and maximum water level of the his- torical ﬂood in 2000, which was chosen as the design event for ﬂood control infrastructures in the delta. p g A large share of the overestimated exposed area of rice crops can be attributed to the simulated inundation extents, although efforts have been made to update, reﬁne, and cali- brate the model (Dung et al., 2011; Manh et al., 2014; Triet et al., 2017). The main source of uncertainty stems from the in- terpolation of 1-D model results to a 2-D raster, which could not be reduced even by the high-resolution lidar DEM. Triet et al. (2017) reported a ﬂood area index (FAI) of 0.64 for the comparison of modelled and observed inundated areas for the whole VMD. The FAI was computed by dividing the sets of pixels presenting the intersection of observed and simulated inundation with the set of pixels presenting the union of ob- served and simulated (Eq. 3 in Aronica et al., 2002). This value increased to 0.74 only if the ﬂood-prone area of the VMD was considered. According to Aronica et al. 4.1 Validation of estimated damage ¯Di = 1 2(Di + Di+1) (4) 1Pi = pi+1 −pi, (5) The damage estimation was validated by comparing the es- timated damage for the ﬂood in 2011 with ofﬁcial damage data. The exposed cropping area was overestimated by 18 %, i.e. 32 500 ha in comparison with a reported area of 27 000 ha (Tinh, 2012). We estimated rice crop losses of USD 42.7 mil- lion. This number is equivalent to 81 % of the reported agri- cultural damages from the National Steering Committee for Flood and Storm Prevention and Control (USD 52.8 mil- lion) (MRC, 2011). Flood damages to other agriculture crops and facilities, e.g. farmhouses, which were included in the reported damages (lumped into a single value categorized as agriculture losses), were not yet incorporated in the pre- sented damage estimation. Considering that paddy rice is the predominant crop in the delta, it is very likely to share a large part of the reported losses. Paddy ﬁelds derived from the land-use LC2014 raster account for 72 % of whole agri- culture land within the focus area of this study (deep- and shallow-inundation areas). Assuming a linear distribution of damages in the lumped ofﬁcial reported damages with land- use proportion, it can be reasoned that the simulated dam- ages are in the range of the reported. However, it has to be acknowledged that spatial distribution and market prices of different crops are likely to be important for the damage esti- mation. In any case, although not the ideal piece of informa- tion, the reported agriculture losses were the only available data with which to evaluate our rice crop damage calculation. (5) where 1P is the increment of annual probability of ex- ceedance = 1(1 −p), with p as the annual probability of non-exceedance. In this work p = 0.90, 0.95, 0.98 and 0.99 according to the selected return periods; D is the calculated damage induced by the given event; i is the numerator of the probability levels considered, and n is the number of proba- bility levels. The average estimated annual damage (EAD) of the four hydrograph shapes was computed as the weighed sum of the EAD values, with the probability of occurrence of the hydro- graph (Pr) as weights (Eq. 6). The average crop risk indicator was computed by dividing the average EAD by the total an- nual rice plantation area. 4.1 Validation of estimated damage EAD = 4 X i=1 Pr(shp = shpi)EADi (6) (6) 4 Results and discussion 1Pi and ¯Di are calculated as follows: 1Pi and ¯Di are calculated as follows: N. V. K. Triet et al.: Towards risk-based ﬂood management in highly productive paddy rice cultivation and its damage: and its damage: ﬂo ﬂood for three scenarios (current situation, expansion and re- moval of high dykes). EAD = n X i=1 1Pi · ¯Di. (3) (3) Nat. Hazards Earth Syst. Sci., 18, 2859–2876, 2018 N. V. K. Triet et al.: Towards risk-based ﬂood management in highly productive paddy rice cultivation N. V. K. Triet et al.: Towards risk-based ﬂood management in highly productive paddy rice cultivation 2867 3.5 Estimation of risk variation as a result of two land-use scenarios (2002), who suggested that a FAI higher than 0.7 is considered ac- ceptable for an inundation simulation model, it can be con- cluded that the performance of the inundation model for the VMD is acceptable for the ﬂood-prone area of the VMD, where the bulk of ﬂood damages occur. The ﬂood propagation model simulated the 16 synthetic ﬂoods for these two dyke scenarios, while the lower bound- ary conditions were preserved as in 2011. Figure 6 exemplar- ily illustrates the simulated inundation extent for the 10-year Nat. Hazards Earth Syst. Sci., 18, 2859–2876, 2018 4.2 Flood hazard assessment A small share of the overestimation of the 2011 ﬂood might stem from the land-use data. Considering the rapid expansion of the triple-season rice areas in the delta (Le et al., 2018), it can be expected that the used land-use prod- uct of 2014 overestimates the spatial coverage of triple- season rice paddies, possibly resulting in an overestimation of the ﬂood damage. Also, land-use data have a resolution of 250 × 250 m; therefore the majority of inland canals (width 10–30 m) were likely classiﬁed as rice pixels (see Fig. 3). Considered the channel density in the delta of 14 mha−1 (Hung et al., 2012), not separating these inland water pix- els might contribute with a small share to the overestimation. Additionally, other important factors were not considered, such as dyke failures and local ﬂood management measures, i.e. early harvesting of rice crops despite not being 100 % ripe or local raising of dyke segments with sandbags. The ﬂood event time series at Kratie were transformed into four hazard indicators: maximum water level, date of occur- rence, inundation extent, and depth. The simulated annual maximum water levels (AMWLs) at locations with water level gauges in the VMD are summarized in Table 2. Figure 7 presents the date of occurrence (DO) of the AMWLs at these points. The chosen locations encompass nine gauges in the mainstream Mekong (i.e. Mekong River and Bassac River), and seven inland gauges in the two most important ﬂood- plains, i.e. LXQ (Long Xuyen Quadrangle) and PoR (Plain of Reeds). Inundated areas were calculated for the three pe- riods speciﬁed in Table 1 and aggregated to the ﬂood-prone area of the VMD on the basis of the four hydrograph shapes (Fig. 8). Some of these error sources might be resolved by further reﬁning the model. For instance, the land-use data set can be improved by considering the inland canals when the crop areas are extracted. Enhancing model performance is, how- ever, not that straightforward considering the huge amount of data required for the large-scale model domain. Despite these deﬁciencies, the ﬂood damage assessment proposed in this study can produce reliable results, particularly when the typically large errors in ﬂood damage estimation are taken as a reference (e.g. Schröter et al., 2014). Thus, the proposed method is judged to be appropriate to estimate ﬂood hazard and risk to rice cropping in the VMD. N. V. K. Triet et al.: Towards risk-based ﬂood management in highly productive paddy rice cultivation 2868 An Giang Dong Thap Expansion of AWC in An Giang & Dong Thap (c) ¬ Inundation depth No inundation < 0.5 m 0.5 ÷ 1.0 m 1.0 ÷ 1.5 m 1.5 ÷ 2.0 m 2.0 ÷ 2.5 m > 2.5 m Study area (a) No AWC in An Giang & Dong Thap An Giang Dong Thap ¬ An Giang Dong Thap Current situation (b) ¬ An Giang Dong Thap Expansion of AWC in An Giang & Dong Thap (c) ¬ 0 50 100 25 km Scale: Figure 6. Simulated maximum inundation extent for a 10-year return period ﬂood (T10shp1) for three land-use scenarios. (a) No plantation of autumn–winter crop in An Giang and Dong Thap, (b) present condition as of 2011, and (c) expansion of high-dyke areas in An Giang and Dong Thap to enlarge triple-season rice crops. Inundation depth No inundation (a) No AWC in An Giang & Dong Thap An Giang Dong Thap ¬ 0 50 100 25 km Scale: Expansion of AWC in An Giang & Dong Thap No AWC in An Giang & Dong Thap (b) (a) (c) Figure 6. Simulated maximum inundation extent for a 10-year return period ﬂood (T10shp1) for three land-use scenarios. (a) No plantation of autumn–winter crop in An Giang and Dong Thap, (b) present condition as of 2011, and (c) expansion of high-dyke areas in An Giang and Dong Thap to enlarge triple-season rice crops. www.nat-hazards-earth-syst-sci.net/18/2859/2018/ www.nat-hazards-earth-syst-sci.net/18/2859/2018/ N. V. K. Triet et al.: Towards risk-based ﬂood management in highly productive paddy rice cultivation Nat. Hazards Earth Syst. Sci., 18, 2859–2876, 2018 et et al.: Towards risk-based ﬂood management in highly productive paddy rice cultivation N. V. K. Triet et al.: Towards risk-based ﬂood management in highly productive paddy rice cultivation 2869 N. V. K. Triet et al.: Towards risk-based ﬂood management in highly productive paddy rice cultivation 2869 Red Magneta Cyan Blue Shape 1, p =21 % Shape 2, p = 22 % Shape 3, p = 34 % Shape 4, p = 23 % Hydrograph shape T = 10 years T = 20 years T = 100 years T = 50 years Flood magnitude Gauge stations Xuan To Tri Ton Tan Hiep Vi Thanh (d) Gauge stations Moc Hoa Hung Thanh Kien Binh Tan An (c) Gauge stations Long Xuyen Can Tho Phung Hiep Chau Doc (b) 15 Sep 01 Oct 15 Oct 01 Nov Date Gauge stations Tan Chau Vam Nao Cao Lanh My Thuan (a) 15 Sep 01 Oct 15 Oct 01 Nov Date 15 Sep 01 Oct 15 Oct 01 Nov Date 15 Sep 01 Oct 15 Oct 01 Nov Date Figure 7. Date of occurrence (DO) of the annual maximum water level at key monitoring gauges in the Vietnamese Mekong Delta (a) stations in the Mekong branch, (b) stations in the Bassac branch, (c) stations in the Plain of Reeds, and (d) stations in the Long Xuyen Quadrangle. The four hydrograph shapes (indicated by colours) are shown in combination with different ﬂood magnitudes (indicated by markers). Gauge stations Long Xuyen Can Tho Phung Hiep Chau Doc (b) 15 Sep 01 Oct 15 Oct 01 Nov Date 15 Sep 01 Oct 15 Oct 01 Nov Date Gauge stations Tan Chau Vam Nao Cao Lanh My Thuan (a) D t (b) Gauge stations Xuan To Tri Ton Tan Hiep Vi Thanh (d) g 5 Sep 01 Oct 15 Oct 01 Nov Gauge stations Moc Hoa Hung Thanh Kien Binh Tan An (c) 15 Sep 01 Oct 15 Oct 01 Nov Date D t (d) (c) Figure 7. Date of occurrence (DO) of the annual maximum water level at key monitoring gauges in the Vietnamese Mekong Delta (a) stations in the Mekong branch, (b) stations in the Bassac branch, (c) stations in the Plain of Reeds, and (d) stations in the Long Xuyen Quadrangle. The four hydrograph shapes (indicated by colours) are shown in combination with different ﬂood magnitudes (indicated by markers). plain compartments, and coincides with the period of highest tidal levels in the year (October–November). et et al.: Towards risk-based ﬂood management in highly productive paddy rice cultivation Therefore, the highest AMWL is caused by the hydrodynamic interaction between the upstream and downstream boundaries, and pre- ceding inundation dynamics. Our simulation thus provides numerical evidence to conﬁrm the statement in Tri (2012) and Triet et al. (2017) that the superposition of river ﬂood peaks with high tide periods results in substantial backwater effects and higher water levels up to the border with Cambo- dia. 50 100 20 10 0 250 500 750 1000 1250 1500 1750 Return period [years] Inundated area [10³ ha] Fitted curves Shape 1, p = 21 % Shape 2, p = 22 % Shape 3, p = 34 % Shape 4, p = 23 % Annual inundation August inundation July inundation Simulated values Figure 8. Frequency distributions of the maximum inundated area in July (squares), in August (diamonds), and over the whole year (circles) corresponding to the four ﬂood patterns (different colours). 50 100 20 10 0 250 500 750 1000 1250 1500 1750 Return period [years] Inundated area [10³ ha] Fitted curves Shape 1, p = 21 % Shape 2, p = 22 % Shape 3, p = 34 % Shape 4, p = 23 % Annual inundation August inundation July inundation Simulated values The date of occurrence (DO) of the AMWL in the VMD is less sensitive to changes in ﬂood peak discharge and shape of hydrograph than the actual AMWL (see Fig. 7). The DO can be divided into two groups. The ﬁrst group is composed of stations with prevailing tidal inﬂuence, e.g. Can Tho, My Thuan, and Vi Thanh. For this group AMWL occurs in late October, similarly to the period of maximum tidal levels in 2011, the downstream boundary conditions of the ﬂood prop- agation model. The second group contains gauges further north or gauges far from the main rivers, where the tidal in- ﬂuence is largely reduced. These gauges have the DO in the ﬁrst half of October. Figure 8. Frequency distributions of the maximum inundated area in July (squares), in August (diamonds), and over the whole year (circles) corresponding to the four ﬂood patterns (different colours). The hydrograph shape also inﬂuences the AMWL: higher AMWL were obtained for events with shp1 and shp3. The shape shp3 resulted in higher AMWL in the DSR, while shp1 yielded higher AMWL in the SSR of the delta. In contrast to this, shp2 and shp4 caused lower water levels. 4.2 Flood hazard assessment The simulation results show that the AMWLs in the VMD vary substantially depending on ﬂood magnitude (TF) and hydrograph shapes. We estimated a relative change of 10 %– 20 % (40–60 cm) in simulated AMWL for an event with T100 compared to T10 in the deep-submergence region (DSR), and 4 %–8 % (5–10 cm) in the shallow-submergence region (SSR). The minor increase in the SSR can be attributed to the strong tidal inﬂuence at these stations. Hung et al. (2012) and Triet et al. (2017) reported a tidal inﬂuence of 70 %– 80 % to the river ﬂow and water level at Can Tho and My Thuan during the ﬂood season. Towards the northern part of the VMD the tidal inﬂuence reduces to below 2 % at Tan Chau and Chau Doc at the border with Cambodia (Hung et al., 2012). Nat. Hazards Earth Syst. Sci., 18, 2859–2876, 2018 www.nat-hazards-earth-syst-sci.net/18/2859/2018/ Table 2. Simulated annual maximum water level at key gauge stations in the Vietnamese Mekong Delta in correspondence with T -year ﬂood event. Simulation Mekong River Bassac River Plain of Reeds Long Xuyen Quadrangle year ﬂood with shp4. This result proves the necessity to in- corporate the temporal evolution of ﬂood events into ﬂood hazard and risk assessments in the MD. Our estimation of in- undated areas from the T10 to the T100 event changed from 5 % to 10 % of the size of the ﬂood-prone region of the VMD (2.0 million ha) in July, from 30 % to 50 % in August, and from 60 % to 75 % for the annual maximum extent. This means that even for the 100-year ﬂood, 25 %–40 % of the ﬂood-prone region was cut off from inundation by the imple- mentation of high dykes, initiated after the ﬂood in 2000. Table 2. Simulated annual maximum water level at key gauge stations in the Vietnamese Mekong Delta in correspondence with T -year ﬂood event. Table 2. Simulated annual maximum water level at key gauge stations in the Vietnamese Mekong Delta in correspondence with T -year ﬂood event. Simulation Mekong River Bassac River Plain of Reeds Long Xuyen Quadrangle Simulation Mekong River Bassac River Plain of Reeds Long Xuyen Quadrangle Inland stations Tan Chau Vam Nao Cao Lanh My Thuan Chau Doc Long Xuyen Can Tho Moc Hoa Hung Thanh Kien Binh Tan An Xuan To Tri Ton Tan Hiep Vi Thanh Phung Hiep [1]* [5] [3] [7] [2] [4] [6] [14] [13] [15] [16] [8] [9] [10] [11] [12] T10Shp1 4.94 3.71 2.69 2.04 4.71 2.78 2.27 3.20 3.29 2.09 1.58 4.69 2.90 1.80 1.01 2.01 T20Shp1 5.15 3.88 2.83 2.07 4.92 2.89 2.30 3.45 3.51 2.30 1.61 4.90 3.05 1.91 1.04 2.03 T50Shp1 5.37 4.06 2.98 2.09 5.12 3.02 2.33 3.71 3.74 2.49 1.64 5.12 3.20 2.02 1.08 2.05 T100Shp1 5.50 4.17 3.07 2.11 5.26 3.10 2.35 3.87 3.89 2.61 1.67 5.25 3.29 2.09 1.11 2.07 T10Shp2 4.54 3.41 2.53 1.99 4.33 2.66 2.22 2.75 2.91 1.76 1.50 4.31 2.75 1.66 0.96 1.98 T20Shp2 4.72 3.55 2.64 2.02 4.51 2.74 2.25 2.99 3.11 1.94 1.54 4.49 2.89 1.74 0.99 2.00 T50Shp2 4.92 3.70 2.76 2.05 4.70 2.84 2.28 3.24 3.32 2.15 1.58 4.68 3.02 1.83 1.02 2.02 T100Shp2 5.05 3.81 2.84 2.06 4.82 2.91 2.30 3.39 3.45 2.27 1.60 4.81 3.11 1.91 1.03 2.03 T10Shp3 4.93 3.75 2.79 1.95 4.68 2.87 2.21 3.22 3.29 2.09 1.50 4.66 3.05 1.83 0.94 1.95 T20Shp3 5.13 3.91 2.92 1.98 4.87 2.98 2.25 3.46 3.51 2.29 1.53 4.85 3.19 1.95 0.96 1.97 T50Shp3 5.35 4.09 3.06 2.01 5.08 3.10 2.28 3.72 3.75 2.50 1.56 5.06 3.34 2.07 0.99 1.99 T100Shp3 5.49 4.20 3.17 2.04 5.21 3.19 2.33 3.88 3.89 2.62 1.60 5.19 3.43 2.14 1.02 2.03 T10Shp4 4.54 3.44 2.55 1.96 4.31 2.67 2.19 2.73 2.87 1.72 1.47 4.28 2.77 1.65 0.94 1.96 T20Shp4 4.73 3.59 2.67 1.99 4.49 2.77 2.22 2.99 3.08 1.90 1.51 4.46 2.92 1.74 0.96 1.98 T50Shp4 4.94 3.76 2.80 2.02 4.69 2.88 2.25 3.26 3.32 2.13 1.55 4.66 3.07 1.85 0.99 2.00 T100Shp4 5.09 3.87 2.89 2.04 4.82 2.95 2.27 3.42 3.47 2.27 1.57 4.79 3.16 1.93 1.01 2.01 * The numbers below the station names refer to the codes in Fig. 1b. Deep-inundation regions are indicated by plain text, shallow-inundation regions are indicated in bold. Stations are listed from upstream to downstream and grouped according to their location as Mekong River, Bassac River, Plain of Reeds, Long Xuyen Quadrangle, and inland stations. www.nat-hazards-earth-syst-sci.net/18/2859/2018/ N. V. K. Triet et al.: Towards risk-based ﬂood management in highly productive paddy rice cultivation N. V. K. Triet et al.: Towards risk-based ﬂood management in highly productive paddy rice cultivation 2870 4.3 Exposed rice cropping area and ﬂood damage Exposed areas and ﬂood damage (D) to rice crop were cal- culated on a pixel basis and then aggregated to the eight provinces located in the study area (Fig. 9 and Table 3). The average damage ranged from USD 39.0 million for a 10- year ﬂood to USD 75.0 million for a 100-year ﬂood. These numbers account for 0.23 %–0.45 % of the total gross do- mestic product (GDP) of the eight provinces in 2011. Since such assessments are not available for neighbouring deltas in South Asia, e.g. Chao Phraya in Thailand or Irrawaddy in Myanmar, a comparison of these deltas is not possible. Our worst scenario, i.e. T100shp3, resulted in damages of USD 115.7 million. This value is less than half of the re- ported overall damage of the ﬂood in 2000 (USD 250 mil- lion; reported in MRC (2012), scaled to USD 500 million with 2011 price levels by Chinh et al., 2016), which is con- sidered a 20-year ﬂood in the MD (Le et al., 2007). Al- though the damage ﬁgures from the event in 2000 were the overall damages, of which agricultural damages were an un- known part, this indicates that a large reduction in ﬂood losses can be linked to the ﬂood management and adapta- tion measures being implemented in the VMD following the Decision No. 99/1996 from the Government of Vietnam (The Government of Viet Nam, 1996). Note that the plan was ini- tiated in 1996, but was implemented to a large extent after the ﬂood in 2000 had occurred. The hydrograph shape has a substantial effect on damage to rice crops in the VMD. The shape shp3 resulted in a high amount of damage, roughly 1.5 times higher than the aver- age damage. Flood hydrograph shp2 closely matched the av- erage damage (85 %–90 %), while shp1 and shp4 resulted in approximately 60 %–70 % of the average damage (Fig. 9b). These ﬁndings support our hazard and risk assessment ap- proach and point to the relevance of the temporal evolution of the ﬂood event for damage estimation. For example, the to- tal ﬂood damage from T10shp3 (USD 57.8 million) was about 10 % higher than the T100shp1 event (USD 51.0 million). et et al.: Towards risk-based ﬂood management in highly productive paddy rice cultivation We found a dif- ference in AMWL ranges from 9 cm at SSR to 45 cm at the DSR induced by the shapes shp2 and shp4 compared to the other two hydrograph shapes. These results are explained by the date of Qmax at Kratie: shp1 and shp3 have the second and higher peak in mid-September (shp3) or mid-October (shp1), about 1 month later than the other FP. When this peak is routed to the VMD, it already meets partly ﬁlled ﬂood- Figure 8 illustrates the frequency distribution of maximum inundation extent for three periods: July, August, and the whole year. This indicator varies strongly depending on the hydrograph shapes. A 10-year ﬂood event with hydrograph shape shp1 results in the same maximum extent as the 100- www.nat-hazards-earth-syst-sci.net/18/2859/2018/ Nat. Hazards Earth Syst. Sci., 18, 2859–2876, 2018 N. V. K. Triet et al.: Towards risk-based ﬂood management in highly productive paddy rice cultivation T bl 3 Fl d d i d h i l l d d f h l d the extremely high damages during the ﬂood in 2000, which had a ﬁrst peak at Tan Chau (point 1 in Fig. 1) on 2 August, i.e. 1 month earlier than usual (Xo et al., 2015). In contrast, damage from late ﬂood events, i.e. shp1 or shp4, are limited to the AWC in the DSR due to failure of dyke structure, or ﬂood levels overtopping the current dyke height, similar to the ﬂood damages recorded in 2011. the extremely high damages during the ﬂood in 2000, which had a ﬁrst peak at Tan Chau (point 1 in Fig. 1) on 2 August, i.e. 1 month earlier than usual (Xo et al., 2015). In contrast, damage from late ﬂood events, i.e. shp1 or shp4, are limited to the AWC in the DSR due to failure of dyke structure, or ﬂood levels overtopping the current dyke height, similar to the ﬂood damages recorded in 2011. ences, with ∼16 % of the total ﬂood damage calculated for Long An located in the Plain of Reeds. This is 10–15 times higher than the share of Kien Giang (D in Kien Giang is less than 2 % of the total D), although the rice cropping area is larger in Kien Giang (Bouvet and Le Toan, 2011; Nguyen et al., 2015). The remaining SSR provinces accounted for about a quar- ter of the total D, which can be linked to their smaller rice cultivation areas in combination with their ﬂood control mea- sures. Our calculated ﬂood damage to rice crop for Can Tho is, on average, in the range USD 2.9–6.3 million from the 10-year to the 100-year ﬂood. These ﬁgures amount to about 60 % of the estimated urban damages of Can Tho (USD 5.0- 9.7 million) for events with similar magnitudes (Chinh et al., 2017). Since Can Tho has the highest urban/rural area ratio compared to the other delta provinces, it is likely that agri- Aggregating the mean D values per province showed that the two northern provinces, An Giang and Dong Thap, ac- counted for about two-thirds of the total D of the VMD, with ﬂood damage in An Giang being about 1.5 times higher than in Dong Thap. This difference increases up to a factor of 3 in the case of late ﬂood peaks (Table 3). This can be explained by the larger areas with triple rice crops in An Giang. N. V. K. Triet et al.: Towards risk-based ﬂood management in highly productive paddy rice cultivation T bl 3 Fl d d i d h i l l d d f h l d N. V. K. Triet et al.: Towards risk-based ﬂood management in highly productive paddy rice cultivation 2871 N. V. K. Triet et al.: Towards risk-based ﬂood management in highly productive paddy rice cultivation 2871 Table 3. Flood damage to rice crop aggregated to the province level and converted to a percentage of the total damage. Simulation Return period An Giang Dong Thap Long An Kien Giang Can Tho Hau Giang Tien Giang Vinh Long T10shp1 10 year 50.5 18.0 13.6 0.2 6.2 1.8 0.9 8.9 T10shp2 30.3 22.7 21.1 1.3 11.2 2.5 3.6 7.2 T10shp3 36.3 29.8 18.9 0.5 6.5 1.6 1.7 4.7 T10shp4 42.9 23.3 14.6 0.2 7.3 2.0 1.0 8.8 T20shp1 20 year 54.1 16.9 12.1 0.2 6.4 1.7 1.0 7.5 T20shp2 27.7 21.7 20.9 2.1 13.3 2.9 5.0 6.3 T20shp3 37.5 27.7 19.9 0.8 6.7 1.5 2.0 3.9 T20shp4 41.2 25.1 14.4 0.2 7.7 2.1 1.2 8.1 T50shp1 50 year 61.0 14.1 10.4 0.2 5.7 1.5 1.3 5.7 T50shp2 25.1 19.7 21.2 2.9 14.3 3.4 7.7 5.9 T50shp3 40.6 24.6 19.5 1.0 7.0 1.6 2.3 3.2 T50shp4 43.4 25.2 13.3 0.2 7.6 2.0 1.6 6.6 T100shp1 100 year 62.2 13.7 10.0 0.2 5.9 1.5 1.3 5.1 T100shp2 26.8 18.0 19.6 4.2 14.4 3.4 8.4 5.2 T100shp3 40.1 23.8 20.2 1.3 7.2 1.7 2.9 2.9 T100shp4 44.5 24.7 13.6 0.3 7.6 2.0 1.6 5.8 10 20 50 100 0 25 50 75 100 125 150 Return period [years] Flood damage [million US$] (b) 10 20 50 100 0 25 50 75 100 Return period [years] Exposed area [10³ ha] (a) Simulated values Fitted curves Shape 1, p = 21 % Shape 2, p = 22 % Shape 3, p = 34 % Shape 4, p = 23 % Mean Figure 9. (a) Total exposed areas of rice crop in the Vietnamese Mekong Delta to ﬂoods of different return periods, calculated for the second and third rice crops and aggregated to the whole year. (b) Total damage for ﬂoods of different return periods. 10 20 50 100 0 25 50 75 100 125 150 Return period [years] Flood damage [million US$] (b) Figure 9. (a) Total exposed areas of rice crop in the Vietnamese Mekong Delta to ﬂoods of different return periods, calculated for the second and third rice crops and aggregated to the whole year. (b) Total damage for ﬂoods of different return periods. Nat. Hazards Earth Syst. Sci., 18, 2859–2876, 2018 Long Xuyen Quadrangle The results provide evidence that rice cropping in the VMD is most vulnerable to ﬂooding stemming from the early ﬂood pulse in August–September when the total plantation area is expected to be at its maximum. During this period damage occurs to the second crop (SAC) in both DSR and SSR, and the AWC in the SSR. This ﬁnding is in line with www.nat-hazards-earth-syst-sci.net/18/2859/2018/ Nat. Hazards Earth Syst. Sci., 18, 2859–2876, 2018 Nat. Hazards Earth Syst. Sci., 18, 2859–2876, 2018 N. V. K. Triet et al.: Towards risk-based ﬂood management in highly productive paddy rice cultivation 2871 Table 3. Flood damage to rice crop aggregated to the province level and converted to a percentage of the total damage. Simulation Return period An Giang Dong Thap Long An Kien Giang Can Tho Hau Giang Tien Giang Vinh Long T10shp1 10 year 50.5 18.0 13.6 0.2 6.2 1.8 0.9 8.9 T10shp2 30.3 22.7 21.1 1.3 11.2 2.5 3.6 7.2 T10shp3 36.3 29.8 18.9 0.5 6.5 1.6 1.7 4.7 T10shp4 42.9 23.3 14.6 0.2 7.3 2.0 1.0 8.8 T20shp1 20 year 54.1 16.9 12.1 0.2 6.4 1.7 1.0 7.5 T20shp2 27.7 21.7 20.9 2.1 13.3 2.9 5.0 6.3 T20shp3 37.5 27.7 19.9 0.8 6.7 1.5 2.0 3.9 T20shp4 41.2 25.1 14.4 0.2 7.7 2.1 1.2 8.1 T50shp1 50 year 61.0 14.1 10.4 0.2 5.7 1.5 1.3 5.7 T50shp2 25.1 19.7 21.2 2.9 14.3 3.4 7.7 5.9 T50shp3 40.6 24.6 19.5 1.0 7.0 1.6 2.3 3.2 T50shp4 43.4 25.2 13.3 0.2 7.6 2.0 1.6 6.6 T100shp1 100 year 62.2 13.7 10.0 0.2 5.9 1.5 1.3 5.1 T100shp2 26.8 18.0 19.6 4.2 14.4 3.4 8.4 5.2 T100shp3 40.1 23.8 20.2 1.3 7.2 1.7 2.9 2.9 T100shp4 44.5 24.7 13.6 0.3 7.6 2.0 1.6 5.8 10 20 50 100 0 25 50 75 100 125 150 Return period [years] Flood damage [million US$] (b) 10 20 50 100 0 25 50 75 100 Return period [years] Exposed area [10³ ha] (a) Simulated values Fitted curves Shape 1, p = 21 % Shape 2, p = 22 % Shape 3, p = 34 % Shape 4, p = 23 % Mean Figure 9. (a) Total exposed areas of rice crop in the Vietnamese Mekong Delta to ﬂoods of different return periods, calculated for the second and third rice crops and aggregated to the whole year. (b) Total damage for ﬂoods of different return periods. 4.5 Uncertainties, limitation and future research directions One of the major sources of uncertainties in our estimation of D and EAD is associated with the inundation maps. The process of interpolating from 1-D water levels to 2-D inun- dation raster inherits uncertainties from the hydraulic model and from the DEM used for interpolation (Brandt, 2016). A full 2-D modelling approach might enhance the quality of ﬂood inundation mapping, but this comes at a cost: the set- up of a full 2-D hydraulic model on that scale is challeng- ing because of the high density of man-made channels and hydraulic infrastructure, which need to be implemented in the model with high accuracy to substantially improve the simulations. Additionally, model runtime becomes critical in detailed large-scale 2-D hydraulic simulations within a risk assessment requiring a large number of model simulations. Large-scale approaches building on coarse-resolution mod- elling with sub-grid parameterization (Sampson et al., 2015) still cannot provide sufﬁcient accuracy to properly map the hydraulic dynamics in such a complex system with ﬂat to- pography, where details matter a lot. However, reﬁning this approach in high resolution, including all relevant hydraulic structures, and implementation in a highly parallelized en- vironment (e.g. on GPUs) could provide a viable path for reducing the uncertainties in hydraulic modelling of the MD. The second development scenario – introducing ﬂoodwa- ter to the paddy ﬁelds after the SAC and no cultivation of AWC in An Giang and Dong Thap – resulted in smaller D and EAD values, as expected. Abandoning AWC cultivation would result in an EAD decrease of about 40 % (Fig. 10b). The majority of these changes in both scenarios stems from changes in An Giang and Dong Thap. The other provinces account for 5 %–15 % of the changes in EAD only. The full ﬂood control measures (high dykes) supporting the expansion of AWC areas in An Giang and Dong Thap after the ﬂood in 2000 have been continuously debated. Triet et al. (2017) revealed that the change from low-dyke to high- dyke systems in this upstream part of the delta increased the inundation hazard in downstream areas, e.g. by an increase of 9–13 cm in AMWL at Can Tho and My Thuan. 4.4 Rice cropping ﬂood risk For the current land use, the EAD for rice cropping in the whole delta amounts to USD 4.5 million (see Fig. 10b), with an average crop risk of USD 1.0–4.6 for each unit of land [ha] (see Fig. 11a). The highest risk was calculated for the provinces located in the deep-submergence region (DSR), except for Kien Giang, where the EAD was very low. p g y An expansion of areas with triple rice cropping in An Gi- ang and Dong Thap, as deﬁned in the expansion develop- ment scenario, would increase the exposure to ﬂooding. Fig- ure 10b shows that this expansion of AWC would triple the EAD to above USD 15.0 million. The expansion of triple rice cropping also means that the current low-dyke system must be raised to a higher design level in order to support the cultivation of AWC during September–November. This, in turn, would lead to higher inundation hazards and risks in downstream provinces (Triet et al., 2017). Figure 11c shows both effects: a substantial increase in EAD in Dong Thap and An Giang, and a slight increase in EAD in the downstream provinces resulting from a higher-inundation hazard caused by the high-dyke development. N. V. K. Triet et al.: Towards risk-based ﬂood management in highly productive paddy rice cultivation 2872 N. V. K. Triet et al.: Towards risk-based ﬂood management in highly productive paddy rice cultivation culture losses have an equal or higher share in ﬂood damages compared to urban losses in the other provinces of the delta. Minderhoud et al., 2017). Expansion of AWC in the northern part of the delta is an option for countering such losses and ensuring food security. Our results could support an evalu- ation of the costs and beneﬁts of further high-dyke devel- opment and triple rice cropping expansion and thus provide important information for future ﬂood management and land- use planning in the delta. Additionally, the damage and risks maps can serve as a basis for ﬂood management. They could support the agricultural insurance, initiated in 2011 by the de- cision of the Prime Minister of Vietnam. In this programme the insurance premium depends on the rice yield only, and the spatial pattern of the ﬂood hazard is not considered (The Government of Viet Nam, 2011). N. V. K. Triet et al.: Towards risk-based ﬂood management in highly productive paddy rice cultivation T bl 3 Fl d d i d h i l l d d f h l d This third crop in the DSR is particularly vulnerable to inundation in October–November (see Fig. 3b). Damages for the other two provinces in the DSR also showed remarkable differ- www.nat-hazards-earth-syst-sci.net/18/2859/2018/ Nat. Hazards Earth Syst. Sci., 18, 2859–2876, 2018 4.5 Uncertainties, limitation and future research directions 4.2 2.6 3.4 1.3 3.3 2.8 1.0 An Giang Tien Giang Hau Giang Dong Thap Long An Vinh Long Can Tho Kien Giang Current land-use 0.1 ¬ (a) 12.0 2.9 12.0 1.7 3.5 2.9 1.1 An Giang Tien Giang Hau Giang Dong Thap Long An Vinh Long Can Tho Kien Giang Expansion of third rice crop in An Giang and Dong Thap ¬ (c) 0.2 1.6 2.3 2.1 1.1 3.3 3.0 0.9 An Giang Tien Giang Hau Giang Dong Thap Long An Vinh Long Can Tho Kien Giang No third rice crop in An Giang and Dong Thap ¬ (b) 0.1 No data < 1.0 1–5 5–10 10–15 Average rice risk [$ ha a ]: 0 km 50 100 Scale: -1 Figure 11. Flood risk for rice crops in the Vietnamese Mekong Delta. Speciﬁc loss is calculated in USD/ha/a for three land-use scenarios: (a) current land use, (b) no autumn–winter crop (AWC) in An Giang and Dong Thap, and (c) expansion of the AWC in these two provinces. 12.0 2.9 12.0 1.7 3.5 2.9 1.1 An Giang Tien Giang Hau Giang Dong Thap Long An Vinh Long Can Tho Kien Giang Expansion of third rice crop in An Giang and Dong Thap ¬ (c) 0.2 1–5 5–10 10–15 Current land-use Expansion of third rice crop in An Giang and Dong Thap Expansion of third rice crop in An Giang and Dong Thap No third rice crop in An Giang and Dong Thap 4.2 2.6 3.4 1.3 3.3 2.8 1.0 An Giang Tien Giang Hau Giang Dong Thap Long An Vinh Long Can Tho Kien Giang Current land-use 0.1 ¬ (a) Averag 0 km 50 100 Scale: (b) (a) (c) Figure 11. Flood risk for rice crops in the Vietnamese Mekong Delta. Speciﬁc loss is calculated in USD/ha/a for three land-use scenarios: (a) current land use, (b) no autumn–winter crop (AWC) in An Giang and Dong Thap, and (c) expansion of the AWC in these two provinces. tem in the MD is well adapted to frequently occurring ﬂoods (in fact these ﬂoods are the basis for the current practice of paddy rice cropping), this effect is likely not as important as in the European studies listed above. ture type and area, plantation calendar, and their vulnerabil- ity to inundation. 4.5 Uncertainties, limitation and future research directions Howie (2005) and Käkönen (2008) challenged the claim that farm- ers could have greater beneﬁt by being able to add another harvest to the cropping system, because of more investment cost for mineral fertilizers to counter the losses of natural fertilization by deposited sediment (Manh et al., 2014), to- gether with negative social and environmental consequences. The proﬁtability of triple rice farming was reported to reduce from initially 57 % to 6 % after 15 years compared to double rice counterparts due to higher production costs (Tran et al., 2018). These arguments and ﬁndings might make the expan- sion of AWC less attractive. Another uncertainty source is the land-use maps used to quantify ﬂood exposure of rice crops. The land-use raster was produced using satellite data from 2014; thus it is somewhat outdated considering the dynamics of agricultural land-use change in the MD. The area of triple-season rice has very likely increased from 2014 to the present. An updated and higher-resolution land-use data set would certainly provide more up-to-date results. Another uncertainty source is the limited number of return periods used to calculate EAD. Ward et al. (2011) showed that the number and choice of the selected return periods can introduce a signiﬁcant bias in the EAD estimates. They also pointed out the importance of considering damages to fre- quently occurring low damage ﬂoods, in line with the ﬁnd- ings of Merz et al. (2009). However, as the agricultural sys- On the other hand, rice cultivation areas in the southern part of the delta are likely to decrease due to increased salin- ity intrusion following higher sea levels (Smajgl et al., 2015; Hak et al., 2016) and land subsidence (Laura et al., 2014; www.nat-hazards-earth-syst-sci.net/18/2859/2018/ Nat. Hazards Earth Syst. Sci., 18, 2859–2876, 2018 N. V. K. Triet et al.: Towards risk-based ﬂood management in highly productive paddy rice cultivation N. V. K. Triet et al.: Towards risk-based ﬂood management in highly productive paddy rice cultivation 2873 Figure 10. (a) Total ﬂood damage for different return periods corresponding to two land-use scenarios. (b) Average crop risk as speciﬁc loss for each hydrograph shape. Figure 10. (a) Total ﬂood damage for different return periods corresponding to two land-use scenarios. (b) A for each hydrograph shape. Figure 10. (a) Total ﬂood damage for different return periods corresponding to two land-use scenarios. (b) Average crop risk as speciﬁc loss for each hydrograph shape. 4.5 Uncertainties, limitation and future research directions Such risk assessments could then be used for scenario planning by varying land-use types and cropping patterns or changing boundary conditions by climate change and upstream developments. In addition, dedicated efforts for validation and development of crop damage curves for the VMD based on recorded ﬂood damages at the plot scale would increase the credibility of the presented risk analysis. Our study is validated against large scale, aggregated dam- age data only, and it is open to ensuring that small-scale vari- ations, which could be important for local adaptation mea- sures, are represented sufﬁciently well. Our work does not consider ﬂood damage to other agricul- ture crops (e.g. orchard farms) or land-use types (e.g. shrimp farms). Although these production types are smaller than rice cropping in terms of area, they generate much higher eco- nomic value per cultivation unit. Therefore, it is highly rec- ommended to include these crops in future studies on agricul- tural ﬂood risk in the VMD. To facilitate such assessments, efforts need to be made to collect data on crop and aquacul- Nat. Hazards Earth Syst. Sci., 18, 2859–2876, 2018 Nat. Hazards Earth Syst. Sci., 18, 2859–2876, 2018 5 Conclusions such a scheme, a reliable seasonal forecasts of the expected ﬂoods are required. Our estimation of ﬂood damage (D) can be used as reference for developing such management plans based on a thorough cost–beneﬁt analysis including a quan- titative consideration of the beneﬁts of natural fertilization vs. mineral fertilizers. (3) The current pilot programme on agriculture insurance should be revised, at least for rice crop- ping in the VMD. Insurance premiums are preferably calcu- lated based on the average rice yield per province. Using our spatially explicit results would include the actual ﬂood haz- ard of the insured area. However, the damages should be up- dated to current economic values, as the values of 2011 were used. such a scheme, a reliable seasonal forecasts of the expected ﬂoods are required. Our estimation of ﬂood damage (D) can be used as reference for developing such management plans based on a thorough cost–beneﬁt analysis including a quan- titative consideration of the beneﬁts of natural fertilization vs. mineral fertilizers. (3) The current pilot programme on agriculture insurance should be revised, at least for rice crop- ping in the VMD. Insurance premiums are preferably calcu- lated based on the average rice yield per province. Using our spatially explicit results would include the actual ﬂood haz- ard of the insured area. However, the damages should be up- dated to current economic values, as the values of 2011 were used. A top-down approach to estimate ﬂood damages and risks to rice cropping in the ﬂood-prone areas of the Vietnamese Mekong Delta (VMD) is presented. The work was moti- vated by recent publications stating that extreme ﬂoods in the Mekong are likely to occur more frequently in the 21st century (Delgado et al., 2010; Hirabayashi et al., 2013), but also by the perceived need to shift ﬂood management towards a risk-based approach. The presented quantiﬁcation of ﬂood risks to rice crops, the predominant land use in the region, is the ﬁrst step in this direction, as a large-scale ﬂood risk assessment has not been implemented to date. This work is thus the very ﬁrst publication on a large-scale ﬂood risk as- sessment for the agricultural sector in the VMD. Finally, our inundation hazard maps can be used to quan- tify ﬂood damages and risks to other agricultural crops and land use in the MD if appropriate land-use maps and dam- age models are available. 5 Conclusions In a similar manner changes in ﬂood hazard and risk inﬂicted by impacts of climate change, sea level rise, the pronounced deltaic land subsidence, land- use changes, and upstream hydropower development can be quantiﬁed systematically. These issues will be addressed in future work. The results showed that the timing of the ﬂoods, the high tides and the cropping calendar are crucial factors for agri- cultural crop damage. Although the cropping calendars are adapted to the general ﬂood dynamics in the different areas of the MD, large damages might still occur in the case of extreme events. A reliable seasonal forecast of the annual ﬂoods would thus be very helpful for a risk-based adaptive ﬂood management of agricultural production. The study sug- gests that ﬂood mitigation measures by the government and farmers, e.g. shifting of the cropping calendar and construc- tion of dykes and sluice gates, before and after the histori- cal ﬂood in 2000 have greatly reduced potential agricultural ﬂood damage. Data availability. The data used in this paper are not publicly accessible; however, the authors can be contacted by email (triet@gfzpotsdam.de or heiko.apel@gfz-potsdam.de) for help in contacting the persons or authorities to acquire such data and how this should be acknowledged. The risk indicators, expected annual damage (EAD), and average crop risk per province can serve as the basis on which to develop spatially explicit ﬂood management and mitiga- tion plans for the delta. The crop risk maps, corresponding to two land-use change scenarios which are frequently used in the public and academic discussion, could be used as input for a cost–beneﬁt analysis to evaluate the alternative of en- largement of the third rice crop in the two northern provinces. Author contributions. All authors contributed to the preparation of this paper. Competing interests. The authors declare that they have no conﬂict of interest. Based on our ﬁndings, the following suggestions can be made to support ﬂood management in the region: (1) appro- priate maintenance is necessary for the ﬂood control systems, with a strong emphasis on the low-dyke systems providing protection against the early ﬂood wave before September. (2) The rice cropping scheme referred to as “ba nam tam vu”, meaning eight rice crops every 3 years, should be reviewed. N. V. K. Triet et al.: Towards risk-based ﬂood management in highly productive paddy rice cultivation 2874 www.nat-hazards-earth-syst-sci.net/18/2859/2018/ www.nat-hazards-earth-syst-sci.net/18/2859/2018/ Nat. Hazards Earth Syst. Sci., 18, 2859–2876, 2018 5 Conclusions According to this scheme, sluice gates will be opened to al- low ﬂoodwater to inundate the compartments in order to re- plenish the natural fertilization with deposited sediment at least once every 3 consecutive years. However, the study of Manh et al. (2014) revealed that during low ﬂood years the estimated deposited sediment in the VMD was ∼14 times smaller than in years of extreme ﬂoods. Thus, opening ﬂood compartments during low ﬂood year might result in little sed- iment deposition in paddy ﬁelds. We propose to open the ﬂood compartments protected with high dykes in An Giang and Dong Thap during extreme events (i.e. larger than a 10- year return period). However, for a proper implementation of Special issue statement. This article is part of the special issue “Flood risk assessment and management”. It is a result of the EGU General Assembly 2018, Vienna, Austria, 8–13 April 2018. Acknowledgements. The work leading to this publication was supported by the German Academic Exchange Service (DAAD) with funds from the German Federal Ministry of Education and Research (BMBF). We acknowledge Akihiko Kotera at Vietnam Japan University for providing processed EVI data. The authors want to specially thank Pham The Vinh and various colleagues at SIWRR for their support in providing the DEM and survey data. We would like to thank Jorge Ramirez and the two anonymous referees who reviewed the manuscript for their comments and suggestions that helped to improve the paper. The article processing charges for this open-access publication were covered by a Research Author contributions. All authors contributed to the preparation of this paper. 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https://openalex.org/W2900192320	https://hrcak.srce.hr/file/297875	English	null	A preliminary study for early breast cancer detection with microwaves	Tehnički glasnik	2,018	cc-by	3,011	1 INTRODUCTION Another method used in breast cancer detection is Ultrasound imaging (USI) in which transient waves are used. In USI, imaging is done by collecting back-reflections from the sound waves sent into the tissue. USI consists of disadvantages due to the pressure application with measuring probe and the necessity of using coupling gel to increase the amount of wave penetrating into the tissue by preventing the reflection of sound waves. The breast cancer, which is especially common among women, occurs on the breast tissue covering the wide area between collarbone and armpit and extending on the chest muscles including the chest. Breast cancer emerges from occurrences of mutations and abnormal changes on genes that control the growth and regeneration of cells. Healthy cell, which has the ability of knowing the way and time of division, multiplies uncontrollably by losing this ability due to gene problems and generates cancerous tissues. Unhealthy tissue formation usually occurs at the tip of the breast lobules or milk channels [1]. Microwave imaging is seen as an alternative method to the above mentioned X-mammography, MRI and USG methods [6-9]. The advantage of microwave imaging is that it allows imaging without the need for any compression or pressure application at lower frequencies than X-rays. With increasing momentum in recent years, microwave imaging located at the center of interest of researchers is still in the research and development phase and prototypes have begun to be created by various research groups [10, 11]. A study in 2009 has shown that deaths in Turkey are mostly due to cardiovascular disorders and cancer and also women with cancer have the most breast cancer [2, 3]. Again according to the same research, deaths caused by cancer are expected to rise to the first place from 2015. Early diagnosis is important for reduction of cancer-related deaths and treatment. While early stage diagnosis is to help of the cancer treatment, delay of diagnosis reduces chances of treatment [4]. In the literature, there are some theoretical and simulation studies. Li et al. [12] presented co-focal microwave imaging algorithm. In the presented study [12], malignant tumor with a diameter of 2mm at the bottom of 3.1 cm deep is placed to developed two-dimensional, anatomically realistic, MRI-derived FDTD (finite difference time Domain, finite differences in time domain) model and developed image creation algorithm is applied. A PRELIMINARY STUDY FOR EARLY BREAST CANCER DETECTION WITH MICROWAVES Emine AVŞAR AYDIN Abstract: Breast cancer is the most common type of cancer in female all over the world. Early detection and treatment gives a chance to overcome this cancer. In breast cancer detection, there are many methods such as X-ray mammography, Magnetic Resonance Imaging, and Ultrasound Imaging. However, existing these methods have limitations such as X- rays, disturbing pressure on the breast, and high cost of devices, etc. Because of all the reasons mentioned, microwave breast imaging has the potential to overcome from some of the limitations of conventional breast cancer screening systems. The physical basis breast cancer detection by microwaves depends on the difference between the dielectric properties of normal and malignant breast tissue. Microwave breast cancer imaging is also a noninvasive method and it has low cost. Therefore, microwave imaging technology for breast cancer detection has attracted much attention by many researchers in these days. By using Computer Simulation Technology Microwave Studio and Antenna Magus Software, breast model with tumor and antennas were generated in this study. The presence of the tumor was investigated using a receiver and a transmitting antenna. While the transmitting antenna was stationary, the receiving antenna was moved to different positions. S11 (return loss) results were evaluated. This study is a preliminary study to determine the location and characteristic features of the tumor. Furthermore, this study will show that scanning methods will determine the location and size of the tumor at higher accuracy and the reconstruction of the reflected waves will allow to clearly determining the location of the tumor. Keywords: antenna design; breast cancer; computer simulation technology (CST); dielectric properties; microwaves ISSN 1846-6168 (Print), ISSN 1848-5588 (Online) https://doi.org/10.31803/tg-20180316133537 ISSN 1846-6168 (Print), ISSN 1848-5588 (Online) https://doi.org/10.31803/tg-20180316133537 Preliminary communication 2.2 Breast Model As shown in Fig. 2, the breast has hemispherical model and the dimensions of the breast tissues and tumor are shown in Tab. 2. The dielectric properties of each tissue are shown in Tab. 2 [11]. One of the dielectric properties is the conductivity (σ), other is the relative permittivity (ɛr). 2 MATERIAL AND METHOD Figure 2 Breast structure in CST Microwave Studio Table 2 Features of breast structure Tissue Portion Size Breast Diameter 100 mm Skin Thickness 2 mm Tumor Radius 5 mm Table 3 Dielectric properties of breast tissues and tumor Tissue Portion Conductivity (S/m) Relative Permittivity Skin 1.49 37.9 Fat 0.14 5.14 Tumor 1.4 50 Figure 2 Breast structure in CST Microwave Studio Figure 2 Breast structure in CST Microwave Studio The physical basis for microwave imaging lies in the significant contrast in the dielectric properties between the normal breast tissue and the malignant tissue at microwave frequencies [15]. Microwave imaging (MWI) techniques consist of tomography based and radar based imaging. Communication of through breast is saved by microwave tomography. At the same time, an electrical property map of the region of interest is created using radar-based imaging [16, 17]. By transmitting electromagnetic waves through the female breast, the scattered field is obtained on the breast in MWI method. While the transmitting antennas are fed by the electromagnetic signals, the receiving antennas collect the electromagnetic signals and define by scattering parameters "S-parameters". Figure 2 Breast structure in CST Microwave Studio Figure 2 Breast structure in CST Microwave Studio Table 2 Features of breast structure Tissue Portion Size Breast Diameter 100 mm Skin Thickness 2 mm Tumor Radius 5 mm Figure 1 Antenna structure in Antenna Magus Software Table 3 Dielectric properties of breast tissues and tumor Tissue Portion Conductivity (S/m) Relative Permittivity Skin 1.49 37.9 Fat 0.14 5.14 Tumor 1.4 50 Emine AVŞAR AYDIN: A PRELIMINARY STUDY FOR EARLY BREAST CANCER DETECTION WITH MICROWAVES are collected and a filtered imaging algorithm is applied to these data. Simulation results confirm with measurement results [14]. in Tab. 1. On the other hand, microstrip patch antenna with breast model was designed in CST and simulation was performed in CST Design Studio. In this article, the presence of the tumor was investigated using a receiver and a transmitting antenna. While the transmitting antenna was stationary, the receiving antenna was moved to different positions. This study is a preliminary study to determine the location and characteristic features of the tumor. 3 RESULTS In this article, the presence of the tumor was investigated using a receiver and a transmitting antenna. As shown in Figure 3 that while the transmitting antenna was stationary, the receiving antenna was moved to different positions. Figure 1 Antenna structure in Antenna Magus Software Figure 3 Data collection process using the receiving circular patch antenna moving vertically along two red dots antenna as the transmitting antenna is stationary Table 1 Features of used antenna Parameter Values Dielectric substrate 2.0 Antenna length 54.2428 mm Antenna width 54.2428 mm Antenna height 1 mm Diameter of patch antenna 18.08095 mm Feed-pin radius 0.25 mm Coaxial diameter 1.15128 mm Table 1 Features of used antenna Parameter Values Dielectric substrate 2.0 Antenna length 54.2428 mm Antenna width 54.2428 mm Antenna height 1 mm Diameter of patch antenna 18.08095 mm Feed-pin radius 0.25 mm Coaxial diameter 1.15128 mm 1 INTRODUCTION As a result of the application, the tumor is found to be 6 mm in diameter and 3.3 cm deep. Widespread and primary method used for the screening of breast cancer is X-ray mammography which uses low power X-rays. The breast is compressed for better imaging and exposed to ionizing X-rays even if low-power in X-ray mammography. This method is painful due to compression and there is a possibility of damaging the tissue because it uses ionizing X-rays. [5]. The method presented by Xie et al. [13] as multistatic adaptive microwave imaging (MAMI) demonstrates high resolution and low side lobe performance. The characteristics of the method in presented study are shown on a three dimensional breast model simulated by the FDTD method. Magnetic resonance imaging (MRI) is an alternative method to X-ray mammography in breast cancer imaging. MRI overcomes the disadvantages of X-ray mammography and can produce successful results about the presence of the tumor in the tissue. But it cannot demonstrate the same success in terms of whether the tumor is good or bad. [5]. Another study in the literature is by Zhou [14]. In microwave imaging studies based on broadband profiles, electric field data scattered from multiple objects in a target TEHNIČKI GLASNIK 12, 2(2018), 109-112 109 Emine AVŞAR AYDIN: A PRELIMINARY STUDY FOR EARLY BREAST CANCER DETECTION WITH MICROWAVES 2.1 Antenna Model Figure 5 The magnitude of two signals (with tumor and without tumor) Figure 6 Comparison of two S11 (with and without tumors) the antenna is placed away from the axis the tumor [18] 4 CONCLUSION In this study, a microwave imaging system was preferred to detect breast cancer. CST software was used to create a life- like breast phantoms and a circular pin-fed linearly polarized microstrip patch antenna which generates a microwave signal. The receiver antenna was moved up from the bottom of the breast structure to generate scatter signals. This movement repeated for breast model with tumor and without tumor. The difference between tumor and without tumor showed that microwave imaging system is successful for breast cancer detection. In future work, a viable 3D image reconstruction process will be applied and will be repeated experimentally at the same time. This study will show that scanning methods will determine the location and size of the tumor at higher accuracy and the reconstruction of the reflected waves will allow to clearly determining the location of the tumor. 5 REFERENCES [1] Meme Kanseri, Wikipedia, http://tr.wikipedia.org/wiki/Meme _kanseri, Feb. 2014. recognized. a) b) c) d) e) f) Figure 4 The S11 results (return loss) in frequency domain for each different position In a study, the S11 simulations with and without tumors were compared at some antenna positions [18]. The positions where the antenna is on the axis of a tumor, S11 of the two simulations with tumor and without tumor were differentiated, i th b b d f 6 t 8 GH (Fi 6) Figure 5 The magnitude of two signals (with tumor and without tumo Figure 5 The magnitude of two signals (with tumor and without tumor) Figure 6 Comparison of two S11 (with and without tumors) the antenna is placed away from the axis the tumor [18] Figure 6 Comparison of two S11 (with and without tumors) the antenna is placed away Figure 6 Comparison of two S11 (with and without tumors) the antenna is placed away from the axis the tumor [18] [1] Meme Kanseri, Wikipedia, http://tr.wikipedia.org/wiki/Meme _kanseri, Feb. 2014. 4 CONCLUSION In this study, a microwave imaging system was preferred to detect breast cancer. CST software was used to create a life- like breast phantoms and a circular pin-fed linearly polarized microstrip patch antenna which generates a microwave signal. The receiver antenna was moved up from the bottom of the breast structure to generate scatter signals. This movement repeated for breast model with tumor and without tumor. The difference between tumor and without tumor showed that microwave imaging system is successful for breast cancer detection. In future work, a viable 3D image reconstruction process will be applied and will be repeated experimentally at the same time. This study will show that scanning methods will determine the location and size of the tumor at higher accuracy and the reconstruction of the reflected waves will allow to clearly determining the location of the tumor. f) Figure 4 The S11 results (return loss) in frequency domain for each different position In a study, the S11 simulations with and without tumors were compared at some antenna positions [18]. The positions where the antenna is on the axis of a tumor, S11 of the two simulations with tumor and without tumor were differentiated, in the same sub-band of 6 to 8 GHz, (Fig. 6). 2.1 Antenna Model Figure 3 Data collection process using the receiving circular patch antenna moving vertically along two red dots antenna as the transmitting antenna is stationary A circular pin-fed linearly polarized microstrip patch antenna was preferred in this paper. This antenna was put into Computer Simulation Technology (CST) platform from Antenna Magus Software, a tool for modeling and designing antennas (Fig. 1). The reason for choosing this antenna in this study is its low cost, low profile, lightweight, and high efficiency [17]. Features of the antenna are shown As shown in Fig. 3, the receiver antenna was moved up from the bottom of the breast structure to generate scatter signals. S11 (return loss) results were obtained at each antenna position from the bottom of breast structure to top. Graphics (a, b, c, d, e, and f) in Fig. 4 represent different positions of receiving antenna. When the antenna is in the same position as TECHNICAL JOURNAL 12, 2(2018), 109-112 110 Emine AVŞAR AYDIN: A PRELIMINARY STUDY FOR EARLY BREAST CANCER DETECTION WITH MICROWAVES Figure 5 The magnitude of two signals (with tumor and without tumor) Figure 6 Comparison of two S11 (with and without tumors) the antenna is placed away from the axis the tumor [18] Fi 5 Th it d f t i l ( ith t d ith t t ) the tumor or different position, the results vary. For instance; b graphic shows that the antenna is in the same positon as the tumor and also is different from others. On the other hand, Fig. 5 shows two signals at the same position from two simulation models (one of them is with tumor, other is without tumor). It is clear that two signals’ magnitude can be recognized. tumor). It is clear that two signals magnitude can be recognized. a) b) c) d) e) f) Figure 4 The S11 results (return loss) in frequency domain for each different position In a study, the S11 simulations with and without tumors were compared at some antenna positions [18]. The positions where the antenna is on the axis of a tumor, S11 of the two simulations with tumor and without tumor were differentiated, in the same sub-band of 6 to 8 GHz, (Fig. 6). Author’s contact: [5] Hassan, A. M. & El-Shenawee, M. (2011). Review of electromagnetic techniques for breast cancer detection. IEEE Reviews in Biomedical Engineering, 4, 103-118. https://doi.org/10.1109/RBME.2011.2169780 Emine AVŞAR AYDIN Adana Science and Technology University, Department of Aeronautical Engineering Adana Bilim ve Teknoloji Üniversitesi, Gültepe Mh., Sarıçam/Adana, Turkey eaydin@adanabtu.edu.tr rasvaenime@gmail.com Emine AVŞAR AYDIN Adana Science and Technology University, Department of Aeronautical Engineering Adana Bilim ve Teknoloji Üniversitesi, Gültepe Mh., Sarıçam/Adana, Turkey eaydin@adanabtu.edu.tr rasvaenime@gmail.com g [6] Xu, M., Thulasiraman, P., & Noghanian, S., (2011). Microwave Tomography for Breast Cancer Detection on Cell Broadband Engine Processors. J. Parallel Distrib. Comput., 72, 1106-1116. https://doi.org/10.1016/j.jpdc.2011.10.013 [7] Santorelli, A. & Popovic, M. (2011). SAR Distribution in Microwave Breast Screening: Results with TWTLTLA Wideband Antenna. Intelligent Sensors, Sensor Networks and Information Processing (ISSNIP) / Adelaide, 11-16. https://doi.org/10.1109/ISSNIP.2011.6146531 [8] Lazaro, A., Girbau, D., & Villarino, R. (2010). Simulated and Experimental Wavelet-based Detection of Breast Tumor using a UWB Radar. 40th European Microwave Conference / Paris, 373-376. [9] Zhang, D. & Mase, A. (2011). Ultrashort-Pulse Radar System for Breast Cancer Detection Experiment: Imaging in Frequency Band. Microwave Conference Proceedings (CJMW) / Hangzhou, 1-3. [10] Meaney, P. M., Fanning, M. W., Li, D., Poplack, S. P., & Paulsen K. D. (2000). A Clinical Prototype for Active Microwave Imaging of the Breast. IEEE Transactions on Microwave Theory and Techniques, 48(11), 1841-1853. https://doi.org/10.1109/22.883861 [11] Klemm, M., Craddock, I. J., Leendertz, J. A., Preece, A., Gibbins, D. R., Shere, M., & Benjamin, R. (2010). Clinical Trials of a UWB Imaging Radar for Breast Cancer. Antennas and Propagation (EuCAP), 2010 Proceedings of the Fourth European Conference on. [12] Li, X. & Hagness S. C. (2001). A Confocal Microwave Imaging Algorithm for Breast Cancer Detection. IEEE Microwave and Wireless Components Letters, 11(3), 130-132. https://doi.org/10.1109/7260.915627 [13] Xie, Y., Guo, B., Xu, L., Li, J., & Stoica, P. (2006). Multistatic Adaptive Microwave Imaging for Early Breast Cancer Detection. IEEE Transactions on Biomedical Engineering, 53(8), 1647-1657. https://doi.org/10.1109/TBME.2006.878058 [14] Zhou, Y. (2010). Microwave Imaging based on Wideband Range Profiles. Progress in Electromagnetics Research Letters, 19, 57-65. https://doi.org/10.2528/PIERL10100608 [15] Xie, Y., Guo, B., Xu, L., Li, J., & Stoica, P. (2006). Multistatic adaptive microwave imaging for early breast cancer detection. IEEE Trans. Biomed. Eng., 53(8), 1647- 1657. https://doi.org/10.1109/TBME.2006.878058 [16] Bahramiabarghouei, H., Porter, E., Santorelli, A., Gosselin, B., Popovi, M., & Rusch, L. A. (2015). Flexible 16 antenna array for microwave breast cancer detection. IEEE Trans. Biomed. Eng., 62(10), 2516-2525. 5 REFERENCES TEHNIČKI GLASNIK 12, 2(2018), 109-112 111 Emine AVŞAR AYDIN: A PRELIMINARY STUDY FOR EARLY BREAST CANCER DETECTION WITH MICROWAVES / Khartoum, Sudan. https://doi.org/10.1109/ICCCCEE.2017.7866083 [18] Chouiti, S. M., Merad, L., & Meriah, S. M. A Microwave Imaging Technique Implementation for Early Detection of Breast Tumors. Advances in Circuits, Systems, Signal Processing and Telecommunications, pp. 233-236. / Khartoum, Sudan. https://doi.org/10.1109/ICCCCEE.2017.7866083 [18] Chouiti, S. M., Merad, L., & Meriah, S. M. A Microwave Imaging Technique Implementation for Early Detection of Breast Tumors. Advances in Circuits, Systems, Signal Processing and Telecommunications, pp. 233-236. [2] Kanser Kayıtçılığı, Türkiye Halk Sağlığı Kurumu Kanser Daire Başkanlığı, http://www.kanser.gov.tr/daire-faaliyetleri/ kanser-kayitciligi/108-t%C3%BCrkiyede-kanser-kayitcigi. html#sthash.rW1OWNl4.dpuf, Dec. 2013. / Khartoum, Sudan. https://doi.org/10.1109/ICCCCEE.2017.7866083 [18] Chouiti, S. M., Merad, L., & Meriah, S. M. A Microwave Imaging Technique Implementation for Early Detection of Breast Tumors. Advances in Circuits, Systems, Signal Processing and Telecommunications, pp. 233-236. p [3] Gültekin, M. (2013). Kanser Dairesi Faaliyetleri. [4] Kanser Türleri: Meme Kanseri, Türkiye Halk Sağlığı Kurumu Kanser Daire Başkanlığı, http://www.kanser.gov.tr/kanser/ kanser-turleri/49-memekanseri.html#sthash.RFWV7MIA. dpuf (2013). TECHNICAL JOURNAL 12, 2(2018), 109-112 Author’s contact: https://doi.org/10.1109/TBME.2015.2434956 [17] Eltigani, F. M., Yahya, M. A. A., & Osman, M. E. (2017). 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https://openalex.org/W2884511978	https://www.um.edu.mt/library/oar/bitstream/123456789/55622/1/Relationship_of_Hyperglycaemia_Hypoglycaemia_and_Glucose_Variability.pdf	English	null	Relationship of Hyperglycaemia, Hypoglycaemia, and Glucose Variability to Atherosclerotic Disease in Type 2 Diabetes	Journal of diabetes research	2,018	cc-by	7,418	Hindawi Journal of Diabetes Research Volume 2018, Article ID 7464320, 9 pages https://doi.org/10.1155/2018/7464320 Correspondence should be addressed to Stephen Fava; stephen.fava@um.edu.mt Correspondence should be addressed to Stephen Fava; stephen.fava@um.edu.mt Correspondence should be addressed to Stephen Fava; stephen.fava@um.edu.mt Received 2 January 2018; Revised 1 June 2018; Accepted 24 June 2018; Published 22 July 2018 Academic Editor: Andrea Flex Academic Editor: Andrea Flex Copyright © 2018 Caroline Jane Magri et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Objective. Type 2 diabetes mellitus (T2DM) is known to be associated with increased cardiovascular risk. The aim of this study was therefore to investigate the independent eﬀects of hyperglycaemia, hypoglycaemia, and glucose variability on microvascular and macrovascular disease in T2DM. Methods. Subjects with T2DM of <10 years duration and on stable antiglycaemic treatment underwent carotid intima-media thickness (CIMT), ankle-brachial index (ABI), albumin-creatinine ratio (ACR), and HbA1c measurement, as well as 72-hour continuous glucose monitoring. Macrovascular disease was deﬁned as one or more of the following: history of ischaemic heart disease (IHD), cerebrovascular accident (CVA), ABI < 0.9, or abnormal CIMT. Results. The study population comprised 121 subjects with T2DM (89 males : 32 females). The mean age was 62.6 years, and the mean DM duration was 3.7 years. Macrovascular disease was present in 71 patients (58.7%). In multivariate logistic regression analysis, body surface area (BSA) (OR 18.88 (95% CI 2.20–156.69), p = 0 006) and duration of blood glucose (BG) < 3.9 mmol/L (OR 1.12 (95% CI 1.014–1.228), p = 0 024) were independent predictors of macrovascular disease. BSA (OR 12.6 (95% CI 1.70–93.54), p = 0 013) and duration of BG < 3.9 mmol/L (OR 1.09 (95% CI 1.003–1.187), p = 0 041) were independent predictors of abnormal CIMT. Area under the curve for BG > 7.8 mmol/L (β = 15.83, p = 0 005) was the sole independent predictor of albuminuria in generalised linear regression. Conclusions. This study demonstrates that hypoglycaemia is associated with the occurrence of atherosclerotic disease while hyperglycaemia is associated with microvascular disease in a Caucasian population with T2DM of recent duration. Caroline Jane Magri ,1,2 Dillon Mintoff ,3 Liberato Camilleri ,4 Robert G. Xuereb,2 Joseph Galea,5 and Stephen Fava 5 Caroline Jane Magri ,1,2 Dillon Mintoff ,3 Liberato Camilleri ,4 Robert G. Xuereb,2 Joseph Galea,5 and Stephen Fava 5 1Department of Cardiology, Mater Dei Hospital and University of Malta, Msida, Malta 2Department of Cardiology, Mater Dei Hospital, Msida, Malta 3Mater Dei Hospital, Msida, Malta 4Statistics & Operations Research, Faculty of Science, University of Malta, Msida, Malta 5Mater Dei Hospital and University of Malta, Msida, Malta Caroline Jane Magri ,1,2 Dillon Mintoff ,3 Liberato Camilleri ,4 Robert G. Xuereb,2 Joseph Galea,5 and Stephen Fava 5 1Department of Cardiology, Mater Dei Hospital and University of Malta, Msida, Malta 2Department of Cardiology, Mater Dei Hospital, Msida, Malta 3M t D i H it l M id M lt 1Department of Cardiology, Mater Dei Hospital and University of Malta, Msida, Malta 2Department of Cardiology, Mater Dei Hospital, Msida, Malta p 4Statistics & Operations Research, Faculty of Science, University of Malta, Msida, Malta 5Mater Dei Hospital and University of Malta, Msida, Malta 4Statistics & Operations Research, Faculty of Science, University of Malta, Msida, Malta 5Mater Dei Hospital and University of Malta, Msida, Malta 4Statistics & Operations Research, Faculty of Science, University of Malta, Msida, Malta 5Mater Dei Hospital and University of Malta, Msida, Malta 1. Introduction associated with a twofold increased risk of cardiovascular dis- ease in type 2 diabetes [5]. Furthermore, both hyperglycae- mia and hypoglycaemia following acute coronary syndrome have been associated with increased mortality [6, 7] The tra- ditional explanations of these observations are the proar- rhythmogenic eﬀect of hypoglycaemia as a consequence of catecholamine release and QTc prolongation [8, 9] and the unavailability of an energy substrate to the myocardium dur- ing hypoglycaemia leading to an ischaemia equivalent [10]. However, the increased mortality extends beyond the hypo- glycaemic episode itself. Although a hypoglycaemic episode is known to predict future ones [11], this is unlikely to be Diabetes is known to be associated with both microvascular and macrovascular disease [1]. Whilst good glycaemic con- trol has been consistently shown to reduce microvascular dis- ease, most intervention studies have failed to document a reduction in macrovascular disease outcomes [2, 3]. One possible explanation for this discrepancy is the det- rimental eﬀect of hypoglycaemia. Östgren and colleagues have recently reported a U-shaped relationship of HbA1c with cardiovascular events, including mortality [4]. A recent meta-analysis has also conﬁrmed that hypoglycaemia is Journal of Diabetes Research Journal of Diabetes Research 2 of Malta Research Ethics Committee. All participants gave written informed consent. the sole mechanism. Another possible contributory mecha- nism that has not been well investigated is the possibility that hypoglycaemia may accelerate atherosclerosis, as suggested by some animal studies. For example, Yasunari and colleagues reported that repeated hypoglycaemia worsens injury- provoked intimal thickening in a male Goto-Kakizaki rat carotid artery [12], whilst Jin and colleagues reported that hypoglycaemiainducedmonocyteadhesiontorataorticendo- thelium [13]. Furthermore, a small study found higher carotid and femoral intima-media thickness in 25 subjects with type 1 diabetes and with repeated hypoglycaemic episodes when comparedto20subjectswithtype1diabetesbutwithouthypo- glycaemia [14]. However, continuous glucose monitoring was not performed, and therefore, hyperglycaemic episodes were not captured. Since the two groups had similar HbA1c, it is likely that the hypoglycaemic group also had more hyper- glycaemia, which might conceivably have contributed to increased intima-media thickness. 2.1. Clinical and Laboratory Measurements. The presence of history of ischaemic heart disease and of cerebrovascular dis- ease was ascertained by use of a questionnaire and by review of medical notes. Height and weight were measured using a calibrated balance and a stadiometer, with the subjects wear- ing light clothing and without shoes. Body mass index (BMI) was calculated as weight divided by height squared. 1. Introduction Waist circumference was measured to the nearest 0.5 cm in the hor- izontal plane at the midpoint between the lowest rib and the iliac crest [17]. The waist index was calculated as waist cir- cumference (cm) divided by 94 for men and 80 for women [18] Oﬃce blood pressure was measured in the supine posi- tion after 5 min of rest. All subjects underwent routine blood investigations in the fasting state on the day of the clinical examination. No medication was taken on the morning of the examina- tion. Patients who were treated with insulin discontinued injections after 22:00 on the day preceding the examina- tion. Estimated glomerular ﬁltration rate (eGFR) was calcu- lated using the Modiﬁed Diet in Renal Disease (MDRD) formula (National Kidney Foundation Calculator for Health- care Professionals). Haemoglobin A1c (measured using high- performance liquid chromatography) and fructosamine were taken as markers of glycaemic control. Urine albumin was measured by an immunoturbidometric technique (Roche Diagnostics, Mannheim, Germany). Creatinine was mea- sured by a kinetic colorimetric test using the Jaﬀe reaction (Roche Diagnostics). Albumin-creatinine ratio (ACR) was assessed in all patients as a marker of microvascular disease. Body surface area was provided by the Esaote machine used to perform CIMT by inputting weight and height. Another possible explanation of the failure of clinical tri- als to demonstrate a beneﬁcial eﬀect of glycaemic control is that none of them targeted reducing ﬂuctuations in blood glucose. Indeed, many forms of treatment may increase blood glucose ﬂuctuations. Acute ﬂuctuations in blood glu- cose have been shown to be associated with increased mortal- ity [15]. This may be mediated by endothelial dysfunction, oxidative stress, and endothelial cell apoptosis [16]. Such eﬀects of glucose ﬂuctuations could predispose to both microvascular and macrovascular disease. The aim of this study was therefore to investigate the independent eﬀects of hyperglycaemia, hypoglycaemia, and glucose variability on microvascular and macrovascu- lar disease in type 2 diabetes. Urinary albumin-creatinine served as a marker of microvascular disease. Ankle-brachial index, carotid intima-media thickness, and documented car- diovascular disease served as markers of macrovascular disease. 72-hour continuous glucose monitoring was used to assess hypoglycaemia, hyperglycaemia, and glucose var- iability. HBA1c and fructosamine served as additional gly- caemic indexes. 2.2. Continuous Glucose Monitoring. Seventy-two-hour sub- cutaneous continuous glucose monitoring (CGM) was per- formed in all study participants on an ambulatory basis using the iPro2 continuous glucose-monitoring system (Medtronic MiniMed, Northridge, CA, USA). 1. Introduction Participants were advised to pursue their normal daily living without alterations in their usual diet, exercise, and medications taken. Data from the iPro2 were uploaded online via the CareLink iPro software. The following parameters were extracted for each patient: mean blood glucose (MBG), standard deviation of blood glucose (SD), the highest and lowest blood glucose values, the number of high and low excursions, the area under the curve when the blood glucose was higher than 7.8 mmol/L (AUC >7.8), the area under the curve when the blood glucose was lower than 3.9 mmol/L (AUC <3.9), the duration (%) when the blood glucose was higher than 7.8 mmol/L, the duration (%) when the blood glucose was lower than 3.9 mmol/L, and the duration (%) when the blood glucose was between 3.9 mmol/L and 7.8 mmol/L. 3. Results 3.1. Characteristics of Study Population. The study popula- tion comprised 121 T2DM subjects of Caucasian origin. The baseline characteristics are outlined in Table 1, whilst continuous glucose-monitoring data are shown in Table 2. Patients had reasonably well-controlled diabetes with a median HbA1c of 6.8% (45 mmol/mol), a median fasting plasma glucose of 7.08 mmol/L, and a mean blood glucose (MBG) during CGM of 7.9 mmol/L. Hyperglycaemic epi- sodes were more prevalent than hypoglycaemic episodes as shown by the number of high and low excursions, the AUC>7.8 mmol/L and AUC< 3.9 mmol/L, as well as dura- tion of BG >7.8 mmol/L, between 3.9 and 7.8 mmol/L, and <3.9 mmol/L. However, none of the study participants reported hypoglycaemic episodes, indicating that all these episodes were subclinical. There were no patients with missing data. 2.4. Clinical Deﬁnitions. eGFR was deﬁned as being low if it was <60mL/min/1.73m2. PAD was deﬁned as an ABI of ≤0.9. The mean arterial pressure was the average arterial pressure during a single cardiac cycle and pulse pressure the diﬀerence between the systolic and diastolic blood pres- sure readings. Macrovascular disease was deﬁned as the pres- ence of one or more of the following: history of ischaemic heart disease (IHD) or cerebrovascular accident (CVA), ABI <0.9, or abnormal CIMT. 3.2. Predictors of Macrovascular Disease. Seventy-two sub- jects exhibited macrovascular disease. Univariate followed by multivariate analysis was performed to identify predictors of macrovascular disease in the study population. The results of univariate analysis are shown in Tables 3–5. In univariate analysis, the following variables had a p value < 0.1 and were thus included in the multivariate model: body surface area (BSA), smoking pack years, HDL cholesterol, triglyc- erides, ESR, lowest blood glucose (BG) value, AUC for BG <3.9 mmol/L, and duration of BG< 3.9 mmol/L. In multi- variate logistic regression analysis, BSA (OR 18.88 (95% CI 2.20–156.69), p = 0 006) and duration of BG< 3.9 mmol/L (OR 1.12 (95% CI 1.014–1.228), p = 0 024) were shown to be independentpredictorsofmacrovasculardisease.Therelation between presence of cardiovascular disease and duration of BG <3.9 mmol/L is shown in Figure 1. 2.5. Statistical Analysis. All data were analysed using SPSS ver- sion 24.0 for Windows. Results are presented as mean ± stan- dard deviation (SD) or median (interquartile range (IQR)). Continuous variables were checked for normality of distri- bution using the Kolmogorov-Smirnov method. 2. Methods The study was cross-sectional in nature; data collection was performed over a one-year period, from April 2015 to April 2016. Patients were randomly selected from the list of outpa- tients attending the Diabetes Centre, Mater Dei Hospital, Malta, during the year 2014. The inclusion criteria were sub- jects who were diagnosed with type 2 DM according to World Health Organization criteria within the last ten years and were on stable antiglycaemic treatment. Patients suﬀer- ing from dementia or mental illnesses with the consequent inability to give an informed consent with regard to partici- pation in the study were excluded from the study. Other exclusion criteria included recent admission to hospital with a diabetes-related complication such as hyperglycaemic hyperosmolar ketoacidosis, liver failure, or kidney failure, as well as any other factors that result in glycaemic ﬂuctua- tions. The study protocol was approved by the University 2.3. Carotid Intima-Media Thickness Measurement. Carotid intima-media thickness (CIMT) was assessed in both com- mon carotid arteries in each participant using the Esaote Journal of Diabetes Research 3 Quality intima media thickness (QIMT®). The common carotid artery was utilised since, according to the Mannheim consensus, it increases the accuracy and reproducibility of the measurements obtained. The Esaote QIMT utilises a radio frequency signal to enable measurement of the CIMT with high spatial resolution. The QIMT tool automatically provides accurate measurements of all the parameters which are independent from both the investigator and device set- tings. Results were categorised as normal or abnormal using age-speciﬁc cut-oﬀs, which have been validated in large worldwide databases [19]. Furthermore, all study partici- pants were screened for the presence of carotid plaques in view that the combination of plaque area and thickness have greater value in predicting CVD compared to the thickness alone [17]. Patients with an increased CIMT as assessed by QIMT and/or presence of carotid plaque were categorised as having an abnormal CIMT. 2.6. Role of Funding Source. The study sponsor had no role in study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to sub- mit the paper for publication. The authors had full access to the data. 3. Results Generalised linear regression model revealed AUC for BG> 7.8 mmol/L (β = 15 83, p = 0 005) to be the sole independent predictor of albuminuria. Table 1: Continued. Patient characteristics (n = 121) Values Non-HDL cholesterol (mmol/L)† 2.8 (2.26–3.82) Triglycerides (mmol/L)† 1.36 (0.97–1.7) Alkaline phosphatase (mmol/L)† 69 (55–83.5) Alanine transaminase (mmol/L)† 23 (18–32.5) Uric acid (μmol/L)∗ 313.05 ± 79.98 Erythrocyte sedimentation rate (mm 1st hr)† 10.5 (6–18) Albumin-creatinine ratio (mg/mmol)† 7.08 (1–23.34) Fasting plasma glucose (mmol/L)† 7.08 (6.11–8.18) Fructosamine (μmol/L)† 278 (257–303) Glycated haemoglobin (%)† 6.8 (6.3–7.6) Values are expressed as number (%) of patients, ∗mean ± SD, or †median (IQR). Table 2: Baseline continuous glucose-monitoring ﬁndings of study population. Patient characteristics (n = 121) Values Highest value (mmol/L) 13.2 (11.3–15.5) Lowest value (mmol/L) 3.95 (2.93–5.1) Mean blood glucose (mmol/L) 7.35 (6.63–8.65) Standard deviation (mmol/L) 1.9 (1.5–2.3) Number of high excursions 8 (5–10) Number of low excursions 0 (0–2) AUC above 7.8 mmol/L 0.63 (0.23–1.43) AUC below 3.9 mmol/L 0 (0–0.02) Duration during 72 hr period with BG above 7.8 mmol/L (%) 37 (19.25–60.75) Duration during 72 hr period with BG within 3.9–7.8 mmol/L (%) 60 (37.5–77) Duration during 72 hr period with BG below 3.9 mmol/L (%) 0 (0–4) Values are expressed as median (IQR). AUC: area under curve. Table 1: Continued. Patient characteristics (n = 121) Values Non-HDL cholesterol (mmol/L)† 2.8 (2.26–3.82) Triglycerides (mmol/L)† 1.36 (0.97–1.7) Alkaline phosphatase (mmol/L)† 69 (55–83.5) Alanine transaminase (mmol/L)† 23 (18–32.5) Uric acid (μmol/L)∗ 313.05 ± 79.98 Erythrocyte sedimentation rate (mm 1st hr)† 10.5 (6–18) Albumin-creatinine ratio (mg/mmol)† 7.08 (1–23.34) Fasting plasma glucose (mmol/L)† 7.08 (6.11–8.18) Fructosamine (μmol/L)† 278 (257–303) Glycated haemoglobin (%)† 6.8 (6.3–7.6) Values are expressed as number (%) of patients, ∗mean ± SD, or †median (IQR). Table 1: Baseline demographic and clinical ﬁndings of study population. Table 1: Continued. 3. Results BMI, WI, MBG, SD, AUC for BG >7.8 mmol/L, duration of BG> 7.8 mmol/L, duration of BG between 3.9 and 7.8 mmol/L, and highest BG value recorded. Generalised linear regression model revealed AUC for BG> 7.8 mmol/L (β = 15 83, p = 0 005) to be the sole independent predictor of albuminuria. 3. Results Patient characteristics (n = 121) Values Age (years)† 64 (57–68) Male : female (n (%)) 89 (73.6) : 32 (26.4) Diabetes duration (years)† 3 (2–5) Smoking (current/ex/ nonsmokers) (n (%)) 22 (18.2)/41 (33.9)/58 (47.9 Hypertension (n (%)) 80 (66.1) Hyperlipidaemia (n (%)) 87 (71.9) Ischaemic heart disease (n (%)) 15 (12.4) Peripheral arterial disease (n (%)) 7 (5.8) Cerebrovascular accident/transient ischaemic attack (n (%)) 3 (2.5) Abnormal carotid intima-media thickness (n (%)) 64 (52.9) Macrovascular disease (n (%)) 72 (59.5) Chronic kidney disease (n (%)) 9 (7.4) On metformin (n (%)) 109 (90.1) On sulphonylurea (n (%)) 36 (29.8) On gliptins (n (%)) 6 (5) On insulin (n (%)) 6 (5) On angiotensin-converting enzyme inhibitor/on angiotensin receptor blocker (n (%)) 77 (63.6) On β-blocker (n (%)) 22 (18.2) On calcium channel blocker (n (%)) 23 (19) On diuretic (n (%)) 33 (23.7) On aspirin (n (%)) 38 (31.4) On statin (n (%)) 81 (66.9) On ﬁbrate (n (%)) 7 (5.8) On allopurinol (n (%)) 5 (4.1) Body mass index (kg/m2)† 31.22 (27.94–34.53) Body surface area (m2)∗ 1.89 ± 0.21 Waist index∗ 1.19 ± 0.14 Pulse rate (bpm)† 68 (61.5–78.5) Mean systolic pressure (mmHg)∗ 144.19 ± 17.82 Mean diastolic pressure (mmHg)∗ 84.60 ± 8.76 Pulse pressure (mmHg)∗ 61.53 ± 13.66 Mean arterial pressure (mmHg)† 107 (93–113) White cell count (×109/L)† 7.16 (6.26–8.69) Haemoglobin (g/dL)† 14.35 (13.33–15.18) Platelet count (×109/L)† 240.5 (206–284.75) Red blood cell distribution width (%)† 13.1 (12.53–13.7) Mean platelet volume (fL)† 10.9 (10.2–11.6) Estimated glomerular ﬁltration rate (mL/min/1.73 m2)∗ 91.22 ± 24.61 Total cholesterol (mmol/L)† 4.12 (3.55–5.15) HDL cholesterol (mmol/L)† 1.24 (1.06–1.51) LDL cholesterol (mmol/L)† 2.19 (1.67–3.19) Table 2: Baseline continuous glucose-monitoring ﬁndings of study population. Table 2: Baseline continuous glucose-monitoring ﬁndings of study population. Patient characteristics (n = 121) Values Highest value (mmol/L) 13.2 (11.3–15.5) Lowest value (mmol/L) 3.95 (2.93–5.1) Mean blood glucose (mmol/L) 7.35 (6.63–8.65) Standard deviation (mmol/L) 1.9 (1.5–2.3) Number of high excursions 8 (5–10) Number of low excursions 0 (0–2) AUC above 7.8 mmol/L 0.63 (0.23–1.43) AUC below 3.9 mmol/L 0 (0–0.02) Duration during 72 hr period with BG above 7.8 mmol/L (%) 37 (19.25–60.75) Duration during 72 hr period with BG within 3.9–7.8 mmol/L (%) 60 (37.5–77) Duration during 72 hr period with BG below 3.9 mmol/L (%) 0 (0–4) Values are expressed as median (IQR). AUC: area under curve. 3. Results Compari- sons of continuous variables between two groups were made using independent sample t-test for normally distributed var- iables.Fornonparametricvariables,theMann–WhitneyUtest was used for comparison of 2 groups. Categorical variables were compared using the χ2 test. The Pearson test was used to test correlation of normally distributed variables, and the Spearman test was used for comparison of non-normally dis- tributed variables. Univariate followed by multivariate analyses (logistic regression analyses) were performed to identify independent determinants of both the occurrence of macrovascular disease and abnormal CIMT in the study population. Variables were entered into the regression model if their p value was <0.1 in univariate analysis. Predictors were removed from the model if their p value exceeded 0.05. 3.3. Predictors of Abnormal Carotid Intima-Media Thickness. In the study population, 64 subjects had abnormal CIMT (composite of increased CIMT and presence of carotid plaque). In univariate analysis, the following variables had a p value <0.1: age, DM duration, smoking pack years, BSA, white cell count, eGFR, ESR, and duration of BG<3.9mmol/L. These variables were consequently included in the multi- variate model. With regard to ACR, univariate followed by multi- variate analysis was again performed with variables with a p value <0.1 in univariate analysis being included in the mul- tivariate model; however, a generalised linear model was per- formed as multivariate analysis in view that ACR exhibited a gamma distribution (a right-skewed distribution). All tests were two-sided, and a p value of <0.05 was considered to be statistically signiﬁcant. Binary logistic regression analysis revealed that BSA (OR 12.6 (95% CI 1.70–93.54), p = 0 013) and duration of BGM <3.9 mmol/L (OR 1.09 (95% CI 1.003–1.187), p = 0 041) were independent predictors of abnormal CIMT. The relation between abnormal CIMT and duration of BG <3.9 mmol/L is shown in Figure 1. Sample size was determined so as to have statistical power of 90% to detect a moderate eﬀect size (f 2 = 0 15) at α = 0 05. 3.4. Predictors of Albuminuria. The following had p value < 0.1 in univariate analysis: low eGFR (<60mL/min/1.73m2), white cell count, HDL cholesterol, triglycerides, uric acid, Journal of Diabetes Research Journal of Diabetes Research 4 BMI, WI, MBG, SD, AUC for BG >7.8 mmol/L, duration of BG> 7.8 mmol/L, duration of BG between 3.9 and 7.8 mmol/L, and highest BG value recorded. MAP (mmHg)† Values are expressed as number (%) of patients, ∗mean ± SD, or †median (IQR). ACEI: angiotensin-converting enzyme inhibitor; ARB: angiotensin receptor blocker; BMI: body mass index; BSA: body surface area; CCB: calcium channel blocker; CKD: chronic kidney disease; CVD: cardiovascular disease; DM: diabetes mellitus; DBP: diastolic blood pressure; MAP: mean arterial pressure; SBP: systolic blood pressure. Values are expressed as number (%) of patients, ∗mean ± SD, or †median (IQR). ACEI: angiotensin-converting enzyme inhibitor; ARB: angiotensin receptor blocker; BMI: body mass index; BSA: body surface area; CCB: calcium channel blocker; CKD: chronic kidney disease; CVD: cardiovascular disease; DM: diabetes mellitus; DBP: diastolic blood pressure; MAP: mean arterial pressure; SBP: systolic blood pressure. associated with ACR. These observations may be related to the short median duration of diabetes in our patients. Our ﬁndings are consistent with clinical trial data which have shown that lowering blood glucose for a few years is eﬀective in reducing microvascular but not macrovascular disease [2, 3]. In the UKPDS, it took 10 years of intensive control and a further 10 years of follow-up to demonstrate a beneﬁcial eﬀect of lowering blood glucose on macrovascu- lar outcomes [20]. Another reason could be the relatively good glycaemic control in our cohort. These ﬁndings are consistent with those of the Diabetes Control and Complications Trial (DCCT), which reported that the rate of severe hypoglycaemic episodes in subjects with type 1 diabetes was associated with increased coro- nary calcium score in those with a HbA1c below 7.5% (58 mmol/L) [18]. It is, however, possible that severe hypo- glycaemia was acting as a marker for increased glucose ﬂuctuations. Since we performed CGM, we can exclude this possibility in our study. Furthermore, whilst the DCCT data showed an association of severe hypoglycaemia with increased coronary calcium score in type 1 diabetes, the present study shows a relationship of mild asymptomatic hypoglycaemia, as detected by CGM, to atherosclerosis in type 2 diabetes. These ﬁndings are consistent with those of the Diabetes Control and Complications Trial (DCCT), which reported that the rate of severe hypoglycaemic episodes in subjects with type 1 diabetes was associated with increased coro- nary calcium score in those with a HbA1c below 7.5% (58 mmol/L) [18]. It is, however, possible that severe hypo- glycaemia was acting as a marker for increased glucose ﬂuctuations. Since we performed CGM, we can exclude this possibility in our study. 4. Discussion The present study found that the duration of blood glu- cose <3.9 mmol/L was signiﬁcantly and independently asso- ciated with abnormal CIMT and with the composite of abnormal CIMT, ABI, coronary artery disease, and cerebro- vascular disease in subjects with type 2 diabetes. This sug- gests that hypoglycaemia may predispose to atherosclerosis. Journal of Diabetes Research 5 Table 3: Demographic and clinical ﬁndings of subjects with and without macrovascular disease. Table 3: Demographic and clinical ﬁndings of subjects with and without macrovascular disease. Variable No macrovascular disease (n = 49) Macrovascular disease (n = 72) p value Age (years)† 66 (62–69) 61 (54.25–67) 0.001 Male : female (n (%)) 34 (69.4) : 15 (30.6) 55 (76.4) : 17 (23.6) 0.41 Diabetes duration (years)† 3.5 (2–5) 3 (2–4) 0.23 Smoking (current/ex/nonsmokers) (n (%)) 4 (8.1)/19 (38.8)/26 (53.1) 18 (25.0)/22 (30.6)/32 (44.4) 0.06 Hypertension (n (%)) 29 (59.18) 51 (70.83) 0.24 Hyperlipidaemia (n (%)) 36 (73.47) 51 (70.83) 0.84 CKD (n (%)) 2 (4.08) 7 (9.72) 0.31 Metformin (n (%)) 44 (89.80) 65 (90.28) 1.00 Sulphonylurea (n (%)) 14 (28.57) 50 (69.44) 0.84 Gliptins (n (%)) 4 (8.16) 2 (2.77) 0.22 Insulin (n (%)) 1 (2.04) 5 (6.94) 0.40 ACEI/ARB (n (%)) 29 (59.18) 48 (66.67) 0.44 β-blocker (n (%)) 7 (14.29) 15 (20.83) 0.47 CCB (n (%)) 11 (22.45) 12 (16.67) 0.48 Diuretic (n (%)) 14 (28.57) 19 (26.39) 0.84 Aspirin (n (%)) 9 (18.36) 29 (40.28) 0.02 Statin (n (%)) 29 (59.18) 52 (72.22) 0.17 Fibrate (n (%)) 6 (12.24) 1 (1.39) 0.02 Allopurinol (n (%)) 2 (4.08) 3 (4.17) 1.00 BMI (kg/m2)† 31.22 (27.67–33.97) 31.31 (27.9–35.19) 0.35 BSA (m2)∗ 1.83 ± 0.20 1.93 ± 0.21 0.007 Waist index∗ 1.17 ± 0.15 1.20 ± 0.13 0.22 Pulse rate (bpm)† 65 (61–80.25) 71 (62–78) 0.84 Mean SBP (mmHg)∗ 146 ± 18.28 142.96 ± 17.54 0.36 Mean DBP (mmHg)∗ 85.66 ± 7.34 83.88 ± 9.58 0.25 Pulse pressure (mmHg)∗ 62.18 ± 15.6 61.07 ± 12.22 0.68 MAP (mmHg)† 106 (97.5–112.5) 90.75 (107.5–113) 0.77 Values are expressed as number (%) of patients, ∗mean ± SD, or †median (IQR). ACEI: angiotensin-converting enzyme inhibitor; ARB: angiotensin receptor blocker; BMI: body mass index; BSA: body surface area; CCB: calcium channel blocker; CKD: chronic kidney disease; CVD: cardiovascular disease; DM: diabetes mellitus; DBP: diastolic blood pressure; MAP: mean arterial pressure; SBP: systolic blood pressure. MAP (mmHg)† Table 4: Laboratory ﬁndings of subjects with and without macrovascular disease. Values are expressed as ∗mean ± SD or †median (IQR). Abbreviations: ALP: alkaline phosphatase; ALT: alanine transaminase; eGFR: estimated glomerular ﬁltration rate; ESR: erythrocyte sedimentation rate; FPG: fasting plasma glucose; HbA1c: glycated haemoglobin; MPV: mean platelet volume; RDW: red blood cell distribution width. Table 5: Continuous glucose-monitoring ﬁndings of subjects with and without macrovascular disease. Table 5: Continuous glucose-monitoring ﬁndings of subjects with and without macrovascular disease. us glucose-monitoring ﬁndings of subjects with and without macrovascular disease. Table 5: Continuous glucose-monitoring ﬁndings of subjects with and without macrovascular disease. Variable No macrovascular disease (n = 49) Macrovascular disease (n = 72) p value Highest value (mmol/L)† 13.4 (11.3–15.85) 13.0 (11.3–15) 0.35 Lowest value (mmol/L)† 4.4 (3.2–5.25) 3.7 (2.8–5) 0.09 Mean blood glucose (mmol/L)† 7.6 (6.85–9.0) 7.3 (6.6–8.4) 0.3 Standard deviation (mmol/L)† 3.0 (1.3–2.3) 1.8 (1.5–2.4) 0.9 Number of high excursions† 8 (6–10) 8 (5–10) 0.49 Number of low excursions† 0 (0–2) 1 (0–2) 0.10 AUC above 7.8 mmol/L† 0.64 (0.24–1.76) 0.57 (0.21–1.35) 0.39 AUC below 3.9 mmol/L† 0 (0–0.01) 0 (0–0.03) 0.05 Duration during 72 hr period with BG above 7.8 mmol/L (%)† 37 (18.5–66.5) 35 (20–58) 0.39 Duration during 72 hr period with BG within 3.9–7.8 mmol/L (%)† 58 (31.5–79) 60 (41–77) 0.63 Duration during 72 hr period with BG below 3.9 mmol/L (%)† 0 (0-1) 1 (0–5) 0.02 Values are expressed as number (%) of patients or †median (IQR). AUC: area under curve. Values are expressed as number (%) of patients or †median (IQR). AUC: area under curve. interleukin-6 [26, 28]. Many of these eﬀects may extend to beyond the hypoglycaemic period [26]. Furthermore, Fadini and colleagues have recently reported that hypoglycaemia may impair endothelial progenitor cell response [29]. Inter- estingly, Peña and colleagues have reported that hypoglycae- mia, but not glucose variability, is associated with endothelial dysfunction in children with type 1 diabetes [30]. as sulphonylureas [22, 23]and insulin [24] which are prone to cause hypoglycaemia have also been associated with increased mortality and cardiovascular events by some authors. Hypoglycaemia can promote atherogenesis in a number of ways. MAP (mmHg)† Furthermore, whilst the DCCT data showed an association of severe hypoglycaemia with increased coronary calcium score in type 1 diabetes, the present study shows a relationship of mild asymptomatic hypoglycaemia, as detected by CGM, to atherosclerosis in type 2 diabetes. Our data suggest that hypoglycaemia may have a more rapid eﬀect in promoting atherosclerosis than mild hypergly- caemia or glucose ﬂuctuations. They may help explain the relationship between hypoglycaemia and adverse cardio- vascular outcomes even in the short term and why these deleterious eﬀects extend well beyond the period of hypo- glycaemia itself. They may also explain why too aggressive lowering of blood glucose in the Action to Control Car- diovascular Risk in Diabetes (ACCORD) trial was associ- ated with increased mortality [21] and why agents such We did not ﬁnd signiﬁcant associations of any of the hyperglycaemic indexes (including HbA1c, fructosamine, AUC > 7.8 mmol/L, and duration >7.8 mmol/L) or of glucose ﬂuctuations (as assessed by standard deviation of blood glu- cose during CGM) with macrovascular disease. However, duration of blood glucose >7.8 mmol/L was independently Journal of Diabetes Research 6 Table 4: Laboratory ﬁndings of subjects with and without macrovascular disease. Variable No macrovascular disease (n = 49) Macrovascular disease (n = 72) p value White cell count (×109/L)† 7.09 (5.88–7.87) 7.29 (6.42–9.24) 0.13 Haemoglobin (g/dL)† 14.2 (13.3–15.1) 14.4 (13.6–15.2) 0.48 Platelet count (×109/L)† 242 (204.5–283.5) 239 (206–287) 0.78 RDW (%)† 13 (12.5–13.45) 13.1 (12.6–13.8) 0.13 MPV (fL)† 10.95 (10.23–11.5) 10.9 (10.1–11.6) 0.53 eGFR (mL/min/1.72 m2)∗ 87.76 ± 22.21 93.58 ± 26.0 0.19 Total cholesterol (mmol/L)† 4.14 (3.65–5.25) 4.1 (3.53–5.11) 0.80 HDL cholesterol (mmol/L)† 1.32 (1.1–1.58) 1.24 (1.04–1.46) 0.07 LDL cholesterol (mmol/L)† 2.3 (1.65–3.36) 2.18 (1.7–3.13) 0.81 Non-HDL cholesterol (mmol/L)† 2.74 (2.18–3.86) 2.83 (2.29–3.78) 0.67 Triglycerides (mmol/L)† 1.13 (0.91–1.64) 1.47 (1.14–1.85) 0.03 ALP (mmol/L)† 66 (52–93) 69 (59.25–80) 0.89 ALT (mmol/L)† 22 (17.5–32) 24 (18–32.75) 0.6 Uric acid (μmol/L)∗ 319.56 ± 83.19 308.65 ± 78.03 0.47 ESR (mm 1st hr)† 13 (7–23) 9 (5–17) 0.08 FPG (mmol/L)† 7.14 (6.02–8.53) 7.08 (6.23–8.05) 0.55 Fructosamine (μmol/L)† 281 (259.25–305) 277 (256–303.5) 0.77 HbA1c (%)† 7.0 (6.3–8.1) 6.7 (6.3–7.4) 0.23 HbA1c (mmol/L)† 53 (45–65) 50 (45–57) 0.23 Values are expressed as ∗mean ± SD or †median (IQR). Abbreviations: ALP: alkaline phosphatase; ALT: alanine transaminase; eGFR: estimated glomerular ﬁltration rate; ESR: erythrocyte sedimentation rate; FPG: fasting plasma glucose; HbA1c: glycated haemoglobin; MPV: mean platelet volume; RDW: red blood cell distribution width. Disclosure The study was presented in abstract form at the EuroPrevent meeting held in Ljubljana, Slovenia, April 19–21, 2017. Conflicts of Interest Figure 1: Box-and-whisker plot demonstrating relation of duration of glucose < 3.9 mmol/L (expressed as percentage of the entire 72 hr period) and macrovascular disease and carotid intima-media thickness. ∗denoting outliers. The authors have no conﬂict of interest to declare. Data Availability The data used to support the ﬁndings of this study are avail- able from the corresponding author upon request. MAP (mmHg)† Acute hypoglycaemia has been shown to result in decreased nitric oxide bioavailability, increased oxidative stress [25], platelet activation [26], and release of proinﬂam- matory [27], proatherogenic, and prothrombotic cytokines such as PAI-1, VEGF, vascular adhesion molecules, and Body surface area was also found to be independently associated with atherosclerotic markers. This is probably related to insulin resistance and/or hyperinsulinaemia. Journal of Diabetes Research 7 30 25 20 15 10 5 0 Absent Present Duration with BG < 3.9 mmol/L (%) Macrovascular disease Duration with BG < 3.9 mmol/L (%) 30 25 20 15 10 5 0 Carotid intima-media thickness Normal Abnormal p = 0.041 p = 0.024 Figure 1: Box-and-whisker plot demonstrating relation of duration of glucose < 3.9 mmol/L (expressed as percentage of the entire 72 hr period) and macrovascular disease and carotid intima-media thickness. ∗denoting outliers. patients are less likely to have variation in their glycaemic parameters over the course of their disease. Thirdly, we per- formed blind, rather than real-time, CGM so that patients did not alter their behaviour as result of the glucose readings. Additionally, patients were instructed not to change their usual routines. In this way, the CGM data are more likely to be representative of the patients’ usual glycaemic proﬁle. Furthermore, many glycaemic indices such as HbA1c [32] and hypoglycaemia [11] have been shown to exhibit signiﬁ- cant temporal tracking. Another limitation is that in view of the cross-sectional nature of our study, we cannot prove causal relationships. It is possible that those with macrovas- cular disease were treated more aggressively, hence causing more hypoglycaemia. However, this would not explain the observed relationship with CIMT, which is a subclinical marker of atherosclerosis. 30 25 20 15 10 5 0 Absent Present Duration with BG < 3.9 mmol/L (%) Macrovascular disease p = 0.024 In conclusion, the study suggests that mild asymptomatic hypoglycaemia in subjects with type 2 diabetes is associated with atherosclerotic disease. This cannot be explained by an association of hypoglycaemia with hyperglycaemia or glucose ﬂuctuations. Hyperglycaemic load, as measured by AUC for BG >7.8 mmol/L, was associated with microalbuminuria. Duration with BG < 3.9 mmol/L (%) 30 25 20 15 10 5 0 Carotid intima-media thickness Normal Abnormal p = 0.041 Acknowledgments The study was sponsored by the Dean’s Funds of the Univer- sity of Malta Medical School. The patients with macrovascular disease were on average younger than those without macrovascular disease. This could be due to the cross-sectional nature of our study. Macrovascular disease shortens life expectancy resulting in a lower average age of subjects with macrovascular disease compared to subjects without. Furthermore, macrovascular disease was associated with hypoglycaemia, which is also known to decrease survival [21]. Duration of diabetes was similar in both groups, meaning that those with macrovascu- lar disease had younger onset type 2 diabetes, which might be associated with higher mortality [31]. References F. 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Osonoi et al., “Repetitive hypogly- cemia increases circulating adrenaline level with resultant worsening of intimal thickening after vascular injury in male Goto-Kakizaki rat carotid artery,” Endocrinology, vol. 155, no. 6, pp. 2244–2253, 2014. [24] S. E. Holden, S. Jenkins-Jones, C. L. Morgan, G. Schernthaner, and C. J. Currie, “Glucose-lowering with exogenous insulin monotherapy in type 2 diabetes: dose association with all- cause mortality, cardiovascular events and cancer,” Diabetes, Obesity and Metabolism, vol. 17, no. 4, pp. 350–362, 2015. [13] W. L. Jin, K. Azuma, T. Mita et al., “Repetitive hypoglycaemia increases serum adrenaline and induces monocyte adhesion to the endothelium in rat thoracic aorta,” Diabetologia, vol. 54, no. 7, pp. 1921–1929, 2011. [25] J. Wang, A. Alexanian, R. Ying et al., “Acute exposure to low glucose rapidly induces endothelial dysfunction and mitochondrial oxidative stress: role for AMP kinase,” Arterio- sclerosis, Thrombosis, and Vascular Biology, vol. 32, no. 3, pp. 712–720, 2012. [14] M. Giménez, R. Gilabert, J. Monteagudo et al., “Repeated epi- sodes of hypoglycemia as a potential aggravating factor for preclinical atherosclerosis in subjects with type 1 diabetes,” Diabetes Care, vol. 34, no. 1, pp. 198–203, 2011. [26] N. G. Joy, D. B. Tate, L. M. Younk, and S. N. Davis, “Eﬀects of acute and antecedent hypoglycemia on endothelial function and markers of atherothrombotic balance in healthy humans,” Diabetes, vol. 64, no. 7, pp. 2571–2580, 2015. [15] J. G. Timmons, S. G. Cunningham, C. A. R. Sainsbury, and G. C. Jones, “Inpatient glycemic variability and long-term mortality in hospitalized patients with type 2 diabetes,” Journal of Diabetes and its Complications, vol. 31, no. 2, pp. 479–482, 2017. [27] L. Razavi Nematollahi, A. E. Kitabchi, F. B. Stentz et al., “Pro- inﬂammatory cytokines in response to insulin-induced hypo- glycemic stress in healthy subjects,” Metabolism, vol. 58, no. 4, pp. 443–448, 2009. [16] N. Wu, H. Shen, H. Liu, Y. Wang, Y. Bai, and P. Han, “Acute blood glucose ﬂuctuation enhances rat aorta endothelial cell apoptosis, oxidative stress and pro-inﬂammatory cytokine expression in vivo,” Cardiovascular Diabetology, vol. 15, no. 1, p. 109, 2016. [28] B. Kiec-Wilk, B. Matejko, U. [31] S. Zoungas, M. Woodward, Q. Li et al., “Impact of age, age at diagnosis and duration of diabetes on the risk of macrovascu- lar and microvascular complications and death in type 2 diabe- tes,” Diabetologia, vol. 57, no. 12, pp. 2465–2474, 2014. children with type 1 diabetes,” Diabetes Technology & Thera- peutics, vol. 14, no. 6, pp. 457–462, 2012. References Razny et al., “Hypoglycemic epi- sodes are associated with inﬂammatory status in patients with type 1 diabetes mellitus,” Atherosclerosis, vol. 251, pp. 334– 338, 2016. [29] G. P. Fadini, M. Albiero, S. Vigili de Kreutzenberg, and A. Avogaro, “Hypoglycemia aﬀects the changes in endothelial progenitor cell levels during insulin therapy in type 2 diabetic patients,” Journal of Endocrinological Investigation, vol. 38, no. 7, pp. 733–738, 2015. [17] M. F. Piepoli, A. W. Hoes, S. Agewall et al., “2016 European guidelines on cardiovascular disease prevention in clinical practice: the sixth joint task force of the European Society of Cardiology and other societies on cardiovascular disease pre- vention in clinical practice (constituted by representatives of 10 societies and by invited experts). Developed with the special contribution of the European Association for Cardiovascular [30] A. S. Peña, J. J. Couper, J. Harrington et al., “Hypoglycemia, but not glucose variability, relates to vascular function in Journal of Diabetes Research 9 9 children with type 1 diabetes,” Diabetes Technology & Thera- peutics, vol. 14, no. 6, pp. 457–462, 2012. [31] S. Zoungas, M. Woodward, Q. Li et al., “Impact of age, age at diagnosis and duration of diabetes on the risk of macrovascu- lar and microvascular complications and death in type 2 diabe- tes,” Diabetologia, vol. 57, no. 12, pp. 2465–2474, 2014. [32] J. A. Edge, T. James, and B. Shine, “Persistent individual track- ing within overall improvement in HbA1c in a UK paediatric diabetes clinic over 15 years,” Diabetic Medicine, vol. 27, no. 11, pp. 1284–1288, 2010. References Stem Cells International Hindawi www.hindawi.com Volume 2018 Hindawi www.hindawi.com Volume 2018 MEDIATORS INFLAMMATION of Endocrinology International Journal of Hindawi www.hindawi.com Volume 2018 Hindawi www.hindawi.com Volume 2018 Disease Markers Hindawi www.hindawi.com Volume 2018 BioMed Research International Oncology Journal of Hindawi www.hindawi.com Volume 2013 Hindawi www.hindawi.com Volume 2018 Oxidative Medicine and Cellular Longevity Hindawi www.hindawi.com Volume 2018 PPAR Research Hindawi Publishing Corporation http://www.hindawi.com Volume 2013 Hindawi www.hindawi.com The Scientific World Journal Volume 2018 Immunology Research Hindawi www.hindawi.com Volume 2018 Journal of Obesity Journal of Hindawi www.hindawi.com Volume 2018 Hindawi www.hindawi.com Volume 2018 Computational and Mathematical Methods in Medicine Hindawi www.hindawi.com Volume 2018 Behavioural Neurology Ophthalmology Journal of Hindawi www.hindawi.com Volume 2018 Diabetes Research Journal of Hindawi www.hindawi.com Volume 2018 Hindawi www.hindawi.com Volume 2018 Research and Treatment AIDS Hindawi www.hindawi.com Volume 2018 Gastroenterology Research and Practice Hindawi www.hindawi.com Volume 2018 Parkinson’s Disease Evidence-Based Complementary and Alternative Medicine Volume 2018 Hindawi www.hindawi.com Submit your manuscripts at www.hindawi.com Hindawi www.hindawi.com Volume 2018 MEDIATORS INFLAMMATION of Endocrinology International Journal of Hindawi www.hindawi.com Volume 2018 Hindawi www.hindawi.com Volume 2018 Disease Markers Hindawi Publishing Corporation http://www.hindawi.com Volume 2013 Hindawi www.hindawi.com The Scientific World Journal Volume 2018 Immunology Research Hindawi www.hindawi.com Volume 2018 Journal of Diabetes Research Journal of Hindawi www.hindawi.com Volume 2018 Hindawi www.hindawi.com Volume 2018 Gastroenterology Research and Practice Submit your manuscripts at www.hindawi.com The Scientific World Journal Hindawi Publishing Corporation Hindawi The Scientific World Journal Diabetes Research Journal of Hindawi Endocrinology International Journal of Hindawi www.hindawi.com Volume 2018 Submit your manuscripts at www.hindawi.com Hindawi www.hindawi.com Volume 2018 BioMed Research International Submit your manuscripts at www.hindawi.com Stem Cells International Hindawi www.hindawi.com Volume 2018 Oncology Journal of Hindawi www.hindawi.com Volume 2013 Hindawi www.hindawi.com Volume 2018 Oxidative Medicine and Cellular Longevity Obesity Journal of Hindawi www.hindawi.com Volume 2018 Hindawi www.hindawi.com Volume 2018 Computational and Mathematical Methods in Medicine Hindawi www.hindawi.com Volume 2018 Behavioural Neurology Ophthalmology Journal of Hindawi www.hindawi.com Volume 2018 Hindawi www.hindawi.com Volume 2018 Research and Treatment AIDS Hindawi www.hindawi.com Volume 2018 Parkinson’s Disease Evidence-Based Complementary and Alternative Medicine Volume 2018 Hindawi www.hindawi.com Stem Cells International Hindawi www.hindawi.com Volume 2018 Oncology Journal of Hindawi www.hindawi.com Volume 2013 Hindawi www.hindawi.com Volume 2018 Oxidative Medicine and Cellular Longevity Obesity Journal of Hindawi www.hindawi.com Volume 2018 Hindawi www.hindawi.com Volume 2018 Computational and Mathematical Methods in Medicine Hindawi www.hindawi.com Volume 2018 Behavioural Neurology Ophthalmology Journal of Hindawi www.hindawi.com Volume 2018 Hindawi www.hindawi.com Volume 2018 Research and Treatment AIDS Hindawi www.hindawi.com Volume 2018 Parkinson’s Disease Evidence-Based Complementary and Alternative Medicine Volume 2018 Hindawi www.hindawi.com Obesity Journal of Hindawi www.hindawi.com Volume 2018

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